Thermal sterilisation of an antisense oligonucleotide

Abstract

Thermal sterilisation of an antisense oligonucleotide This invention relates to a method for sterilising nusinersen or a salt thereof, comprising a step of heating an aqueous solution of nusinersen or a salt thereof. It also provides a sterile pharmaceutical compositions obtainable by the method for the treatment of spinal muscular atrophy (SMA).

Description

[0001] Thermal sterilisation of an antisense oligonucleotide

[0002] This invention relates to thermal sterilisation of an antisense oligonucleotide, and particularly a method for the sterilisation of nusinersen or a salt thereof.

[0003] The effective sterilisation of drug products is important from both a medical and an economic perspective. From a medical perspective, sterilisation is critical in ensuring the drug is not contaminated with microorganisms that might represent an infection risk to the patient. The growth of microorganisms can also lead to the degradation of the active pharmaceutical ingredient thereby reducing efficacy and possibly resulting in the production of toxic byproducts. Economically, sterilisation improves the shelf-life of drug products leading to less waste and the ability to replace expired products on a less frequent basis. This can also allow for storage under ambient conditions rather than requiring refrigeration which is associated with increased costs, particularly during transport.

[0004] Multiple methods are available for the preparation of sterile drug products. For parenteral drug products, the two most common methods are terminal sterilisation and membrane filtration. In terminal sterilisation, heat or ionising radiation is used in order to destroy contaminant microorganisms following filling of the drug product into the final container. In membrane filtration, the product stream is filtered through a semi-permeable membrane (pore size <0.22 pm) and filled aseptically into pre-sterilised containers.

[0005] The use of terminal sterilisation is considered to provide greater assurance of sterility than membrane filtration and the current recommendation of the European Medicine Agency (EMA) is that steam sterilisation is carried out for at least 15 minutes at a temperature of at least 121 °C. However, exposure to the conditions used in steam sterilisation can result in unfavourable changes in the drug profile owing to increased degradation or alteration of the physical and chemical properties of the sterilised compound. This is a particular issue for antisense oligonucleotide compounds.

[0006] In this regard, steam sterilisation has been reported to be unsuitable for the sterilisation of antisense oligonucleotides, such as volanesorsen (Waylivra®, EMA / 180717 / 2019), inotersen (Tegsedi®, EMA / 411876 / 2018) and mipomersen (Kynamro®, EMA / 305826 / 2013). In the case of inotersen, a significant increase in degradation products was observed after treatment at 121°C for 8 minutes. Similarly, for mipomersen it was observed that treatment at 121°C for 15 minutes resulted in unacceptable levels of degradation of the active ingredient and increase of impurities. Accordingly, the approved pharmaceutical products are sterilised by membrane sterilisation only and are recommended for storage under refrigerated conditions (2-8°C). Nusinersen (marketed as Spinraza®) is an antisense oligonucleotide compound approved in the United States, Europe and other countries globally for the treatment of spinal muscular atrophy (SMA) in paediatric and adult patients. Nusinersen has the sequence 3’UCACUUUCAUAAUGCUGG5’, wherein each nucleoside has a 2’-O-(2-methoxyethyl)- (MOE) modification, each inter-nucleoside linkage is a thiophosphate, each C is 5-methyl- cytidine and each U is 5-methyl-uracil. The finished nusinersen drug product is marketed as a single use, preservative-free, isotonic solution comprising nusinersen in the form of its sodium salt, formulated in an artificial cerebrospinal fluid (aCSF), which is intended for administration by intrathecal injection. The currently recommended dosage is 12 mg, however clinical trials into higher dosage amounts are ongoing (NCT04089566).

[0007] At present, the finished product is manufactured by sterile filtration followed by aseptic filling since based on the known temperature sensitive nature of oligonucleotides, it was considered unfeasible to perform thermal sterilisation. This is in line with the guidance provided by the EMA (CPMP / QWP / 054 / 98). It was the recommendation of the EMA that the product be stored under refrigerated conditions (2-8°C).

[0008] As such, there remains a need in the art for improved methods to reliably ensure the sterility of pharmaceutical compositions of nusinersen, which result in a drug product that can be stored for long time periods under ambient conditions.

[0009] The present invention provides a method for sterilising nusinersen or a salt thereof, comprising a step of heating an aqueous solution of nusinersen or a salt thereof.

