Lyophilized formulation of AAV drug product

Abstract

The present disclosure provides a stable lyophilized pharmaceutical composition comprising recombinant adeno-associated virus (rAAV) particles.In certain embodiments, the composition contains, in addition to rAAV particles, a buffer, one or more salts, surfactants, bulking agents, and sugars.The pharmaceutical composition of the present disclosure exhibits a significant degree of rAAV stability upon stress and storage.

Description

[Technical field]

[0001] The present invention relates to stable lyophilized compositions of recombinant adeno-associated virus (rAAV) products and methods for producing such compositions. [Background technology]

[0002] In recent years, gene therapy has attracted significant interest among biopharmaceutical companies and emerged as a transformative strategy that can provide new treatment options for numerous genetic diseases. This is due to advances in virology, immunology, vector engineering technology, vector manufacturing science, and other related fields that continue to provide improved vectors that have overcome the safety issues associated with earlier gene delivery vectors such as retroviruses. Currently, more than 700 gene therapy clinical trials are ongoing or completed worldwide. Adeno-associated viruses (AAVs), non-enveloped single-stranded DNA viruses, have emerged as an attractive class of therapeutic agents to deliver genetic material into host cells for gene therapy due to their ability to transduce a wide range of species and tissues in vivo, low risk of immunotoxicity, and mild innate and adaptive immune responses.

[0003] AAV gene therapy products are typically administered by injection, formulated as liquid solutions, and stored frozen at low temperatures (e.g., below −65°C). For any injectable liquid formulation, it is usually the preferred dosage form because it is easy to manufacture and convenient for the end user. However, maintaining the stability of AAV products in an aqueous environment can be difficult due to poor structural stability. Thus, freezing is commonly used to improve the long-term storage stability of AAV, which minimizes the rate of degradation due to physical and chemical instability, but freezing poses real challenges to supply chain and inventory management. In a typical drug product (DP) manufacturing process, AAV may be subjected to freeze-thaw when the drug substance (DS) is stored frozen and thawed prior to preparation of the DP fill-finish. A minimum of one freeze-thaw is also required before administering the AAV DP in the clinic. The DP manufacturing process, subsequent packaging and labeling, as well as clinical applications, require short-term storage of AAV in a liquid state under ambient conditions. Product degradation, such as AAV aggregation or loss of titer, can also occur during freeze-thaw and during long-term storage at elevated temperatures. Aggregation can reduce viral titers, decrease product efficacy, increase the load of potentially immunogenic viral proteins, affect biodistribution, and lead to inconsistent in vivo activity of AAV products. Therefore, maintaining the stability and activity of AAV products during manufacture and storage is critical for the successful development of AAV as a therapeutic agent. Summary of the Invention

[0004] In one aspect, the disclosure provides a pharmaceutical formulation comprising: (i) recombinant adeno-associated virus (rAAV) particles; (ii) a buffering agent at a concentration of about 1 mM to about 20 mM; (iii) a first salt at a concentration of about 10 mM to about 50 mM; (iv) a second salt at a concentration of about 20 mM to 100 mM; (v) a sugar at a concentration of about 1% w / v to about 10% w / v; (vi) a bulking agent at a concentration of about 1% w / v to about 6% w / v; and (vii) a surfactant at a concentration of about 0.001% w / v to about 0.1% w / v; and having a pH of about 6.8 to about 7.8.

[0005] In some embodiments, the buffer is a Tris buffer or a sodium phosphate buffer. In some cases, the buffer is a Tris buffer. In some embodiments, the concentration of the buffer is 5 mM to 15 mM. In some cases, the concentration of the buffer is 10 mM ± 2 mM.

[0006] In some embodiments, the first salt is sodium chloride, potassium chloride, or magnesium chloride. In some cases, the first salt is sodium chloride. In some embodiments, the concentration of the first salt is 20 mM to 40 mM. In some cases, the concentration of the first salt is 30 mM ± 5 mM.

[0007] In some embodiments, the second salt is arginine hydrochloride. In some embodiments, the concentration of the second salt is 70 mM to 90 mM. In some cases, the concentration of the second salt is 80 mM ± 5 mM. In some embodiments, the concentration of the second salt is about 20 mM to about 60 mM. In some cases, the concentration of the second salt is 40 mM ± 5 mM. In some embodiments, the concentration of the second salt is 30 mM to 90 mM.

