Method for preparing oligonucleotide compositions using ultrafiltration / dialysis filtration

Abstract

This invention provides a method for preparing a composition containing an oligonucleotide. [Solution] A method for preparing a composition containing oligonucleotides, comprising subjecting an aqueous solution of oligonucleotides to ultrafiltration / dialysis filtration (UF / DF) to form a retaining solution containing oligonucleotides, wherein the ultrafiltration / dialysis filtration (UF / DF) is performed using an aqueous buffer containing one or more salts.

Description

[Technical Field]

[0001] Cross-reference of related applications This application is a U.S. provisional application filed on February 21, 2020, under Section 119 of the U.S. Patent Act. This asserts the benefit of the filing date of Patent No. 62 / 979,687, and the entire content of this asserts the reference This is incorporated herein by means of.

[0002] This application relates to biopharmaceutical technology in general, and more specifically to oligonucleotide composition. Using filtration technology that can control the salt content in the material, high-purity oligonucleotide compositions are produced. Regarding the method of preparation. The method disclosed herein involves the use of ultrafiltration / diafiltration (UF / DF). , additional processing commonly used in the manufacture of freeze-dried (solid) active pharmaceutical ingredients (APIs) It can be linked to freeze-drying in a way that does not require performing steps. [Background technology]

[0003] Oligonucleotides are short-chain DNA or RNA oligonucleotides that can be chemically synthesized for research and medical purposes. It's a smear. Oligonucleotides are usually identified by adding nucleotide residues in a stepwise manner. It is prepared by generating a sequence of oligonucleotides in the desired sequence. Following the completion of development, the target oligonucleotide typically fails to produce the sequence and other pros. It is obtained as a mixture with ses and product-related impurities.

[0004] Oligonu for therapeutic use, such as commercially available oligonucleotides approved for use by the FDA. The preparation of creotides is further complicated by stringent commercial specifications and formulation verification requirements. Appropriate purification and formulation techniques for oligonucleotides ensure the chemical composition and stability of the product, and It is necessary to take the administration method into consideration.

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

[0006] Spinraza® (nusinersen) is an antisense oligonucleotide (ASO) drug used for the treatment of spinal muscular atrophy (SMA), a rare neuromuscular disor der . The commercially available Spinraza® formulation is a lyophilized API obtained from a solvent-intensive process. It is necessary to integrate an aqueous-based platform process, and ultima tely, a liquid drug substance is produced via ultrafiltration / diafiltration (UF / DF) using a lyophilized API having a specific salt (e.g., sodium and acetate) content This approach will minimize formulation validation and meet existing commercial specifications without the need for adding off-platform liquid reduction steps and / or additional equipment . SUMMARY OF THE INVENTION

[0007] This disclosure describes methods for concentrating and buffer exchanging oligonucleotides using ultrafiltration / diafiltration (UF / DF) to obtain aqueous oligonucleotide solutions suitable for lyophilization without additional (intervening) processing steps Figure 1 shows that the method of the present disclosure is a method that does not require performing solvent-based precipitation that generally precedes the lyophilization step, and provides lyophilized AP I ​Using UF / DF in the I platform process, the aqueous-based platform shows how processes can be integrated.

[0008] In particular, the methods disclosed herein control the sodium content before and after lyophilization and the sodium content after lyophilization, and the acetate content after lyophilization in oligonucleotide APIs to meet predetermined specifications for sodium and acetate. This is achieved by controlling components (e.g., salts) in the UF / DF aqueous buffer. The methods described herein also allow control of the membrane permeation flux and the retentate concentration of oligonucleotides while performing the UF / DF step within the manufacturer's recommended transmembrane pressure (TMP) conditions.

[0009] One aspect of the present disclosure relates to a method of preparing a composition comprising an oligonucleotide, the method comprising subjecting an aqueous solution of an oligonucleotide to ultrafiltration / diafiltration (UF / DF) to form a retentate 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, wherein the composition is obtained by one of the methods described herein.

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

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

[0013] Additional objects, advantages, and other features of the present disclosure will be set forth in part in the description that follows, and in part will ​This will become apparent to those skilled in the art through the following considerations, or can be learned from the practice of this disclosure. The benefits of the disclosure can be realized and obtained, as specifically set forth in the attached claims. To be understood, this disclosure allows for other and different embodiments, and several of them. The details thereof may be changed in various obvious ways without all deviations from this disclosure. It is possible. In this regard, the explanation in this specification is essentially illustrative and limited. It should be understood that there is no such thing. [Brief explanation of the drawing]

[0014] [Figure 1] This disclosure describes an exemplary integration of solvent-intensive processes with aqueous-based processes. [Figure 2] This graph shows the decrease in permeate flux during buffer exchange when UF / DF is performed at different ammonium acetate concentrations. [Figure 3] This graph shows the effect of the DF buffer on the second steady-state permeation flux as the conductivity of the DF buffer increases. [Figure 4] This graph shows the maximum ASO concentration in the retaining solution as a percentage of the total acetate concentration in the UF / DF buffer. [Figure 5] This graph shows the trend of sodium and ammonium content after freeze-drying (after freezing). [Figure 6] This graph shows the sodium (Na) content in the retaining solution after UF / DF, relative to the percentage (%) of sodium acetate (NaOAc) in UF buffers containing different amounts of ammonium acetate (NH4OAc). [Figure 7] This is a photograph of an exemplary freeze-drying chamber with LyoGuard trays. [Figure 8] These are photographs of exemplary freeze-dried ASO material in trays and bags. [Figure 9]This graph shows how the permeation flux decreases as the concentration of oligonucleotides in the retention solution increases when UF / DF is performed in water. [Figure 10] This graph shows the mass percentage (%) of acetic acid (OAc) remaining in the solid API relative to the total concentration of acetic acid (OAc) in the UF buffer. [Figure 11] This document compares an exemplary aqueous-based platform process with an exemplary freeze-drying API platform process that includes 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 is the use of ultrafiltration / diafiltration (UF / DF) for oligonucleotides. Traditionally used to prepare the solid form of rheotide active pharmaceutical ingredients (APIs). This is a method for integrating into a freeze-drying API platform process. Embodiments of this disclosure are Antisense oligonucleotides such as Spinraza® (nusinersen) This includes methods for preparing oligonucleotides for therapeutic use.

