Method for preparing highly concentrated liquid drug substance

Abstract

Provided herein is a method for preparing high-concentration liquid composition comprising oligonucleotide compounds.In an exemplary embodiment, the method includes: exchanging the oligonucleotide compounds in starting solution with diafiltration (DF) solution to obtain intermediate solution, the concentration of the oligonucleotide compounds in starting solution is 140mg / mL or less, and the DF solution comprises one or more salts; concentrating the oligonucleotide compounds in intermediate solution with ultrafiltration to obtain high-concentration liquid composition, the concentration of the oligonucleotide compounds in high-concentration liquid composition is greater than about 150mg / mL.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS The benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 313,840, filed February 25, 2022, is claimed herein, the disclosure of which is incorporated herein by reference.

[0002] Incorporation by Reference of Electronically Submitted Materials The computer readable nucleotide / amino acid sequence listing submitted contemporaneously herewith is incorporated by reference in its entirety and is identified as follows: 20.5 KB file entitled "A-2897-WO01-SEC_Seq_Listing.xml", created on February 20, 2023. [Background technology]

[0003] Oligonucleotide drugs are on the rise, as the number of oligonucleotides approved for commercial therapeutic use has more than doubled in the past five years, and the number of oligonucleotide programs in development has tripled in the past decade (Muslehiddinoglu et al., Nucleic Acid Therapeutics 30(4);189-197(2020)). All currently marketed oligonucleotide drugs are formulated for parenteral administration, including intravitreal, intravenous, intrathecal, intramuscular, or subcutaneous administration. In many cases, the oligonucleotide drug substance (DS) used to manufacture drug product (DP) is a lyophilized powder. Lyophilized drug substances are easy to transport and store, have a low risk of microbial growth, and exhibit stability of more than 3 years under refrigerated and frozen conditions. However, lyophilization is an energy-intensive, time-consuming, and costly process. In terms of batch production and cycle time, lyophilization can add up to 5 days to the manufacturing process, thereby increasing the overall process inefficiency. Additionally, the overall complexity of the manufacturing process increases when lyophilization is included in the process, as lyophilized drug products require reconstitution, dissolution, compounding and dilution with formulation buffers (Muslehiddinoglu et al., 2020, supra).

[0004] A flow diagram of the manufacturing process of conventional oligonucleotide compounds from powder (lyophilized) DS is shown in Figure 1A. Oligonucleotide DS are manufactured as lyophilized powders that can be stored at 2-8 °C or -20 °C. During the DP manufacturing process, the DS powder is thawed to a controlled room temperature. After thawing, the powder is weighed and transferred to a compounding vessel for further reconstitution with formulation buffer to the desired concentration, followed by thorough mixing to produce the final bulk drug product suitable for filtration and filling operations.

[0005] Instead of lyophilized powders, aqueous solutions of oligonucleotide DS have been proposed as a more efficient manufacturing-to-administration route (Muslehiddinoglu et al., 2020, supra). A liquid DS solution allows for an easier DP manufacturing process flow, including thawing of the frozen DS solution, followed by mixing, filtration and filling processes, without the need for powder handling and reconstitution. A flow diagram of an exemplary manufacturing process for oligonucleotide compounds from liquid drug substances is shown in Figure 1B. In addition to simplifying the overall manufacturing process, manufacturing costs will also be reduced.

[0006] Solution DS and solution DP are not without challenges as stability concerns and microbial contamination risks may arise. Transporting large volumes of solution can also lead to complex supply chain issues, especially if the solution is stored frozen. In addition, the manufacture of solution DS or solution DP typically involves ultrafiltration / diafiltration (UF / DF). Challenges associated with UF / DF include low flow rates, long process times, mechanical recovery and recovery losses, operator-intensive intervention or handling, low mass transfer rates, poor energy efficiency, and hydraulic limitations in the concentrator. Although UF / DF can achieve active pharmaceutical ingredient (API) concentrations of 40-150 mg / mL in solution (Muslehiddinoglu et al., 2020, supra), the maximum concentration of oligonucleotide compounds achievable by UF / DF is limited by oligonucleotide-membrane interactions that result in membrane fouling. The higher the target API concentration, the more susceptible it is to membrane fouling, which slows the movement of water and smaller ions through the membrane, thereby lengthening the time to reach the desired concentration, if not completely preventing the achievement of the target API concentration. Increasing the molecular weight cut-off (MWCO) of the membrane used in UF / DF can facilitate the process, but can also lead to reduced oligonucleotide recovery. Such challenges can limit the attainment of high-concentration oligonucleotide DPs produced using liquid DS. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Muslehiddinoglu et al.,Nucleic Acid Therapeutics 30(4);189-197(2020) Summary of the Invention [Problem to be solved by the invention]

[0008] Therefore, there is a need for a new process for preparing oligonucleotide compounds, such as oligonucleotide DS, as a liquid composition (e.g., solution) with high concentration.This process results in the preparation of the liquid composition that contains, for example, oligonucleotide compounds at a concentration of more than about 150mg / mL.Ideally, this process avoids the need to freeze-dry, and thus avoids the need to reconstitute and dissolve DS powder. [Means for solving the problem]

[0009] The data presented herein supports the feasibility of the method for preparing high-concentration liquid compositions comprising oligonucleotide compounds.As detailed herein, the method achieves the preparation of liquid compositions comprising oligonucleotide compounds at a concentration as high as about 150 mg / mL, and in various examples, the method can obtain even higher concentrations of oligonucleotide compounds.As described herein, the method can obtain liquid compositions comprising oligonucleotide compounds at a concentration of, for example, more than about 150 mg / mL, more than about 160 mg / mL, more than about 170 mg / mL, more than about 180 mg / mL, more than about 190 mg / mL, more than about 200 mg / mL, more than about 210 mg / mL, more than about 220 mg / mL, or more than about 230 mg / mL.

[0010] Without being bound by any particular theory, obtaining such a high concentration of liquid composition is at least partially due to the solution in which the oligonucleotide compound is present during ultrafiltration, which solution relates to the diafiltration (DF) solution used in the present method in an exemplary embodiment. Without being bound by any particular theory, the solution in which the oligonucleotide compound is present during ultrafiltration or the DF solution used in diafiltration contains one or more salts that interact with the oligonucleotide compound in such a way that the oligonucleotide compound is stabilized and / or its hydrodynamic diameter is increased. Without being bound by any particular theory, the salt of the solution, for example the DF solution, interacts with the oxygen atom and / or sulfur atom of the oligonucleotide compound, for example, by hydrogen bonding, metal coordination, electrostatic interaction. Without being bound by any particular theory, the greater the interaction between the oligonucleotide compound and the salt, the higher the concentration of the oligonucleotide compound can be obtained. Such interaction is related to the absence or minimization of fouling of the membrane used in the present method.

[0011] Thus, provided herein is a method for preparing a high concentration liquid composition comprising an oligonucleotide compound, the liquid composition comprising an oligonucleotide compound at a concentration of greater than about 150 mg / mL, greater than about 160 mg / mL, greater than about 170 mg / mL, greater than about 180 mg / mL, greater than about 190 mg / mL, greater than about 200 mg / mL, greater than about 210 mg / mL, greater than about 220 mg / mL. In an exemplary embodiment, the method for preparing a high concentration liquid composition comprises (a) preparing an oligonucleotide compound in a first solution comprising one or more salts, the concentration of the oligonucleotide compound in the first solution being 140 mg / mL or less, and the total salt concentration of the first solution being about 25 mM to about 800 mM, and (b) concentrating the first solution by ultrafiltration to obtain a high concentration liquid composition comprising an oligonucleotide compound at a concentration of greater than about 150 mg / mL. In various aspects, the first solution is prepared by diafiltration with a DF solution. In various aspects, the first solution is the same as the DF solution, except that the first solution contains the oligonucleotide compound and the DF solution does not contain the oligonucleotide compound. In various examples, the exchange of a starting solution containing about 140 mg / mL or less of the oligonucleotide compound with a DF solution containing one or more salts and having a total salt concentration of 25 mM to about 800 mM is achieved by diafiltration. Thus, in an exemplary embodiment, a method for preparing a high concentration liquid composition includes (a) exchanging the oligonucleotide compound in the starting solution with a DF solution by diafiltration to obtain an intermediate solution, the concentration of the oligonucleotide compound in the starting solution being 140 mg / mL or less and the DF solution containing one or more salts, and (b) concentrating the oligonucleotide compound in the intermediate solution by ultrafiltration to obtain a high concentration liquid composition, the concentration of the oligonucleotide compound in the high concentration liquid composition being about 150 mg / mL or more. In an exemplary example, the total salt concentration of the DF solution is about 25 mM to about 800 mM. Optionally, the total salt concentration of the DF solution is from about 25 mM to about 500 mM. In an illustrative example, the method includes ultrafiltration by continuous diafiltration and / or tangential flow filtration.In various aspects, the methods use polyethersulfone (PES) or stabilized cellulose membranes for diafiltration and / or ultrafiltration. Optionally, the membranes have a molecular weight cut-off (MWCO) of less than 10 kDa, and in illustrative examples, the MWCO is about 5 kDa or about 3 kDa.

[0012] Also provided herein is a method for concentrating a first solution containing a low concentration of oligonucleotide compounds to obtain a second solution containing a high concentration of oligonucleotide compounds by ultrafiltration, wherein the oligonucleotide compound concentration of the second solution is greater than about 150mg / mL, greater than about 160mg / mL, greater than about 170mg / mL, greater than about 180mg / mL, greater than about 190mg / mL, greater than about 200mg / mL, greater than about 210mg / mL, greater than about 220mg / mL.In exemplary embodiments, the oligonucleotide compound concentration of the first solution is less than about 140mg / mL, less than about 130mg / mL, less than about 120mg / mL, less than about 110mg / mL, less than about 100mg / mL, less than about 90mg / mL, less than about 80mg / mL, less than about 70mg / mL, less than about 60mg / mL, or less than about 50mg / mL.In various embodiments, the second solution is the retentate obtained during ultrafiltration. In various embodiments, the retentate is obtained after 1 hour or 2 hours of ultrafiltration. In an exemplary example, the oligonucleotide compound is double-stranded. In an exemplary example, the total salt concentration of the first solution is about 25 mM to about 800 mM. Optionally, the total salt concentration of the first solution is about 25 mM to about 500 mM or about 25 mM to about 250 mM. In various embodiments, the first solution contains only one salt. Optionally, the one salt is an inorganic salt, such as any of the inorganic salts described herein. In various embodiments, the first solution does not contain any acetate salt. In an exemplary example, the concentration method is performed after diafiltration with a DF solution. In various embodiments, the first solution is the same as the DF solution, except that the first solution contains the oligonucleotide compound and the DF solution does not contain the oligonucleotide compound. In various examples, diafiltration is performed with a DF solution containing one or more salts, and the total salt concentration of the DF solution is about 25 mM to about 800 mM. Optionally, the total salt concentration of the DF solution is about 25 mM to about 500 mM or about 25 mM to about 250 mM. In an exemplary embodiment, the concentration method is performed prior to diafiltration with the DF solution. In various examples, the oligonucleotide compound is a double-stranded oligonucleotide compound, optionally a siRNA.

[0013] Further provided herein is a method for preparing a solution API comprising an oligonucleotide compound API, wherein the oligonucleotide compound API is present in the solution at a concentration of more than 150 mg / mL. In various aspects, the oligonucleotide compound API is double-stranded, optionally siRNA. In an exemplary embodiment, the method for preparing a solution API comprises the method disclosed herein for concentrating a first solution comprising a low concentration of an oligonucleotide compound by ultrafiltration to obtain a second solution comprising a high concentration of an oligonucleotide compound. In various examples, the method for preparing a solution API comprises (i) synthesizing an oligonucleotide compound or a strand thereof, for example by solid phase synthesis; (ii) performing one or more rounds of chromatography, diafiltration and annealing; and (iii) concentrating by ultrafiltration according to the method disclosed herein for concentrating a first solution comprising a low concentration of an oligonucleotide compound by ultrafiltration to obtain a second solution comprising a high concentration of an oligonucleotide compound. In various embodiments, the oligonucleotide compound concentration of the second solution is greater than about 150 mg / mL, greater than about 160 mg / mL, greater than about 170 mg / mL, greater than about 180 mg / mL, greater than about 190 mg / mL, greater than about 200 mg / mL, greater than about 210 mg / mL, greater than about 220 mg / mL, and the oligonucleotide compound concentration of the first solution is less than about 140 mg / mL, less than about 130 mg / mL, less than about 120 mg / mL, less than about 110 mg / mL, less than about 100 mg / mL, less than about 90 mg / mL, less than about 80 mg / mL, less than about 70 mg / mL, less than about 60 mg / mL, or less than about 50 mg / mL.In various embodiments, the second solution is the retentate obtained during ultrafiltration.In various embodiments, the retentate is obtained after 1 hour or 2 hours of ultrafiltration. In the exemplary embodiment of the method for preparing solution API comprising oligonucleotide compound API, oligonucleotide API is not lyophilized at any point during the method.Advantageously, the method for preparing solution API is a method that does not have any lyophilization.Therefore, the method for preparing solution API of the present disclosure does not have lyophilization, providing a more time-efficient and cost-effective method for preparing oligonucleotide compound API.In various examples, the solution API prepared by the method disclosed herein is directly used to prepare a solution DP that includes an oligonucleotide compound API. Optionally, the solution API is sterile filtered and filled into a vial or a prefilled syringe or an automatic injection device. Thus, a method is provided for producing a DP, such as a solution DP, that includes a solution API that is composed of an oligonucleotide compound API. In various embodiments, the method for producing a DP does not include any lyophilization. In various embodiments of the method for producing a DP, lyophilization of the oligonucleotide compound API is not performed at any point during the method.

[0014] In an exemplary embodiment, the method of the present disclosure results in a high concentration liquid composition, and the concentration of oligonucleotide compounds in the high concentration liquid composition is about 150 mg / mL or more. In an exemplary embodiment, the high concentration liquid composition obtained by the method disclosed herein comprises more than 80% of the oligonucleotide compounds of the starting solution. In an exemplary example, the method achieves a recovery rate of 70% or 80% of the oligonucleotide compounds of the starting solution. In an exemplary embodiment, the membrane flux is substantially maintained during the method, for example, during diafiltration and / or ultrafiltration. In various examples, the membrane flux is substantially maintained for at least 6 hours, at least 8 hours, or more than 9 hours. In an exemplary embodiment, little to no membrane fouling occurs during the method, for example, during diafiltration and / or ultrafiltration. In an exemplary example, a high concentration liquid composition comprising an oligonucleotide compound at a concentration of about 150 mg / mL or more is obtained, and the membrane flux is reduced to 50% or less of the steady state membrane flux, and this membrane flux is optionally maintained for at least 4 hours or more.