[0008] In some embodiments, the sugar is sucrose or trehalose. In some cases, the sugar is sucrose. In some embodiments, the concentration of the sugar is 3% w / v to 7% w / v. In some cases, the concentration of the sugar is 5% w / v ± 1% w / v.

[0009] In some embodiments, the bulking agent is mannitol or glycine. In some cases, the bulking agent is mannitol. In some embodiments, the bulking agent has a concentration of 2% w / v to 5% w / v. In some cases, the bulking agent has a concentration of 3.3% w / v ± 0.5% w / v.

[0010] In some embodiments, the surfactant is a polysorbate or a poloxamer. In some cases, the polysorbate is polysorbate 80 (PS80). In some cases, the poloxamer is poloxamer 188 (P188). In some embodiments, the concentration of the surfactant is 0.001% w / v to 0.1% w / v. In some cases, the concentration of the surfactant is 0.01% w / v ± 0.005% w / v.

[0011] In some embodiments, the pH of the formulation is 7.0 to 7.6. In some cases, the pH of the formulation is 7.3±0.1.

[0012] In some embodiments, the ratio of bulking agent to sugar is 1:1 to 1:3. In some embodiments, the ratio of bulking agent to sugar is 1:1 to 1:7. In some cases, the ratio of mannitol to sucrose is 1:1 to 1:3. In some cases, the ratio of mannitol to sucrose is 1:1 to 1:7. In some embodiments, the ratio of mannitol to sucrose is 3.3:5. In some embodiments, the ratio of the first salt to the second salt is 1:1 to 1:4. In some cases, the ratio of sodium chloride to arginine hydrochloride is 1:1 to 1:4. In some embodiments, the ratio of sodium chloride to L-arginine hydrochloride is 3:8.

[0013] In some embodiments, the formulation has a total ionic strength of 70 mM to 120 mM. In some cases, the total ionic strength is 80 mM ± 5 mM. In some cases, the total ionic strength is 110 mM ± 10 mM.

[0014] In some embodiments, the pharmaceutical formulation comprises between 1×10E10 vg / mL and 1×10E15 vg / mL. In some cases, the pharmaceutical formulation comprises between 1×10E12 vg / mL and 1×10E14 vg / mL. In some cases, the pharmaceutical formulation comprises about 1×10E13 vg / mL.

[0015] In one aspect, the disclosure provides a pharmaceutical formulation comprising (i) recombinant adeno-associated virus (rAAV) particles at a concentration of 1×10E10 vg / mL to 1×10E15 vg / mL, (ii) phosphate buffer at a concentration of 10 mM±1 mM, (iii) sodium chloride at a concentration of 30 mM±3 mM, (iv) L-arginine hydrochloride at a concentration of 80 mM±8 mM, (v) sucrose at a concentration of 5% w / v±0.5% w / v, (vi) mannitol at a concentration of 3.3% w / v±0.3% w / v, (vii) poloxamer 188 at a concentration of 0.005% w / v±0.001% w / v, and having a pH of 7.3±0.2. In some cases, the pharmaceutical formulation comprises 1×10E12 vg / mL to 1×10E14 vg / mL. In some cases, the pharmaceutical formulation contains about 1×10E13 vg / mL.

[0016] In one aspect, the disclosure provides a pharmaceutical formulation comprising (i) recombinant adeno-associated virus (rAAV) particles at a concentration of 1×10E10 vg / mL to 1×10E15 vg / mL, (ii) Tris buffer at a concentration of 10 mM±1 mM, (iii) sodium chloride at a concentration of 30 mM±3 mM, (iv) L-arginine hydrochloride at a concentration of 80 mM±8 mM, (v) sucrose at a concentration of 5% w / v±0.5% w / v, (vi) mannitol at a concentration of 3.3% w / v±0.3% w / v, (vii) poloxamer 188 at a concentration of 0.01% w / v±0.002% w / v, and having a pH of 7.3±0.2. In some cases, the pharmaceutical formulation comprises 1×10E12 vg / mL to 1×10E14 vg / mL. In some cases, the pharmaceutical formulation contains about 1×10E13 vg / mL.