[0016] Water-based platform processes generally involve one or two chromatography processes. The process includes a separation step and a deprotection step, and involves concentrating the oligonucleotide of interest into the intrathecal cavity. (IT) Ultrafiltration / diafiltration (UF / D) performs buffer exchange to liquid formulations suitable for administration. The process ends in step F). This platform is used to obtain liquid active pharmaceutical ingredients from UF / DF operations. It is then delivered to a parenteral filling facility for final dilution, filtration, and filling. In contrast, frozen Dry API platform processes often utilize solvent-based purification processes. Figure 11 shows a ready-to-fill liquid containing Spinraza® (nusinersen). A water-based platform process used to prepare body morphology, and this product A freeze-drying API platform used to prepare freeze-dried (solid) forms of [the product]. This shows the difference from Rothes. The base platform process is a liquid form of Spinraza that can be filled immediately. Ion exchange chromatography steps and This is followed by an ultrafiltration / diafiltration (UF / DF) step. In contrast, freeze-dried APIs The platform process involves ethanol precipitation to obtain a lyophilized (solid) formulation. The process includes a den step, followed by freeze-drying and compounding.

[0017] Currently used to prepare commercially available Spinraza (registered trademark) (nusinersen) The freeze-drying API platform process uses an aqueous-based ethanol precipitation step. By replacing the UF / DF step used in the platform process Making changes would be beneficial. Firstly, for both environmental and regulatory reasons, this commercial product It would be beneficial to eliminate the use of organic solvents in the final preparation. Secondly, UF / DF is Typically, it can be used to precisely control the salt content of processed products, thus allowing for more accurate control. The controlled salt content can be obtained by using UF / DF instead of solvent precipitation. This is possible. However, as explained below, the implementation of UF / DF and large-scale freeze-drying is required. Due to the above limitations, these processes cannot be completed in Spinraza without including additional steps (registration It has been impossible to successfully integrate trademarks into commercial-scale preparations until now.

[0018] The main difficulty in integrating UF / DF into large-scale freeze-drying is the aqueous retention solution provided by UF / DF. This is caused by the salt content and / or oligonucleotide content of Spinraza. (Registered Trademark) (Nusinersen) has a relatively low salt content (approximately 5% by weight in the freeze-dried product). It is necessary to have such a low salt content (and When attempting to perform UF / DF with an aqueous solution having low conductivity, the permeation flux through the membrane is reduced. This causes the low permeate flux to be partially formed at the liquid surface retained by the UF / DF film. This occurs due to undesirable "caking." Low permeation flux results in the desired oligonucleotides being lost. It reduces both the rate of oxide formation and the concentration of oligonucleotides in the holding solution. UF / To properly operate DF and reach the ideal ASO concentration for lyophilization, use UF / DF buffer. A minimum level of conductivity is required inside. On the other hand, the salt content of the UF / DF buffer should be appropriately high. When increased to the level necessary to enable permeation flow rate (oligonucleotides in the holding liquid) (The concentration of salts becomes appropriately high), resulting in too much salt being present in the resulting retention solution. This requires an additional step(s) to remove any excess salt.

[0019] The method disclosed herein addresses these problems by controlling the salt concentration and salt content in the buffer solution. This method can overcome the problem by integrating the aqueous purification process with the freeze-drying step. Without adding any additional steps and / or apparatus, the predetermined sodium and acetate Create a solid API with specifications. The UF / DF process involves UF / DF operations (flux and concentration). The operability (degrees), and the composition of the solid API after the product has been freeze-dried following UF / DF. It can be determined by both. Not only the target oligonucleotide, but also the purification process. The purification process intermediate, which contains various molecular species involved, reaches the target sodium content after freeze-drying. The process is carried out and, after freeze-drying, concentrated and treated by UF / DF to satisfy the acetate specifications. A UF / DF process is developed. The UF / DF process of this disclosure is negatively charged A pair of io along the phosphorothioate or phosphorodiester oligonucleotide skeleton By controlling the average number of sodium cations occupying the position, the total sodium content This achieves control over the film permeation flux required for large-scale manufacturing processes. By ensuring the maximum retention fluid concentration is met, the efficient operation of the UF / DF process is facilitated. do.

[0020] Unless otherwise defined, all technical and scientific terms used herein are those of the United States. It has the same meaning as commonly understood by the person concerned. In case of conflict, including definition, The specifications prevail.

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

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

[0023] The use of "a" or "an" in this specification to describe various elements and components is simply This is for convenience and to give a general meaning to this disclosure. This statement is one or fewer. It should be interpreted as including at least one, and the singular means that it does not. It includes multiple people unless it is clear that they are being referred to.

[0024] When a specific quantity or value is used, if it is equivalent to or substantially the same as that specific quantity or value Those skilled in the art will understand that it can include a small deviation from a particular quantity or value. It should be understood that in some embodiments, a certain quantity or value is a certain quantity or It includes ±10% of the value. In some embodiments, a specific quantity or value is a specific quantity or This includes ±5% of the value.

[0025] One aspect of this disclosure relates to oligonucleotides such as Spinraza® (Nusinersen). This relates to a method for preparing a composition containing a rheotide. This method involves the aqueous solubility of oligonucleotides. The liquid is subjected to ultrafiltration / dialysis filtration (UF / DF) to form a retaining solution containing oligonucleotides. Ultrafiltration / diafiltration (UF / DF) includes the use of aqueous solutions containing one or more salts. It is executed using a buffer.

[0026] In some embodiments, one or more salts in the aqueous buffer finalize the composition of the retaining solution. It can be formulated in a way that controls it precisely. For example, in some embodiments, the aqueous buffer is The holding solution generated by UF / DF may be formulated to control the sodium content. This indirectly controls the sodium content of the freeze-dried product. Such control is In an aqueous buffer solution containing a sodium salt and an antagonist salt having a cation different from sodium, This is made possible by the presence of multiple salts of the compound. Antagonistic salts include, for example, ammonium and dimethylammonium. Ammonium, trimethylammonium, potassium, lithium, rubidium, copper, silver, and Examples include salts having different cations, such as other suitable monovalent cations. Antagonistic salts are volatile. It may be a volatile salt, a non-volatile salt, or a combination thereof.