[0015] The present disclosure also provides a high-concentration liquid composition prepared by the method of the present disclosure.In various examples, the high-concentration liquid composition comprises an oligonucleotide compound at a concentration of about 150mg / mL or more, for example, about 175mg / mL, about 180mg / mL, about 185mg / mL, about 190mg / mL, about 195mg / mL, about 200mg / mL, about 205mg / mL, about 210mg / mL, about 215mg / mL, about 220mg / mL, about 225mg / mL, about 230mg / mL, about 235mg / mL, about 240mg / mL, about 245mg / mL, about 250mg / mL or more.Frozen preparations are also provided by storing the liquid composition of the present disclosure at a temperature below 0°C.The frozen preparation in exemplary embodiments is not a lyophilized preparation or a freeze-dried preparation.

[0016] The present disclosure further provides a method for manufacturing a drug comprising oligonucleotide compound.In an exemplary embodiment, the method includes: carrying out the method of the present disclosure to obtain a high-concentration liquid composition comprising more than about 150mg / mL of oligonucleotide compound; formulating the high-concentration liquid composition with a pharma-ceutically acceptable excipient; and filling the formulated high-concentration liquid composition into a container.

[0017] Further provided is a method for treating a subject having a disease. In an exemplary embodiment, the method comprises administering to the subject a liquid composition of the present disclosure, such as a liquid DP, in an amount effective to treat the disease of the subject. Optionally, the drug is administered by injection or infusion.

[0018] There is further provided a use of the highly concentrated liquid composition of the present disclosure in the manufacture of a medicament for treating a disease in a subject. [Brief description of the drawings]

[0019] [Figure 1A] FIG. 1 is a flow diagram of a pharmaceutical manufacturing process using powdered drug substances. [Figure 1B] FIG. 1 is a flow diagram of a pharmaceutical manufacturing process with bulk liquid drug substance. [Diagram 2]1 is a diagram of an exemplary method for preparing a highly concentrated liquid composition comprising an oligonucleotide compound, with the direction of solution movement indicated by the solid black arrows. [Diagram 3] Schematic diagram of the structure of a model oligonucleotide compound. The top strand, listed in the 5' to 3' direction, is the sense strand (SEQ ID NO: 3), and the bottom strand, listed in the 3' to 5' direction, is the antisense strand (SEQ ID NO: 4). The black circles represent nucleotides with a 2'-O-methyl modification, the white circles represent nucleotides with a 2'-deoxy-2'-fluoro ("2'-fluoro") modification, and the grey circles represent deoxyadenosine nucleotides linked to adjacent nucleotides via a 3'-3' bond (i.e., inverted). The grey lines connecting the circles represent phosphodiester bonds, while the black lines connecting the circles represent phosphorothioate bonds. A trivalent GalNAc moiety having the structure shown is represented by R1 and is covalently attached to the 5' end of the sense strand by a phosphorothioate bond. [Figure 4] Chromatograms showing membrane flux (top) and pressure (bottom) across a 5 kDa Hydrosart stabilized cellulose membrane in an Ambr cross-flow setup using two different DF solutions: (i) 1x PBS and (ii) a buffer solution containing 20 mM phosphate buffer with 40 mM NaCl. [Diagram 5] 1 is a graph of the unfolding temperature of oligonucleotide compounds placed in DF solutions composed of various salt concentrations. [Figure 6] 1 is a graph of the hydrodynamic diameter of oligonucleotide compounds placed in DF solutions composed of various salt concentrations. [Figure 7]FIG. 1 is a simplified schematic diagram of an exemplary manufacturing process for a double-stranded oligonucleotide compound comprising a sense strand and an antisense strand. Each strand is synthesized separately, followed by purification, buffer exchange and / or concentration by chromatography and / or UF / DF, before annealing the sense and antisense strands. After annealing, the double-stranded oligonucleotide compound is buffer exchanged and concentrated by UF / DF to obtain a high concentration solution of drug substance (double-stranded oligonucleotide compound), where the concentration of the double-stranded oligonucleotide compound is greater than 150 mg / mL. This high concentration solution of drug substance is used to prepare a DP, which is sterile filtered and filled into a container. Optionally, the high concentration solution of drug substance is exchanged with a formulation buffer before sterile filtration and filling. Optionally, the DP is stored after sterile filtration and filling. Lyophilization is not performed at any point during this process. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Provided herein is a method for preparing a liquid composition containing an oligonucleotide compound at a high concentration. In an exemplary embodiment, the method involves preparing a liquid composition containing an oligonucleotide compound at a high concentration, for example, greater than or about 150 mg / mL, and includes (a) preparing a first solution containing an oligonucleotide compound and one or more salts, wherein the concentration of the oligonucleotide compound in the first solution is less than or equal to 140 mg / mL, and the total salt concentration of the first solution is about 25 mM to about 800 mM, and (b) concentrating the first solution by ultrafiltration to obtain a liquid composition containing an oligonucleotide compound at a concentration of greater than about 150 mg / mL. In various aspects, the first solution is prepared by diafiltration, exchanging the oligonucleotide compound in the starting solution into the first solution or diafiltration (DF) solution to obtain an intermediate solution, which is then concentrated by ultrafiltration to obtain a liquid composition containing an oligonucleotide compound at a high concentration. In various aspects, the first solution is the same as the DF solution, except that the first solution contains the oligonucleotide compound and the DF solution does not contain the oligonucleotide compound. The first solution can be the same as any DF solution described herein, but also contains a low concentration (e.g., less than about 140 mg / mL) of oligonucleotide compound.In an exemplary embodiment, the method includes: (a) exchanging the oligonucleotide compound in the starting solution with a diafiltration (DF) solution by diafiltration to obtain an intermediate solution, wherein the concentration of the oligonucleotide compound in the starting solution is 140 mg / mL or less, and the DF solution contains one or more salts; and (b) concentrating the oligonucleotide compound in the intermediate solution by ultrafiltration to obtain a high concentration liquid composition, wherein the concentration of the oligonucleotide compound in the high concentration liquid composition is about 150 mg / mL or more.In exemplary embodiments, the high concentration liquid composition obtained by the method disclosed herein comprises a concentration of about 150 mg / mL or more, such as about 160 mg / mL or more, about 170 mg / mL or more, about 180 mg / mL or more, about 190 mg / mL or more, about 200 mg / mL or more, about 210 mg / mL or more, about 220 mg / mL or more, about 230 mg / mL or more, about 240 mg / mL or more, about 250 mg / mL or more, about 260 mg / mL or more, about 270 mg / mL or more, about 280 mg / mL or more, about 290 mg / mL or more, about 300 mg / mL or more. In exemplary embodiments, the method disclosed herein achieves a high recovery rate of the oligonucleotide compounds present in the starting solution. In various examples, the method achieves a recovery rate of at least 70% or at least 80% of the oligonucleotide compounds of the starting solution. In various embodiments, the method achieves a recovery rate of at least 85% (e.g., at least 90%, at least 95%, at least 98%) of the oligonucleotide compounds present in the starting solution. In exemplary embodiments, the concentrated liquid composition obtained by the method disclosed herein comprises more than 80% of the oligonucleotide compounds present in the starting solution. In various examples, the concentrated liquid composition obtained by the method disclosed herein comprises more than 85% (e.g., more than 90%, more than 95%, more than 98%) of the oligonucleotide compounds present in the starting solution. Without being bound by theory, the method disclosed herein achieves such high levels of oligonucleotide compound recovery and such high concentrations of oligonucleotide compounds in the resulting liquid composition because diafiltration and / or ultrafiltration occurs without substantial membrane fouling. In various examples, membrane flux is substantially maintained during diafiltration and / or ultrafiltration. In various embodiments, membrane flux is substantially maintained for at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours or more. In various aspects, membrane flux reaches a steady state level during diafiltration and / or ultrafiltration and declines to 50% or less (optionally 60% or less, 70% or less, 80% or less, 90% or less).Advantageously, the disclosed method is more efficient given that the membrane flux is substantially maintained without substantial membrane fouling. In various aspects, the maintained membrane flux and the absence of substantial membrane fouling leads to a more concentrated liquid composition in a shorter time compared to other methods that are slowed by substantial membrane fouling. Thus, the disclosed method is advantageously characterized by a short process time to obtain a concentrated liquid composition. In various aspects, the process time to obtain a concentrated liquid composition is less than 36 hours, less than 32 hours, less than 30 hours, less than 28 hours, less than 26 hours, less than 24 hours, less than 22 hours, less than 20 hours, less than 18 hours, less than 16 hours, less than 14 hours, less than 12 hours, or less than 10 hours. Without being bound by any particular theory, the method disclosed herein provides an efficient method for buffer-exchanging a starting solution containing oligonucleotide compounds into a DF solution and concentrating the oligonucleotide compounds in the DF solution without any membrane fouling, without significant loss of oligonucleotide compounds (e.g., with good oligonucleotide compound recovery rate), and / or within a relatively short period of time (e.g., about 10 hours or less). Advantageously, the method disclosed herein avoids the need for energy-intensive and time-consuming lyophilization of oligonucleotide compounds, and is therefore more efficient.

[0021] In an exemplary embodiment, the method includes exchanging the oligonucleotide compound in the starting solution into a diafiltration (DF) solution by diafiltration to obtain an intermediate solution. In various aspects, the oligonucleotide compound starts from a starting solution that includes a first component or set of components, and the method includes transferring the oligonucleotide compound from the starting solution to a diafiltration solution that is different in composition from the starting solution and includes a different component or set of components. This exchange from the starting solution to the diafiltration solution in various aspects is known in the art as "buffer exchange" and is accomplished by diafiltration. The term "diafiltration" or "DF" refers to a process for exchanging macromolecular products from one solution or buffer to another solution or buffer. DF is often used for buffer exchange. In various aspects, diafiltration is performed to exchange the starting solution into the DF solution (so that the oligonucleotide compound is efficiently transferred from the starting solution to the DF solution) to obtain an intermediate solution. In various aspects, the intermediate solution includes the oligonucleotide compound and the DF solution. Optionally, the intermediate solution includes at least 70% of the amount of the oligonucleotide compound present in the starting solution. In various embodiments, intermediate solution comprises at least 75%, at least 80%, at least 85% or at least 90% of the amount of oligonucleotide compounds present in starting solution.In various examples, diafiltration achieves at least 70% or at least 80% recovery of the oligonucleotide compounds of starting solution.In various embodiments, the method achieves at least 85% (e.g., at least 90%, at least 95%, at least 98%) recovery of the oligonucleotide compounds present in starting solution.

[0022] In an exemplary embodiment, the method includes concentrating the oligonucleotide compound in the intermediate solution. In an exemplary embodiment, the method includes increasing the concentration of the oligonucleotide compound in the intermediate solution by concentrating, and the concentration is achieved by ultrafiltration. The term "ultrafiltration" or "UF" refers to a process that uses a membrane to separate macromolecular compounds from small molecular components (e.g., water, cations, anions) to desalt or concentrate a solution containing the macromolecular compounds.

[0023]

[0024] The method disclosed herein for preparing high-concentration liquid oligonucleotide compounds utilizes a starting solution and a DF solution. In various embodiments, the starting solution contains a lower concentration of oligonucleotide compounds than that of the high-concentration liquid composition. In various embodiments, the concentration of oligonucleotide compounds in the starting solution is less than 150 mg / mL, less than 140 mg / mL, less than 130 mg / mL, less than 120 mg / mL, less than 110 mg / mL, less than 100 mg / mL, less than 90 mg / mL, less than 80 mg / mL, less than 70 mg / mL, less than 60 mg / mL, or less than 50 mg / mL (e.g., less than 45 mg / mL, less than 40 mg / mL, less than 35 mg / mL, less than 30 mg / mL, less than 25 mg / mL, less than 20 mg / mL, less than 15 mg / mL, less than 10 mg / mL, less than 5 mg / mL). In various embodiments, the concentration of the oligonucleotide compound in the starting solution is from about 30 mg / mL to about 140 mg / mL, from about 30 mg / mL to about 130 mg / mL, from about 30 mg / mL to about 120 mg / mL, from about 30 mg / mL to about 110 mg / mL, from about 30 mg / mL to about 100 mg / mL, from about 30 mg / mL to about 90 mg / mL, from about 30 mg / mL to about 80 mg / mL, from about 30 mg / mL to about 70 mg / mL, from about 30 mg / mL to about 60 mg / mL, from about 30 mg / mL to about 50 mg / mL, The concentration of the oligonucleotide compound in the starting solution is about 30 mg / mL to about 40 mg / mL, about 40 mg / mL to about 140 mg / mL, about 50 mg / mL to about 140 mg / mL, about 60 mg / mL to about 140 mg / mL, about 70 mg / mL to about 140 mg / mL, about 80 mg / mL to about 140 mg / mL, about 90 mg / mL to about 140 mg / mL, about 100 mg / mL to about 140 mg / mL, about 110 mg / mL to about 140 mg / mL, about 120 mg / mL to about 140 mg / mL, or about 130 mg / mL to about 140 mg / mL. In various embodiments, the concentration of the oligonucleotide compound in the starting solution is about 30 mg / mL to about 100 mg / mL.In various embodiments, the concentration of the oligonucleotide compound in the starting solution is from about 50 mg / mL to about 140 mg / mL, from about 50 mg / mL to about 130 mg / mL, from about 50 mg / mL to about 120 mg / mL, from about 50 mg / mL to about 110 mg / mL, from about 50 mg / mL to about 100 mg / mL, from about 50 mg / mL to about 90 mg / mL, from about 50 mg / mL to about 80 mg / mL, from about 50 mg / mL to about 70 mg / mL. , about 50 mg / mL to about 60 mg / mL, about 60 mg / mL to about 140 mg / mL, about 70 mg / mL to about 140 mg / mL, about 80 mg / mL to about 140 mg / mL, about 90 mg / mL to about 140 mg / mL, about 100 mg / mL to about 140 mg / mL, about 110 mg / mL to about 140 mg / mL, about 120 mg / mL to about 140 mg / mL, or about 130 mg / mL to about 140 mg / mL. In various embodiments, the concentration of the oligonucleotide compound in the starting solution is about 50 mg / mL to about 100 mg / mL. In an exemplary embodiment, the starting solution includes water or a buffer solution. In an exemplary embodiment, the starting solution does not include a buffer solution. Advantageously, the method of the present disclosure is not limited to the components of the starting solution.

