Method for preparing oligonucleotide compositions using ultrafiltration / dialysis filtration

Abstract

The present specification discloses a method for preparing a composition comprising oligonucleotides.The disclosed method comprises subjecting an aqueous solution of oligonucleotides to ultrafiltration / diafiltration (UF / DF) to form a retentate comprising oligonucleotides, and the ultrafiltration / diafiltration (UF / DF) is carried out using an aqueous buffer comprising one or more salts.The present specification also discloses the oligonucleotide-containing composition obtained by these methods.

Description

[Technical Field]

[0001] Cross-reference of related applications This application asserts the benefit as of the filing date of U.S. Provisional Application No. 62 / 979,687, filed on 21 February 2020 under Section 119 of the U.S. Patent Act, the entirety of which is incorporated herein by reference.

[0002] This application relates to biopharmaceutical technology in general, and more specifically to a method for preparing high-purity oligonucleotide compositions using a filtration technique that can control the salt content in the oligonucleotide composition. The method of this disclosure can combine the use of ultrafiltration / diafiltration (UF / DF) with lyophilization in a manner that does not require performing additional processing steps commonly used in the production of lyophilized (solid) active pharmaceutical ingredients (APIs). [Background technology]

[0003] Oligonucleotides are short-chain DNA or RNA oligomers that can be chemically synthesized for research and medical purposes. Oligonucleotides are typically prepared by stepwise adding nucleotide residues to generate a specific sequence. Following the completion of the synthesis of the oligonucleotide in the desired sequence, the target oligonucleotide is typically obtained as a mixture along with failed sequences and other process and product-related impurities.

[0004] The preparation of therapeutic oligonucleotides, including commercially available oligonucleotides approved for use by the FDA, is further complicated by stringent commercial specifications and formulation validation requirements. Appropriate purification and formulation techniques for therapeutic oligonucleotides must take into account the chemical composition and stability of the product, as well as the method of administration.

[0005] Therapeutic oligonucleotides are typically prepared using either an aqueous-based platform process or a lyophilized API platform process, depending on the required formulation. Lyophilized (solid) formulations are potentially preferred over liquid formulations for some products due to their stability profile, ease of storage, and ease of handling.

[0006] Spinraza® (nusinersen) is an antisense oligonucleotide (ASO) drug used to treat spinal muscular atrophy (SMA), a rare neuromuscular disorder. The commercially available Spinraza® formulation is a lyophilized API obtained from a solvent-intensive process. An aqueous-based platform process needs to be integrated, ultimately resulting in a liquid active pharmaceutical ingredient produced via ultrafiltration / diafiltration (UF / DF) using a lyophilized API with specific salt (e.g., sodium and acetate) content. This approach would minimize formulation validation and meet existing commercial specifications without requiring additional liquid reduction steps and / or equipment beyond the platform. [Overview of the project]

[0007] This disclosure describes a method for concentrating and buffer-exchanging oligonucleotides using ultrafiltration / dialysis filtration (UF / DF) to obtain an aqueous oligonucleotide solution suitable for lyophilization without additional (intervening) processing steps. Figure 1 illustrates how the method of this disclosure can be integrated into an aqueous-based platform process using UF / DF in a lyophilization API platform process, in a manner that typically eliminates the need to perform solvent-based precipitation preceding the lyophilization step.

[0008] In particular, the methods disclosed herein can control the sodium content before and after lyophilization and the acetate content after lyophilization in an oligonucleotide API to meet the specified sodium and acetate specifications. This is achieved by controlling the components (e.g., salts) in the UF / DF aqueous buffer. The methods described herein can also control the membrane permeate flux and the retained liquid concentration of the oligonucleotide while performing the UF / DF step within the transmembrane pressure (TMP) conditions recommended by the manufacturer.

[0009] One aspect of the present disclosure relates to a method for preparing a composition comprising an oligonucleotide, the method comprising subjecting an aqueous solution of the oligonucleotide to ultrafiltration / diafiltration (UF / DF) to form a retained liquid comprising the oligonucleotide, wherein the ultrafiltration / diafiltration (UF / DF) is performed using an aqueous buffer comprising one or more salts.

[0010] Another aspect of the present disclosure relates to a composition comprising an oligonucleotide, the composition being obtainable by one of the methods described herein.

[0011] In some embodiments, the composition is in the form of an aqueous solution comprising an oligonucleotide.

[0012] In some embodiments, the composition is in the form of a lyophilized composition comprising an oligonucleotide.

[0013] Additional objects, advantages, and other features of the present disclosure will in part be set forth in the description that follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned from the practice of the present disclosure. The advantages of the present disclosure may be realized and attained as particularly pointed out in the appended claims. As will be understood, the present disclosure is capable of other and different embodiments, and some of the details thereof are capable of modification in various obvious respects without departing from the present disclosure. In this regard, the description herein is to be regarded as illustrative in nature and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] [Figure 1] Describes an exemplary integration of a solvent-intensive process with the aqueous-based process of the present disclosure. [Figure 2] Graph showing the decline in permeate flux during buffer exchange when UF / DF is performed at different ammonium acetate concentrations. [Figure 3] Graph showing the DF buffer effect on the second steady-state permeate flux when the conductivity of the DF buffer increases. [Figure 4] Graph showing the maximum ASO concentration in the retention fluid with respect to the total concentration of acetate in the UF / DF buffer. [Figure 5] Graph showing the trend after lyophilization (post-freezing) of sodium and ammonium contents. [Figure 6] Graph showing the sodium (Na) content in the retention fluid after UF / DF with respect to the percentage (%) of sodium acetate (NaOAc) in the UF buffer containing different amounts of ammonium acetate (NH4OAc). [Figure 7] Photograph of an exemplary lyophilization chamber with a LyoGuard tray. [Figure 8] Photograph of an exemplary lyophilized ASO material in the tray and bag. [Figure 9] Graph showing how the permeate flux decreases as the concentration of oligonucleotide in the retention fluid increases when UF / DF is carried out in water. [Figure 10] Graph showing the mass percentage (%) of acetic acid (OAc) remaining in the solid API with respect to the total concentration of acetic acid (OAc) in the UF buffer. [Figure 11] Shows a comparison between an exemplary aqueous-based platform process and an exemplary lyophilized API platform process including an ethanol precipitation step. [Figure 12]This shows how sodium (Na+) and ammonium (NH4+) ions may occupy different counterion positions along the negatively charged phosphorothioate oligonucleotide skeleton. [Modes for carrying out the invention]

[0015] Disclosed herein are methods for integrating the use of ultrafiltration / diafiltration (UF / DF) into lyophilized API platform processes traditionally used to prepare the solid form of oligonucleotide active pharmaceutical ingredients (APIs). Embodiments of this disclosure include methods for preparing oligonucleotides for therapeutic applications, such as the antisense oligonucleotide Spinraza® (nusinersen).