[0027] In some embodiments, the use of at least one antagonistic salt is necessary for the method of the present disclosure. By controlling the average number of sodium cations occupying the counterion positions of the ligonucleotide skeleton, This makes it possible. Figure 12 shows sodium (Na + ) ions and ammonium (NH4 + ) How ions negatively charged phosphorothioates or phosphorodiester oligos This indicates how many different counterion positions can be occupied along the nucleotide backbone. In that embodiment, the present disclosure involves introducing one or more antagonistic salts into an aqueous buffer. Therefore, the salt content in the oligonucleotide holding solution generated by UF / DF is controlled. The present disclosure provides a method for doing so. In other words, the method of this disclosure provides an aqueous solution of oligonucleotides The solution is subjected to ultrafiltration / dialysis filtration (UF / DF) to form a retaining solution containing oligonucleotides. Ultrafiltration / diafiltration (UF / DF) includes the use of aqueous solutions containing sodium salts and antagonist salts. This is carried out using a buffer solution. 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 cation and its antagonist cation reach equilibrium at the counterion position of the oligonucleotide. Oligonucleotides in solution are not completely sodium-conjugated, that is, oligo The result was that the nucleotide pair-ion position was not completely occupied by the sodium cation. See Figure 12. This equilibrium ratio can be expressed as follows:

number

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

[0030] In some embodiments, one or more salts contained in the aqueous buffer are at least one It may contain one volatile salt. The UF / DF process utilizing the volatile salts of this disclosure is a desired The maximum ASO concentration in the retaining solution can be achieved by controlling the thorium content. Depending on the group properties, a volatile antagonistic salt is used to control the total salt content in the freeze-dried product (total salt content of the holding solution). It is possible to reduce the content (compared to the amount contained). In volatile antagonistic salts, the volatile antagonistic catio of the salt The base exists in equilibrium with its corresponding volatile conjugate base. (The opposing cation species (corresponding conjugate) Due to the volatility of the neutral form (with the active base), it can be removed by sublimation during freeze-drying.

[0031] Ammonium acetate (NH4OAc) is a volatile compound used in some embodiments of the present disclosure. An example of a sex-antagonistic salt is shown below, as is the ammonium cation (NH4 + ) is anmoni It exists in equilibrium with a(NH3) and acetate anion (AcO - ) is in equilibrium with acetic acid (AcOH). It exists in that state. [ka]

[0032] In the equilibrium state shown above, the protonated ammonium cation reacts with the acetate anion. It acts as a proton source for conversion to acetic acid, which is volatile and can be removed by freeze-drying. This is possible. Proton transfer from ammonium to acetate allows both species to become neutral. They become volatile, making it easier to remove both species during freeze-drying.

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

[0034] By using volatile antagonist salts such as ammonium acetate shown above, after UF / DF Next, the retaining solution is prepared, while maintaining the sodium content in the retaining solution, and a considerable amount of volatile compounds are added. The anti-salt solution can be removed and freeze-dried. Therefore, the retaining liquid after UF / DF is also possible. It has a total salt content that is significantly lower than the total salt content inside, while based on the composition of the aqueous buffer solution To produce a freeze-dried oligonucleotide composition having a controlled sodium content. This can be achieved by using volatile antagonist salts. This feature allows the method of this disclosure to This process removes antagonistic salts to trace amounts while producing a solid oligonucleotide with a predetermined sodium content. It is possible to generate a rheotide API.

[0035] In some embodiments, the antagonist salt is a non-volatile salt that is not removed by freeze-drying. For example, an aqueous buffer solution can be sodium acetate, sodium chloride, sodium bromide or Sodium salts such as sodium iodide, as well as potassium acetate, potassium chloride, and potassium bromide. Alternatively, it may contain non-volatile antagonistic salts such as potassium iodide. Other non-volatile antagonistic salts include, for example, For example, potassium salts, lithium salts (e.g., lithium acetate, lithium chloride, lithium bromide) (e.g., lithium iodide), rubidium salts (e.g., rubidium acetate, rubidium chloride, rubidium bromide) Pidium (or rubidium iodide), copper salts (e.g., copper acetate, copper chloride, copper bromide or iodine), Examples include copper(II) oxides and silver salts (e.g., silver acetate, silver chloride, silver bromide, or silver iodide).

[0036] In the method disclosed herein, the composition of the aqueous buffer is as follows: in the holding solution after UF / DF and after lyophilization. The wide range of sodium content in our products, from essentially zero sodium content to completely sodium content. The target sodium content is far greater than the equivalent amount of sodium-modified ASO. It can be controlled to do so.

[0037] In some embodiments, the aqueous buffer is sodium acetate, ammonium acetate, and vinegar. It comprises at least one salt selected from potassium acid. In some embodiments, aqueous sulfate The liquid contains sodium acetate and ammonium acetate. In some embodiments, an aqueous solution is used. The buffer solution contains sodium acetate and potassium acetate. In some embodiments, an aqueous buffer solution is used. The solution contains sodium acetate, ammonium acetate, and potassium acetate.

[0038] In some embodiments, the sodium content in the oligonucleotide-containing holding solution (for example) (For example, sodium concentration) is the total concentration of salt in aqueous buffer solution, with at least one sodium concentration. This is controlled by adjusting the proportion of the salt. In other embodiments, oligonucleotides in the holding liquid The proportion of sodium cations occupying the counterion position of the creotide is the total amount of the salt in the aqueous buffer solution. This is controlled by adjusting the ratio of at least one sodium salt to the concentration.

[0039] In some embodiments, the molar ratio of the sodium salt to the antagonist salt contained in the aqueous buffer. This is 1:100~100:1, or 1:20~20:1, or 1:10~10:1. Or 1:1~19:1, or 5:1~19:1, or 12:1~15:1, or It falls within the range of 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 solution comprises sodium acetate and ammonium acetate. The molar ratio of sodium acetate to ammonium acetate in aqueous buffer solution is 1:100~1 00:1, or 1:20~20:1, or 1:10~10:1, or 1:1~19 :1, or 5:1~19:1, or 12:1~15:1, or 5:1~10:1, Or it is in the range of 5:1 to 6:1, or 5:1 to 6:1.8. In some embodiments In this case, the molar ratio of sodium acetate to ammonium acetate is 17:3. In this embodiment, the aqueous buffer solution contains 34 mM sodium acetate and 6 mM ammonium acetate Contains um.