[0017] In some embodiments, the rAAV particles are of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-DJ, AAV-DJ / 8, AAV-Rh10, AAV-retro, AAV-PHP.B, AAV8-PHP.eB, or AAV-PHP.S. In some embodiments, the rAAV particles are of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In some cases, the rAAV particles are of serotype AAV1. In some cases, the rAAV particles are of serotype AAV5. In some cases, the rAAV particles are of serotype AAV8. In some cases, the rAAV particles are of serotype AAV9.

[0018] In some embodiments, the formulation has an average particle size of about 20 nm to about 40 nm as determined by dynamic light scattering.

[0019] In some embodiments, the formulation comprises a population of rAAV particles that contain at least 95% intact capsids as determined by anion exchange chromatography.

[0020] In one aspect, the present disclosure provides a stable lyophilized pharmaceutical composition formed by lyophilizing a pharmaceutical formulation as described above or discussed herein.

[0021] In some embodiments, the lyophilized pharmaceutical composition has a moisture content of 1.5% or less. In some embodiments, the lyophilized pharmaceutical composition has a moisture content of 1.0% or less. In some embodiments, the lyophilized pharmaceutical composition has a moisture content of 0.5% to 0.8%, or 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, or 0.8%. In any of the various embodiments, the moisture content may be determined by Karl Fischer (KF) titration.

[0022] In some embodiments, the lyophilized pharmaceutical composition can be reconstituted in 30 seconds or less. In some embodiments, the lyophilized pharmaceutical composition can be reconstituted in 20 seconds or less. In some embodiments, the lyophilized pharmaceutical composition can be reconstituted in 15 seconds or less.

[0023] In one aspect, the present disclosure provides a reconstituted pharmaceutical product formed by reconstituting a lyophilized pharmaceutical composition as described above or discussed herein.

[0024] In some embodiments, the reconstituted pharmaceutical product comprises an average particle size of about 20 nm to about 40 nm as determined by dynamic light scattering.

[0025] In some embodiments, the reconstituted pharmaceutical product comprises a population of rAAV particles comprising at least 95% complete capsids as determined by anion exchange chromatography. In some embodiments, the reconstituted pharmaceutical product comprises a population of rAAV particles comprising a percentage of complete capsids as determined by anion exchange chromatography, and the percentage of complete capsids in the reconstituted pharmaceutical product is within 2% of the percentage of complete capsids in the pharmaceutical formulation prior to lyophilization. In some cases, the percentage of complete capsids in the reconstituted pharmaceutical product is within 1% of the percentage of complete capsids in the pharmaceutical formulation prior to lyophilization. In some cases, the percentage of complete capsids in the reconstituted pharmaceutical product is within 0.5% of the percentage of complete capsids in the pharmaceutical formulation prior to lyophilization.

[0026] In some embodiments, the reconstituted pharmaceutical product comprises a vector genome titer as determined by droplet digital polymerase chain reaction, and the vector genome titer in the reconstituted pharmaceutical product is within 10% of the vector genome titer present in the pharmaceutical formulation prior to lyophilization. In some cases, the vector genome titer in the reconstituted pharmaceutical product is within 7% of the vector genome titer present in the pharmaceutical formulation prior to lyophilization.

[0027] In any of the various embodiments described above or discussed herein, the reconstituted pharmaceutical product may be formed by reconstituting a lyophilized pharmaceutical composition after one month of storage at a temperature between 2-8° C. In some embodiments, the reconstituted pharmaceutical product may be formed by reconstituting a lyophilized pharmaceutical composition after three months of storage at a temperature between 2-8° C. In some embodiments, the reconstituted pharmaceutical product may be formed by reconstituting a lyophilized pharmaceutical composition after six months of storage at a temperature between 2-8° C.

[0028] In one aspect, the disclosure provides a container housing a pharmaceutical formulation as described above or discussed herein.

[0029] In one aspect, the present disclosure provides a container housing a lyophilized pharmaceutical composition as described above or discussed herein.

[0030] In one aspect, the present disclosure provides a container containing a reconstituted pharmaceutical product as described above or discussed herein.

[0031] In any of the various embodiments, the container may be a glass vial.

[0032] In one aspect, the disclosure provides a kit comprising a container as described above or discussed herein and instructions for use.

[0033] In one aspect, the present disclosure provides a method of producing a stable lyophilized pharmaceutical composition as described above or discussed herein, the method comprising performing a lyophilization process on a pharmaceutical formulation as described above or discussed herein.