[0016] Aqueous-based platform processes typically involve one or two chromatographic separation and deprotection steps, culminating in an ultrafiltration / diafiltration (UF / DF) step that concentrates the oligonucleotide of interest and performs buffer exchange to a liquid formulation suitable for intrathecal (IT) administration. This platform delivers the liquid API obtained from the UF / DF operation to a parenteral filling facility for final dilution, filtration, and filling. In contrast, lyophilized API platform processes often leverage solvent-based purification processes. Figure 11 illustrates the difference between an aqueous-based platform process used to prepare a ready-to-fill liquid form of Spinraza® (nusinersen) and a lyophilized API platform process used to prepare a lyophilized (solid) form of the product. As shown in Figure 11, the aqueous-based platform process for Spinraza® includes an ion exchange chromatography step followed by an ultrafiltration / diafiltration (UF / DF) step to obtain Spinraza® (nusinersen) in a ready-to-fill liquid form. In contrast, the lyophilization API platform process includes an ethanol precipitation step, followed by lyophilization and compounding, to obtain a formulation in lyophilized (solid) form.

[0017] It would be beneficial to modify the lyophilization API platform process currently used to prepare commercially available Spinraza® (nusinersene) by replacing the ethanol precipitation step with the UF / DF step used in the aqueous-based platform process. Firstly, for both environmental and regulatory reasons, it would be beneficial to eliminate the use of organic solvents in the final preparation of this commercial product. Secondly, since UF / DF can typically be used to precisely control the salt content of the processed product, a more precisely controlled salt content can be achieved by using UF / DF instead of solvent precipitation. However, as described below, the implementation limitations of UF / DF and large-scale lyophilization have made it impossible to successfully integrate these processes into the commercial-scale preparation of Spinraza® without including additional steps.

[0018] The difficulty in integrating UF / DF into large-scale lyophilization arises primarily from the salt and / or oligonucleotide content in the aqueous holding solution used by UF / DF. Spinraza® (nusinersen) requires a relatively low salt content (approximately 5% by weight sodium content in the lyophilized product), and attempting to perform UF / DF with an aqueous solution having such a low salt content (and low conductivity) results in a low permeation flux through the membrane. This low permeation flux is partly due to undesirable "caking" that forms on the holding solution surface of the UF / DF membrane. Low permeation flux reduces both the rate of oligonucleotide generation and the concentration of oligonucleotides in the holding solution. Minimal conductivity is required in the UF / DF buffer for UF / DF to operate correctly and reach the ideal ASO concentration for lyophilization. On the other hand, if the salt content of the UF / DF buffer is increased to a level necessary to enable a sufficiently high permeation flow rate (i.e., the concentration of oligonucleotides in the retention solution becomes sufficiently high), the resulting retention solution will contain too much salt, requiring an additional step(s) to remove the excess salt.

[0019] The method of this disclosure can overcome these problems by controlling the salt concentration and salt content in the buffer. This method integrates an aqueous purification process with a lyophilization step to create a solid API with predetermined specifications of sodium and acetate without adding additional steps and / or equipment. The UF / DF process can be determined by both the operability of the UF / DF operation (flux and concentration) and the composition of the solid API after the UF / DF product has been lyophilized. A UF / DF process is developed in which purification process intermediates, including not only the target oligonucleotide but also various molecular species involved in the purification process, are concentrated and treated by UF / DF so that the target sodium content is achieved and the acetate specifications are met after lyophilization. The UF / DF process of this disclosure achieves control of the total sodium content by controlling the average number of sodium cations occupying counterion positions along the negatively charged phosphorothioate or phosphorodiester oligonucleotide skeleton. The novel method also facilitates efficient operation of the UF / DF process by meeting the minimum membrane permeation flux and maximum retention solution concentration required for large-scale manufacturing processes.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. In case of any conflict, this specification shall prevail, including the definitions.

[0021] Unless otherwise specified, all percentages, parts, ratios, etc., are based on weight.

[0022] Where a quantity, concentration, or other value or parameter is given as a range, or as a list of upper and lower limits, this is understood to specifically disclose all ranges consisting of any pair of any upper and lower limits, regardless of whether the ranges are disclosed separately. Where numerical ranges are enumerated herein, unless otherwise specified, such ranges are intended to include their endpoints, as well as all integers and fractions within that range. The scope of this disclosure is not intended to be limited to any specific values ​​enumerated in the definition of a range.

[0023] The use of “a” or “an” in this specification to describe various elements and components is solely for convenience and to give a general meaning to this disclosure. This description should be read as including one or at least one, and the singular includes the plural unless it is clearly implied otherwise.

[0024] When a specific quantity or value is used, it should be understood that it should include a small deviation from that quantity or value, which a person skilled in the art would understand to be equivalent to or substantially the same as that quantity or value. In some embodiments, the specific quantity or value includes ±10% of the specific quantity or value. In some embodiments, the specific quantity or value includes ±5% of the specific quantity or value.

[0025] One aspect of the present disclosure relates to a method for preparing a composition comprising an oligonucleotide such as Spinraza® (nusinersen). The method comprises subjecting an aqueous solution of the oligonucleotide to ultrafiltration / dialysis (UF / DF) to form a retention solution comprising the oligonucleotide, the ultrafiltration / dialysis (UF / DF) being performed using an aqueous buffer containing one or more salts.

[0026] In some embodiments, one or more salts in the aqueous buffer can be formulated in a way that ultimately controls the composition of the retention solution. For example, in some embodiments, the aqueous buffer can be formulated to control the sodium content of the retention solution produced by UF / DF, thereby indirectly controlling the sodium content of the lyophilized product. Such control is made possible by the presence of multiple salts in the aqueous buffer, including sodium salts and antagonistic salts having cations different from sodium. Antagonistic salts include salts having different cations such as ammonium, dimethylammonium, trimethylammonium, potassium, lithium, rubidium, copper, silver, or other suitable monovalent cations. The antagonistic salts can be volatile salts, non-volatile salts, or combinations thereof.

[0027] In some embodiments, the use of at least one antagonistic salt allows the method of the present disclosure to control the average number of sodium cations occupying the counterion positions of the oligonucleotide backbone. Figure 12 shows sodium (Na) + ) ions and ammonium (NH4 + This shows how ions can occupy different counterion positions along the negatively charged phosphorothioate or phosphorodiester oligonucleotide skeleton. Thus, in some embodiments, the disclosure provides a method for controlling the salt content in an oligonucleotide holding solution produced by UF / DF by introducing one or more antagonistic salts into an aqueous buffer. In other words, the method of the disclosure comprises subjecting an aqueous solution of oligonucleotides to ultrafiltration / diafiltration (UF / DF) to form a holding solution containing the oligonucleotide, the ultrafiltration / diafiltration (UF / DF) being performed using an aqueous buffer containing a sodium salt and an antagonist salt. In some embodiments, the antagonist salt is a potassium salt. In other embodiments, the antagonist salt is an ammonium salt.

[0028] After buffer exchange occurs in an aqueous solution containing both sodium cations and antagonistic cations, the sodium cations and antagonistic cations reach equilibrium at the counterion positions of the oligonucleotide, resulting in the oligonucleotide in solution not being completely sodiumized, i.e., the oligonucleotide counterion positions not being completely occupied by sodium cations. See Figure 12. This equilibrium ratio can be expressed as follows:

number

[0029] The properties of antagonistic salts can affect not only the composition of the retaining liquid after the UF / DF process, but also the final composition of the solid product after freeze-drying. For example, if the antagonistic salt is a volatile salt, it is possible to reduce the total salt content of the freeze-dried product (relative to the total salt content of the retaining liquid) without affecting the sodium content.