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

[0042] In some embodiments, the pH of the aqueous buffer is 4.0 to 10.0, or 4.5 to 9. 0.5, or 5.0-9.0, or 5.0-8.5, or 5.0-8.0, or 5 0.5~9.0, or 5.5~8.5, or 5.5~8.0, or 5.5~7.5, Or 6.0~9.0, or 6.0~8.5, or 6.0~7.5, or 6.0~ 7.0, or 6.5-9.0, or 6.5-8.5, or 6.5-8.0, or It falls within the range of 6.5 to 7.5, or 6.9 to 7.5.

[0043] In some embodiments, the aqueous buffer does not contain sodium salts, thereby UF / DF The subsequent retention solution does not contain sodium. In other embodiments, the aqueous buffer does not contain any antagonistic salts. No.

[0044] In some embodiments, the method of the present disclosure involves lyophilizing a target oligonucleotide. The process may include a step of freeze-drying the UF / DF holding solution to produce the final product. This results in volatile UF / DF buffer components (e.g., volatile antagonist salts such as ammonium acetate). This can remove it. The freeze-drying step can be carried out as a single freeze-drying, Alternatively, it can be implemented as multiple freeze-drying processes carried out using a single freeze-drying apparatus or multiple freeze-drying apparatuses. It can be done.

[0045] In some embodiments, the proportion of one or more antagonistic salts in the freeze-dried composition is , less than the proportion of one or more antagonistic salts contained in the retention solution after UF / DF. As explained above, volatile antagonist salts such as ammonium acetate in aqueous buffer solution then These can be removed (partially or completely) during freeze-drying.

[0046] The method disclosed herein also involves adjusting the pH of the retaining solution after UF / DF before freeze-drying. It may contain a tep. In some embodiments, the pH of the retaining solution is 5.0 to 9.0, or 5.0-8.5, or 5.0-8.0, or 5.5-9.0, or 5.5-8.5 , or 5.5-8.0, or 5.5-7.5, or 6.0-9.0, or 6.0 ~8.5, or 6.0~7.5, or 6.0~7.0, or 6.5~9.0, also The pH is adjusted to a range of 6.5-8.5, 6.5-8.0, or 6.5-7.5. In one embodiment, the pH of the holding solution is adjusted to a pH in the range of 6.9 to 7.5.

[0047] The method disclosed herein allows for very precise control of the proportion of sodium in the freeze-dried composition. This is possible. In some embodiments, the weight percentage of sodium in the freeze-dried composition The amount of the lyophilized material is 0% to 100%, or 0% to 50%, or 1% of the total weight of the freeze-dried composition. %~25%, or 1%~10%, or 2%~10%, or 1%~5%, or 5% ~10%, or 4.3%~6.1%, or 4.8%~5.4%, or 4.9%~5 It is in the range of 0.0%. In some embodiments, the weight of sodium in the freeze-dried composition The concentration is 5.2% ± 0.9%. In some embodiments, oligonucleotides are used. This is nusinersene, and the weight percentage of sodium in the lyophilized composition of nusinersene. The percentage is 5.2% ± 0.9%.

[0048] With respect to the method disclosed herein, the concentration of oligonucleotides in the retaining solution after UF / DF is aqueous. By adjusting the total concentration of salts in the buffer (and consequently the conductivity), it can be controlled indirectly. It can be controlled. When UF / DF is performed using deionized water, the sodium can be completely removed. The sodium content of the converted ASO is retained, but the oligosaccharides in pure water can be directly freeze-dried. Performing UF / DF on nucleotides is limited by permeation flux and maximum retention solution concentration. It is not possible. UF / DF treatment of oligonucleotides in water causes gelation of the film surface or concentration fractionation. It was found that this is limited by polar phenomena, which reduces the membrane permeation flux and makes it difficult to achieve. The maximum holding solution concentration is only 30-40 g / L.

[0049] Operate UF / DF correctly and achieve the ideal ASO concentration for freeze-drying (i.e., at least 5 To reach 0 g / L, a minimum amount of salt concentration (and conductivity) is required in the UF / DF buffer. I discovered something. 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, a continuous UF / DF process is performed using different concentrations of acetic acid. When using ammonium, when using a lower concentration of ammonium acetate The permeate flux decreases dramatically, and the diavolume of UF / DF increases. As this progressed, a more dramatic decrease in the permeation flux was observed. As shown in Figure 3, total salt The concentration is proportional to the membrane permeation flux at a given TMP.

[0050] Based on this observation, the permeate flux of the UF / DF process and the retaining liquid after UF / DF are determined. To control the concentration of oligonucleotides, the total salt concentration in the aqueous buffer can be used. We discovered that it is possible. As illustrated in the study in Figure 4, the concentration of acetate in aqueous buffer solution We discovered that increasing this can increase the concentration of ASO in the final retaining solution. The increase in total salt concentration in the aqueous buffer solution was due to the permeate flux and the highest concentration of oligonucleotides in the retention solution. Because it results in a large increase in both concentration without the need for additional steps (i.e., solvent removal) To enable imitation freeze-drying, the desired permeation flux and preferably high retention liquid concentration are achieved. The method described herein can be used for this purpose.

[0051] In some embodiments, the total concentration of one or more salts in the aqueous buffer is 1 mM to 5 mM. 00mM, or 10mM-200mM, or 20mM-100mM, or 30mM The concentration is in the range of ~60 mM, or 35 mM to 45 mM. In some embodiments, aqueous solvents are used. The total concentration of one or more salts in the cinder is 40 mM.

[0052] When using acetates as components of an aqueous buffer solution, these salts are removed at trace levels during freeze-drying. It may be necessary to remove even this much. As illustrated in the study in Figure 10, acetate in solid API It has also been found that the content is proportional to the total salt concentration (and therefore total acetate concentration) in the aqueous buffer solution. Therefore, the total acetate content of the UF / DF buffer is less than the trace amount of acetate in the solid API. It can be controlled to reduce it to a certain level. In some embodiments, ammonium acetate When using volatile acetates such as ammonium, the volatile acetates are removed during freeze-drying, so The final acetate content can be further reduced.

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

[0054] In some embodiments, the aqueous buffer solution comprises sodium acetate and potassium acetate, and vinegar The total concentration of sodium sulfate and potassium acetate is 1 mM to 500 mM, or 10 mM to 20 mM. 0mM, or 20mM-100mM, or 30mM-60mM, or 35mM-4 It is in the range of 5 mM. In some embodiments, sodium acetate and acetic acid in an aqueous buffer. The total potassium concentration is 40 mM.