[0010] It was surprisingly found by the present inventors that aqueous solutions of nusinersen can be stably heated to the temperatures required for effective microbial sterilisation without degradation of the active pharmaceutical ingredient. It was further found that when nusinersen is thermally sterilised by the method according to the present invention, the resulting composition is stable under both ambient and accelerated storage conditions. It is therefore considered preparing pharmaceutical compositions in this way will have an excellent degree of sterility and long term shelf life.

[0011] Accordingly, the present invention further provides the sterile nusinersen composition obtainable by the method, and its use in a method of treating SMA in a human patient.

[0012] The present invention will now be described in detail.

[0013] The invention relates to a method for sterilising nusinersen or a salt thereof, comprising a step of heating an aqueous solution of nusinersen or a salt thereof. Thermal sterilisation is the primary method of terminal sterilisation used in the preparation of pharmaceutical products. Alternative methods of terminal sterilisation include chemical sterilisation and exposure to ionising radiation.

[0014] Heat can be applied using two different methods, namely dry heat using hot air with a moisture content so low as to have insignificant biological activity or moist heat using saturated steam or water heated to the sterilisation temperature (autoclaving). Autoclaving is more effective than the application of dry heat and so allows for effective sterilisation to be achieved more rapidly. Accordingly, it is preferred that the heating of the aqueous solution of nusinersen or a salt thereof is carried out by autoclaving.

[0015] It is important that the heating temperature are selected in order to ensure a sufficient level of sterility without resulting in the degradation of the active ingredient.

[0016] A temperature of at least 110°C is considered to be ideal for ensuring effective sterilisation of microorganisms. Accordingly, it is preferred that the heating is at a temperature of 110-150°C, more preferably the heating is at a temperature of 120-140°C.

[0017] The heating time should also be selected in order to achieve an optimum balance of properties in the final solution. It is preferred that the heating is carried out for 2-180 minutes, more preferably for 5-30 minutes.

[0018] It is considered beneficial to select the heating temperature in combination with the heating time. In this regard, it is common to consider the lethality of the process in terms of the equivalent time in minutes at a temperature of 121 °C delivered by the process to the load in its container with reference to microorganisms possessing a z-value of 10 (Fo). The z-value refers to the change in temperature required to alter the D-value by a factor of 10, where the D-value refers to the duration required to reduce the number of viable organisms to 10 per cent of the original number.

[0019] An Fo value of >8 is commonly targeted, however an Fo value of <8 minutes may be suitable when heating is performed following sterile filtration and aseptic filling. Equation 1 can be used to determine a suitable heating duration (tT), wherein T is the heating temperature: (Equation 1 )

[0020] For example an Fo value of >8 can be achieved by exposure to a temperature of at least 121 °C for at least 8 minutes or by a temperature of 125°C for at least 5 minutes. It is considered that the skilled person would be capable of selecting a suitable combination of temperature and time in order to achieve a desired level of sterility. Ideally, the temperature is maintained for a longer time period in order to ensure maximal sterilisation of the solution and it has been unexpectedly found that nusinersen is stable when exposed to conditions equivalent to a temperature of at least 121 °C for at least 15 minutes as recommended in the EMA guidelines. Accordingly, in a preferred embodiment the aqueous solution of nusinersen is heated to a temperature of 121 °C for 15 minutes.

[0021] As noted hereinabove, it is possible to combine thermal sterilisation with other sterilisation methods in order to ensure an optimum degree of sterility is achieved. Sterile filtration can be used to remove microbial contaminants by passing the solution through a suitable membrane. It is therefore preferred that the method further comprises the step of sterile filtration of the aqueous solution of nusinersen or a salt thereof.

[0022] Filters used in the sterile filtration step can be made of any suitable membrane material, for example polysulfone (PS / PSF), polyethersulfone (PES), polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE) or nylon. It is recommended that in order to remove microbial contaminants a pore size of <0.22 pm is used, with pore sizes of 0.20 pm and 0.10 pm commonly used in the art. In a preferred embodiment the pore size is 0.20 pm.

[0023] The step of sterile filtration may be performed either before or after the step of heating the aqueous solution of nusinersen or a salt thereof. It is beneficial to perform the step of heating after the step of sterile filtration, so that the heating step ensures the sterility assurance level of the filtered solution. As such, in a preferred embodiment the step of sterile filtration is performed prior the step of heating the aqueous solution of nusinersen or a salt thereof.