[0030] In some embodiments, one or more salts in the aqueous buffer may include at least one volatile salt. The UF / DF process utilizing the volatile salts of this disclosure can achieve a maximum ASO concentration in the retention solution with a desired sodium content. Due to the acid-base properties of the volatile salts, it is possible to reduce the total salt content in the lyophilized product (compared to the total salt content in the retention solution) using volatile antagonistic salts. In volatile antagonistic salts, the volatile antagonistic cation of the salt exists in equilibrium with the corresponding volatile conjugate base. The volatility of the antagonistic cation species (neutral form with the corresponding conjugate base) allows for removal by sublimation during lyophilization.

[0031] Ammonium acetate (NH4OAc) is an example of a volatile antagonist salt used in some embodiments of this disclosure. As shown below, ammonium cation (NH4 + ) exists in equilibrium with ammonia (NH3), and acetate anion (AcO -) exists in equilibrium with acetic acid (AcOH). [ka]

[0032] In the equilibrium shown above, the protonated ammonium cation acts as a proton source for converting the acetate anion to acetic acid, which is volatile and can be removed by freeze-drying. Proton transfer from ammonium to acetate makes both species neutral and volatile, facilitating their removal during freeze-drying.

[0033] Other examples of volatile antagonistic salts include, for example, ammonium salts of formic acid, propionic acid, butyric acid, lactic acid, and carbonate.

[0034] By using volatile antagonist salts such as ammonium acetate as shown above, the retaining solution after UF / DF can then be freeze-dried in a manner that removes a considerable amount of volatile antagonist salt while maintaining the sodium content in the retaining solution. Therefore, it is possible to produce a freeze-dried oligonucleotide composition having a total salt content significantly lower than the total salt content in the retaining solution after UF / DF, while having a sodium content controlled based on the composition of the aqueous buffer, by using volatile antagonist salts. This feature allows the method of this disclosure to produce a solid oligonucleotide API with a predetermined sodium content while removing antagonist salts to trace amounts.

[0035] In some embodiments, the antagonist salt may be a non-volatile salt that is not removed by freeze-drying. For example, the aqueous buffer may contain sodium salts such as sodium acetate, sodium chloride, sodium bromide, or sodium iodide, and non-volatile antagonist salts such as potassium acetate, potassium chloride, potassium bromide, or potassium iodide. Other non-volatile antagonist salts include, for example, potassium salts, lithium salts (e.g., lithium acetate, lithium chloride, lithium bromide, or lithium iodide), rubidium salts (e.g., rubidium acetate, rubidium chloride, rubidium bromide, or rubidium iodide), copper salts (e.g., copper acetate, copper chloride, copper bromide, or copper iodide), and silver salts (e.g., silver acetate, silver chloride, silver bromide, or silver iodide).

[0036] In the method of the present disclosure, the composition of the aqueous buffer can be controlled to target a wide range of sodium content in the retention solution after UF / DF and in the product after lyophilization, from essentially zero sodium content to a sodium content far greater than the equivalent amount of fully sodiumized ASO.

[0037] In some embodiments, the aqueous buffer comprises at least one salt selected from sodium acetate, ammonium acetate, and potassium acetate. In some embodiments, the aqueous buffer comprises sodium acetate and ammonium acetate. In some embodiments, the aqueous buffer comprises sodium acetate and potassium acetate. In some embodiments, the aqueous buffer comprises sodium acetate, ammonium acetate, and potassium acetate.

[0038] In some embodiments, the sodium content (e.g., sodium concentration) in the oligonucleotide-containing retention solution is controlled by adjusting the ratio of at least one sodium salt to the total concentration of salts in the aqueous buffer. In other embodiments, the proportion of sodium cations occupying the counterion positions of oligonucleotides in the retention solution is controlled by adjusting the ratio of at least one sodium salt to the total concentration of salts in the aqueous buffer.

[0039] In some embodiments, the molar ratio of sodium salt to antagonistic salt in the aqueous buffer solution is in the range of 1:100 to 100:1, or 1:20 to 20:1, or 1:10 to 10:1, or 1:1 to 19:1, or 5:1 to 19:1, or 12:1 to 15:1, or 5:1 to 10:1, or 5:1 to 6:1, or 5:1 to 6:1.8.

[0040] In some embodiments, the aqueous buffer contains sodium acetate and ammonium acetate, and the molar ratio of sodium acetate to ammonium acetate in the aqueous buffer is in the range of 1:100 to 100:1, or 1:20 to 20:1, or 1:10 to 10:1, or 1:1 to 19:1, or 5:1 to 19:1, or 12:1 to 15:1, or 5:1 to 10:1, or 5:1 to 6:1, or 5:1 to 6:1.8. In some embodiments, the molar ratio of sodium acetate to ammonium acetate is 17:3. In some embodiments, the aqueous buffer contains 34 mM sodium acetate and 6 mM ammonium acetate.

[0041] In some embodiments, the aqueous buffer contains sodium acetate and potassium acetate, and the molar ratio of sodium acetate to potassium acetate in the aqueous buffer is in the range of 1:100 to 100:1, or 1:20 to 20:1, or 1:10 to 10:1, or 1:1 to 19:1, or 5:1 to 19:1, or 12:1 to 15:1, or 5:1 to 10:1, or 5:1 to 6:1, or 5:1 to 6:1.8. In some embodiments, the molar ratio of sodium acetate to potassium acetate is 17:3. In some embodiments, the aqueous buffer contains 34 mM sodium acetate and 6 mM potassium acetate.

[0042] In some embodiments, the pH of the aqueous buffer is in the range of 4.0 to 10.0, or 4.5 to 9.5, or 5.0 to 9.0, or 5.0 to 8.5, or 5.0 to 8.0, or 5.5 to 9.0, or 5.5 to 8.5, or 5.5 to 7.5, or 6.0 to 9.0, or 6.0 to 8.5, or 6.0 to 7.5, or 6.0 to 7.0, or 6.5 to 9.0, or 6.5 to 8.5, or 6.5 to 8.0, or 6.5 to 7.5, or 6.9 to 7.5.

[0043] In some embodiments, the aqueous buffer does not contain sodium salts, and therefore the retention solution after UF / DF is sodium-free. In other embodiments, the aqueous buffer does not contain any antagonistic salts.

[0044] In some embodiments, the method of the present disclosure may include the step of lyophilizing a UF / DF holding solution to produce a lyophilized composition containing a target oligonucleotide. Lipodrying can remove volatile UF / DF buffer components (e.g., volatile antagonist salts such as ammonium acetate). The lypodrying step may be carried out as a single lypodry or as multiple lypodryings performed in a single lypodrying apparatus or multiple lypodrying apparatuses.

[0045] In some embodiments, the proportion of one or more antagonistic salts in the lyophilized composition is less than the proportion of one or more antagonistic salts in the retaining solution after UF / DF. For example, as described above, volatile antagonistic salts such as ammonium acetate in the aqueous buffer can then be removed (partially or completely) during lyophilization.

[0046] The method of the present disclosure may also include the step of adjusting the pH of the retention solution after UF / DF before freeze-drying. In some embodiments, the pH of the retention solution is adjusted to a pH in the range of 5.0 to 9.0, or 5.0 to 8.5, or 5.0 to 8.0, or 5.5 to 9.0, or 5.5 to 8.5, or 5.5 to 7.5, or 6.0 to 9.0, or 6.0 to 8.5, or 6.0 to 7.5, or 6.0 to 7.0, or 6.5 to 9.0, or 6.5 to 8.5, or 6.5 to 8.0, or 6.5 to 7.5. In one embodiment, the pH of the retention solution is adjusted to a pH in the range of 6.9 to 7.5.