[0025] The exchange solution, or DF solution, which in some instances may also be referred to as an exchange buffer or diafiltration buffer, comprises at least one salt. In various embodiments, the total salt concentration of the DF solution is from about 25 mM to about 800 mM, or from about 50 mM to about 800 mM. In exemplary embodiments, the total salt concentration of the DF solution is about 50 mM to about 750 mM, about 50 mM to about 700 mM, about 50 mM to about 650 mM, about 50 mM to about 600 mM, about 50 mM to about 550 mM, about 50 mM to about 500 mM, about 50 mM to about 450 mM, about 50 mM to about 400 mM, about 50 mM to about 350 mM, about 50 mM to about 300 mM, about 50 mM to about 250 mM, about 50 mM to about 200 mM, about 50 mM to about 150 mM, about 50 mM to about 100 mM, about 50 mM to about 75 mM, , about 75 mM to about 800 mM, about 100 mM to about 800 mM, about 150 mM to about 800 mM, about 200 mM to about 800 mM, about 250 mM to about 800 mM, about 300 mM to about 800 mM, about 350 mM to about 800 mM, about 400 mM to about 800 mM, about 450 mM to about 800 mM, about 500 mM to about 800 mM, about 550 mM to about 800 mM, about 600 mM to about 800 mM, about 650 mM to about 800 mM, about 700 mM to about 800 mM, or about 750 mM to about 800 mM. In an exemplary embodiment, the total salt concentration is less than 500 mM. Optionally, the total salt concentration is about 25 mM to about 500 mM, about 25 mM to about 400 mM, about 25 mM to about 300 mM, about 25 mM to about 250 mM, about 25 mM to about 200 mM, or about 25 mM to about 150 mM. In various embodiments, the DF solution comprises, consists essentially of, or consists of an inorganic salt comprising a monovalent cation, and the total salt concentration of the DF solution is about 25 mM to about 500 mM, about 25 mM to about 400 mM, about 25 mM to about 300 mM, about 25 mM to about 250 mM, about 25 mM to about 200 mM, or about 25 mM to about 150 mM. In various embodiments, the total salt concentration is about 75 mM to about 500 mM, about 75 mM to about 400 mM, about 75 mM to about 300 mM, about 75 mM to about 250 mM, about 75 mM to about 200 mM, or about 75 mM to about 150 mM.In various embodiments, the DF solution comprises, consists essentially of, or consists of an inorganic salt comprising a monovalent cation, and the total salt concentration of the DF solution is about 75 mM to about 500 mM, about 75 mM to about 400 mM, about 75 mM to about 300 mM, about 75 mM to about 250 mM, about 75 mM to about 200 mM, or about 75 mM to about 150 mM. In various examples, the total salt concentration of the DF solution is about 125 mM to about 500 mM. Optionally, the total salt concentration of the DF solution is about 150 mM to about 500 mM, about 175 mM to about 500 mM, about 200 mM to about 500 mM, about 225 mM to about 500 mM, about 250 mM to about 500 mM, about 275 mM to about 500 mM, about 300 mM to about 500 mM, about 325 mM to about 500 mM, about 350 mM to about 500 mM, about 375 mM to about 500 mM, about 400 mM to about 500 mM, about 425 mM to about 500 mM, about 450 mM to about 500 mM, about 475 mM In an exemplary embodiment, the total salt concentration of the DF solution is about 140 mM to about 300 mM. Optionally, the total salt concentration of the DF solution is about 140 mM to about 280 mM, about 140 mM to about 260 mM, about 140 mM to about 240 mM, about 140 mM to about 220 mM, about 140 mM to about 200 mM, about 140 mM to about 180 mM, about 140 mM to about 160 mM, about 160 mM to about 300 mM, about 180 mM to about 300 mM, about 200 mM to about 300 mM, about 220 mM to about 300 mM, about 240 mM to about 300 mM, about 260 mM to about 300 mM, or about 280 mM to about 300 mM.In various examples, the total salt concentration of the DF solution is about 50 mM to about 900 mM, about 50 mM to about 875 mM, about 50 mM to about 850 mM, about 50 mM to about 800 mM, about 50 mM to about 775 mM, about 50 mM to about 750 mM, or about 50 mM to about 700 mM, for example, about 60 mM to about 700 mM, about 70 mM to about 700 mM, about 80 mM to about 700 mM, or about 80 mM to about 700 mM. M, about 90mM to about 700mM, about 100mM to about 700mM, about 110mM to about 700mM, about 120mM to about 700mM, about 130mM to about 700mM, about 140 mM~about 700mM, about 150mM~about 700mM, about 160mM~about 700mM, about 170mM~about 700mM, about 180mM~about 700mM, about 190mM~about 70 0mM, approximately 200mM to approximately 700mM, approximately 210mM ​​to approximately 700mM, approximately 220mM to approximately 700mM, approximately 230mM to approximately 700mM, approximately 240mM to approximately 700mM, approximately 250mM to about 700mM, about 260mM to about 700mM, about 270mM to about 700mM, about 280mM to about 700mM, about 290mM to about 700mM, about 300mM about 310 mM to about 700 mM, about 320 mM to about 700 mM, about 330 mM to about 700 mM, about 340 mM to about 700 mM, about 350 mM to about 700 mM, about 360 mM to about 700 mM, about 370 mM to about 700 mM, about 380 mM to about 700 mM, about 390 mM to about 700 mM, and about 400 mM to about 700 mM. In various examples, the total salt concentration of the DF solution is about 100 mM to about 900 mM, about 200 mM to about 900 mM, about 300 mM to about 900 mM, about 400 mM to about 900 mM, about 500 mM to about 900 mM, about 600 mM to about 900 mM, about 700 mM to about 900 mM, or about 800 mM to about 900 mM.In various examples, the total salt concentration of the DF solution is from about 100 mM to about 700 mM, optionally from about 100 mM to about 650 mM, from about 100 mM to about 600 mM, from about 100 mM to about 550 mM, from about 100 mM to about 500 mM, from about 100 mM to about 450 mM, from about 100 mM to about 400 mM, from about 100 mM to about 350 mM, from about 100 mM to about 300 mM, from about 100 mM to about 250 mM, from about 100 mM to about 200 mM, The total salt concentration of the DF solution is, in various embodiments, about 100 mM to about 600 mM. Optionally, the total salt concentration of the DF solution is about 125 mM to about 300 mM, optionally about 125 mM to about 200 mM, or about 125 mM to about 150 mM. In an exemplary embodiment, the total salt concentration of the DF solution is about 130 mM to about 140 mM. In various examples, the DF solution is substantially free of potassium. Optionally, the DF solution includes a molar ratio of salt (e.g., first salt) and potassium of about 100:1.

[0026] In an exemplary embodiment, the first solution or DF solution comprises at least one salt, and the total salt concentration of the DF solution is about 25 mM to about 800 mM, or about 50 mM to about 800 mM. Optionally, the total salt concentration of the DF solution is less than 500 mM, for example, about 25 mM to about 500 mM. In an exemplary example, the DF solution comprises, consists essentially of, or consists of an inorganic salt. As used herein, the term "inorganic salt" refers to any inorganic compound having an ionic bond that does not include a carbon-hydrogen bond. Inorganic salts include, but are not limited to, transition metal salts, alkali metal salts, post-transition metal salts, metalloid salts, alkaline earth metal salts, lanthanide salts, ammonium salts, halogen inorganic salts, reactive nonmetal salts, and xenon salts. Suitable inorganic salts include any of the inorganic salts listed in the catalog at https: / / www.thermofisher.com / search / browse / category / us / en / 80013663 / inorganic-salts that are not toxic to or destabilize oligonucleotide compounds. In exemplary embodiments, the inorganic salt is an alkali metal salt, an alkaline earth metal salt, or an ammonium salt. In various embodiments, the alkali metal salt is any inorganic salt that includes an element in Group 1a of the periodic table. Examples of alkali metal salts include sodium chloride, lithium chloride, potassium chloride, sodium bromide, potassium hydrogen phosphate, sodium hydroxide, potassium carbonate, cesium hydroxide, lithium hydroxide, potassium hydroxide, sodium dihydrogen phosphate, sodium acetate, sodium fluoride, potassium titanium oxide, sodium carbonate, lithium amide, and the like. In various embodiments, the alkaline earth metal salt is any inorganic salt that includes an element in Group 2 of the periodic table.Examples of alkaline earth metal salts include calcium chloride, magnesium chloride, magnesium oxide, calcium carbonate, strontium fluoride, barium sulfate, calcium chromate, DL-calcium glycerate dihydrate, barium manganate, magnesium silicate, calcium hydride, calcium nitrate tetrahydrate, strontium chloride hexahydrate, magnesium hydroxide carbonate pentahydrate, strontium bromide, calcium sulfate dihydrate, magnesium niobium oxide, methyl magnesium chloride, magnesium thiosulfate, magnesium perchlorate, calcium perchlorate, barium acetate, etc. In an illustrative example, the ammonium salt is any inorganic salt having an ammonium cation. Examples of ammonium salts include ammonium bicarbonate, ammonium persulfate, ammonium carbonate, ammonium hydroxide, ammonium formate, ammonium cobalt(II) phosphate, ammonium copper(II) sulfate, ammonium sulfamate, ammonium oxalate, ammonium perchlorate, ammonium nitrate, ammonium bromide, ammonium dichromate, ammonium dihydrogen phosphate, etc. In various aspects, the inorganic salt comprises a monovalent cation. Optionally, the inorganic salt comprises an alkali metal, optionally sodium, potassium or lithium. In various embodiments, the inorganic salt comprises a halogen counterion, optionally chloride or bromide. In various examples, the inorganic salt is sodium chloride, sodium bromide, potassium chloride or lithium chloride. In some embodiments, the monovalent cation is an ammonium cation, and optionally the inorganic salt is ammonium chloride. In alternative embodiments, the inorganic salt comprises a divalent cation. Optionally, the divalent cation is an alkaline earth metal, optionally magnesium or calcium. In an exemplary embodiment, the inorganic salt is magnesium chloride or calcium chloride. In an exemplary embodiment, the DF solution comprises, consists essentially of, or consists of an organic salt. As used herein, the term "organic salt" refers to a salt containing an organic ion that contains at least one carbon-hydrogen bond and is usually composed only of covalent bonds. Organic salts generally contain only one or more of carbon, hydrogen, oxygen, sulfur, nitrogen and phosphorus atoms.In an illustrative example, the organic salt comprises a quaternary ammonium cation. Optionally, the organic salt is choline chloride or benzyltrimethylammonium chloride. In various embodiments, the total salt concentration of the DF solution is about 25 mM to about 500 mM, about 25 mM to about 250 mM, or about 25 mM to about 150 mM, and the DF solution comprises, consists essentially of, or consists of an inorganic salt comprising a divalent cation, optionally calcium chloride or magnesium chloride. In various examples, the total salt concentration of the DF solution is about 75 mM to about 300 mM, about 75 mM to about 250 mM, or about 75 mM to about 200 mM, and the DF solution comprises, consists essentially of, or consists of an inorganic salt comprising a monovalent cation. In an illustrative example, the DF solution comprises about 75 mM to about 300 mM sodium chloride, sodium bromide, lithium chloride, potassium chloride, or ammonium chloride. In various examples, the total salt concentration of the DF solution is from about 100 mM to about 300 mM, and the DF solution comprises, consists essentially of, or consists of an organic salt, such as CCl or BTMACl.

[0027] In various embodiments, the total salt concentration of the DF solution depends on the number of net negative charges of the oligonucleotide compound. For example, if the oligonucleotide contains 41 net negative charges, the total salt concentration is about 50 mM to about 800 mM, or any of the total salt concentrations of the DF solution described herein. In various examples, the total salt concentration of the DF solution is expressed as the concentration (e.g., mM) per negative charge of the oligonucleotide compound. Thus, in various examples, if the oligonucleotide compound contains 41 net negative charges, the concentration per net negative charge can be multiplied by 41. For example, in various embodiments, the total salt concentration of the DF solution per negative charge is about 3.0 mM to 18.0 mM. Optionally, the total salt concentration of the DF solution per negative charge is about 3.0 mM to 17.0 mM, about 3.0 mM to 16.0 mM, about 3.0 mM to 15.0 mM, about 3.0 mM to 14.0 mM, about 3.0 mM to 13.0 mM, about 3.0 mM to 12.0 mM, about 3.0 mM to 11.0 mM, about 3.0 mM to 10.0 mM, about 3.0 mM to 9.0 mM, about 3.0 mM to 8.0 mM, about 3.0 mM to 7.0 mM, about 3.0 mM to 6.0 mM, about 3.0 mM to 5.0 mM, about 3.0 mM to 4.0 mM, about 4.0 mM to 18.0 mM, about 5.0 mM to 18.0 mM, about 6.0 mM to 18.0 mM, about 7.0 mM to 18.0 mM, about 8.0 mM to 18.0 mM, about 9.0 mM to 18.0 mM, about 10.0 mM to 18.0 mM, about 11.0 mM to 18.0 mM, about 12.0 mM to 18.0 mM, about 13.0 mM to 18.0 mM, about 14.0 mM to 18.0 mM, about 15.0 mM to 18.0 mM, about 16.0 mM to 18.0 mM, or about 17.0 mM to 18.0 mM. In various embodiments, the total salt concentration of the DF solution per negative charge of the oligonucleotide compound is at least about 3.5 mM, at least about 3.6 mM, at least about 3.7 mM, at least about 3.8 mM, at least about 3.9 mM, or at least about 4.0 mM. In exemplary embodiments, the DF solution comprises one or two salts. In various examples, the DF solution comprises three or more salts, optionally three, four, five, six or more salts.