[0024] Further measures may be used to ensure optimum sterility of the solution during storage. Accordingly, it is preferred that the method further comprises the step of aseptically filling the sterilised aqueous solution of nusinersen or a salt thereof into a pre-sterilised container. Suitable containers include vials and syringes. It is preferred that the volume filled into each container is 1-20 ml_, preferably 5-6 ml_. Using the method of the present invention it is possible to store the sterilised solution of nusinersen or a salt thereof in a multi-dose container. It is preferred that the step of aseptic filling is performed prior to the step of heating the aqueous solution of nusinersen or a salt thereof. When the step of sterile filtration is also used, it is preferred that the step of aseptic filling is performed after the step of sterile filtration. Accordingly, in a preferred embodiment the method for the sterilisation of nusinersen or a salt thereof according to the present invention comprises the steps of:

[0025] (i) sterile filtration of an aqueous solution of nusinersen or a salt thereof;

[0026] (ii) aseptic filling of the solution of step (i) into a pre-sterilised container; and

[0027] (iii) heating the aqueous solution of nusinersen or a salt thereof stored within the container of step (ii).

[0028] Using the method of the present invention it is possible to achieve a sterility assurance level (SAL) of <106in the final solution of nusinersen or a salt thereof. This refers to the probability of micro-organisms surviving the sterilisation process, whereby an SAL of 106refers to a probably of not more than 1 non-sterile item in 1 x 106sterilised items of the final product.

[0029] Using the method of the present invention it is possible to ensure that at least 95% of the nusinersen or salt thereof remains in the final solution following sterilisation, preferably at least 99%. Using the method of the present invention it is further possible to ensure a percentage increase in degradation products in the final solution following sterilisation of 10% or less, preferably 5% or less. The relative amount of nusinersen or a salt thereof, and of its degradation products, can be measured using techniques such as HPLC.

[0030] Nusinersen is known for use in the treatment of SMA. In this regard, the method of the present invention is suitable for preparing a composition of nusinersen or a salt thereof for use as a pharmaceutical composition.

[0031] Accordingly, the aqueous solution of nusinersen or a salt thereof may comprise a pharmaceutically relevant concentration of nusinersen. It is preferred that nusinersen or salt thereof is present in the aqueous solution at a concentration equivalent of 1-20 mg / mL of basic nusinersen. The approved Spinraza® product contains a dose equivalent to 12 mg of basic nusinersen in a total volume of 5 mL, which represents a concentration of 2.4 mg / mL. Moreover, clinical trials are ongoing into the use of higher doses of nusinersen for the treatment of SMA, in particular doses equivalent to 28 mg and 50 mg of basic nusinersen. When formulated to deliver a dosage volume of 5 mL, this represents concentrations of 5.6 mg / mL and 10 mg / mL, respectively. The concentration of the aqueous solution of nusinersen is therefore more preferably 2.4 mg / mL, 5.6 mg / mL or 10 mg / L nusinersen basic equivalent.

[0032] Pharmaceutical compositions of nusinersen are primarily intended for intrathecal delivery. It is therefore important that compositions produced by the method of the present invention are compatible with physiological conditions. As such, it is preferred that the pH of the aqueous solution of nusinersen or a salt thereof is 7.0-7.5, preferably 7.2. The pH can be adjusted prior to sterilising by the addition of sodium hydroxide and hydrochloric acid as required. In an embodiment of the present invention, the heating temperature is 121°C and the pH is 7.2. It is further important that the solution contains adequate sodium chloride to be isotonic and physiological levels of electrolytes such a potassium, calcium and magnesium. Accordingly, it is preferred that the aqueous solution in which the nusinersen or salt thereof is dissolved is phosphate buffer saline (PBS) or artificial cerebrospinal fluid (aCSF).

[0033] A preferred aCSF composition comprises:

[0034] 0.100 mg / mL disodium hydrogen phosphate;

[0035] 0.050 mg / mL sodium dihydrogen phosphate dihydrate;

[0036] 8.770 mg / mL sodium chloride;

[0037] 0.220 mg / mL potassium chloride;

[0038] 0.210 mg / mL calcium chloride dihydrate; and 0.160 mg / mL magnesium chloride hexahydrate.

[0039] It is further preferred that the aqueous solution of nusinersen or a salt thereof is free from preservatives.

[0040] Antisense oligonucleotides such as nusinersen are commonly formulated in their salt forms. Examples of suitable salts include sodium and potassium, with the approved form of nusinersen formulated as its sodium salt. Accordingly, it is preferred that the nusinersen is in the form of its sodium salt. With regards to the concentration of nusinersen present in the solution, 2.40 mg / mL basic nusinersen is equivalent to 2.53 mg / mL nusinersen sodium, a concentration of 5.6 mg / mL is equivalent to 5.90 mg / mL nusinersen sodium and a concentration of 10 mg / mL is equivalent to 10.54 mg / mL nusinersen sodium.