[0047] The methods of this disclosure allow for very precise control of the proportion of sodium in the lyophilized composition. In some embodiments, the weight percentage of sodium in the lyophilized composition is in the range of 0% to 100%, or 0% to 50%, or 1% to 25%, or 1% to 10%, or 2% to 10%, or 1% to 5%, or 5% to 10%, or 4.3% to 6.1%, or 4.8% to 5.4%, or 4.9% to 5.0% relative to the total weight of the lyophilized composition. In some embodiments, the weight percentage of sodium in the lyophilized composition is 5.2% ± 0.9%. In some embodiments, the oligonucleotide is nusinersene, and the weight percentage of sodium in the lyophilized composition of nusinersene is 5.2% ± 0.9%.

[0048] With respect to the method of this disclosure, the concentration of oligonucleotides in the retention solution after UF / DF can be indirectly controlled by adjusting the total concentration of salts (and consequently the conductivity) in the aqueous buffer. Performing UF / DF using deionized water preserves the sodium content of the fully sodiumized ASO, but performing UF / DF of oligonucleotides in pure water that can be directly freeze-dried is not possible due to limitations in permeation flux and maximum retention solution concentration. It has been found that UF / DF treatment of oligonucleotides in water is limited by gelation or concentration polarization phenomena at the membrane surface, which reduces the membrane permeation flux and limits the maximum achievable retention solution concentration to only 30-40 g / L.

[0049] We found that a minimum salt concentration (and conductivity) in the UF / DF buffer is required for successful operation of UF / DF and to reach the ideal ASO concentration for lyophilization (i.e., at least 50 g / L). The effects of salt concentration and conductivity on permeation flux are shown in Figures 2 and 3. As shown in the study in Figure 2, when a continuous UF / DF process was carried out using different concentrations of ammonium acetate, it was observed that the permeation flux decreased dramatically with lower concentrations of ammonium acetate, and further dramatically as the diavolume of the UF / DF increased. As shown in Figure 3, the total salt concentration is proportional to the membrane permeation flux at a given TMP.

[0050] Based on this observation, we discovered that the total salt concentration in the aqueous buffer can be used to control the permeate flux of the UF / DF process and the concentration of oligonucleotides in the retaining solution after UF / DF. As illustrated in the study in Figure 4, we found that the final retaining solution ASO concentration can be increased by increasing the acetate concentration in the aqueous buffer. Since increasing the total salt concentration in the aqueous buffer results in an increase in both the permeate flux and the maximum concentration of oligonucleotides in the retaining solution, the method of this disclosure can be used to achieve the desired permeate flux and preferably high retaining solution concentration to enable large-scale lyophilization without additional (i.e., solvent removal) steps.

[0051] In some embodiments, the total concentration of one or more salts in the aqueous buffer is in the range of 1 mM to 500 mM, or 10 mM to 200 mM, or 20 mM to 100 mM, or 30 mM to 60 mM, or 35 mM to 45 mM. In some embodiments, the total concentration of one or more salts in the aqueous buffer is 40 mM.

[0052] When acetates are used as components of the aqueous buffer, it may be necessary to remove these salts to trace levels during lyophilization. As illustrated in the study in Figure 10, it has also been found that the acetate content in the solid API is proportional to the total salt concentration (and therefore total acetate concentration) in the aqueous buffer. Thus, the total acetate content of the UF / DF buffer can be controlled to reduce the acetate in the solid API to trace levels. In some embodiments, using volatile acetates such as ammonium acetate can further reduce the final acetate content because the volatile acetates are removed during lyophilization.

[0053] In some embodiments, the aqueous buffer contains sodium acetate and ammonium acetate, and the total concentration of sodium acetate and ammonium acetate in the aqueous buffer is in the range of 1 mM to 500 mM, or 10 mM to 200 mM, or 20 mM to 100 mM, or 30 mM to 60 mM, or 35 mM to 45 mM. In some embodiments, the total concentration of sodium acetate and ammonium acetate in the aqueous buffer is 40 mM.

[0054] In some embodiments, the aqueous buffer contains sodium acetate and potassium acetate, and the total concentration of sodium acetate and potassium acetate is in the range of 1 mM to 500 mM, or 10 mM to 200 mM, or 20 mM to 100 mM, or 30 mM to 60 mM, or 35 mM to 45 mM. In some embodiments, the total concentration of sodium acetate and potassium acetate in the aqueous buffer is 40 mM.

[0055] In some embodiments, the weight percentage of acetate in the lyophilized composition is less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1%, or less than 0.8%, or less than 0.5%, or less than 0.2% based on the total weight of the lyophilized composition. For example, in some embodiments, the weight percentage of acetate in the lyophilized composition ranges from 5% to 0.1%, or from 5% to 0.5%, or from 5% to 1%, or from 3% to 0.5%, or from 3% to 0.2%, or from 2% to 0.5%, or from 2% to 1%, or from 1% to 0.5%, or from 1% to 0.1%, or from 0.8% to 0.1%, or from 0.5% to 0.1%, or from 0.2% to 0.01% based on the total weight of the lyophilized composition.

[0056] The composition and properties of the aqueous buffer can be controlled to maximize the permeate flux of the UF / DF process (see FIGS. 2 and 3). Accordingly, the present disclosure provides a method of controlling the permeate flux of a UF / DF process by adjusting the total concentration of one or more salts in the aqueous buffer or by adjusting the conductivity of the aqueous buffer. In some embodiments, the UF / DF process is at least 1 L·m -2 ·hr -1 , or at least 5 L·m -2 ·hr -1 , or 5 L·m -2 ·hr -1 ~25 L·m -2 ·hr -1 , or 5 L·m -2 ·hr -1 ~20 L·m -2 [[ID=2,6]]·hr -1 , or 5 L·m -2 ·hr -1 ~15 L·m -2 ·hr -1 , or 10 L·m -2 ·hr -1 ~25 L·m -2 ·hr -1 , or 8 L·m -2 ·hr -1 ~16 L·m -2 ·hr -1 and is performed at a permeate flux of.

[0057] The method of the present disclosure can also be implemented so that the UF / DF process can achieve high diamond capacity levels (see Figure 2). While increasing the number of diamond capacities passing through the film during the UF / DF process, the method of the present disclosure can still maintain an acceptable level of permeation flux, thereby maximizing the overall efficiency and productivity of the process. In some embodiments, the UF / DF process is performed with at least 3, or at least 4, or at least 5, or 3–10, or 5–10, or 5–8 diamond capacities.

[0058] The method of the present disclosure significantly increases the concentration of oligonucleotides in the retention solution after UF / DF, enabling direct freeze-drying of the retention solution after UF / DF without performing an additional water removal (i.e., concentration) step. In some embodiments, the concentration of oligonucleotides in the retention solution is at least 20 g / L, at least 30 g / L, at least 40 g / L, at least 50 g / L, or in the range of 30 g / L to 150 g / L, or in the range of 50 g / L to 150 g / L, or in the range of 60 g / L to 125 g / L, or in the range of 70 g / L to 125 g / L, or in the range of 70 g / L to 100 g / L, or in the range of 80 g / L to 90 g / L.