[0028] In various examples, the DF solution includes two or more salts, one salt, hereinafter referred to as the "first salt", is present in the DF solution at a substantially higher concentration than the other salts of the DF solution. Alternatively, the DF solution includes only one salt, which may be considered the "first salt". In various embodiments, the DF solution includes a first salt and at least one other salt. In various embodiments, the concentration of the first salt is at least 2 times, at least 3 times, at least 4 times, or at least 5 times the concentration of another salt present in the DF solution. In various embodiments, the concentration of the first salt is about 10 times the concentration of another salt present in the DF solution. In various embodiments, the concentration of the first salt is at least 2 times, at least 3 times, at least 4 times, or at least 5 times the concentration of all other salts present in the DF solution. In various embodiments, the concentration of the first salt is about 10 times the concentration of all other salts present in the DF solution. In various embodiments, the DF solution includes a buffer. In various embodiments, the buffer has a pH of less than 7. In various embodiments, the buffer comprises a weak acid, a conjugate base, and a salt. In various embodiments, the buffer is phosphate buffered saline (PBS). The concentration of the first salt in the DF solution is based on the concentration of the oligonucleotide compound present in the starting solution in some embodiments. For example, the molar ratio of the oligonucleotide compound in the starting solution to the first salt in the DF solution is about 1:2 to about 1:100, or about 1:2 to about 1:90, or about 1:2 to about 1:80. In various embodiments, the molar ratio of the oligonucleotide compound in the starting solution to the first salt in the DF solution is about 1:3 to about 1:80, or about 1:3 to about 1:75, or about 1:3 to about 1:70. Optionally, the molar ratio of the oligonucleotide compound in the starting solution to the first salt in the DF solution is about 1:5 to about 1:65.For example, the molar ratio may be from about 1:5 to about 1:60, from about 1:5 to about 1:55, from about 1:5 to about 1:50, from about 1:5 to about 1:45, from about 1:5 to about 1:40, from about 1:5 to about 1:35, from about 1:5 to about 1:30, from about 1:5 to about 1:25, from about 1:5 to about 1:20, from about 1:5 to about 1:15, from about 1:5 to about 1:10, from about 1:6 to about 1:65, from about 1:7 to about 1:65, from about 1: 8 to about 1:65, about 1:9 to about 1:65, about 1:10 to about 1:65, about 1:15 to about 1:65, about 1:20 to about 1:65, about 1:25 to about 1:65, about 1:30 to about 1:65, about 1:35 to about 1:65, about 1:40 to about 1:65, about 1:45 to about 1:65, about 1:50 to about 1:65, about 1:55 to about 1:65, or about 1:60 to about 1:65. In various examples, the concentration of the first salt is greater than 50% of the total salt concentration of the DF solution, optionally greater than 55%, or greater than 60%, or greater than 65%, or greater than 70%, or greater than 75%, or greater than 80% of the total salt concentration. In various examples, the concentration of the first salt is greater than 80% of the total salt concentration of the DF solution, optionally greater than 85% or greater than 90% of the total salt concentration.

[0029] The first salt can be any salt. In various aspects, the first salt comprises a monovalent cation. Optionally, the monovalent cation is sodium. In various aspects, the first salt is sodium chloride. In various examples, the first salt comprises a divalent cation. Optionally, the divalent cation is magnesium or calcium. Optionally, the first salt is magnesium chloride or calcium chloride.

[0030] The DF solution in illustrative examples comprises an amount of the first salt that increases the hydrodynamic diameter of the oligonucleotide compound by at least 1.5 times, or at least 2 times, or at least 3 times, compared to the hydrodynamic diameter of the oligonucleotide compound in water. Optionally, the DF solution comprises an amount of the first salt that increases the hydrodynamic diameter of the oligonucleotide compound by 2 times, compared to the hydrodynamic diameter of the oligonucleotide compound in water. In various examples, the DF solution comprises an amount of the first salt that increases the hydrodynamic diameter of the oligonucleotide compound by about 5 nm to about 6 nm. In various embodiments, the DF solution comprises an amount of the first salt that increases the melting temperature of the oligonucleotide compound by at least 1.5 times or at least 2 times, compared to the melting temperature of the oligonucleotide compound in water. For example, the melting temperature is increased by about 20 degrees or about 30 degrees.

[0031] Also provided herein is a method for concentrating a first solution containing a low concentration of oligonucleotide compounds to obtain a second solution containing a high concentration of oligonucleotide compounds by ultrafiltration, wherein the oligonucleotide compound concentration of the second solution is greater than about 150mg / mL, greater than about 160mg / mL, greater than about 170mg / mL, greater than about 180mg / mL, greater than about 190mg / mL, greater than about 200mg / mL, greater than about 210mg / mL, greater than about 220mg / mL.In exemplary embodiments, the oligonucleotide compound concentration of the first solution is less than about 140mg / mL, less than about 130mg / mL, less than about 120mg / mL, less than about 110mg / mL, less than about 100mg / mL, less than about 90mg / mL, less than about 80mg / mL, less than about 70mg / mL, less than about 60mg / mL, or less than about 50mg / mL.In various embodiments, the second solution is the retentate obtained during ultrafiltration. In an exemplary embodiment, the retentate is obtained within 12 hours of ultrafiltration. In an exemplary embodiment, the duration of ultrafiltration is less than 12 hours, less than 10 hours, less than 8 hours, less than 6 hours, less than 4 hours, less than 2 hours, or less than 1 hour. In various embodiments, the retentate is obtained after 1 hour or 2 hours of ultrafiltration, and the concentration of the oligonucleotide compound is greater than 150 mg / mL, optionally greater than 175 mg / mL, greater than 200 mg / mL, or greater than 225 mg / mL. In an exemplary embodiment, the oligonucleotide compound is double-stranded. In an exemplary embodiment, the total salt concentration of the first solution is about 25 mM to about 800 mM. Optionally, the total salt concentration of the first solution is about 25 mM to about 500 mM, or about 25 mM to about 250 mM. In various embodiments, the first solution contains only one salt. Optionally, the one salt is an inorganic salt, such as any of the inorganic salts described herein. In various embodiments, the first solution does not contain any acetate salts. In an exemplary embodiment, the concentration method is performed after diafiltration with a DF solution. In various embodiments, diafiltration is performed with a DF solution that includes one or more salts, and the total salt concentration of the DF solution is about 25 mM to about 800 mM. Optionally, the total salt concentration of the DF solution is about 25 mM to about 500 mM or about 25 mM to about 250 mM. In an exemplary embodiment, the concentration method is performed before diafiltration with a DF solution.In various examples, the oligonucleotide compound is a double-stranded oligonucleotide compound, optionally a siRNA.

[0032] Further steps The methods disclosed herein include additional steps in various aspects. For example, in some aspects, the methods include one or more upstream or downstream steps involved in preparing a drug substance and / or a drug product. Optionally, the downstream steps are any one of the downstream process steps described herein or known in the art. In exemplary embodiments, the methods include steps for producing oligonucleotide compounds, including, for example, conventional solid-phase nucleic acid synthesis. Oligonucleotide compounds can be constructed on a suitable nucleic acid synthesizer utilizing standard nucleotide or nucleoside precursors (e.g., phosphoramidites). Automated nucleic acid synthesizers are commercially available from several vendors, including DNA / RNA synthesizers from Applied Biosystems (Foster City, CA), MerMade synthesizers from BioAutomation (Irving, TX), and OligoPilot synthesizers from GE Healthcare Life Sciences (Pittsburgh, PA). Oligonucleotides can be synthesized via phosphoramidite chemistry using 2' silyl protecting groups together with acid labile dimethoxytrityl (DMT) at the 5' position of ribonucleosides. Final deprotection conditions are known that do not significantly degrade the RNA product. All syntheses can be performed on large, medium or small scale by any automated or manual synthesizer. Synthesis can also be performed on multiple well plates, columns or glass slides. The various synthetic steps can be performed in alternative sequences or orders to obtain the desired compounds.Other synthetic chemistry transformations, protecting groups (e.g., for hydroxyl, amino, etc. present in bases) and protecting group techniques (protection and deprotection) useful in the synthesis of oligonucleotides are known in the art and include, for example, those described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P.G.M. Huts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) and subsequent editions thereof.

[0033] In exemplary embodiments, the methods disclosed herein include a step for separating and / or purifying an oligonucleotide compound. "Purifying" or "purification" refers to a process that reduces and preferably eliminates the amount of a substance that differs from a target molecule (e.g., an oligonucleotide compound) from a final liquid composition or preparation. Purification may also refer to removing one or more impurities. The term "impurity" refers to a substance that has a structure that differs from a target molecule, and the term may include a single unwanted substance or a combination of several unwanted substances. Impurities may include materials or reagents used in the method to generate an oligonucleotide compound, as well as fragments or other unwanted derivatives or forms of an oligonucleotide compound. In certain embodiments, the impurities include one or more oligonucleotides that are shorter in length than the target oligonucleotide compound. In these and other embodiments, the impurities include one or more failure sequences. Failure sequences may arise during the synthesis of a target oligonucleotide and may result from failed coupling reactions during the stepwise addition of nucleotide monomers to an oligonucleotide chain. The product of an oligonucleotide synthesis reaction is often a heterogeneous mixture of oligonucleotides of various lengths, including the target oligonucleotide and various failure sequences that are shorter in length than the target oligonucleotide (i.e., truncated versions of the target oligonucleotide). In some embodiments, the impurities include one or more process-related impurities. Depending on the synthesis method of producing the oligonucleotide compound, such process-related impurities may include, but are not limited to, nucleotide monomers, protective groups, salts, enzymes, and endotoxins. In an exemplary embodiment, the method includes one or more chromatography steps to separate molecular species of a mixture that includes the oligonucleotide compound. In an exemplary embodiment, the chromatography is analytical chromatography. In another exemplary embodiment, the chromatography is preparative chromatography. In an exemplary embodiment, each molecular species of the mixture is separated by the time at which it elutes from the matrix. In various examples, each molecular species of the mixture elutes at a time that is distinct from the time at which different molecular species elutes.In various embodiments, molecular species are separated by reversed-phase high performance liquid chromatography (RP-HPLC). Reversed-phase chromatography, e.g., RP-HPLC, is described in great detail in the prior art. See, e.g., Reversed Phase Chromatography: Principles and Methods, ed. AA, Amersham Biosciences, Buckinghamshire, England (1999).

[0034] In various embodiments, the method of the present disclosure includes one or more steps to prepare the high-concentration liquid composition obtained by the method of the present disclosure into a pharmaceutical product. In various embodiments, the method includes one or more formulation steps. Formulation is the process of transferring a drug substance into a formulated pharmaceutical product. In various embodiments, the method includes one or more formulation steps to make the liquid composition obtained by the method disclosed herein into a form suitable for clinical administration from the environment, solvent, or other physical conditions.

[0035] In various embodiments, the method for preparing a high-concentration liquid composition comprising more than 150 mg / mL of oligonucleotide compound further comprises a further diafiltration or buffer exchange step, in which the oligonucleotide compound is exchanged into a new solution, such as a formulation buffer. In an illustrative example, the method comprises: (i) exchanging the oligonucleotide compound in the starting solution into a diafiltration (DF) solution by diafiltration to obtain an intermediate solution, in which the concentration of the oligonucleotide compound in the starting solution is 140 mg / mL or less, and the DF solution comprises one or more salts, and the total salt concentration of the DF solution is about 25 mM to about 800 mM, optionally less than 500 mM; (ii) concentrating the oligonucleotide compound in the intermediate solution by ultrafiltration to obtain a high-concentration liquid composition, in which the concentration of the oligonucleotide compound in the high-concentration liquid composition is about 150 mg / mL or more; and (iii) exchanging the oligonucleotide compound in the high-concentration liquid composition into a second DF solution by diafiltration. In various embodiments, the second DF solution is a formulation buffer, and the method achieves a high concentration liquid formulation containing more than 150 mg / mL of oligonucleotide compound. In various embodiments, the method further comprises storing the high concentration liquid formulation containing more than 150 mg / mL of oligonucleotide compound at a temperature below 0° C. to obtain a frozen preparation.

[0036] In various embodiments, the method includes performing one or more washes to release any oligonucleotide compounds from the membrane. In various embodiments, the method includes removing a concentrated liquid composition from a collection vessel, such as a retentate vessel, and then performing one or more washes to release any oligonucleotide compounds from the membrane. In various examples, the oligonucleotide compounds obtained from the one or more washes are combined with the concentrated liquid composition obtained from a collection vessel, such as a retentate vessel. In various embodiments, the oligonucleotide compounds obtained from the one or more washes combined with the concentrated liquid composition obtained from a collection vessel, such as a retentate vessel, show a recovery rate of more than 70%, such as more than 70% of the oligonucleotide compounds of the starting solution. In various examples, the oligonucleotide compounds obtained from the one or more washes combined with the concentrated liquid composition obtained from a collection vessel, such as a retentate vessel, are configured at a concentration of about 150 mg / mL or more.

[0037] In an exemplary embodiment, the additional step does not involve lyophilization or freeze-drying of the oligonucleotide compound. Advantageously, the method of the present invention provides a highly concentrated liquid composition comprising at least 150 mg / mL of oligonucleotide compound in a manner that does not require and avoids lyophilization or freeze-drying of the oligonucleotide compound.

[0038] In various embodiments, the oligonucleotide compound is a double-stranded oligonucleotide compound comprising an antisense strand and a sense strand. In various embodiments, the method of the present disclosure includes annealing the sense strand and the antisense strand to obtain a ds oligonucleotide compound, and then preparing a concentrated liquid composition comprising the ds oligonucleotide compound as described herein by (i) exchanging the ds oligonucleotide compound in the starting solution with a diafiltration (DF) solution by diafiltration to obtain an intermediate solution, the concentration of the oligonucleotide compound in the starting solution being 140 mg / mL or less, and the DF solution comprising one or more salts, and the total salt concentration of the DF solution being about 25 mM to about 800 mM, optionally less than 500 mM, and (ii) concentrating the ds oligonucleotide compound in the intermediate solution by ultrafiltration to obtain a concentrated liquid composition. In various embodiments, the starting solution is the solution obtained from annealing. In various embodiments, the ds oligonucleotide compound is present in the starting solution at a concentration of about 50 mg / mL.

[0039] Liquid compositions and frozen preparations The present disclosure also provides a liquid composition prepared by the method of the present disclosure. In various embodiments, the liquid composition is equivalent to a high concentration liquid composition, such as a final retentate. In various examples, the liquid composition prepared by the method of the present disclosure comprises more than or about 150 mg / mL of oligonucleotide compound. Optionally, the concentration of the oligonucleotide compound in the liquid composition is at least 155 mg / mL, at least 160 mg / mL, at least 165 mg / mL, at least 170 mg / mL, at least 175 mg / mL, at least 180 mg / mL, at least 185 mg / mL, at least 190 mg / mL, at least 195 mg / mL, or at least 200 mg / mL. In various examples, the oligonucleotide compound is present in the liquid composition at a concentration of at least 210 mg / mL, at least 220 mg / mL, at least 230 mg / mL, or more. The present disclosure also provides a frozen preparation produced by storing the liquid composition of the present disclosure at a temperature below 0°C. In an exemplary embodiment, the frozen preparation is not a lyophilized or freeze-dried preparation.