[0055] In some embodiments, the weight percentage of acetate in the freeze-dried composition is the weight percentage of the freeze-dried composition. less than 5%, or less than 4%, or less than 3%, or less than 2%, or 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 composition. For example, in some embodiments, the weight percentage of acetate in the lyophilized composition is based on the total weight of the lyophilized composition 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%.

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

[0057] The method disclosed herein also enables the UF / DF process to achieve high diamond capacity levels. This can be done (see Figure 2). Diacapacitance passing through the film during the UF / DF process While increasing the number of processes, the permissible level of transmission flux remains, and the method of this disclosure processes It enables maximizing the overall efficiency and productivity of the system. In some embodiments, U The F / DF process consists of at least 3, or at least 4, or at least 5, It runs with a diamond capacity of 3-10, 5-10, or 5-8.

[0058] The method disclosed herein significantly increases the concentration of oligonucleotides in the retention solution after UF / DF. Therefore, without performing an additional water removal (i.e., concentration) step, the retained liquid after UF / DF is directly... Freeze-drying is enabled. In some embodiments, the concentration of oligonucleotides in the holding solution The concentration is at least 20g / L, at least 30g / L, at least 40g / L, at 50g / L, or in the range of 30g / L to 150g / L, or 50g / L to 150g / L The range of 60g / L to 125g / L, or the range of 70g / L to 125g / L It is within the range of 70g / L to 100g / L, or 80g / L to 90g / L.

[0059] The method disclosed herein utilizes any suitable UF / DF filter film known in the art. It can be used. For example, in some embodiments, the UF / DF process is 1kD a~10kDa, or 1kDa~7kDa, or 1kDa~5kDa, or 2kD This is carried out using a membrane with a molecular weight cutoff (MWCO) of a to 4 kDa. In that embodiment, the membrane has a MWCO of 3 kDa.

[0060] In some embodiments, the UF / DF step uses tangential flow filtration. It is then executed.

[0061] The method disclosed herein involves 10-50 nucleotides, 10-30 nucleotides, 10-25 nucleotides Rheotide, 10-20 nucleotides, 16-30 nucleotides, 16-25 nucleotides , or any oligonucleotide having 16-20 nucleotides (antisense oligonucleotide) It can be applied to (such as nucleotides). In some embodiments, oligonucleotides D is nusinersene. In some embodiments, the lyophilized oligonucleotide composition The product is Spinraza (registered trademark).

[0062] In some embodiments, the methods disclosed herein are not limited to specific process steps. or limited to excluding specific process steps. For example, several implementations In terms of form, the method involves performing at least one ultrafiltration / diafiltration (UF / DF) on the retained fluid. To obtain the holding solution, then perform lyophilization at least once to obtain the oligonucleotide. The method includes obtaining a freeze-dried composition. In other embodiments, the method involves a single ultrafiltration / dialysis filter. Perform a freeze-drying (UF / DF) to obtain a retaining solution, and then freeze-dry the retaining solution at least once. The process involves carrying out the procedure to obtain a freeze-dried composition containing an oligonucleotide. Further other embodiments In this case, the method involves performing a single ultrafiltration / diafiltration (UF / DF) to obtain the retained solution, and then The holding liquid is freeze-dried once to obtain a freeze-dried composition containing an oligonucleotide. It consists of and .

[0063] In some embodiments, the method disclosed herein involves freeze-drying the holding liquid to form a freeze-dried composition. Before obtaining the result, the retaining solution undergoes (i) additional filtration, (ii) additional buffer exchange, and (iii) additional This may be carried out in such a way that it is not subjected to concentration and / or (iv) further purification. In one embodiment, the method of the present disclosure involves freeze-drying the holding liquid to obtain a freeze-dried composition, The retaining solution undergoes (i) additional filtration, (ii) additional buffer exchange, (iii) additional concentration, and ( iv) It may be carried out in such a way that it is not subjected to any further purification. Several methods In terms of form, the retention liquid produced from the UF / DF step is frozen directly without any additional steps. It will be dried.

[0064] Another aspect of this disclosure relates to compositions obtained using the methods described herein. In one embodiment, the composition is Spinraza® (nusinersen), etc. Contains oligonucleotides. The compositions of this disclosure contain oligonucleotides after UF / DF. It may be in the form of an aqueous solution such as a retaining solution, or freeze-dried containing an oligonucleotide. It may be in the form of a solid or semi-solid material, such as a composition. [Examples]

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

[0066] Example 1. UF / DF in water Figure 9 shows a typical UF / DF process using pure water instead of the aqueous buffer solution of this disclosure. How does the permeation flux change over time as the concentration of oligonucleotides in the holding solution increases? This summarizes the experimental results of studies conducted to determine whether or not something is affected by the target. This study uses a water-soluble solution containing 10 g / L of Spinraza® (nusinersen). The liquid (labeled "Alpha Syn" in Figure 9) is subjected to UF / DF using pure water. The process involves both the permeation flux passing through the membrane and the concentration of oligonucleotides in the retaining solution. The method is measured over time. The concentration of oligonucleotides is measured by spectroscopic measurement before and after the procedure. Measurements were taken after the experiment. Since the total mass of ASO in the system is known, the changes in retention amount were measured. The concentration was estimated.

[0067] After replacing the buffer in Alpha-Syn ASO with water, use a buffer containing only water. We conducted an experiment to determine the maximum ASO concentration that could be achieved by concentrating the holding solution. F / DF enrichment is performed at a transmembrane pressure (TMP) of 20 psi and 1.5 LMM (liters / minute / meter). - 2 The procedure was performed using a cross-flow. The concentration step was performed using a 10 g / L retaining solution concentration. The process starts with a permeate flux of 7 LMH, and the retaining liquid concentration increases during the process (and retaining liquid As the volume decreased, the permeation flux rapidly decreased (Figure 9). The permeation flux was approximately 32 g. The ASO concentration in the holding solution of / L decreased to 1LMH. This experiment was conducted using UF / containing only water. It is impossible to achieve high concentrations (≧50g / L) of ASO using DF buffer. To prove it.