[0034] In some embodiments, the freeze-drying process includes (i) freezing, (ii) annealing, (iii) primary drying, and (iv) secondary drying. In some cases, freezing is performed at a temperature of -45°C ± 5°C for 60 minutes ± 10 minutes. In some cases, freezing is performed both before and after annealing. In some cases, annealing is performed at a temperature of -20°C ± 5°C for 6 hours ± 30 minutes. In some cases, primary drying is performed at a temperature of -26°C ± 5°C for 50 hours ± 10 hours. In some cases, secondary drying is performed at a temperature of 35°C ± 5°C for between 5 hours and 20 hours. In some cases, secondary drying is performed at a temperature of 35°C ± 5°C for 15 hours ± 5 hours.

[0035] In various embodiments, any of the features or components of the embodiments described above or discussed herein may be combined, and such combinations are encompassed within the scope of the present disclosure. Any specific value described above or discussed herein may be combined with another associated value described above or discussed herein to recite a range having values ​​representing the upper and lower limits of the range, and such ranges are encompassed within the scope of the present disclosure.

[0036] Other embodiments will be apparent from consideration of the detailed description that follows. [Brief description of the drawings]

[0037] [Figure 1A] Figures 1A and 1B show an AAV capsid containing a heterologous nucleic acid molecule (e.g., a therapeutic gene or gene of interest (GOI)) (Figure 1A), as well as an empty capsid, a partially full capsid, and a full capsid (Figure 1B). [Figure 1B] Same as above. [Figure 2-1]Figure 2 shows the size distribution profiles of seven formulations evaluated in a lyophilization feasibility study (Example 2). The left solid peak in all panels except F7 represents the particle size before lyophilization, and the right dashed peak in all panels except F7 represents the particle size after lyophilization. In the F7 panel, the two peaks overlap each other. [Figure 2-2] Same as above. [Diagram 3] FIG. 3 shows a particle size comparison of AAV8 and a placebo formulation (without AAV) and demonstrates the presence of nanobubbles in the reconstituted composition. [Figure 4] Figure 4 shows the effect of surfactant concentration in the AAV formulation on AAV titer recovery. As shown, polysorbate 80 and poloxamer 188 showed comparable recoveries at 0.005% w / v in the formulations tested, and increasing the concentration of surfactant (P188) to 0.01% w / v improved recovery. Further increasing the surfactant concentration to 0.2% w / v did not significantly change the recovery from that observed at 0.01% w / v. [Diagram 5] Figure 5 shows the cake structure of each of the two formulations in the absence (BA1) and presence (BA2) of mannitol after lyophilization. The BA2 formulation results in an elegant cake appearance without the defects resulting from the BA1 formulation. [Figure 6A] Figures 6A and 6B show the results of thermal characterization of AAV8 formulations: Figure 6A shows the characterization of the collapse temperature Tc by lyophilization microscopy, and Figure 6B shows the glass transition temperature Tg' by DSC in formulation F7. [Figure 6B] Same as above. [Figure 7A] Figures 7A, 7B, and 7C show the in-solution stability results of AAV8 formulations at 25° C. AAV8 vectors were formulated in F7 and stored at 25° C. for 2 days. Figure 7A shows particle size analysis by DLS, Figure 7B shows viral genome titer analysis by ddPCR, and Figure 7C shows analysis of capsid purity by percentage of complete capsids using AEX-UPLC. [Figure 7B] Same as above. [Figure 7C]Same as above. [Figure 8A] Figures 8A, 8B, and 8C show storage stability results at 2-8 °C for lyophilized AAV8 products in F7 formulation. Results for the formulation before lyophilization are shown as light colored dots, and results for the reconstituted product after lyophilization are shown as dark colored dots. Figure 8A shows particle size analysis by DLS, Figure 8B shows viral genome titer analysis by ddPCR, and Figure 8C shows analysis of capsid purity by percentage of complete capsids using AEX-UPLC. [Figure 8B] Same as above. [Figure 8C] Same as above. [Figure 9A] Figures 9A and 9B show the results of salt screening for formulation preparation. Figure 9A shows the formulation composition of a formulation containing 1 x 1013 vg / mL AAV8, 10 mM Tris, 30 mM NaCl, 80 mM L-Arg HCl, 5% w / v sucrose, 3.3% w / v mannitol, 0.01% w / v P188, pH 7.3 (control), and formulations F8-F10 containing various types and concentrations of salt. Figure 9B shows the recovery of genome titer after lyophilization assessed by comparison of viral genome titer in the formulation before lyophilization and in the product reconstituted after lyophilization. [Figure 9B] Same as above. [Figure 10A] Figures 10A, 10B, and 10C show the in-solution stability results of AAV8 formulations at 25° C. AAV8 vectors were formulated in a formulation containing 1×10 vg / mL AAV8, 10 mM Tris, 30 mM NaCl, 80 mM L-Arg HCl, 5% w / v sucrose, 3.3% w / v mannitol, 0.01% w / v P188, pH 7.3, and then stored at 25° C. for 2 days. Figure 10A shows particle size analysis by DLS, Figure 10B shows viral genome titer analysis by ddPCR, and Figure 10C shows capsid purity analysis by percent complete capsids using AEX-UPLC. [Figure 10B] Same as above. [Figure 10C] Same as above. [Figure 11A]Figures 11A, 11B, and 11C show the results of stability of lyophilized AAV8 products in formulation (1x1013 vg / mL AAV8, 10mM Tris, 30mM NaCl, 80mM L-Arg HCl, 5% w / v sucrose, 3.3% w / v mannitol, 0.01% w / v P188, pH 7.3) at 25°C. The results of the formulation before lyophilization are shown as light dots, and the results of the reconstituted product after lyophilization are shown as dark dots. Figure 11A shows particle size analysis by DLS, Figure 11B shows viral genome titer analysis by ddPCR, and Figure 11C shows analysis of capsid purity by percentage of intact capsids using AEX-UPLC. [Figure 11B] Same as above. [Figure 11C] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting, since the scope of the present invention is limited only by the appended claims.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.As used herein, the term "about" when used in relation to a specific referenced numerical value means that the value can vary by 1% or less from the referenced value.For example, as used herein, the expression "about 100" includes 99 and 101, and all values ​​therebetween (for example, 99.1, 99.2, 99.3, 99.4, etc.).