[0040] Advantageously, the liquid composition prepared by the method is storage stable and / or the liquid composition is stable to one or more freeze-thaw cycles. In various embodiments, after storage at a temperature of 0° C. or less, the liquid composition comprises at least 95% of the oligonucleotide compound as measured by HPLC. In various embodiments, after storage at a temperature of 0° C. or less for at least about 1 week, at least about 2 weeks, or at least about 4 weeks, the liquid composition comprises at least 95% of the oligonucleotide compound as measured by HPLC. In illustrative examples, after storage at a temperature of 0° C. or less for at least about 1 week, or about 2 weeks, or about 4 weeks, and subsequently thawed to 2-8° C., the liquid composition comprises at least 95% of the oligonucleotide compound as measured by HPLC. In illustrative examples, after one or more freeze-thaw cycles, the liquid composition comprises at least 95% of the oligonucleotide compound as measured by HPLC.

[0041] In various aspects, the oligonucleotide compound of the liquid composition has not been subjected to lyophilization or freeze-drying. In aspects of the liquid composition of the present disclosure, the oligonucleotide compound has not been lyophilized or freeze-dried. The liquid composition is not a reconstituted lyophilized oligonucleotide compound drug substance or drug product. Optionally, throughout the manufacturing process, the oligonucleotide compound is always in solution or frozen solution.

[0042] Manufacturing method The present disclosure further provides a method for preparing a solution drug comprising an oligonucleotide compound, i.e., a solution API (a solution of an oligonucleotide API). In an exemplary embodiment, the preparation method disclosed herein does not include any lyophilization. In an exemplary example of the preparation method disclosed herein, the API is not subjected to lyophilization. In an exemplary embodiment, the method includes carrying out the method disclosed herein to obtain a high-concentration liquid composition comprising more than about 150 mg / mL of an oligonucleotide compound, formulating the high-concentration liquid composition with a pharma- ceutically acceptable excipient, and filling the formulated high-concentration liquid composition into a container. The pharma- ceutically acceptable excipient can be any of the excipients described in The Handbook of Pharmaceutical Excipients, Third Edition, AH Kibbe (Pharmaceutical Press, London, UK, 2000) or Remington's Pharmaceutical Sciences, Sixteenth Edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980), each of which is incorporated by reference in its entirety.

[0043] In an exemplary embodiment, the method includes (a) preparing an oligonucleotide compound in a first solution containing one or more salts, the concentration of the oligonucleotide compound in the solution being 140 mg / mL or less, and the total salt concentration of the solution being about 25 mM to about 800 mM; and (b) concentrating the first solution by ultrafiltration to obtain a high concentration liquid composition containing an oligonucleotide compound at a concentration of about 150 mg / mL or more. In various aspects, the first solution is prepared by diafiltration with a DF solution. In various examples, the diafiltration achieves the exchange of a starting solution containing about 140 mg / mL or less of the oligonucleotide compound into a DF solution containing one or more salts and having a total salt concentration of 25 mM to about 800 mM. Thus, in an exemplary embodiment, a method for preparing a high-concentration liquid composition includes (a) exchanging the oligonucleotide compounds in a starting solution with a DF solution by diafiltration to obtain an intermediate solution, where the concentration of the oligonucleotide compounds in the starting solution is 140 mg / mL or less, and the DF solution includes one or more salts; and (b) concentrating the oligonucleotide compounds in the intermediate solution by ultrafiltration to obtain a high-concentration liquid composition, where the concentration of the oligonucleotide compounds in the high-concentration liquid composition is about 150 mg / mL or more. In an exemplary example, the total salt concentration of the DF solution is about 25 mM to about 800 mM. Optionally, the total salt concentration of the DF solution is about 25 mM to about 500 mM. In an exemplary example, the method includes continuous diafiltration and / or ultrafiltration by tangential flow filtration. In various aspects, the method uses polyethersulfone (PES) membranes or stabilized cellulose membranes for diafiltration and / or ultrafiltration. Optionally, the membrane has a molecular weight cut off (MWCO) of less than 10 kDa, and in illustrative examples, the MWCO is about 5 kDa or about 3 kDa.

[0044] In an exemplary embodiment, the method includes concentrating a first solution comprising a low concentration of oligonucleotide compounds by ultrafiltration to obtain a second solution comprising a high concentration of oligonucleotide compounds, and the oligonucleotide compound concentration of the second solution is greater than about 150 mg / mL, greater than about 160 mg / mL, greater than about 170 mg / mL, greater than about 180 mg / mL, greater than about 190 mg / mL, greater than about 200 mg / mL, greater than about 210 mg / mL, or greater than about 220 mg / mL. In an exemplary embodiment, the oligonucleotide compound concentration of the first solution is less than about 140 mg / mL, less than about 130 mg / mL, less than about 120 mg / mL, less than about 110 mg / mL, less than about 100 mg / mL, less than about 90 mg / mL, less than about 80 mg / mL, less than about 70 mg / mL, less than about 60 mg / mL, or less than about 50 mg / mL. In various embodiments, the second solution is the retentate obtained during ultrafiltration. In various embodiments, the retentate is obtained after 1 hour or 2 hours of ultrafiltration. In an exemplary embodiment, the oligonucleotide compound is double-stranded. In an exemplary embodiment, the total salt concentration of the first solution is about 25 mM to about 800 mM. Optionally, the total salt concentration of the first solution is about 25 mM to about 500 mM or about 25 mM to about 250 mM. In various embodiments, the first solution contains only one salt. Optionally, the one salt is an inorganic salt, such as any of the inorganic salts described herein. In various embodiments, the first solution does not contain any acetate salt. In an exemplary embodiment, the concentration method is performed after diafiltration with a DF solution. In various embodiments, diafiltration is performed with a DF solution containing one or more salts, and the total salt concentration of the DF solution is about 25 mM to about 800 mM. Optionally, the total salt concentration of the DF solution is about 25 mM to about 500 mM or about 25 mM to about 250 mM. In an exemplary embodiment, the concentration method is performed prior to diafiltration with the DF solution. In various examples, the oligonucleotide compound is a double-stranded oligonucleotide compound, optionally a siRNA.

[0045] Further provided herein is a method for preparing a solution API comprising an oligonucleotide compound API, wherein the oligonucleotide compound API is present in the solution at a concentration of more than 150 mg / mL. In various aspects, the oligonucleotide compound API is double-stranded, optionally siRNA. In an exemplary embodiment, the method for preparing a solution API comprises the method disclosed herein for concentrating a first solution comprising a low concentration of an oligonucleotide compound by ultrafiltration to obtain a second solution comprising a high concentration of an oligonucleotide compound. In various examples, the method for preparing a solution API comprises (i) synthesizing an oligonucleotide compound or a strand thereof, for example by solid phase synthesis; (ii) performing one or more rounds of chromatography, diafiltration and annealing; and (iii) concentrating by ultrafiltration according to the method disclosed herein for concentrating a first solution comprising a low concentration of an oligonucleotide compound by ultrafiltration to obtain a second solution comprising a high concentration of an oligonucleotide compound. In various embodiments, the oligonucleotide compound concentration of the second solution is greater than about 150 mg / mL, greater than about 160 mg / mL, greater than about 170 mg / mL, greater than about 180 mg / mL, greater than about 190 mg / mL, greater than about 200 mg / mL, greater than about 210 mg / mL, greater than about 220 mg / mL, and the oligonucleotide compound concentration of the first solution is less than about 140 mg / mL, less than about 130 mg / mL, less than about 120 mg / mL, less than about 110 mg / mL, less than about 100 mg / mL, less than about 90 mg / mL, less than about 80 mg / mL, less than about 70 mg / mL, less than about 60 mg / mL, or less than about 50 mg / mL.In various embodiments, the second solution is the retentate obtained during ultrafiltration.In various embodiments, the retentate is obtained after 1 hour or 2 hours of ultrafiltration. In the exemplary embodiment of the method for preparing solution API comprising oligonucleotide compound API, oligonucleotide API is not lyophilized at any point during the method.Advantageously, the method for preparing solution API is a method that does not have any lyophilization.Therefore, the method for preparing solution API of the present disclosure does not have lyophilization, providing a more time-efficient and cost-effective method for preparing oligonucleotide compound API.In various examples, the solution API prepared by the method disclosed herein is directly used to prepare a solution DP that includes an oligonucleotide compound API. Optionally, the solution API is sterile filtered and filled into a vial or a prefilled syringe or an automatic injection device. Thus, a method is provided for producing a DP, such as a solution DP, that includes a solution API that is composed of an oligonucleotide compound API. In various embodiments, the method for producing a DP does not include any lyophilization. In various embodiments of the method for producing a DP, lyophilization of the oligonucleotide compound API is not performed at any point during the method.

[0046] In various examples, the manufacturing method complies with the current good manufacturing practice (cGMP) recommended by the US Food and Drug Administration. Thus, the manufacturing method disclosed herein results in a cGMP grade drug containing a high concentration (greater than about 150 mg / mL) of oligonucleotide compound, and the drug is a liquid composition. In various aspects, the manufacturing method includes carrying out the method disclosed herein to obtain a cGMP grade high concentration liquid composition containing more than about 150 mg / mL of oligonucleotide compound. In an exemplary aspect, the drug is a clinical grade high concentration liquid composition containing more than about 150 mg / mL of oligonucleotide compound. In an embodiment of the manufacturing method disclosed herein, the oligonucleotide compound is not subjected to lyophilization or freeze drying. In an embodiment of the manufacturing method disclosed herein, the oligonucleotide compound is not lyophilized or freeze dried. Optionally, throughout the manufacturing method, the oligonucleotide compound is always in solution or frozen solution.

[0047] In various embodiments, the oligonucleotide compound is a double-stranded (ds) oligonucleotide compound comprising an antisense strand and a sense strand. In various embodiments, the method of manufacture includes annealing the sense strand and the antisense strand to obtain a ds oligonucleotide compound, and then preparing a high-concentration liquid composition as described herein by (i) exchanging the ds oligonucleotide compound in the starting solution with a diafiltration (DF) solution by diafiltration to obtain an intermediate solution, the concentration of the oligonucleotide compound in the starting solution being 140 mg / mL or less, and the DF solution comprising one or more salts, and the total salt concentration of the DF solution being about 25 mM to about 800 mM, optionally less than 500 mM, and (ii) concentrating the ds oligonucleotide compound in the intermediate solution by ultrafiltration to obtain a high-concentration liquid composition.

[0048] In various aspects, the method further comprises completing the final retentate, and optionally filling the completed final retentate into a container. In various aspects, the method further comprises sterile filtering the drug substance prior to storage. In various examples, the method is completely devoid of lyophilization of the oligonucleotide compound.

[0049] Oligonucleotides and Oligonucleotide Compounds As used herein, oligonucleotide compounds refer to a class of biochemical entities that include one or more oligonucleotides, where an oligonucleotide is an oligomer or polymer of nucleotides or modified nucleotides. For example, an oligonucleotide may include ribonucleotides, deoxyribonucleotides, modified nucleotides, or a combination thereof. Optionally, an oligonucleotide includes nucleotides or modified nucleotides linked to each other by phosphodiester bonds and / or modified internucleotide bonds. In various examples, an oligonucleotide compound includes only one oligonucleotide, while in other examples, an oligonucleotide compound includes two, three, four or more oligonucleotides. An oligonucleotide can be, for example, a duplex, triplex, or quadruplex, including two, three, and four oligonucleotides, respectively. In various aspects, an oligonucleotide compound includes two or more oligonucleotides, each linked to one by a non-covalent bond. In various aspects, an oligonucleotide compound is a duplex that includes the oligonucleotide of its complementary strand, where each oligonucleotide is linked to the other via hydrogen bonds. In various examples, an oligonucleotide compound is single-stranded or double-stranded.

[0050] The oligonucleotide component of the oligonucleotide compound can be several nucleotides long, up to several hundred nucleotides long, for example, about 10 nucleotides to about 300 nucleotides long, about 12 nucleotides to about 100 nucleotides long, about 15 nucleotides to about 250 nucleotides long, about 20 nucleotides to about 80 nucleotides long, about 15 nucleotides to about 30 nucleotides long, about 18 nucleotides to about 26 nucleotides long, or about 19 nucleotides to about 23 nucleotides long. In some embodiments, the oligonucleotide component of the oligonucleotide compound used in the method of the invention is about 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides long. In one embodiment, the oligonucleotide is about 19 nucleotides long. In another embodiment, the oligonucleotide is about 20 nucleotides long. In yet another embodiment, the oligonucleotide is about 21 nucleotides long. In yet another embodiment, the oligonucleotide is about 23 nucleotides long.

[0051] The oligonucleotide compound may be a natural oligonucleotide isolated from a cell or organism. For example, the oligonucleotide compound may be derived from genomic DNA or a fragment of genomic DNA, particularly telomeric or promoter regions, or may be derived from messenger RNA (mRNA) or a fragment of mRNA, particularly 5' or 3' untranslated regions. In some embodiments, the oligonucleotide compound is a synthetic oligonucleotide compound produced by chemical synthesis or in vitro enzymatic methods. In some embodiments, the oligonucleotide is a single-stranded RNA or DNA. In some embodiments, the oligonucleotide compound is a small hairpin RNA (shRNA), a precursor miRNA (pre-miRNA), an anti-miRNA oligonucleotide (e.g., antagomir and antimiR), or an antisense oligonucleotide. In other embodiments, the oligonucleotide compound is a double-stranded RNA molecule or an RNA interference agent, such as a small interfering RNA (siRNA), a microRNA (miRNA), or a miRNA mimic.

[0052] In certain embodiments, the oligonucleotide compound is a therapeutic oligonucleotide designed to target a gene or RNA molecule associated with a disease or disorder. For example, in one embodiment, the oligonucleotide compound is an antisense oligonucleotide (ASO) that comprises a single-stranded sequence that is complementary to a region of a target gene or mRNA sequence. A first sequence is "complementary" to a second sequence if the oligonucleotide comprising the first sequence can hybridize to the oligonucleotide comprising the second sequence under certain conditions to form a double-stranded region. "Hybridize" or "hybridization" typically refers to the pairing of complementary polynucleotides via hydrogen bonds (e.g., Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonds) between complementary bases in two oligonucleotides. A first sequence is considered to be fully complementary (100% complementary) to a second sequence if the oligonucleotide comprising the first sequence base pairs with the oligonucleotide comprising the second sequence over the entire length of one or both nucleotide sequences without any mismatches.