[0059] The method of this disclosure can utilize any suitable UF / DF filter membrane known in the art. For example, in some embodiments, the UF / DF process is carried out using a membrane having a molecular weight cutoff (MWCO) of 1 kDa to 10 kDa, or 1 kDa to 7 kDa, or 1 kDa to 5 kDa, or 2 kDa to 4 kDa. In some embodiments, the membrane has an MWCO of 3 kDa.

[0060] In some embodiments, the UF / DF step is performed using tangential flow filtration.

[0061] The methods of this disclosure may be applied to any oligonucleotide (such as antisense oligonucleotides) having 10-50 nucleotides, 10-30 nucleotides, 10-25 nucleotides, 10-20 nucleotides, 16-30 nucleotides, 16-25 nucleotides, or 16-20 nucleotides. In some embodiments, the oligonucleotide is nusinersene. In some embodiments, the lyophilized oligonucleotide composition is Spinraza®.

[0062] In some embodiments, the methods of the present disclosure are not limited to specific process steps or are limited to excluding specific process steps. For example, in some embodiments, the method comprises performing at least one ultrafiltration / diafiltration (UF / DF) to obtain a retaining solution, and then performing at least one lyophilization of the retaining solution to obtain a lyophilized composition containing oligonucleotides. In other embodiments, the method comprises performing a single ultrafiltration / diafiltration (UF / DF) to obtain a retaining solution, and then performing at least one lyophilization of the retaining solution to obtain a lyophilized composition containing oligonucleotides. In yet another embodiment, the method comprises performing a single ultrafiltration / diafiltration (UF / DF) to obtain a retaining solution, and then performing a single lyophilization of the retaining solution to obtain a lyophilized composition containing oligonucleotides.

[0063] In some embodiments, the method of the present disclosure may be carried out such that the retaining solution is not subjected to (i) additional filtration, (ii) additional buffer exchange, (iii) additional concentration, and / or (iv) additional purification before being lyophilized to obtain a lyophilized composition. In other embodiments, the method of the present disclosure may be carried out such that the retaining solution is not subjected to any of (i) additional filtration, (ii) additional buffer exchange, (iii) additional concentration, and (iv) additional purification before being lyophilized to obtain a lyophilized composition. In some embodiments, the retaining solution produced from the UF / DF step is lyophilized directly without any additional steps.

[0064] Another aspect of this disclosure relates to compositions obtained using the methods described herein. In some embodiments, the compositions include oligonucleotides such as Spinraza® (nusinersene). The compositions of this disclosure may be in the form of aqueous solutions, such as post-UF / DF retention solutions, or in the form of solid or semi-solid materials, such as lyophilized compositions, that include oligonucleotides. [Examples]

[0065] Materials and methods KrosFlo KR2i TFF System (Spectrum Labs) and Pellicon 3 (0.11m 2 UF / DF experiments were conducted using a 3kDa) regenerated cellulose membrane cassette. Laboratory-scale freeze-drying was performed using LyoStar 2, and production-scale freeze-drying was performed using LyoStar 3.

[0066] Example 1. UF / DF in water Figure 9 summarizes the experimental results of a study conducted to determine how the permeate flux of a typical UF / DF process using pure water instead of the aqueous buffer of this disclosure is affected over time as the concentration of oligonucleotides in the retention solution increases. In this study, an aqueous solution containing 10 g / L of Spinraza® (nusinersen) (labeled "Alpha Syn" in Figure 9) was subjected to a UF / DF process using pure water, and both the permeate flux through the membrane and the concentration of oligonucleotides in the retention solution were measured over time. The oligonucleotide concentration was measured by spectroscopic measurements before and after the operation. Since the total mass of ASO in the system was known, the concentration was estimated based on the change in retention amount.

[0067] Experiments were conducted to determine the maximum ASO concentration achievable by buffering Alpha-Syn ASO with water and then concentrating the retention solution using a buffer containing only water. UF / DF concentration was performed at a transmembrane pressure (TMP) of 20 psi and 1.5 LMM (liters / minute / meter).2 The process was carried out using a cross-flow. The concentration step was started with a holding solution concentration of 10 g / L and a permeate flux of 7 LMH, and the permeate flux rapidly decreased as the holding solution concentration increased during the process (and the holding solution volume decreased) (Figure 9). The permeate flux decreased to 1 LMH at an ASO concentration of approximately 32 g / L in the holding solution. This experiment demonstrates that high concentrations (≥50 g / L) of ASO are not achievable using UF / DF buffer containing only water.

[0068] The study in Figure 9 shows that performing the UF / DF process of oligonucleotides in water is limited by gelation or concentration polarization phenomena on the membrane surface, which significantly reduces the membrane permeation flux, resulting in a maximum achievable retention solution concentration of 30–40 g / L. Since a concentration of 30–40 g / L is not high enough to achieve the desired cake structure during freeze-drying, oligonucleotides after UF / DF in water are typically subjected to additional unit operations to reduce their volume.

[0069] Example 2. Effect of salt concentration in aqueous buffer on permeate flux The results of Example 1 demonstrated that buffer exchange to water at ideal API concentrations is not feasible due to gelation or concentration polarization phenomena on the membrane surface, which lead to a decrease in membrane permeation flux. However, we found that introducing salt additives to the aqueous buffer could increase the permeation flux during buffer exchange. Experiments attempting buffer exchange to water showed that high conductivity correlated with high permeation flux, indicating that adding salt content to the diafiltration buffer is an effective way to increase permeation flux.

[0070] Ammonium acetate was selected as an experimental additive to increase conductivity and, therefore, permeate flux. Both the ammonium and acetate species are compatible with the freeze-drying API platform process and thus do not introduce any new substances into the overall manufacturing process, and both species are known to be volatile in a neutral state. Furthermore, the pH of ammonium acetate is within the desired range (6.9–7.7) based on the desired pH of the API product.

[0071] Three experimental studies were conducted to map the relationship between ammonium acetate concentration and permeate flux. All three experiments used 105 g / L ASO, 710 mM NaCl, and 25 mM Tris at pH 7.2 as starting materials. All three experiments were conducted at a transmembrane pressure (TMP) of 35 psi and a cross-flow of 3 LMM (liters / minute / meter). 2 ), and 120g / m² 2 A membrane load was used.

[0072] In this study, three aqueous ammonium acetate buffer solutions were prepared with ammonium acetate concentrations of 50 mM, 100 mM, and 200 mM. The permeation flux obtained during buffer exchange with an aqueous solution of Spinraza® (nusinersen) was measured. The results (Figure 2) show the steady-state permeation flux for each condition after approximately 3 volumes, and the correlation between ammonium acetate concentration and steady-state permeation flux.

[0073] As shown in Figure 2, the decrease in permeate flux occurring throughout the UF / DF process was observed to be directly proportional to the concentration of ammonium acetate in the aqueous buffer. Using a concentration of 200 mM ammonium acetate, the permeate flux remained at 10 L·m even after 5 dia volume of buffer had passed through the membrane. -2 · hr -1 This enables a UF / DF process to maintain a high permeation flux. The study in Figure 2 shows that by controlling the salt concentration in the aqueous buffer, the UF / DF method of this disclosure enables achieving a sufficiently high concentration of oligonucleotides in the retaining solution after UF / DF, making direct lyophilization of the retaining solution possible.