[0068] The study in Figure 9 shows that performing the UF / DF process of oligonucleotides in water is possible on the membrane surface. This shows that it is limited by gelation or concentration polarization phenomena, thereby the membrane permeation flux The concentration decreases significantly, and the maximum achievable retention solution concentration becomes 30-40 g / L. The concentration of water is not high enough to achieve the desired cake structure during freeze-drying. The oligonucleotides after UF / DF are typically subjected to additional unit operations to reduce their volume. It can be applied.

[0069] Example 2. Effect of salt concentration in aqueous buffer on permeate flux The results of Example 1 showed that buffer exchange to water at an ideal API concentration resulted in a decrease in membrane permeation flux. We demonstrated that this is impossible due to gelation or concentration polarization phenomena on the connected membrane surface. However, introducing salt additives into aqueous buffer solution may increase the permeate flux during buffer exchange. We discovered that there is a high conductivity and high permeation flux. In experiments where we attempted to exchange the buffer solution with water, we found that high conductivity corresponds to high permeation flux. Correlating with this, adding salt content to the dialysis filtration buffer is an effective way to increase permeate flux. It demonstrated that it is the law.

[0070] Ammonium acetate is used as an experimental additive to increase conductivity and therefore the permeation flux. I selected ammonium. Both the ammonium and acetate species are freeze-dried on the API platform. It is compatible with the existing process, and therefore introduces the new material into the entire manufacturing process. There is no such thing as both species being volatile in a neutral state. The pH of the nium is within the desired range (6.9-7.7) based on the desired pH of the API product. It is located there.

[0071] To map the relationship between ammonium acetate concentration and permeate flux, three experimental studies were conducted. The experiment was conducted. All three experiments used 105 g / L ASO4 and 710 mM Na4 as starting materials. The solution contained Cl and 25 mM Tris at pH 7.2, and in all three experiments, the pressure was 35 psi. Transmembrane pressure (TMP), 3 LMM cross-flow (liters / min / meter) 2 ), and 1 20g / m 2 A membrane load was used.

[0072] In this study, the concentrations of ammonium acetate were set to 50 mM, 100 mM, and 200 mM. Three types of aqueous ammonium acetate buffer solutions were prepared, and Spinraza (registered trademark) ( The permeate flux obtained during buffer exchange using an aqueous solution of nusinersen was measured. Results (Figure 2) shows the steady-state permeate flux and ammonium acetate concentration under each condition after approximately 3 Dia volume. This shows the correlation between the degree and the steady-state permeation flux.

[0073] As shown in Figure 2, the decrease in permeation flux that occurs throughout the UF / DF process is due to aqueous buffer. It was observed that the concentration was directly proportional to the concentration of ammonium acetate inside. Using a high concentration of 5 dia volume buffer, even after the buffer has passed through the membrane, it remains at 10 L·m. -2 · hr -1 This enables UF / DF processes to maintain ultratransmission flux. The study in Figure 2 shows By controlling the salt concentration in the aqueous buffer solution, direct freeze-drying of the holding solution can be achieved. This disclosure aims to achieve a sufficiently high concentration of oligonucleotides in the retention solution after UF / DF. This demonstrates the possibility of using the UF / DF method.

[0074] Figure 3 shows how the conductivity of the aqueous buffer affects the permeate flux in the UF / DF process. This summarizes the experimental results of related studies conducted to determine the conductivity of the buffer solution. By investigating the relationship between this and the permeation flux, we determined the minimum salt content required for the UF / DF process. The study found conductivity values ​​of approximately 5.1 mS / cm, 9.8 mS / cm, and 18.9 mS / cm. Three aqueous buffer solutions were prepared, set to m, and Spinraza(registered trademark) (Nusine The permeation flux obtained from buffer exchange performed with an aqueous solution of lucene was measured. The conductivity was Orion Versa Star Pro, Advanced Electroche Measurements were taken using a mistry meter.

[0075] As shown in Figure 3, the permeation flux is directly proportional to the conductivity of the aqueous buffer solution, and therefore the permeation flux The increase was found to be linearly related to the increase in buffer conductivity. The results of this study suggest that vinegar This demonstrates that ammonium ammonium acid is an effective enhancer of permeate flux within the appropriate ASO concentration range. The comparison of steady-state flux (Figure 2) and buffer conductivity (Figure 3) in this study is linear. This indicates the relationship and, therefore, targets a specific flux (Figure 3) that is important for controlling UF / DF operations. He demonstrated his abilities.

[0076] Example 3. Correlation between the total concentration of salts in aqueous buffer and the maximum oligonucleotide concentration in the retention solution. Figure 4 shows the total concentration of salts in the aqueous buffer, and the oligonucleotides in the retention solution after UF / DF. This summarizes the experimental results of studies conducted to determine how it affects the final concentration. In this study, the total concentration of acetate was increased from approximately 4 mM to approximately 200 mM. Many aqueous buffer solutions (200 mM ammonium acetate; 50 mM ammonium acetate and 50 mM M sodium acetate; 75 mM ammonium acetate and 25 mM sodium acetate; 90 mM ammonium acetate Ammonium acetate and 10 mM sodium acetate; 7.5 mM ammonium acetate and 42.5 Prepare mM sodium acetate (4 mM ammonium acetate and 36 mM sodium acetate). The gradual grading was performed using an aqueous solution of Spinraza (registered trademark) (nusinersen) up to a volume of 8 dia. The concentration of the retained fluid after UF / DF was measured for the fluid exchange. UF / DF was measured in 8 days. After running until the volume is reached, the retained liquid volume is reduced until the permeate flux drops to <2 LMH, and the final concentration is performed. We implemented the steps.

[0077] As shown in Figure 4, the concentration of ligonenucleotides in the retention solution after UF / DF is in the aqueous buffer solution. It was observed that the conductivity of the UF / DF buffer increased in direct proportion to the total concentration of acetate in the buffer. (Total salt concentration) facilitates higher permeation flux, while a higher achievable maximum retention liquid The concentration was also simplified. This relationship is linear, and by manipulating the UF / DF buffer salt concentration... Furthermore, it demonstrated the ability to target the maximum achievable retention solution concentration.

[0078] The study in Figure 4, surprisingly, shows that by controlling the concentration of salt in the aqueous buffer solution, the retention solution This demonstrates that the concentration of oligonucleotides can also be controlled, and provides a method to significantly increase the concentration of the retention solution. This allows the UF / DF process to be performed, thereby allowing the retaining liquid to be (additional st Solid API can be formed by direct freeze-drying (without a lid).