[0041] The present invention further relates to a sterile nusinersen composition produced by the method of the invention, for use in the treatment of spinal muscular atrophy (SMA) in a human patient. SMA can refers to Type I SMA (infantile-onset SMA), Type II SMA or Type III SMA (later-onset SMA) or Type IV SMA (adult-onset SMA). The human patient may be presy mptomatic for SMA.

[0042] The present invention will now be described with reference to the following examples which are not intended to be limiting.

[0043] Examples

[0044] Example 1

[0045] Preparation of a nusinersen composition

[0046] Four batches of composition comprising nusinersen sodium were prepared with the content of the API and each excipient as defined in Table 1. Batches were subject to different methods of sterilisation as defined in Table 2.

[0047] Table 1

[0048] Table 2

[0049] Batch A - no sterilisation

[0050] Sodium dihydrogen phosphate dihydrate (0.0505 g), disodium hydrogen phosphate (0.1003 g), sodium chloride (8.7700 g), potassium chloride (0.2201 g), calcium chloride dihydrate (0.2101 g) and magnesium chloride hexahydrate (0.1606 g) were added to approximately 300 ml_ of Milli-Q ultrapure water (18.2 MQ.cm at 25°C). The solution was stirred using a magnetic stirrer until a clear solution was obtained. Additional Milli-Q water was added to obtain a total solution mass of 1000.0g and the resultant solution (Veh1 ) was stirred for 5 minutes. This solution was measured to have an osmolarity of 288 mOsm / kg and a pH of 6.94. An amount of 148.7 mg of nusinersen sodium was dissolved in 5 ml_ of Veh1 and transferred to a separate vessel. The original dissolution vessel was washed with Veh1 solution (3 x 10 ml_) and this was added to the API solution. Additional Veh1 solution was added to obtain a total solution mass of 40 g and the solution mixed for 5 minutes. A sample of the solution was assayed and the concentration adjusted by addition of further Veh1 solution (final solution mass of 50.3 g). The final solution was stirred for 5 minutes and the pH corrected with 1% sodium hydroxide to a final pH of 7.2.

[0051] The final solution was neither filtered nor thermally sterilised before filling into 6R vials (5.3 ml_ per vial).

[0052] Batch B - sterile filtration only

[0053] Sodium dihydrogen phosphate dihydrate (0.0806 g), disodium hydrogen phosphate (0.1500 g), sodium chloride (13.1554 g), potassium chloride (0.3306 g), calcium chloride dihydrate (0.3157 g) and magnesium chloride hexahydrate (0.1606 g) were added to approximately 1000 ml_ of Milli-Q ultrapure water (18.2 MQ.cm at 25°C). The solution was stirred using a magnetic stirrer until a clear solution was obtained. Additional Milli-Q water was added to obtain a total solution mass of 1507.2 g and the resultant solution (Veh1 ) was mixed for 5 minutes.

[0054] An amount of 2.339 g of nusinersen sodium was dissolved in 50 ml_ of Veh1 and transferred to a separate vessel containing 600 ml_ of Veh1 . The original dissolution vessel was washed with Veh1 solution (3 x 20 ml_) and this was added to the API solution. The solution mixed for 5 minutes. A sample of the solution was assayed and the concentration adjusted by addition of further Veh1 solution (final solution mass of 804.8 g). The final solution was stirred for 5 minutes and the pH corrected with 1% sodium hydroxide to a final pH of 7.2. The solution was stirred for a further five minutes.

[0055] The final solution was filtered through a 0.2 pm ECV polyethersulfone membrane and filled into 6R vials (5.3 ml_ per vial).

[0056] Batch C - sterile filtration and thermal sterilisation

[0057] Sodium dihydrogen phosphate dihydrate (0.0807 g), disodium hydrogen phosphate (0.1604 g), sodium chloride (14.0322 g), potassium chloride (0.3522 g), calcium chloride dihydrate (0.3363 g) and magnesium chloride hexahydrate (0.2606 g) were added to approximately 1300 ml_ of Milli-Q ultrapure water (18.2 MQ.cm at 25°C). Additional Milli-Q water was added to a final mass of 1608.64 g. The solution was stirred using a magnetic stirrer until a clear solution was obtained (15 mins) (Soli ). An amount of 2.4589 g of nusinersen sodium was dissolved in 70 ml_ of Soli and the resultant solution transferred to a vessel containing to a further 350 ml_ of Solution"! . A sample of the solution was assayed (assay 210.86%) and the concentration adjusted by addition of further Soli to obtain a final concentration of 2.4 mg / mL basic equivalent of nusinersen sodium. The pH was corrected with 1% sodium hydroxide to final pH of 7.2 and stirred for 5 minutes.