[0053] In another embodiment, the oligonucleotide compound is an siRNA or other type of double-stranded RNA interference agent, which comprises an antisense strand and a sense strand, where the antisense strand comprises a sequence that is complementary to a region of a target gene or mRNA sequence. The strand of an siRNA or other type of double-stranded RNA interference agent that comprises a region that has a sequence that is complementary to a target sequence (e.g., a target mRNA) is referred to as "antisense strand". "Sense strand" refers to the strand that comprises a region that is complementary to a region of the antisense strand. In some embodiments, the sense strand can comprise a sequence identical to a region of a target sequence or mRNA sequence.

[0054] The oligonucleotide compound may contain one or more modified nucleotides. "Modified nucleotide" refers to a nucleotide that has one or more chemical modifications at the nucleoside, nucleobase, pentose ring or phosphate group. Such modified nucleotides can include, but are not limited to, nucleotides with 2' sugar modifications (such as 2'-O-methyl, 2'-methoxyethyl, 2'-fluoro, deoxynucleotides), abasic nucleotides, inverted nucleotides (3'-3' linked nucleotides), phosphorothioate linked nucleotides, nucleotides with bicyclic sugar modifications (such as LNA, ENA) and nucleotides that contain base analogs (such as universal bases, 5-methylcytosine, pseudouracil, etc.).

[0055] In certain embodiments, modified nucleotides have modifications of the ribose sugar. These sugar modifications can include modifications of the 2' and / or 5' positions of the pentose ring as well as bicyclic sugar modifications. A 2'-modified nucleotide refers to a nucleotide having a pentose ring that includes a substituent at the 2' position other than OH. Such 2' modifications include 2'-H (e.g., deoxyribonucleotides), 2'-O-alkyl (e.g., O-C1-C2-alkyl ... 10 Or O-C1~C 10Modifications at the 5' position of the pentose ring include, but are not limited to, 5'-methyl (R or S), 5'-vinyl and 5'-methoxy. A "bicyclic sugar modification" refers to a modification of the pentose ring in which two atoms of the ring are linked by a bridge to form a second ring, resulting in a bicyclic sugar structure. In some embodiments, a bicyclic sugar modification comprises a bridge between the 4' and 2' carbons of the pentose ring. Nucleotides containing a sugar moiety having a bicyclic sugar modification are referred to herein as bicyclic nucleic acids or BNAs. Exemplary bicyclic sugar modifications include α-L-methyleneoxy (4'-CH2-O-2') bicyclic nucleic acids (BNAs); β-D-methyleneoxy (4'-CH2-O-2') BNAs (also referred to as locked nucleic acids or LNAs); ethyleneoxy (4'-(CH2)2-O-2') BNAs; aminooxy (4'-CH2-ON(R)-2') BNAs; oxyamino (4'-CH2-N(R)-O-2') BNAs; methyl(methyleneoxy) (4'-CH(CH3)-O-2' ) BNAs (also referred to as constrained ethyl or cEt); methylene-thio (4'-CH2-S-2') BNAs; methylene-amino (4'-CH2-N(R)-2') BNAs; methyl carbocyclic (4'-CH2-CH(CH3)-2') BNAs; propylene carbocyclic (4'-(CH2)3-2') BNAs; and methoxy(ethyleneoxy) (4'-CH(CH2OMe)-O-2') BNAs (also referred to as constrained MOE or cMOE). These and other sugar modified nucleotides that can be incorporated into oligonucleotide compounds are described in U.S. Pat. No. 9,181,551, U.S. Patent Application Publication No. 2016 / 0122761, and Deleavey and Damha, Chemistry and Biology, Vol. 19:937-954, 2012, all of which are incorporated herein by reference in their entireties.

[0056] In some embodiments, the oligonucleotide compound comprises one or more 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-allyl modified nucleotides, bicyclic nucleic acids (BNAs), or combinations thereof. In certain embodiments, the oligonucleotide compound comprises one or more 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, or combinations thereof. In one particular embodiment, the oligonucleotide compound comprises one or more 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, deoxynucleotides, or combinations thereof. In another particular embodiment, the oligonucleotide compound comprises one or more 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, or combinations thereof.

[0057] The oligonucleotide compound may also include one or more modified internucleotide linkages. As used herein, the term "modified internucleotide linkage" refers to an internucleotide linkage other than the natural 3'-5' phosphodiester linkage. In some embodiments, the modified internucleotide linkage is a phosphorus-containing internucleotide linkage, such as phosphotriester, aminoalkylphosphotriester, alkylphosphonate (e.g., methylphosphonate, 3'-alkylenephosphonate), phosphinate, phosphoramidate (e.g., 3'-aminophosphoramidate and aminoalkylphosphoramidate), phosphorothioate (P=S), chiral phosphorothioate, phosphorodithioate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester and boranophosphate. In one embodiment, the modified internucleotide linkage is a 2'-5' phosphodiester linkage. In other embodiments, the modified internucleotide linkage is a non-phosphorus-containing internucleotide linkage, and thus may be referred to as a modified internucleoside linkage. Such non-phosphorus-containing linkages include, but are not limited to, morpholino linkages (formed in part from the sugar portion of the nucleoside); siloxane linkages (-O-Si(H)2-O-); sulfide, sulfoxide and sulfone linkages; formacetyl and thioformacetyl linkages; alkene-containing backbones; sulfamate backbones; methylenemethylimino (-CH2-N(CH3)-O-CH2-) and methylenehydrazino linkages; sulfonate and sulfonamide linkages; amide linkages; and others having mixed N, O, S and CH2 component moieties. In one embodiment, the modified internucleoside linkage is a peptide-based linkage (e.g., aminoethylglycine) to generate peptide nucleic acids or PNAs as described in U.S. Pat. Nos. 5,539,082, 5,714,331 and 5,719,262.Other suitable modified internucleotide and internucleoside linkages that can be incorporated into oligonucleotide compounds are described in U.S. Pat. No. 6,693,187, U.S. Pat. No. 9,181,551, U.S. Patent Application Publication No. 2016 / 0122761, and Deleavey and Damha, Chemistry and Biology, Vol. 19:937-954, 2012, all of which are incorporated by reference in their entireties.

[0058] In certain embodiments, the oligonucleotide compound comprises one or more phosphorothioate internucleotide linkages. The oligonucleotide compound may comprise 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages. In some embodiments, all of the internucleotide linkages in the oligonucleotide compound are phosphorothioate internucleotide linkages. In other embodiments, the oligonucleotide compound may comprise one or more phosphorothioate internucleotide linkages at the 3'-terminus, the 5'-terminus, or both the 3'-terminus and the 5'-terminus. For example, in certain embodiments, the oligonucleotide compound comprises about 1 to about 6 or more (e.g., about 1, 2, 3, 4, 5, 6 or more) consecutive phosphorothioate internucleotide linkages at the 3'-terminus. In other embodiments, the oligonucleotide compound comprises about 1 to about 6 or more (e.g., about 1, 2, 3, 4, 5, 6 or more) consecutive phosphorothioate internucleotide linkages at the 5'-terminus.

[0059] The oligonucleotide compounds used in the methods of the present invention can be easily made using techniques known in the art, for example, using conventional solid-phase nucleic acid synthesis. Oligonucleotides can be constructed on a suitable nucleic acid synthesizer utilizing standard nucleotide or nucleoside precursors (e.g., phosphoramidites). Automated nucleic acid synthesizers are commercially available from several vendors, including the DNA / RNA synthesizer from Applied Biosystems (Foster City, CA), the MerMade synthesizer from BioAutomation (Irving, TX), and the OligoPilot synthesizer from GE Healthcare Life Sciences (Pittsburgh, PA). Oligonucleotides can be synthesized via phosphoramidite chemistry using 2' silyl protecting groups with acid-labile dimethoxytrityl (DMT) at the 5' position of the ribonucleoside. Final deprotection conditions that do not significantly degrade the RNA product are known. All syntheses can be performed on large, medium, or small scale by any automated or manual synthesizer. Synthesis can also be performed in multiple well plates, columns, or glass slides. The 2'-O-silyl group can be removed by exposure to fluoride ions, which can include any source of fluoride ions, such as salts containing fluoride ions paired with inorganic counterions, such as cesium fluoride and potassium fluoride, or salts containing fluoride ions paired with organic counterions, such as tetraalkylammonium fluoride. The deprotection reaction can utilize crown ether catalysts in combination with inorganic fluorides. The preferred fluoride ion source is tetrabutylammonium fluoride or aminohydrofluoride (e.g., mixing aqueous HF with triethylamine in a dipolar aprotic solvent such as dimethylformamide). The various synthetic steps can be carried out in an alternative order or sequence to obtain the desired compound.Other synthetic chemistry transformations, protecting groups (e.g., for hydroxyl, amino, etc. present in bases) and protecting group techniques (protection and deprotection) useful in the synthesis of oligonucleotides are known in the art and include, for example, those described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P.G.M. Huts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) and subsequent editions thereof.

[0060] As can be understood by those skilled in the art, further methods of synthesizing oligonucleotide compounds will be apparent to those skilled in the art. For example, oligonucleotides can be synthesized using enzymes in an in vitro system, such as those described in Jensen and Davis, Biochemistry, Vol. 57: 1821-1832, 2018. Natural oligonucleotides can be isolated from cells or organisms using conventional methods. Custom synthesis of oligonucleotides is also available from several suppliers, including Dharmacon, Inc. (Lafayette, CO), AxoLabs GmbH (Kulmbach, Germany) and Ambion, Inc. (Foster City, CA).

[0061] In various aspects, the oligonucleotide compound comprises or consists of the sequence 5'-UCGUAUAACAAUAAGGGGCUG-3' (SEQ ID NO: 2). In some such embodiments, the oligonucleotide compound comprises or consists of a sequence of modified nucleotides according to the sequence 5'-usCfsgUfaUfaacaaUfaAfgGfgGfcsUfsg-3' (SEQ ID NO: 4), where a, g, c and u are 2'-O-methyl adenosine, 2'-O-methyl guanosine, 2'-O-methyl cytidine and 2'-O-methyl uridine, respectively; Af, Gf, Cf and Uf are 2'-deoxy-2'-fluoro ("2'-fluoro") adenosine, 2'-fluoro guanosine, 2'-fluoro cytidine and 2''-fluoro uridine, respectively; and s is a phosphorothioate linkage. In various examples, the complementary oligonucleotide of the oligonucleotide compound comprises or consists of the sequence 5'-CAGCCCCUUAUUGUUAUACGA-3' (SEQ ID NO:1). In related embodiments, the complementary oligonucleotide comprises or consists of a sequence of modified nucleotides according to the sequence 5'-csagccccuUfAfUfuguuauacgs(invdA)-3' (SEQ ID NO:3), where a, g, c and u are 2'-O-methyl adenosine, 2'-O-methyl guanosine, 2'-O-methyl cytidine and 2'-O-methyl uridine, respectively; Af, Gf, Cf and Uf are 2'-deoxy-2'-fluoro ("2'-fluoro") adenosine, 2'-fluoro guanosine, 2'-fluoro cytidine and 2'-fluoro uridine, respectively; invdA is an inverted deoxyadenosine (a 3'-3' linked nucleotide) and s is a phosphorothioate linkage. In various examples, the oligonucleotide compound is comprised of a duplex comprising a sense strand and an antisense strand.

[0062] In some embodiments, the oligonucleotide compound may include additional components other than the oligonucleotide component. For example, the oligonucleotide component of the oligonucleotide compound may be covalently attached to a sugar, polymer, amino acid, fatty acid, cholesterol moiety, vitamin, steroid, folate moiety, peptide, polypeptide, or protein. In certain embodiments, the oligonucleotide component of the oligonucleotide compound is covalently attached to a targeting moiety that delivers the oligonucleotide compound to a specific tissue or cell type. The targeting moiety may include an antibody or antigen-binding fragment thereof that specifically binds to a receptor expressed on the surface of the target cell type (e.g., hepatocyte). Alternatively, the targeting moiety may include a ligand of a receptor expressed on the surface of the target cell or tissue to which the oligonucleotide compound is delivered. In one such embodiment, the targeting moiety includes a ligand of the asialoglycoprotein receptor (ASGPR) expressed on the surface of hepatocytes. The ligand of the ASGPR may include galactose, galactosamine, or N-acetyl-galactosamine (GalNAc). In certain embodiments, the ASGPR ligand includes a multivalent galactose or GalNAc moiety, such as a trivalent or tetravalent galactose or GalNAc moiety. Such multivalent galactose and multivalent GalNAc moieties are known to those of skill in the art. An exemplary GalNAc moiety that can be covalently attached to an oligonucleotide compound used in the methods of the invention is shown as R1 in Figure 3.

[0063] In certain embodiments, the oligonucleotide compound comprises a sense strand comprising a sequence of modified nucleotides according to SEQ ID NO: 3 and an antisense strand comprising a sequence of modified nucleotides according to SEQ ID NO: 4. The structure of an exemplary oligonucleotide compound is shown in FIG. 3 and further described in the Examples. In an exemplary example, the oligonucleotide compound is double-stranded and / or comprises a double helix structure. Optionally, the oligonucleotide compound is an siRNA. In various examples, the oligonucleotide is double-stranded. In various aspects, the oligonucleotide compound is comprised of a duplex comprising an antisense oligonucleotide and a sense oligonucleotide. In exemplary aspects, each of the antisense oligonucleotide and the sense oligonucleotide independently comprises at least 11 nt, optionally about 15 to about 30 nucleotides, about 18 to about 26 nucleotides, about 19 to about 23 nucleotides, or about 19 to about 21 nucleotides. Optionally, the oligonucleotide compound has a molecular weight of more than about 7,000 daltons, or more than about 10,000 daltons, or more than about 15,000 daltons. In certain embodiments, the oligonucleotide compound is a single-stranded antisense oligonucleotide. In such embodiments, the antisense oligonucleotide comprises from about 15 to about 25 nucleotides, from about 18 to about 22 nucleotides, or about 20 nucleotides.