[0074] Figure 3 summarizes the experimental results of related studies conducted to determine how the conductivity of aqueous buffers affects the permeate flux in the UF / DF process. The relationship between buffer conductivity and permeate flux was investigated to determine the minimum salt content required for the UF / DF process. In this study, three aqueous buffers were prepared with conductivity values ​​of approximately 5.1 mS / cm, 9.8 mS / cm, and 18.9 mS / cm, and the permeate flux obtained during buffer exchange with an aqueous solution of Spinraza® (nusinersen) was measured. Conductivity was measured using an Orion Versa Star Pro, Advanced Electrochemistry Meter.

[0075] As shown in Figure 3, the permeate flux was found to be directly proportional to the conductivity of the aqueous buffer, thereby linearly relating an increase in permeate flux to an increase in buffer conductivity. The results of this study demonstrate that ammonium acetate is an effective enhancer of permeate flux within an appropriate ASO concentration range. The comparison of steady-state flux (Figure 2) and buffer conductivity (Figure 3) in this study showed a linear relationship, thus demonstrating the ability to target specific fluxes (Figure 3) that are important for controlling UF / DF operations.

[0076] Example 3. Correlation between the total concentration of salts in aqueous buffer and the maximum oligonucleotide concentration in the retention solution. Figure 4 summarizes the experimental results of a study conducted to determine how the total concentration of salts in the aqueous buffer affects the final concentration of oligonucleotides in the retention solution after UF / DF. In this study, many aqueous buffers were prepared with increasing total concentrations of acetate from approximately 4 mM to approximately 200 mM (200 mM ammonium acetate; 50 mM ammonium acetate and 50 mM sodium acetate; 75 mM ammonium acetate and 25 mM sodium acetate; 90 mM ammonium acetate and 10 mM sodium acetate; 7.5 mM ammonium acetate and 42.5 mM sodium acetate; 4 mM ammonium acetate and 36 mM sodium acetate), and the retention solution concentration after UF / DF was measured for buffer exchange with an aqueous solution of Spinraza® (nusinersen) up to 8 dia volume. After performing UF / DF up to 8 dia volume, the retention solution volume was subjected to a final concentration step until the permeate flux decreased to <2 LMH.

[0077] As shown in Figure 4, it was observed that the concentration of ligonucleotides in the retention solution after UF / DF was directly proportional to the total concentration of acetate in the aqueous buffer. The increasing conductivity (total salt concentration) of the UF / DF buffer facilitated higher permeation flux, while also facilitating a higher achievable maximum retention solution concentration. This relationship was also linear, demonstrating the ability to target the achievable maximum retention solution concentration by manipulating the UF / DF buffer salt concentration.

[0078] The study in Figure 4 surprisingly demonstrates that the concentration of oligonucleotides in the retention solution can also be controlled by controlling the concentration of salts in the aqueous buffer, enabling the UF / DF process to be performed in a way that significantly increases the retention solution concentration, thereby allowing the retention solution to be directly freeze-dried (without additional steps) to form a solid API.

[0079] The studies shown in Figures 2 and 4 demonstrate that the maximum achievable ASO concentration in the holding solution is directly proportional to the total acetate concentration in both the UF / DF buffer and the permeate flux. A minimum concentration of 80 g / L was targeted to ensure acceptable quality and density of the solid cake, as well as compatibility with the existing freeze-dryer (LyoStar3).

[0080] The total salt concentration of the buffer controls the permeate flux and the achievable maximum ASO concentration in the retaining solution, so that an increase in total salt results in a reproducible increase in flux and maximum retaining solution concentration. This control method allows the desired permeate flux and maximum retaining solution concentration to be targeted and reproducibly achieved.

[0081] Example 4. Method for controlling the sodium content in an oligonucleotide composition after freeze-drying. Figure 5 and Table 1 summarize the experimental results of a study conducted to determine how the molar ratio of sodium acetate and ammonium acetate in aqueous buffer affects the amounts of sodium and ammonium in the composition after freeze-drying. All UF / DF experiments in this study used the following conditions: 35psi TMP 3LMM crossflow 50-275g / m² 2 membrane loading The freeze-drying pool of UF / DF was run in a LyoGuard tray under the following conditions: Initial freezing point: -50°C Primary drying at 23°C and 100 mTorr Secondary drying at 30°C and 100 mTorr.

[0082] In this study, a series of aqueous buffer solutions (see Table 1) were prepared by varying the content of sodium acetate (NaOAc) and ammonium acetate (NH4OAc) so that the molar ratio of sodium to ammonium increased from 0% to 100%, and the mass percentages of sodium and ammonium in the freeze-dried composition were measured.

[0083] Solid API samples after freeze-drying were analyzed for sodium (using inductively coupled plasma emission spectroscopy (ICP-OES)), ammonia (using the NH3BioTest Kit for Cedex Bio HT Analyzer), and acetate content (using LC-UV method (comparison with standard)) (Figure 5). The target mass% for sodium was 5.2 ± 0.9%, and the target mass% for acetate was ≤ 0.8 mass%. Although there was no specification regarding the amount of ammonia present, it was desirable to reduce the amount of residual ammonia as much as possible or to remove it completely from the API. An intermediate ammonia target of 0.5% was selected because it appeared to be the lowest level consistently achievable under the tested conditions.

[0084] [Table 1]

[0085] The trade-off between sodium and ammonia was consistent across the entire range of sodium-to-ammonium ratios in the tested buffer systems (Figure 5), demonstrating the ability to target specific concentrations of sodium and ammonium in the final product (solid API). In all examples in Figure 5, the UF / DF holding solution was concentrated to similar concentrations of 80–85 g / L. Since sodium present in the solution in the UF / DF pool was not removed by lyophilization, the final API sodium value includes sodium present in the buffer introduced into the lyophilization process. If the UF / DF pool has a lower concentration of ASO (larger volume), the sodium value will shift higher, and conversely, if the UF / DF pool can be increased to a higher concentration (smaller volume), the sodium value will shift lower. When targeting specific sodium and ammonia content, any changes in the ASO concentration of the final UF / DF holding solution must be taken into account.

[0086] As shown in Figure 5, we found that the ratio of ammonium to sodium in the UF / DF buffer determined the final sodium and ammonium content in the composition after lyophilization. Lyophilization of the UF / DF pool was performed in a LyoGuard tray using primary drying conditions of 23°C and 100 mTorr, and secondary drying conditions of 30°C and 100 mTorr. Under the lyophilization conditions of this study, it was observed that the final sodium and ammonium content was linearly related to the molar ratios of sodium acetate and ammonium acetate, respectively.

[0087] As shown in the related studies summarized in Figure 6, the sodium content after lyophilization can be controlled by fine-tuning the ratio of UF / DF buffer components (e.g., ammonium to sodium ratio), allowing the solid API to meet specific specifications, such as a critical sodium content of 5.2% ± 0.9%. Although the sodium content results in this study were lower than the target of 5.2 ± 0.9% by mass, the linear trend allows us to predict the necessary UF / DF buffer composition (sodium acetate and ammonium acetate) that would yield a sodium content within the target range. As shown in Figure 6, by extrapolating the data to the center of the target sodium content range, we predicted that manipulating UF / DF with a buffer consisting of 85% sodium acetate and 15% ammonium acetate would yield a product with a sodium content of approximately 5.2% by mass.