[0040] As used herein, the terms "include", "includes", and "including" are intended to be open-ended and are understood to mean "comprise", "comprises", and "comprising", respectively. As used herein, the term "and / or" refers to and includes all possible combinations of one or more of the associated listed items, as well as the absence of a combination when interpreted alternatively ("or"). As used herein, the term "or" refers to any one member of a particular list and includes any combination of the members of that list. As used herein, unless otherwise indicated or clear from the context, in the context described in this disclosure (particularly in the context of the claims below), "a", "an", "the", and similar referents are deemed to include both the singular and the plural.

[0041] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All patents, applications, and non-patent publications mentioned herein are incorporated by reference in their entirety.

[0042] definition "Adeno-associated virus" or "AAV" is a non-pathogenic parvovirus with a single-stranded DNA, non-enveloped genome of approximately 4.7 kb, and an icosahedral conformation. AAV was first discovered in 1965 as a contaminant of adenovirus preparations. AAV belongs to the genus Dependovirus and family Parvoviridae, and requires helper functions from either herpesvirus or adenovirus for replication. In the absence of helper virus, AAV can set up latency by integrating into human chromosome 19 at 19q13.4. The AAV genome consists of two open reading frames (ORFs), one for each of the two AAV genes Rep and Cap. The AAV DNA termini have 145 bp inverted terminal repeats (ITRs), and the 125 terminal bases are palindromic, leading to a characteristic T-shaped hairpin structure.

[0043] "Recombinant viral particle" refers to a viral particle that includes one or more heterologous sequences (eg, nucleic acid sequences not of viral origin) which may be adjacent to at least one viral nucleotide sequence.

[0044] "Recombinant AAV particle" refers to an adeno-associated virus particle that contains one or more heterologous sequences (e.g., nucleic acid sequences not of AAV origin) that may be flanked by at least one, e.g., two, AAV inverted terminal repeats (ITRs). Such rAAV particles can be replicated and packaged when present in a host cell that is infected with a suitable helper virus (or expresses suitable helper functions) and expresses the AAV rep and cap gene products (i.e., AAV Rep and Cap proteins).

[0045] "Virus particle" refers to a viral particle that is composed of at least one viral capsid protein and an encapsidated viral genome.