[0079] The studies shown in Figures 2 and 4 indicate that the maximum achievable ASO concentration in the retention solution is achieved when the UF / DF ratio slows down. This shows that the total acetate concentration is directly proportional to both the saturation fluid and the permeate flux. (Solid cake) The acceptable quality and density, as well as compatibility with existing freeze dryers (LyoStar3) To ensure this, the target was a minimum concentration of 80 g / L.

[0080] The total salt concentration of the buffer controls the permeate flux and the maximum achievable ASO concentration in the retaining solution. Therefore, an increase in total salt yields a reproducible increase in flux and maximum retained solution concentration. This method allows for the reproducible achievement of desired permeation flux and maximum retaining solution concentration. can.

[0081] Example 4. Method for controlling the sodium content in an oligonucleotide composition after freeze-drying. Figure 5 and Table 1 show the molar ratio of sodium acetate and ammonium acetate in aqueous buffer solution. This determines how it affects the amount of sodium and ammonium in the composition after freeze-drying. This document summarizes the experimental results of the research conducted to determine the UF / DF ratio in this study. All tests used the following conditions: 35psi TMP • 3LMM crossflow • 50-275g / m² 2 membrane loading Perform the freeze-drying pool of UF / DF in a LyoGuard tray using the following conditions: Ta: 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, sodium acetate (NaOAc) and ammonium acetate (NH4OAc) The content is changed so that the molar ratio of sodium to ammonium increases from 0% to 100%. The mass percentages of sodium and ammonium in the freeze-dried composition were then measured. A series of aqueous buffer solutions (see Table 1) were prepared.

[0083] The solid API sample after freeze-drying was subjected to sodium (inductively coupled plasma emission spectroscopy (I (Using CP-OES), ammonia (for Cedex Bio HT Analyzer) (Using the NH3BioTest Kit), and acetate content (LC-UV method (standard) We analyzed the results using (comparison with) (Figure 5). The target mass % of sodium was 5.2 ± 0. The target mass percentage for 9% and acetate was ≤0.8% by mass. Regarding the present ammonia Although not specified, the amount of residual ammonia should be reduced as much as possible, or completely removed from the API. This was desired. An intermediate ammonia target of 0.5% was selected, which was under the conditions tested. This is because it appears to be the minimum level that can be consistently achieved.

[0084] [Table 1]

[0085] The trade-off between sodium and ammonia is that sodium in the tested buffer system The ratio to ammonium is consistent across the entire range (Figure 5), and the ammonium content in the final product (solid API) is consistent. The ability to target specific concentrations of thorium and ammonium has been demonstrated. (See all of Figure 5) In this example, the UF / DF holding solution was concentrated to a similar concentration of 80-85 g / L. Because the sodium present in the solution in the DF pool was not removed by freeze-drying, The final API sodium value includes the sodium present in the buffer introduced into the freeze-drying process. It contains um. UF / DF pool is a lower concentration ASO (larger volume) In total, sodium levels shifted to higher levels, and conversely, UF / DF pools shifted to higher concentrations (lower). If the volume can be increased, the sodium level will likely shift to a lower level. If the target sodium and ammonia content is to be met, then the final UF / DF holding solution A All changes in SO concentration must be taken into consideration.

[0086] As shown in Figure 5, the ratio of ammonium to sodium in the UF / DF buffer is To determine the final sodium and ammonium content in the composition after freeze-drying. We discovered that freeze-drying of UF / DF pools can be performed under primary drying conditions of 23°C and 100 mTorr. Furthermore, using secondary drying conditions of 30°C and 100mTorr, LyoGuard The study was conducted using Ray. The final content of sodium and ammonium under freeze-drying conditions in this study. The quantity is linearly related to the molar ratio of sodium acetate and ammonium acetate, respectively. This was observed.

[0087] As shown in the related studies summarized in Figure 6, the ratio of UF / DF buffer components (e.g.: By fine-tuning the ammonium-to-sodium ratio, the sodium content after freeze-drying can be adjusted. It can control certain aspects, such as the important sodium content of the solid API being 5.2% ± 0.9%. It can satisfy the requirements. The sodium content results from this study are 5.2 ± 0.9 of the target. Although lower than mass%, the linear trend resulted in a sodium content within the target range. Predicting the necessary UF / DF buffering compositions (sodium acetate and ammonium acetate) This can be done. As shown in Figure 6, the data can be extrapolated to the center of the target sodium content range. By doing so, a buffer solution consisting of 85% sodium acetate and 15% ammonium acetate is used. By manipulating UF / DF, a product with a sodium content of approximately 5.2% by mass is obtained. This was predicted.

[0088] Example 5. Acetate content in oligonucleotide composition after lyophilization and total content in aqueous buffer Correlation with acetate content Figure 10 shows that the total acetate content in the aqueous buffer corresponds to the amount of acetate in the solid API after freeze-drying. This summarizes the experimental results of studies conducted to determine how it affects things. In this study, a series of aqueous buffer solutions were prepared with total acetate concentrations ranging from 40 mM to 100 mM. (See Examples 2-8 in Table 1) and measure the mass percentage of acetate in the composition after freeze-drying. Determined. As shown in Figure 10, the residual acetic acid content of the API after freeze-drying is determined by the UF / DF buffer. It was found to be proportional to the total content of acetate in the UF / DF buffer matrix. A linear trend was observed between the acetate content and the residual acetate content in the solid API. ≤ 0. All buffer conditions meeting the 8% by mass salt specification include 40 mM total acetate. Ta.

[0089] The residual acetate content is also not affected by the ammonium / sodium cation ratio. Furthermore, based on the volatility of ammonium acetate under freeze-drying conditions, aqueous buffering was discovered. By reducing the total acetate concentration in the liquid, acetic acid can be removed to trace levels after freeze-drying. came.

[0090] Based in part on the above experimental studies, as the total acetate concentration decreases, the UF / DF buffer Optimizing the ratio of sodium acetate to ammonium acetate in the mixture is key to UF / DF. We discovered that it is possible to link aqueous downstream processes, including freeze-drying APIs.