[0058] The final solution was filtered through a 0.2 pm ECV polyethersulfone membrane and filled into 6R vials (5.3 ml_ per vial). The vials were closed with an aluminium cap with septum and steam sterilised at 121 °C for 15 minutes

[0059] Batch D - sterile filtration and thermal sterilisation

[0060] Sodium dihydrogen phosphate dihydrate (0.0501 g), disodium hydrogen phosphate (0.1001 g), sodium chloride (8.7701 g), potassium chloride (0.2202 g), calcium chloride dihydrate (0.2101 g) and magnesium chloride hexahydrate (0.1603 g) were added to 380 ml_ of Milli-Q ultrapure water (18.2 MQ.cm at 25°C). The solution was stirred using a magnetic stirrer until a clear solution was obtained (—15 mins) (Soli).

[0061] An amount of 3.00474 g of API was dissolved in 100 ml_ Soli and stirred for 5 minutes. A sample of the solution was assayed and the concentration adjusted by addition of further Soli to obtain a final concentration of 2.4 mg / mL basic equivalent of nusinersen sodium. The pH was corrected with 1% sodium hydroxide to final pH of 7.2 and stirred for 5 minutes.

[0062] The final solution was filtered through a 0.2 pm ECV polyethersulfone membrane and filled into 6R vials (5.3 mL per vial). The vials were closed with an aluminium cap with septum and steam sterilised at 121 °C for 15 minutes.

[0063] Batch E - sterile filtration and thermal sterilisation

[0064] Sodium dihydrogen phosphate dihydrate (0.0504 g), disodium hydrogen phosphate (0.1002 g), sodium chloride (8.7707 g), potassium chloride (0.2207 g), calcium chloride dihydrate (0.2103 g) and magnesium chloride hexahydrate (0.1602 g) were added to approximately 400 mL of Milli-Q ultrapure water (18.2 MQ.cm at 25°C). The solution was stirred using a magnetic stirrer until a clear solution was obtained. Additional Milli-Q water was added to obtain a total solution mass of 1004.8 g and the resultant solution (Veh1 ) was mixed for 5 minutes.

[0065] An amount of 1 .5766 g of nusinersen sodium was dissolved in 30 mL of Veh1 and transferred to a separate vessel containing 400 mL of Veh1. The original dissolution vessel was washed with Veh1 solution (3 x 10 mL) and this was added to the API solution. The solution mixed for 5 minutes. A sample of the solution was assayed and the concentration adjusted by addition of further Veh1 solution (final solution mass of 533.5 g). The final solution was stirred for 5 minutes and the pH corrected with 1% sodium hydroxide to a final pH of 7.2. The solution was stirred for a further five minutes.

[0066] The final solution was filtered through a 0.2 pm ECV polyethersulfone membrane and filled into 6R vials (5.5 ml_ per vial). A batch of 94 vials were split into two groups: (1 ) 46 vials (E1 ) which were not subject to any further sterilisation steps and (2)48 vials (E2) which were thermally sterilised by steam sterilisation at 121 °C for 15 minutes.

[0067] Example 2

[0068] Effect of thermal sterilisation

[0069] Steam sterilisation can be performed at various conditions according to European Pharmacopeia 11.5 (5.1.1. Methods of Preparation of Sterile Products) and EMA guidelines (EMA / CHMP / CVMP / QWP / 850374 / 2015). A minimum temperature of 110°C and a lethality of Fo ^8 minutes is recommended.

[0070] Samples from batches A, B and E2 were subjected to steam sterilisation under various conditions as presented in Table 3. A comparison of the relative amount of nusinersen and degradation products indicated that the various thermal sterilisation conditions used did not influence the assay of the drug product. In general degradation products are impurities resulting from a chemical change in the active substance generated during the drug product manufacturing process and / or storage. The conditions that induce chemical or physical changes are e.g. light, temperature, pH, solvents, reactive groups in excipients or extractables. In this particular example, the term degradation products refers only to impurities brought about during the sterilisation step.