[0064] In various aspects, the oligonucleotide compounds regulate gene expression through various processes including RNAi, targeted degradation by RNase H-mediated cleavage, splicing regulation, non-coding RNA inhibition, gene activation and programmed gene editing. Optionally, the oligonucleotide compound is any one of those described in Roberts et al., Nature Reviews Drug Discovery 19;673-694 (2020). In exemplary aspects, the oligonucleotide is an ASO. In various examples, the ASO is a single strand of about 18 to about 30 nucleotides. Optionally, the oligonucleotide compound is selected from the group consisting of mipomersen, pegaptanib, defibrotide, patisiran, custorisene, fomivirsen, oblimersen, eteplirsen, nusinersen, peracarsen, inotersen, givosiran, golodirsen and viltolarsen. Optionally, the oligonucleotide compound is any of those described in Table 2 of Roberts et al., 2020, supra, such as inclisiran, chibanisiran, miravirsen, GF012, TF-101, cobomarsen, lemlarsen, SLN124, MTL-CEPBA, subodirsen, fitusiran, lumasiran, butrisiran, levusiran, casimersen, tofersen, etc. In various embodiments, the oligonucleotide is a double-stranded siRNA, such as patisiran, givosiran, or olpaciran.

[0065] Treatment method Further provided is a method of treating a subject having a disease. In an exemplary embodiment, the method includes administering to a subject a liquid composition of the present disclosure in an amount effective to treat the disease of the subject. Optionally, the drug is administered by injection or infusion. As used herein, the term "treat" and its related terms do not necessarily mean 100% or complete treatment. Rather, there are various degrees of treatment that are recognized by those skilled in the art as having potential benefits or therapeutic effects. In this aspect, the treatment method of the present disclosure can provide any amount or level of treatment. Furthermore, the treatment provided by the method of the present disclosure can include treatment of one or more conditions or symptoms or signs of the disease being treated. The treatment provided by the method of the present disclosure can also include slowing the progression of the disease. For example, the method can treat the disease by reducing the signs and symptoms of the disease, stopping or slowing the progression of the disease, reducing the frequency of recurrence of the disease, delaying the onset of the disease, etc. In exemplary embodiments, the method treats by delaying the onset or recurrence of a disease by 1 day, 2 days, 4 days, 6 days, 8 days, 10 days, 15 days, 30 days, 2 months, 4 months, 6 months, 1 year, 2 years, 4 years or more.

[0066] In various aspects, the disease, condition or disorder treated or alleviated according to the method of the present invention is a disease, condition or disorder associated with abnormal target gene expression or activity, for example, where the gene product is overexpressed, resulting in a pathological phenotype.Exemplary target genes targeted by the oligonucleotide compounds contained in the liquid composition include, but are not limited to, LPA, PNPLA3, ASGR1, F7, F12, FXI, APOCIII, APOB, APOL1, TTR, PCSK9, SCAP, MARC1, KRAS, CD274, PDCD1, C5, ALAS1, HAO1, LDHA, ANGPTL3, SERPINA1, AGT, HAMP, LECT2, EGFR, VEGF, KIF11, AT3, CTNNB1, HMGB1, HIF1A and STAT3.Target genes can also include viral genes, such as hepatitis B and hepatitis C virus genes, human immunodeficiency virus genes, herpes virus genes, and the like.In some embodiments, the target gene is a gene encoding human microRNA (miRNA). In some embodiments, the disease is a cardiovascular disease, such as atherosclerosis. In other embodiments, the disease is cancer. In certain other embodiments, the disease is a liver disease, such as non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cirrhosis, etc. In various examples, the subject is an adult human.

[0067] The following examples are presented merely to illustrate the present invention and are not intended to limit the scope of the invention in any way. EXAMPLES

[0068] Example 1 This example describes the evaluation of different membranes used to concentrate a solution containing a drug substance by ultrafiltration / diafiltration (UF / DF).

[0069] The purpose of this study was to identify a membrane with a suitable molecular weight cut-off (MWCO) for concentrating double-stranded siRNA in the retentate. In this study, a double-stranded siRNA molecule was used as a model oligonucleotide compound. Each strand of the siRNA is 21 nucleotides long and contains chemically modified nucleotides. To selectively target the liver, a targeting ligand containing N-acetylgalactosamine is linked to the 5' end of the sense strand. The structure of the siRNA molecule is shown in Figure 3.

[0070] A lab-scale UF / DF system was used to buffer exchange (by diafiltration) and concentrate (by ultrafiltration) the siRNA samples. The system was equipped with one of several different membrane types as described in Table 1. A starting solution containing siRNA (30 mg / mL) and water was exchanged into a diafiltration (DF) containing 12 mM potassium phosphate dibasic (K2HPO4), 8 mM potassium phosphate monobasic (KH2PO4), 40 mM sodium chloride (NaCl) and water for injection. Ultraviolet analysis (260 nm) was performed to measure the concentration of siRNA duplexes in the final retentate. A summary of the membrane MWCO, siRNA duplex concentrations in the starting solution, DF solution and retentate are shown in Table 1. Observational notes from each experiment are also shown in Table 1.

[0071] [Table 1]

[0072] A 10 kDa filter membrane was selected because the siRNA molecules used in the experiments have a molecular weight of 16.3 kDa. However, after UF / DF, most of the siRNA was detected in the permeate, not in the retentate. Therefore, using a membrane with a 10 kDa MWCO did not seem to be a good option. Considering the above results, membranes with smaller MWCOs (e.g., MWCOs less than 10 kDa) were evaluated. Hydrosart membranes with MWCOs of 2 kDa or 5 kDa (each with dimensions of 20 cm 2) were analyzed. As shown in Table 1, the membrane with a 5 kDa MWCO resulted in a higher retentate siRNA concentration compared to the concentration achieved with the membrane with a 2 kDa MWCO. The 2 kDa MWCO membrane also showed a very slow flux (less than 4 mL / min). In both instances, the flux dropped to zero, which prevented further concentration of siRNA in the retentate. Another 5 kDa MWCO membrane (Sartocon Slice 200 Hydrosart membrane, dimension 100 cm 2 ) was evaluated. The retentate siRNA concentration achieved with this membrane was 109 mg / mL. The lower concentration was likely due to the smaller amount of starting solution used in this experiment compared to the amounts of starting solution used with the other 5 kDa MWCO membranes in Table 1. Collectively, the results of this study suggested that the 5 kDa MWCO membranes are most compatible with UF / DF of siRNA molecules.

[0073] In all cases, regardless of the membrane used, a flux reduction was observed that limited the enrichment of siRNA in the retentate. Without being bound to any particular theory, the flux reduction was the result of membrane fouling. To address this issue, further studies were performed.

[0074] Example 2 This example describes the effect of salt concentration of the diafiltration (DF) solution on siRNA concentration in the retentate after UF / DF.

[0075] To investigate the effect of the salt concentration of the DF solution on the siRNA concentration of the retentate, a series of UF / DF experiments were carried out to concentrate the siRNA drug substance solution. Briefly, a high-throughput small-scale UF / DF system equipped with an Ambré crossflow, 5 kDa MWCO Sartorius Hydrosart membrane was used. A starting solution (approximately 25 mL) containing 30 mg / mL of the same siRNA molecule used in Example 1 in water was fed into the UF / DF system. This starting solution was exchanged for 10 diavolumes of DF solution. The DF solutions were 1×PBS, 0.5×PBS or water. 1×PBS contained the following components: 2.67 mM potassium chloride (KCl), 1.47 mM monobasic potassium phosphate (KH2PO4), 137.93 mM sodium chloride (NaCl) and 8.06 mM dibasic sodium phosphate (Na2HPO4-7H2O). 0.5×PBS was made by diluting 1×PBS with an equal volume of water. After diafiltration, the supply of diafiltration solution was stopped and the ultrafiltration process was started to concentrate the siRNA in the retentate. The permeate solution was collected in a container separate from the retentate. The ultrafiltration process was completed when the retentate volume was reduced to approximately 5 mL or when the permeate flow stopped. The retentate was then collected and subjected to ultraviolet analysis (260 nm) to measure the siRNA duplex concentration. Table 2 summarizes the experimental conditions and results.

[0076] [Table 2]

[0077] As shown in Table 2, an increase in the salt concentration of the DF solution was associated with an increase in the siRNA concentration in the retentate. A concentration of approximately 150 mg / mL of siRNA molecules in the retentate was achieved using 1× PBS as the DF solution. The recovery of siRNA molecules in the final retentate was greater than 85%. Also, the flux was maintained for a longer duration when 1× PBS was used as the DF solution (compared to other DF solutions). A sudden increase in flux was observed when the DF solution was water. This sudden change in flux was indicative of membrane rupture caused by high membrane pressure / fouling. These results indicated that a higher salt concentration in the DF solution was associated with a higher siRNA concentration in the retentate and a longer duration for which membrane flux was maintained.

[0078] Example 3 This example describes the evaluation of PES membranes for use in UF / DF.

[0079] UF / DF was carried out essentially as described in Example 2, except that the membrane used was a 5 kDa Sartorius polyethersulfone (PES) membrane. The starting solution contained siRNA molecules (30 mg / mL) in water, and the DF solution contained 1×PBS. Under these conditions, the siRNA concentration in the retentate increased 5-fold from 30 mg / mL to 151.7 mg / mL. The recovery of siRNA molecules in the final retentate was greater than 85%. These results suggest that PES membranes such as stabilized cellulose with a MWCO of 5 kDa are also suitable for UF / DF of siRNA molecules.

[0080] Example 4 In this example, the effect of high salt concentration in the DF solution is described.

[0081] To further evaluate the correlation between the salt concentration of the DF solution and the siRNA concentration of the retentate, the test in Example 2 was performed using DF solutions composed of higher salt concentrations. The sodium chloride concentrations of the DF solutions examined in this study ranged from 40 mM to 690 mM. The DF solution composed of the highest sodium chloride concentration (5xPBS) was prepared by diluting with Dulbecco's 10x phosphate buffered saline (dPBS). The dPBS contained the following: 9.0 mM calcium chloride, 4.9 mM magnesium chloride, 26.7 mM potassium chloride, 14.7 mM potassium phosphate monobasic, 1379.3 mM sodium chloride, and 80.6 mM sodium phosphate dibasic. The DF solution composed of a medium sodium chloride concentration (2xPBS) was also made by diluting dPBS. The DF solution composed of the lowest sodium chloride concentration was a buffer solution containing 20 mM phosphate buffer and 40 mM sodium chloride.

[0082] In this study, a UF / DF system equipped with a PES membrane of 5 kDa MWCO was used. The starting solution contained 50 mg / mL ± 10 mg / mL siRNA (the same molecule as described in Example 1 and Figure 3) in water. Approximately five diafiltration volumes were performed to ensure complete exchange with the DF solution. Process parameters, including the transmembrane pressure (TMP) required to drive the ultrafiltration process, were kept constant throughout all experiments. The retentate siRNA concentration achieved in each experiment was measured using a UV spectrometer at 260 nm. The results are summarized in Table 3.

[0083] [Table 3]

[0084] The recovery of siRNA duplexes in the final retentate for each UF / DF was greater than 80%.

[0085] Figure 4 is a pair of graphs showing exemplary membrane flux (top) and transmembrane pressure (bottom) as a function of time during UF / DF when the DF solution is a buffer solution containing 20 mM phosphate buffer and 40 mM sodium chloride (red) or 1x PBS (blue). As shown in Figure 4, dramatic changes in flux and TMP were observed much earlier in the time course when the DF solution was a low salt concentration (60 mM total salt concentration of formulation buffer - red line) compared to a DF solution with a high salt concentration (150.2 mM total salt concentration of 1x PBS - blue line). This figure confirms that higher salt concentration of the DF solution is associated with a longer time that membrane flux is maintained.

[0086] As shown in Table 3, increasing salt concentration in the DF solution is associated with increasing retentate siRNA concentration. Each UF / DF run using a DF solution composed of a high salt concentration resulted in a retentate siRNA concentration of approximately 150 mg / mL or higher. Interestingly, the 2x PBS DF solution resulted in a higher retentate siRNA concentration than that achieved with the 5x PBS DF solution (approximately 194.3 mg / mL compared to 172.9 mg / mL). Without being bound to any particular theory, it is hypothesized that once the active functional groups of the negatively charged siRNA molecules are saturated by ionic interactions with cations, further increasing the salt concentration does not help to increase the retentate concentration, and the effect of the salt concentration in the DS solution plateaus. Taken together, these results suggest that, in general, higher salt concentrations in the DF solution result in higher retentate siRNA concentrations, and thus increasing the salt concentration in the DF solution is an effective approach to obtain highly concentrated solutions of oligonucleotide compounds.

[0087] Example 5 This example describes the evaluation of divalent cation salts in DF solutions.

[0088] In Examples 2 to 4, sodium chloride salt was present in a predominant amount compared to the amount of other salts present in the DF solution, so the salt concentration of the DF solution was based on the content of this salt. To evaluate the effect of the type of salt in the DF solution on the siRNA concentration in the retentate, a DF solution containing magnesium chloride, a divalent salt, in water was used. The UF / DF conditions were the same as those described in Example 4, except that the DF solution contained 138 mM magnesium chloride in water. The details of this experiment and the siRNA concentration of the resulting retentate are shown in Table 4.

[0089] [Table 4]

[0090] The recovery of siRNA duplexes in the final retentate was greater than 85%. As shown in Table 4, using a DF solution containing 138 mM divalent cation salt resulted in a higher retentate siRNA concentration (approximately 209.1 mg / mL). This retentate siRNA concentration was higher than that achieved with the same concentration of monovalent cation salt, sodium chloride (149.0 mg / mL; see Table 3). Taken together, these results support the positive effect of divalent salts on siRNA concentration during the ultrafiltration process.

[0091] Example 6 In this example, the evaluation of salt concentration in DF solution on the stability and structure of siRNA molecules is described.

[0092] To better understand the relationship between increasing siRNA concentration in the retentate and high salt concentration in the DF solution, selected physical properties of siRNA molecules were evaluated in the presence and absence of salt. The same siRNA molecules described in Example 1 and Figure 3 were used for these experiments. The unfolding temperature (T m ) and hydrodynamic diameter (d h) were measured using a differential scanning calorimeter (DSC) and a Malvern Mastersizer, respectively. The results are shown in Figures 5 and 6. As shown in Figure 5, the unfolding temperature increased with increasing salt concentration. Since the unfolding temperature is an indication of the energy required to denature the siRNA duplex, these data suggested that the stability of the siRNA duplex increased with increasing salt concentration in the DF solution.