[0088] Example 5. Correlation between acetate content in oligonucleotide composition after freeze-drying and total acetate content in aqueous buffer solution Figure 10 summarizes the experimental results of a study conducted to determine how the total acetate content in aqueous buffer affects the amount of acetate in solid API after lyophilization. In this study, a series of aqueous buffers were prepared with total acetate concentrations ranging from 40 mM to 100 mM (see Examples 2-8 in Table 1), and the mass percentage of acetate in the lyophilized compositions was measured. As shown in Figure 10, the residual acetate content of the lyophilized API was found to be proportional to the total acetate content in the UF / DF buffer. A linear trend was observed between the total acetate content in the UF / DF buffer matrix and the residual acetate content in the solid API. All buffer conditions that met the ≤0.8 mass% salt specification contained 40 mM total acetate.

[0089] We found that the residual acetate content was not affected by the ammonium / sodium cation ratio. Furthermore, based on the volatility of ammonium acetate under freeze-drying conditions, we were able to remove acetic acid to trace levels after freeze-drying by reducing the total acetate concentration in the aqueous buffer.

[0090] Based in part on the experimental studies described above, we found that optimizing the ratio of sodium acetate to ammonium acetate in the UF / DF buffer, along with decreasing the total acetate concentration, allows for linking the UF / DF buffer to aqueous downstream processes, including lyophilized APIs.

[0091] Example 6. Large-scale preparation of freeze-dried APIs Large-scale experiments were conducted using the fixed molar ratio of sodium salt in aqueous buffer and the concentration of oligonucleotide in the retaining solution after UF / DF (indicated as "oligonucleotide concentration before lyophilization" in Table 2 below), and the water content, sodium content, and acetate content of the lyophilized composition were measured. In these experiments, the oligonucleotide Spinraza® (nusinersene) was subjected to the UF / DF method of this disclosure at a fixed molar ratio of sodium salt.

[0092] Based on previous experiments, 6 mM ammonium acetate and 34 mM sodium acetate (85% sodium acetate vs. 15% ammonium acetate, total acetate concentration 40 mM) were selected as the buffer matrix that best targeted the endpoints of maximum ASO concentration, sodium content, and acetate content in the retention solution. Conditions were verified at lab scale and repeated at manufacturing (MFG) scale (18 mmol). The UF / DF pool from the manufacturing process was divided, with a portion lyophilized at lab scale (indicated as "lab scale lyophilized" in Table 2) and the remainder lyophilized at manufacturing scale.

[0093] As shown in Table 2, no significant difference was observed between sodium and acetate after freeze-drying, demonstrating that cation control by manipulating the UF / DF buffer is scalable. The maximum achievable UF / DF pool concentration was the same at both scales. The water content of the solid API was slightly higher at the production scale, which was attributed to differences in equipment. Overall, the scaling up of cation and acetate control by manipulating the UF / DF buffer, and the enhancement of permeate flux and retention solution concentration by salt composition, were successful, demonstrating the process's scalability.

[0094] [Table 2]

[0095] Bulk freeze-drying was performed on material obtained from a production-scale run (Example 12 in Table 2) using four LyoGuard trays with a total liquid volume of 6 L. Figure 7 shows the four LyoGuard trays containing the freeze-dried material. The freeze-drying process was modified to facilitate the maximum removal of volatile UF / DF buffer components. This bulk freeze-drying process included freezing at -50°C, primary drying at 23°C, followed by secondary drying at 30°C at a pressure of 150 mTorr. Successful removal of trace amounts of water and volatile buffer components was achieved, meeting API specifications. Figure 8 shows the freeze-dried material in the LyoGuard trays, which were then transferred to storage bags.

[0096] The selected final buffers were 34 mM NaOAc and 6 mM NH4OAc. The conditions and results of the procedure (Example 12 in Table 2) are summarized below. • Solid APINa content: 4.9-5.0% (Target value: 5.2% ± 0.9%) • Concentration after UF / DF: Up to 85g / L liquid API is acceptable. ·Permeation flux: Maintain>10LMH flux • UF / DF process duration: Unit operation completed in 1 day • Final acetate content: Minimum residual acetate after freeze-drying • Stability of composition after freeze-drying: Solid API is stable for 31 days at 25°C.

[0097] While various embodiments of this disclosure are shown and described herein, it will be apparent that such embodiments are provided for illustrative purposes only. Numerous modifications, alterations, and substitutions can be made without departing from this disclosure. Therefore, this disclosure is intended to be limited only by the spirit and scope of the appended claims. Embodiments of the Invention [Aspect 1] A method for preparing a composition comprising an oligonucleotide, the method comprising subjecting an aqueous solution of the oligonucleotide to ultrafiltration / diafiltration (UF / DF) to form a retaining solution comprising the oligonucleotide, wherein the ultrafiltration / diafiltration (UF / DF) is performed using an aqueous buffer containing one or more salts. [Aspect 2] The method according to aspect 1, wherein the total concentration of one or more salts in the buffer solution is in the range of 10 mM to 200 mM, 20 mM to 100 mM, or 30 mM to 60 mM. [Aspect 3] The method according to aspect 2, wherein the total concentration of the one or more salts in the buffer solution is 40 mM. [Aspect 4] The method according to any one of aspects 1 to 3, wherein the aqueous buffer solution comprises at least one salt selected from sodium acetate, potassium acetate, and ammonium acetate. [Aspect 5] The method according to aspect 4, wherein the aqueous buffer solution comprises sodium acetate and ammonium acetate. [Aspect 6] The method according to any one of aspects 1 to 3, wherein the aqueous buffer solution contains sodium acetate and potassium acetate. [Aspect 7] The aqueous buffer solution contains sodium acetate and ammonium acetate, and the total concentration of sodium acetate and ammonium acetate is in the range of 10 mM to 200 mM, or 20 mM to 100 mM, or 30 mM to 60 mM, or 40 nM, or The method according to embodiment 5 or 6, wherein the aqueous buffer solution contains sodium acetate and potassium acetate, and the total concentration of sodium acetate and potassium acetate is in the range of 10 mM to 200 mM, or 20 mM to 100 mM, or 30 mM to 60 mM, or 40 nM. [Aspect 8] The method according to any one of aspects 5 to 7, wherein the molar ratio of sodium acetate to ammonium acetate in the buffer solution, or the molar ratio of sodium acetate to potassium acetate, is in the range of 1:20 to 20:1, or 1:1 to 19:1, or 5:1 to 19:1, or 5:1 to 6:1, or 5:1 to 6:1.8, or 12:1 to 15:1. [Aspect 9] The aqueous buffer solution contains sodium acetate and ammonium acetate, and the molar ratio of sodium acetate to ammonium acetate in the aqueous buffer solution is 17:3, or The method according to embodiment 8, wherein the aqueous buffer solution contains sodium acetate and potassium acetate, and the molar ratio of sodium acetate to potassium acetate in the aqueous buffer solution is 17:3. [Aspect 10] The method according to any one of aspects 1 to 3, wherein the aqueous buffer solution contains 34 mM sodium acetate and 6 mM ammonium acetate, or the aqueous buffer solution contains 34 mM sodium acetate and 6 mM potassium acetate. [Aspect 11] The ultrafiltration / diafiltration (UF / DF) is at least 5 L·m -2 · hr -1 The method according to any one of embodiments 1 to 10, performed with a permeation flux. [Aspect 12] The ultrafiltration / dialysis filtration (UF / DF) is 5 L·m -2 · hr -1 ~25L·m -2 · hr -1 , or 8L·m-2 · hr -1 ~16L·m -2 · hr -1 The method according to embodiment 11, performed with a transmission flux in the range of . [Aspect 13] The method according to any one of aspects 1 to 12, wherein the ultrafiltration / diafiltration (UF / DF) is performed with at least 3, or at least 4, or at least 5, or 3 to 10 dia volumes. [Aspect 14] The method according to any one of aspects 1 to 13, wherein the concentration of the oligonucleotide in the holding solution is at least 50 g / L, or in the range of 70 g / L to 125 g / L, or 80 g / L to 90 g / L. [Aspect 15] The method according to any one of aspects 1 to 14, wherein the ultrafiltration / dialysis filtration (UF / DF) is performed using a membrane having a molecular weight cutoff (MWCO) in the range of 1 kDa to 7 kDa, or 2 kDa to 4 kDa, or 3 kDa. [Aspect 16] The method according to any one of aspects 1 to 15, wherein the ultrafiltration / dialysis filtration (UF / DF) is performed by tangential flow filtration. [Aspect 17] The method according to any one of aspects 1 to 16, further comprising subjecting the holding liquid to freeze-drying in order to form a freeze-dried composition containing the oligonucleotide. [Aspect 18] The method according to aspect 17, wherein the weight percentage of sodium in the freeze-dried composition is in the range of 0% to 100%, 0% to 50%, 2% to 10%, 4.3% to 6.1%, or 4.8% to 5.4%. [Aspect 19] The method according to aspect 18, wherein the weight percentage of sodium in the freeze-dried composition is in the range of 4.9% to 5.0%. [Aspect 20] The method according to aspect 18, wherein the weight percentage of sodium in the freeze-dried composition is 5.2% ± 0.9%. [Aspect 21] The method according to any one of aspects 4 to 20, wherein the weight percentage of acetate in the freeze-dried composition is less than 3%, less than 2%, less than 1%, or less than 0.8%. [Aspect 22] The method according to any one of aspects 1 to 21, wherein the oligonucleotide is an antisense oligonucleotide having 16 to 30 nucleotides or 16 to 20 nucleotides. [Aspect 23] The method according to any one of aspects 1 to 21, wherein the oligonucleotide is nusinersene. [Aspect 24] A composition comprising an oligonucleotide, wherein the composition is obtained by the method described in any one of aspects 1 to 23. [Aspect 25] The composition according to aspect 24, in the form of an aqueous solution containing the oligonucleotide. [Aspect 26] The composition according to aspect 25, which is in the form of a freeze-dried composition containing the oligonucleotide.