[0071]

[0072] Table 3 Example 3

[0073] Stability testing

[0074] Stability tests on samples from batches C-D were initiated according to conditions described in the ICH Q1 A guidelines, as detailed in Table 4. Long-term studies are performed for a minimum of 12 months accelerated studies performed for a minimum of 6 months.

[0075] Table 4 Initial stability testing results

[0076] The relative amount of nusinersen was assayed following 6 months of storage at the conditions listed in Table 5. The assay of the active substance and increase in degradation products presented is relative to the amounts present in the final sterilised product, as measured at the start of the testing procedure.

[0077] Table 5

[0078] The data in T able 5 show an improvement in the relative concentration of nusinersen in solution under both room temperature and accelerated storage conditions, combined with a reduced increase in degradation products. This supports the hypothesis that it is possible to change the storage conditions of pharmaceutical compositions comprising nusinersen from cold storage at 2-8°C to room temperature storage 25°C.

[0079] Example 4

[0080] Further stability testing

[0081] Two further batches of nusinersen (F1 and F2) were prepared in the same way as Example 1, E1 and E2. The vials of batch F1 were not subject any further sterilisation steps, whereas the vials of batch F2 were thermally sterilised by steam sterilisation at 121°C for 15 minutes. Stability testing was initiated according to conditions described in the ICH Q1 A guidelines, with the amount of nusinersen, total impurities and aggregates, as well as solution pH and osmolarity, assayed at the start of the study and after 6 months of storage.

[0082] Table 6 Table 7

[0083] The data in Tables 6 and 7, show that thermal sterilisation has no negative effect on the assay of nusinersen, or on the pH and osmolarity of the formulation. The use of thermal sterilisation was also not observed to result in any increase in impurities. The results are consistent regardless of the storage conditions or time of storage.

[0084] Additionally, a significant decrease in the amount of aggregates was observed in the formulations which underwent thermal sterilisation (0.12% for F2 compared to 0.17% for F1). This indicates that thermal sterilisation has a destructive influence on aggregates, leading to their reduction in the formulation. This is of key importance, given the potential of such aggregates to negatively impact drug safety, efficacy and stability.

Claims

Claims1. A method for sterilising nusinersen or a salt thereof, comprising a step of heating an aqueous solution of nusinersen or a salt thereof.

2. The method according to claim 1 , wherein the heating is carried out by autoclaving.

3. The method according to claim 1 or 2, wherein the heating is at a temperature of 110- 150°C.

4. The method according to any preceding claim, wherein the heating is carried out for 2- 180 minutes.

5. The method according to any preceding claim, wherein the heating is carried out at a temperature of 121 °C for 15 minutes.

6. The method according to any preceding claim, further comprising a step of sterile filtration of the aqueous solution of nusinersen or a salt thereof.

7. The method according to claim 6, wherein the step of sterile filtration is performed prior the step of heating the aqueous solution of nusinersen or a salt thereof.

8. The method of any preceding claim further comprising the step of aseptically filling the sterilised aqueous solution of nusinersen or a salt thereof into a pre-sterilised container.

9. The method according to any previous claim comprising the steps of:(i) sterile filtration of an aqueous solution of nusinersen or a salt thereof;(ii) aseptic filling of the solution of step (i) into a pre-sterilised container; and(iii) heating the aqueous solution of nusinersen or a salt thereof stored within the container of step (ii).

10. The method according to any of preceding claim, wherein the nusinersen or salt thereof is present in the aqueous solution at a concentration equivalent of 1-20 mg / mL basic nusinersen.

11. The method according to any preceding claim, wherein the aqueous solution of nusinersen or a salt thereof has a pH of 7.0-7.5.

12. The method according to any preceding claim, wherein the aqueous solution in which the nusinersen or salt thereof is dissolved is phosphate buffer saline (PBS) or artificial cerebrospinal fluid (aCSF).

13. The method according to claim 12, wherein the nusinersen or salt thereof is dissolved in aCSF, and the aCSF comprises:0.100 mg / mL disodium hydrogen phosphate;0.050 mg / mL sodium dihydrogen phosphate dihydrate;8.770 mg / mL sodium chloride; 0.220 mg / mL potassium chloride;0.210 mg / mL calcium chloride dihydrate; and0.160 mg / mL magnesium chloride hexahydrate.

14. The method according to any preceding claim, wherein the nusinersen is in the form of its sodium salt.

15. A sterile nusinersen composition obtainable by the method of any preceding claim, for use in the treatment of spinal muscular atrophy (SMA) in a human patient.

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