[0046] "Heterologous" means that the entity is derived from a genotypically distinct entity from the rest of the entity to which it is compared or introduced or incorporated. For example, a nucleic acid introduced into a different cell type by genetic engineering techniques is a heterologous nucleic acid (which can encode a heterologous polypeptide when expressed). Similarly, a cellular sequence (e.g., a gene or a portion thereof) incorporated into a viral particle is a heterologous nucleotide sequence with respect to the viral particle.

[0047] The term "empty virus capsid" or "empty capsid" refers to a capsid that does not contain a heterologous nucleic acid molecule (e.g., a therapeutic gene), as depicted in FIG. 1B.

[0048] The term "partially complete viral capsid" or "partially complete capsid" refers to a capsid that contains only some of the heterologous nucleic acid molecule (e.g., a therapeutic gene), as depicted in FIG. 1B.

[0049] The term "complete viral capsid" or "complete capsid" refers to a capsid containing a complete heterologous nucleic acid molecule (e.g., a therapeutic gene or gene of interest), as depicted in FIG. 1B.

[0050] When used in relation to the compositions described herein, the term "pharmaceutical acceptable" refers to molecular entities and other components of such compositions that are physiologically acceptable and do not normally cause undesirable reactions when administered to a subject.Preferably, the term "pharmaceutical acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in mammals, more specifically in humans.A "pharmaceutical acceptable salt" is a salt that can be formulated into a compound or conjugate for pharmaceutical use, including, for example, metal salts (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines, which are safe for administration to a subject (e.g., human) in a drug formulation.

[0051] As used herein, a "physiological concentration" of a salt refers to a salt concentration of about 100 mM to about 200 mM of a pharma- ceutically acceptable salt.

[0052] As used herein, a "subphysiological concentration" of a salt refers to a salt concentration of a pharma- ceutically acceptable salt of less than about 100 mM. In certain embodiments, a subphysiological concentration of a salt is less than about 80 mM of a pharma- ceutically acceptable salt. In other embodiments, a subphysiological concentration of a salt is less than about 70 mM, less than about 60 mM, less than about 50 mM, less than about 40 mM, less than about 30 mM, less than about 20 mM, or less than about 10 mM of a pharma- ceutically acceptable salt.

[0053] As used herein, the term "treat" or "treatment" of a condition, disorder, or pathology includes (1) preventing, delaying, or reducing the occurrence and / or likelihood of at least one clinical or subclinical symptom of the condition, disorder, or pathology in a subject who may be afflicted with or susceptible to the condition, disorder, or pathology, but who has not yet experienced or exhibited a clinical or subclinical symptom of the condition, disorder, or pathology, or (2) inhibiting the condition, disorder, or pathology, i.e., preventing, reducing, or delaying the onset / progression of the disease or its recurrence, or at least one clinical or subclinical symptom thereof, or (3) palliating the disease, i.e., regressing the condition, disorder, or pathology, or at least one clinical or subclinical symptom thereof. When using these terms, 100% or complete treatment is not necessarily meant. Rather, there are various degrees of treatment that a person skilled in the art recognizes as having a potential benefit or therapeutic effect. The benefit to the subject being treated is statistically significant or at least perceptible to the patient or physician.

[0054] As used herein, a "therapeutically effective amount or dose," or a "sufficient amount or dose," refers to a dose that produces the effect for which it is administered. The exact dose will depend on the purpose of the treatment and will be ascertainable by one of ordinary skill in the art using known techniques.

[0055] The terms "patient" and "subject" are used interchangeably and refer to an organism suffering from or prone to suffering from a condition that can be prevented or treated by administering the composition of the present disclosure, including both human and non-human animals.Examples of subjects include, but are not limited to, humans, chimpanzees and other apes and monkey species; livestock animals such as cows, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds including poultry, wild birds, game birds, such as chickens, turkeys and other galliformes, ducks, geese, etc. This term does not imply a specific age. Thus, adults, juveniles and newborns are contemplated.

[0056] As used herein, "storage" means that the formulation is not administered to a subject immediately after preparation, but is maintained under particular conditions (e.g., at a particular temperature, etc.) for a period of time prior to use. For example, liquid or lyophilized formulations can be maintained for days, weeks, months, or years prior to administration to a subject under a variety of temperatures, such as refrigerated (e.g., between 0° and 10° C.) or room temperature (e.g., at temperatures up to 32° C.).