[0091] Example 6. Large-scale preparation of freeze-dried APIs The fixed molar ratio of sodium salt in aqueous buffer solution, and the retaining solution after UF / DF (see Table 2 below). Using the oligonucleotide concentration in the "Alkyl Alkyl Concentration Before Freeze-Drying" label Large-scale experiments were conducted to determine the water content, sodium content, and acetate content of the freeze-dried composition. Measurements were taken. In these experiments, oligonucleotide Spinraza(registered trademark) (Nushi Nelsen 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, 34 mM sodium acetate (85% vinegar) Sodium ammonium acetate to 15% ammonium acetate (total acetic acid concentration 40 mM) in the holding solution, with the highest AS value. A buffer that optimally targets the endpoints of oxygen concentration, sodium content, and acetate content. Selected as the liquid matrix. Conditions were verified at lab scale and then at manufacturing (MFG) scale. The process was repeated with (18 mmol). The UF / DF pool from the manufacturing process was divided, and a portion was used The remaining portion was freeze-dried on a laboratory scale (indicated as "laboratory scale freeze-dried" in Table 2), and the rest was prepared It was freeze-dried using a scale-forming agent.

[0093] As shown in Table 2, no significant difference was observed between sodium and acetate after freeze-drying. The manipulation of UF / DF buffers to control cations is scalable. This was demonstrated. The maximum achievable UF / DF pool concentration was the same at both scales. The moisture content of the body API was observed to be slightly higher at the manufacturing scale, but this is due to differences in equipment. This was due to [unclear]. Overall, cation and acetate control by manipulating UF / DF buffer. The scale-up of the process and the promotion of permeate flux and retention liquid concentration through salt composition were successful. Scalability has been demonstrated.

[0094] [Table 2]

[0095] Bulk freeze-drying is performed using four LyoGuard trays with a total liquid volume of 6L. This was performed on the material obtained from the scaling process (Example 12 in Table 2). Figure 7 shows the freeze-dried material. The image shows four LyoGuard trays containing the materials. The UF / DF buffer components of the volatile substances... To promote maximum removal, the freeze-drying process was modified. This bulk freeze-drying process Seth freezes at -50°C, performs primary drying at 23°C, followed by drying at 30°C under a pressure of 150 mTorr. Includes secondary drying. Successfully removed trace amounts of water and volatile buffering components, API The specifications were met. Figure 8 shows the freeze-dried material in a LyoGuard tray, followed by storage. It was moved to a bag.

[0096] The selected final buffers were 34 mM NaOAc and 6 mM NH4OAc, and the operation was performed. The conditions and results (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] Various embodiments of this disclosure are shown and described herein, but such embodiments may not be It should be clear that these are provided for illustrative purposes only. Without departing from this disclosure, numerous Modifications, alterations, and substitutions may be made. Therefore, this disclosure is in line with the spirit of the attached claims. It is intended to be limited only by its range.

Claims

1. A method for preparing a composition containing an oligonucleotide, wherein the method comprises the oligonucleotide An aqueous solution of creotide is subjected to ultrafiltration / dialysis filtration (UF / DF), and the oligonucleotide The ultrafiltration / dialysis filtration (UF / DF) includes forming a retaining solution containing a d, and the ultrafiltration / dialysis filtration (UF / DF) is one The method is carried out using an aqueous buffer containing multiple salts.

2. The total concentration of one or more salts in the buffer solution is 10 mM to 200 mM, or 20 mM. The method according to claim 1, wherein the concentration is in the range of ~100 mM or 30 mM to 60 mM.

3. Claim 2 states that the total concentration of the one or more salts in the buffer solution is 40 mM. Method of loading.

4. The aqueous buffer solution is selected from sodium acetate, potassium acetate, and ammonium acetate. The method according to any one of claims 1 to 3, comprising at least one salt.

5. The method according to claim 4, wherein the aqueous buffer solution comprises sodium acetate and ammonium acetate. Law.

6. The aqueous buffer solution comprises sodium acetate and potassium acetate, any one of claims 1 to 3. The method described in item 1.

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

8. The molar ratio of sodium acetate to ammonium acetate in the buffer solution, or potassium acetate The molar ratio of sodium acetate to mol is 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 1 The method according to any one of claims 5 to 7, wherein the ratio is in the range of 2:1 to 15:

1.

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

3.

10. 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 The method according to any one of claims 1 to 3.

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

12. Pre-recorded external filtration / dialysis filtration (UF / DF) が、5L・m －２ ・hr －１ ~25L·m －２ ・hr －１ or 8 L·m －２ ・hr －１ to 16 L·m －２ ・hr －１ permeation flow within the range of The method according to claim 11, which is performed in bundles.

13. The ultrafiltration / diafiltration (UF / DF) is performed in at least 3, or at least 4, The operation is performed with a diameter capacity of at least 5, or 3 to 10, as per any of claims 1 to 12. The method described in item 1.

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

15. The ultrafiltration / diafiltration (UF / DF) is 1 kDa to 7 kDa, or 2 kDa to 4 kDa. Using a membrane with a molecular weight cutoff (MWCO) in the kDa range or 3 kDa The method according to any one of claims 1 to 14, which is carried out.

16. The aforementioned ultrafiltration / dialysis filtration (UF / DF) is performed using tangential flow filtration. The method according to any one of claims 1 to 15.

17. To form the freeze-dried composition containing the oligonucleotide, the holding liquid freeze-dries The method according to any one of claims 1 to 16, further comprising drying.

18. The weight percentage of sodium in the freeze-dried composition is 0% to 100%, 0% to 50% %, or in the range of 2% to 10%, or 4.3% to 6.1%, or 4.8% to 5.4%. The method according to claim 17.

19. The weight percentage of sodium in the freeze-dried composition is 4.9% to 5.0% The method according to claim 18, which falls within the range of the present invention.

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

21. The weight percentage of acetate in the freeze-dried composition is less than 3%, less than 2%, less than 1%, The method according to any one of claims 4 to 20, wherein the amount is less than 0.8%.

22. The oligonucleotide has 16 to 30 nucleotides, or 16 to 20 nucleotides. An antisense oligonucleotide having a rheotide, any one of claims 1 to 21 The method described in section [section number].

23. The oligonucleotide is nusinersene, according to any one of claims 1 to 21. The method.

24. A composition comprising oligonucleotides, wherein the composition comprises any one of claims 1 to 23. The composition obtained by the method described in the section.

25. The composition according to claim 24, in the form of an aqueous solution containing the oligonucleotide.

26. The composition according to claim 25, which is a form of a freeze-dried composition containing the oligonucleotide. thing.

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