Claims

1. A method for preparing a composition comprising oligonucleotides, the method comprising subjecting an aqueous solution of the oligonucleotide to ultrafiltration / dialysis filtration (UF / DF) to form a retaining solution comprising the oligonucleotide, wherein the ultrafiltration / dialysis filtration (UF / DF) is performed using an aqueous buffer, and the aqueous buffer comprises sodium acetate and ammonium acetate, or the aqueous buffer comprises sodium acetate and potassium acetate, and the ultrafiltration / dialysis filtration (UF / DF) is performed by tangential flow filtration.

2. The method according to claim 1, wherein the total concentration of sodium acetate and ammonium acetate or sodium acetate and potassium acetate in the buffer solution is in the range of 10 mM to 200 mM, 20 mM to 100 mM, or 30 mM to 60 mM.

3. The method according to claim 2, wherein the total concentration of sodium acetate and ammonium acetate or sodium acetate and potassium acetate in the buffer solution is 40 mM.

4. The method according to any one of claims 1 to 3, wherein the aqueous buffer solution comprises sodium acetate and ammonium acetate.

5. The method according to any one of claims 1 to 3, wherein the aqueous buffer solution comprises sodium acetate and potassium acetate.

6. The aqueous buffer solution contains sodium acetate and ammonium acetate, and the total concentration of sodium acetate and ammonium acetate is in the range of 10 mM to 200 mM, or 20 mM to 100 mM, or 30 mM to 60 mM, or 40 mM, or The method according to claim 4 or 5, wherein the aqueous buffer solution comprises sodium acetate and potassium acetate, and the total concentration of sodium acetate and potassium acetate is in the range of 10 mM to 200 mM, or 20 mM to 100 mM, or 30 mM to 60 mM, or 40 mM.

7. The method according to any one of claims 3 to 6, wherein the molar ratio of sodium acetate to ammonium acetate in the buffer solution, or the molar ratio of sodium acetate to potassium acetate, is in the range of 1:20 to 20:1, or 1:1 to 19:1, or 5:1 to 19:1, or 5:1 to 6:1, or 5:1 to 6:1.8, or 12:1 to 15:

1.

8. The aqueous buffer solution contains sodium acetate and ammonium acetate, and the molar ratio of sodium acetate to ammonium acetate in the aqueous buffer solution is 17:3, or The method according to claim 7, wherein the aqueous buffer solution contains sodium acetate and potassium acetate, and the molar ratio of sodium acetate to potassium acetate in the aqueous buffer solution is 17:

3.

9. The method according to any one of claims 1 to 3, wherein the aqueous buffer solution contains 34 mM sodium acetate and 6 mM ammonium acetate, or the aqueous buffer solution contains 34 mM sodium acetate and 6 mM potassium acetate.

10. The aforementioned ultrafiltration / dialysis filtration (UF / DF) is at least 5 L·m －２ ・hr －１ The method according to any one of claims 1 to 9, performed with a permeation flux.

11. The ultrafiltration / diafiltration (UF / DF) is 5 L·m －２ ·hr －１ ~25 L·m －２ ·hr －１ or 8 L·m －２ ·hr －１ ~16 L·m －２ ·hr －１ The method according to claim 10, which is carried out at a permeate flux within the range of

12. The method according to any one of claims 1 to 11, wherein the ultrafiltration / diafiltration (UF / DF) is performed with a diameter of at least 3, or at least 4, or at least 5, or 3 to 10.

13. The method according to any one of claims 1 to 12, wherein the concentration of the oligonucleotide in the holding solution is at least 50 g / L, or in the range of 70 g / L to 125 g / L, or 80 g / L to 90 g / L.

14. The method according to any one of claims 1 to 13, wherein the ultrafiltration / diafiltration (UF / DF) is performed using a membrane having a molecular weight cutoff (MWCO) in the range of 1 kDa to 7 kDa, or 2 kDa to 4 kDa, or 3 kDa.

15. The method according to any one of claims 1 to 14, further comprising subjecting the retaining liquid to freeze-drying in order to form a freeze-dried composition containing the oligonucleotide.

16. The method according to claim 15, wherein the weight percentage of sodium in the freeze-dried composition is in the range of 0% to 100%, 0% to 50%, 2% to 10%, 4.3% to 6.1%, or 4.8% to 5.4%.

17. The method according to claim 16, wherein the weight percentage of sodium in the freeze-dried composition is in the range of 4.9% to 5.0%.

18. The method according to claim 18, wherein the weight percentage of sodium in the freeze-dried composition is 5.2% ± 0.9%.

19. The method according to any one of claims 15 to 18, wherein the weight percentage of acetate in the freeze-dried composition is less than 3%, less than 2%, less than 1%, or less than 0.8%.

20. The method according to any one of claims 1 to 19, wherein the oligonucleotide is an antisense oligonucleotide having 16 to 30 nucleotides or 16 to 20 nucleotides.

21. The method according to any one of claims 1 to 20, wherein the oligonucleotide is nusinersene.

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