[0057] In a preferred embodiment, the formulations and compositions of the present disclosure are sterile. By "sterile" is meant that the composition is substantially free of immunogenic components, e.g., substantially free of microorganisms (e.g., fungi, bacteria, non-AAV viruses, spore forms, etc.).

[0058] overview The present disclosure provides formulations, such as pharmaceutical formulations and lyophilized compositions, that are suitable for administration to subjects (e.g., upon reconstitution), maintain stability, and are suitable for long-term storage of AAV while minimizing loss of AAV potency.In certain embodiments, the pharmaceutical formulations and compositions provided herein retain significant AAV activity when stored for long periods of time.In certain embodiments, the pharmaceutical formulations and compositions discussed herein reduce or delay degradation and / or aggregation.

[0059] A particular challenge of AAV formulation and product development is that long-term storage of AAV products is often inefficient, resulting in a short shelf life (typically 12-18 months) compared to recombinant protein-based biologics. For example, existing formulations of AAV products (Luxturna® and Zolgensma®) only support a limited shelf life of 12 months at or below -60 °C, and in the case of Zolgensma®, only 14 days of stability at 2-8 °C. Frozen AAV products also pose practical challenges for supply chain and inventory management, especially when low temperatures (e.g., below -65 °C) are required for shipping, distribution, and storage.

[0060] Lyophilized compositions such as those discussed herein address both storage and supply chain management challenges. In particular, the formulations and compositions of the present disclosure utilize carefully selected excipients and concentrations to ensure long-term storage stability in solid state to support shelf life, and room temperature stability (e.g., typically at least 48 hours) in liquid state to support manufacturing operations and clinical / therapeutic use conditions. Furthermore, the lyophilized compositions of the present disclosure provide cake elegance and acceptable reconstitution properties.

[0061] The lyophilization cycle discussed herein includes four stages: freezing, annealing (in the presence of bulking agents such as mannitol or glycine), primary drying, and secondary drying. Pharmaceutical formulations undergoing lyophilization play an important role in determining the quality of the pharmaceutical product because the drying process following freezing can impose various stresses such as cold denaturation, solute (protein) concentration (increase caused by freeze concentration), formation of ice crystals, crystallization of important excipients (e.g., buffers, salts), and pH changes. These stresses, if not carefully considered in the selection of formulation excipients, can induce conformational changes in proteins and ultimately affect the stability of the lyophilized pharmaceutical product.

[0062] There are several challenges with lyophilized AAV products that must be addressed during formulation development. First, AAV formulations require the use of high ionic strength to prevent aggregation, but salts can significantly reduce the collapse temperature (Tc) or glass transition temperature (Tg') of amorphous systems, necessitating the use of lower freezing temperatures in the freezing step, which may result in an insufficient sublimation process. Second, AAV pharmaceutical products are typically formulated at low concentrations in the range of 1E12-1E13 vector genome copies (vg) / mL, which corresponds to approximately 0.01-0.2 mg / mL, resulting in a further reduction in Tg' and the risk of losing AAV due to adsorption to different surfaces. Third, due to the low AAV concentration, there may be a need to include bulking agents in the formulation to prevent AAV loss from the vial during drying and to maintain structural integrity from cake collapse. However, in doing so, the bulking agents may not achieve sufficient crystallization during the annealing step and may continue to crystallize during secondary drying and subsequent storage in the dry solid, thereby affecting the reconstitution as well as the stability of the lyophilized pharmaceutical product.

[0063] These and other problems are addressed by the pharmaceutical formulations and compositions of the present disclosure. In certain embodiments, the present disclosure provides a formulation of AAV comprising a therapeutically effective amount or dose of AAV, one or more buffering agents that provide a suitable pH for the formulation, one or more pharma- ceutically acceptable salts, a stabilizing concentration of one or more sugars and / or sugar alcohols, a non-ionic surfactant, and a bulking agent. Generally, the AAV formulations provided herein are suitable for pharmaceutical administration.

[0064] Adeno-associated virus particles AAV particle can be recombinant AAV (rAAV) particle.rAAV particle comprises AAV vector that encodes heterologous transgene or heterologous nucleic acid molecule.AAV particle can be avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, primate AAV and non-primate AAV.