[0093] As shown in Figure 6, increasing salt concentration (up to about 600 mM) correlates with an increase in hydrodynamic diameter, suggesting that salt interacts with the siRNA molecule in a way that increases the hydrodynamic diameter and gives rise to a structure that differs from that of the siRNA molecule in the absence of any salt. Without being bound to a particular theory, it is hypothesized that the different structure of the siRNA duplex in the presence of salt allows for improved flux through the membrane, resulting in a higher retentate siRNA concentration.

[0094] Example 7 This example describes the evaluation of various inorganic salt solutions for preparing highly concentrated oligonucleotide compound solutions.

[0095] The results of the inventors' previous tests support that a retentate containing a high concentration (greater than 150 mg / mL) of oligonucleotide compounds can be obtained using a salt solution consisting of a total salt concentration of about 138 mM to about 850 mM as the DF solution in UFDF. To better understand the types of salts that can be used for this purpose, solutions containing different inorganic salts were made and used to concentrate (by ultrafiltration) low concentration solutions of oligonucleotide compounds.

[0096] In this study, ultrafiltration and diafiltration were performed using a high-throughput liquid dispensing device equipped with a 3 kDa cellulose membrane. Starting solutions containing 200 mM inorganic salt and siRNA at concentrations less than 140 mg / mL were prepared by adding a small amount of siRNA stock solution to a solution containing inorganic salt. The final siRNA and final inorganic salt concentrations of each starting solution were 84 mg / mL and 200 mM, respectively. An aliquot of each starting solution was placed in a well of a multiwell plate and ultrafiltration was performed. Each starting solution was pressurized through a membrane placed at the bottom of each well, where water and inorganic salt were collected as permeate in a permeate container, leaving siRNA in the well. The volume of each well of the multiwell plate was monitored by a laser-based detector of the device, and if the detected volume was less than a preselected volume, the device was prompted to add DF solution to the well. The DF solution was identical to the starting solution without siRNA. The process continued until all samples reached the target volume. The experiment time was typically about 12 hours. The siRNA used in this study was the same siRNA described in Example 1 (hereafter referred to as "siRNA #1"). Samples were run in triplicate. siRNA concentrations 12 hours after the run were measured using a UV spectrometer at 260 nm. A summary of the solutions and the resulting siRNA concentrations (reported as the average of three data points) are summarized in Table 5. Standard deviations are shown in the right-most column.

[0097] [Table 5]

[0098] As shown in Table 5, each of the starting solutions containing 200 mM inorganic salt and 84 mg / mL siRNA was successfully concentrated to an siRNA concentration of over 150 mg / mL. Starting solutions containing alkali metal salts KCl and LiCl worked well for this purpose. Starting solutions containing sodium bromide also achieved high siRNA concentrations, suggesting that inorganic salts can have counterions other than chloride. Furthermore, ammonium chloride solutions worked well to achieve high siRNA concentration retentates, confirming that inorganic salts other than alkali metal salts can be used to concentrate siRNA.

[0099] The theoretical minimum concentration of each salt required to achieve a retentate oligonucleotide concentration of 150 mg / mL was calculated based on the results shown in Table 5. The theoretical minimum concentrations of KCl and LiCl were 83.3 mM and 111.7 mM, respectively, while the theoretical minimum concentrations of ammonium chloride and sodium bromide were 117.3 mM and 94.8 mM, respectively.

[0100] Example 8 This example describes the evaluation of inorganic salts containing divalent cations for preparing highly concentrated oligonucleotide compound solutions.

[0101] In previous studies (Example 5), a concentration of 209 mg / mL of oligonucleotide compound was achieved by using a DF solution composed of inorganic salts containing divalent cations at a concentration of 138 mM. To determine whether a high concentration of oligonucleotide retentate can be obtained using other inorganic salts containing divalent cations, a solution containing calcium chloride was prepared. The oligonucleotide compound used in this study was siRNA #1. Since low solubility of siRNA was observed in 200 mM calcium chloride, siRNA was prepared in 50 mM calcium chloride. Concentration of a starting solution containing 84 mg / mL siRNA in 50 mM calcium chloride was carried out essentially as described in Example 7. Samples were run in triplicate. The concentration of siRNA #1 after 12 hours of the experiment was measured using a UV spectrometer at 260 nm. An overview of the solutions and the resulting siRNA concentrations (reported as the average of three data points) are summarized in Table 6. Standard deviations are shown in the right-most column.

[0102] [Table 6]

[0103] As shown in Table 6, a starting solution containing 50 mM calcium chloride and 84 mg / mL siRNA was successfully concentrated to an siRNA concentration of over 150 mg / mL. Based on the results shown in Table 6, the theoretical minimum concentration of calcium chloride required to achieve an siRNA concentration of 150 mg / mL was calculated to be 31 mM. These results suggest that the total salt concentration of the DF solution could be as low as 31 mM to achieve a retentate containing approximately 150 mg / mL siRNA.

[0104] Example 9 This example describes the evaluation of organic salt solutions for preparing highly concentrated oligonucleotide compound solutions.

[0105] To determine whether a high concentration of oligonucleotide retentate can be obtained using organic salts, a starting solution containing low concentrations of siRNA was prepared using a solution containing choline chloride (CCl), tetramethylammonium chloride (TMACl) and benzyltrimethylammonium chloride (BTMACl). The siRNA used in this study was siRNA #1. Concentration of a starting solution containing 84 mg / mL siRNA in 200 mM CCl, TMACl or BTMACl was carried out essentially as described in Example 7. Samples were run in triplicate. siRNA concentrations 12 hours after the run were measured using a UV spectrometer at 260 nm. A summary of the solutions and the resulting siRNA concentrations (reported as the average of three data points) are summarized in Table 7. Standard deviations are shown in the right-most column.

[0106] [Table 7]

[0107] As shown in Table 7, only the CCl solution successfully concentrated siRNA to above 150 mg / mL. Based on the results shown in Table 7, the theoretical minimum concentration of each salt required to achieve a retentate oligonucleotide concentration of 150 mg / mL was calculated. The theoretical minimum concentrations of CCl, TMACl, and BTMACl were 102.9 mM, 460.0 mM, and 209.2 mM. This suggests that a higher concentration of BTMACl or TMACl could produce an siRNA concentration of 150 mg / mL.

[0108] Example 10 This example describes studies using different oligonucleotide compounds.

[0109] Previously, the experiments described herein were carried out using siRNA #1. In this study, a different siRNA (siRNA #2) was used. siRNA #2 was a double-stranded siRNA molecule that selectively targets patatin-like phospholipase domain-containing 3 (PNPLA3). The sense strand of siRNA #2 is 21 nucleotides long, and the antisense strand is 23 nucleotides long. Similar to siRNA #1, siRNA #2 contains some modified nucleotides and a targeting ligand that contains N-acetylgalactosamine linked to the 5' end of the sense strand to selectively target the liver.

[0110] A starting solution containing 80 mg / mL siRNA #2 and 200 mM NaCl was prepared, and the concentration of this solution was carried out essentially as described in Example 7. Samples were run in triplicate. siRNA concentrations were measured 12 hours after the run using a UV spectrometer at 260 nm. A summary of the solutions and the resulting siRNA concentrations (reported as the average of three data points) are summarized in Table 8. Standard deviations are shown in the right-most column.

[0111] [Table 8]

[0112] As shown in Table 8, the starting solution containing low concentrations of siRNA #2 was successfully concentrated to greater than 150 mg / mL. Based on the results shown in Table 8, the theoretical minimum concentration of each salt required to achieve a retentate oligonucleotide concentration of 150 mg / mL was calculated. The theoretical minimum concentration of NaCl was 88.3 mM. These results suggest that the total salt concentration of the DF solution could be as low as 88 mM to achieve a retentate containing approximately 150 mg / mL of siRNA.

[0113] Example 11 This example describes an exemplary method for preparing a highly concentrated liquid composition containing an oligonucleotide compound.

[0114] A high-concentration liquid composition containing double-stranded (ds) siRNA is prepared as follows: each of the sense and antisense strands is prepared by solid-phase synthesis. After the synthesized strands are cleaved from the solid support and the heterocyclic bases and backbone are deprotected, a first UF / DF is performed to concentrate the strands and exchange them into a new buffer suitable for chromatography. The buffer-exchanged strands are then subjected to chromatographic separation to remove impurities. A second UF / DF is performed to concentrate the strands and exchange them into a new buffer suitable for the strand annealing process. The sense and antisense strands are combined in the strand annealing process to obtain siRNA. The concentration of ds siRNA after the strand annealing process is typically in the range of 25 mg / mL to 50 mg / mL. A final UF / DF is performed to concentrate the double-stranded siRNA to a concentration of more than 150 mg / mL. This final UF / DF uses a diafiltration solution containing about 50 mM to about 150 mM salts containing divalent cations or about 75 mM to about 500 mM salts containing monovalent cations. The highly concentrated liquid composition containing ds siRNA is either frozen or subjected to a series of filtrations to reduce bioburden and sterilize before being filled into syringes, autoinjectors or vials. The filled syringes, autoinjectors or vials are then stored until shipment. A simplified diagram of the process is shown in Figure 7.

[0115] The above method does not involve a lyophilization step and therefore provides a less energy intensive and more cost-effective method for obtaining liquids, such as aqueous solutions, containing high concentrations of oligonucleotide compound drug substances.

[0116] [Table 9]

[0117] All references cited in this specification, including publications, patent applications, and patents, are herein incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and set forth in its entirety herein.

[0118] Use of the terms "a," "an," and "the" and similar referents with respect to the description of this disclosure (particularly with respect to the claims which follow) should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprise," "have," "include," and "containing" should be construed as open-ended terms including the specified components but not excluding other elements (i.e., meaning "including, but not limited to"), unless otherwise specified.

[0119] The recitation of ranges of values ​​herein, unless otherwise stated herein, is merely intended to serve as a shorthand method of referring individually to each of the separate values ​​falling within that range, and each of the endpoints, and each separate value and endpoint is incorporated herein as if it were individually set forth herein.

[0120] Unless otherwise indicated herein or clearly contradicted by context, all methods described herein can be performed in any suitable order. The use of any examples or representative language (e.g., "etc.") provided herein is intended merely to clarify the disclosure and does not impose limitations on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0121] Preferred embodiments of the disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. Variations of these preferred embodiments will become apparent to those skilled in the art upon reading the above description. The inventors anticipate that those skilled in the art will adopt such variations as necessary, and the inventors intend for the disclosure to be practiced in other forms than as specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements is encompassed in the disclosure in all possible variations thereof unless otherwise indicated herein or clearly contradicted by context.

Claims

1. 1. A method for preparing a highly concentrated liquid composition comprising an oligonucleotide compound, comprising: a. exchanging an oligonucleotide compound in a starting solution with a diafiltration (DF) solution by diafiltration to obtain an intermediate solution, wherein the concentration of the oligonucleotide compound in the starting solution is 140 mg / mL or less, the DF solution comprises one or more salts, and the total salt concentration of the DF solution is from about 25 mM to about 800 mM; b. concentrating the oligonucleotide compound in the intermediate solution by ultrafiltration to obtain a concentrated liquid composition, wherein the concentration of the oligonucleotide compound in the concentrated liquid composition is greater than about 150 mg / mL; A method comprising:

2. 10. The method of claim 1, wherein the DF solution comprises, consists essentially of, or consists of inorganic salts.

3. The method of claim 2 , wherein the inorganic salt comprises a monovalent or divalent cation and / or a halogen counterion.

4. 4. The method of claim 3, wherein the inorganic salt is sodium chloride, sodium bromide, potassium chloride, lithium chloride, or ammonium chloride.

5. 4. The method of claim 3, wherein the inorganic salt is magnesium chloride or calcium chloride.

6. The method of any one of claims 1 to 5, wherein the total salt concentration of the DF solution is from about 25 mM to about 500 mM.

7. 7. The method of claim 6, wherein the total salt concentration of the DF solution is from about 75 mM to about 300 mM or from about 75 mM to about 250 mM.

8. The DF solution is a) about 75 mM to about 300 mM sodium chloride, sodium bromide, lithium chloride, potassium chloride, or ammonium chloride; b) about 75 mM to about 150 mM magnesium chloride, or c) at least about 31 mM and less than 200 mM calcium chloride The method of claim 6, comprising:

9. The method of any one of claims 1 to 5, wherein the DF solution comprises a first salt and at least one other salt.

10. The concentration of the first salt is a) at least twice as high as the concentration of the other salt in the DF solution; b) at least 80% of the total salt concentration of the DF solution; or c) based on the concentration of the oligonucleotide compound present in the starting solution; 10. The method of claim 9.

11. 10. The method of claim 9, wherein the molar ratio of the oligonucleotide compound in the starting solution to the first salt in the DF solution is from about 1:2 to about 1:

100.

12. The method of claim 9 , wherein the first salt comprises a monovalent cation.

13. 13. The method of claim 12, wherein the monovalent cation is sodium.

14. 14. The method of claim 13, wherein the first salt is sodium chloride.

15. The method of claim 9 , wherein the first salt comprises a divalent cation.

16. 16. The method of claim 15, wherein the divalent cation is magnesium.

17. 17. The method of claim 16, wherein the first salt is magnesium chloride.

18. 6. The method of any one of claims 1 to 5, wherein the final retentate contains at least 80% of the amount of the oligonucleotide compounds in the starting solution and / or has a concentration of the oligonucleotide compounds at least 2-fold higher than the concentration of the oligonucleotide compounds in the starting solution.

19. 6. The method of any one of claims 1 to 5, further comprising completing or polishing the final retentate and / or collecting the final retentate and filling it into a container with an overall oligonucleotide recovery of greater than 80%.

20. The method of any one of claims 1 to 5, which does not involve lyophilization of the oligonucleotide compound.

21. The oligonucleotide compound comprises: a) is double-stranded or single-stranded; b) is an siRNA; c) is an antisense oligonucleotide (ASO); d) a double-stranded oligonucleotide compound comprising an antisense oligonucleotide and a sense oligonucleotide, wherein each of the antisense and sense oligonucleotides independently comprises at least 11 nt; or e) has a molecular weight greater than about 7,000 daltons; The method according to any one of claims 1 to 5.

22. 10. A method for producing a drug comprising an oligonucleotide compound, the method comprising the steps of: performing the method of any one of claims 1 to 5 to obtain a high-concentration liquid composition comprising at least 150 mg / mL of said oligonucleotide compound; formulating said high-concentration liquid composition with a pharmaceutically acceptable excipient; and filling said formulated high-concentration liquid composition into a container.

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