[0065] In some embodiments, the AAV particle comprises a heterologous nucleic acid molecule (e.g., a therapeutic gene or a gene of interest). In some embodiments, the heterologous nucleic acid molecule is operably linked to a promoter. Exemplary promoters include, but are not limited to, cytomegalovirus (CMV) immediate early promoter, RSV LTR, MoMLV LTR, phosphoglycerate kinase-1 (PGK) promoter, simian virus 40 (SV40) promoter and CK6 promoter, transthyretin promoter (TTR), TK promoter, tetracycline responsive promoter (TRE), HBV promoter, hAAT promoter, LSP promoter, chimeric liver specific promoter (LSP), E2F promoter, telomerase (hTERT) promoter, cytomegalovirus enhancer / chicken beta-actin / rabbit beta-globin promoter, and elongation factor 1-alpha promoter (EF1-alpha) promoter. In some aspects, the promoter comprises a human beta-glucuronidase promoter or a cytomegalovirus enhancer linked to a chicken beta-actin (CBA) promoter. The promoter can be a constitutive, inducible, or repressible promoter. In some aspects, the present disclosure provides a recombinant vector comprising a nucleic acid encoding a heterologous transgene of the present disclosure operably linked to a CBA promoter. In some cases, a native promoter for the transgene or a fragment thereof is used. A native promoter can be used when it is desired that the expression of the transgene should mimic the native expression. A native promoter can be used when the expression of the transgene must be regulated temporally or developmentally, or in a tissue-specific manner, or in response to a specific transcriptional stimulus. In further aspects, other native expression control elements, such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, can also be used to mimic the native expression.

[0066] In an exemplary embodiment, the pharmaceutical formulation and composition of the present disclosure comprises AAV. The AAV may be of any AAV serotype. In an exemplary embodiment, the AAV is of AAV1 serotype, AAV2 serotype, AAV3 serotype, AAV4 serotype, AAV5 serotype, AAV6 serotype, AAV7 serotype, AAV8 serotype, AAV9 serotype, or AAV10 serotype. In an exemplary embodiment, the AAV is of AAV1 serotype. In an exemplary embodiment, the AAV is of AAV5 serotype. In an exemplary embodiment, the AAV is of AAV8 serotype. In an exemplary embodiment, the AAV is of AAV9 serotype. In some cases, the AAV is AAV11, AAV12, AAV-DJ, AAV-DJ / 8, AAV-Rh10, AAV-retro, AAV-PHP.B, AAV8-PHP.eB, or AAV-PHP.S. In some embodiments, the AAV particles are of serotype AAV1, AAV5, AAV8, or AAV9.

[0067] In exemplary embodiments, the pharmaceutical preparations and compositions comprise high titer AAV products. In exemplary embodiments, the pharmaceutical preparations and compositions comprise at least about 10E10 viral particles (vp), or at least about 10E11 viral particles (vp), or at least about 10E12 viral particles (vp), or at least about 10E13 viral particles (vp). In exemplary embodiments, the pharmaceutical preparations and compositions comprise at least about 10E14 viral particles (vp), or at least about 10E15 viral particles (vp), for example, at least about 2×10E15 viral particles (vp), or at least about 5×10E15 viral particles (vp). The pharmaceutical preparations and compositions also comprise about 10E10 vector genome or viral genome (vg), or at least about 10E11 vector genome or viral genome (vg), or at least about 10E12 vector genome or viral genome (vg), or at least about 10E13 vector genome or viral genome (vg). In exemplary embodiments, the pharmaceutical formulations and compositions comprise at least about 10E14 vector genome or viral genome (vg), or at least about 10E15 vector genome or viral genome (vg), such as at least about 2x10E15 vector genome or viral genome (vg), or at least about 5x10E15 vector genome or viral genome (vg).

[0068] Excipients and pH The pharmaceutical formulations and compositions of the present disclosure (including liquid pharmaceutical formulations, lyophilized pharmaceutical compositions, and reconstituted pharmaceutical products, unless otherwise indicated) contain carefully selected excipients at concentrations that maintain the stability of the AAV particles during manufacture and use, as well as during long-term storage under conditions that facilitate supply chain logistics. In various embodiments, the present disclosure includes liquid pharmaceutical formulations, lyophilized pharmaceutical compositions, and reconstituted pharmaceutical products that contain AAV particles (e.g., as discussed above or herein) in combination with one or more buffers within a defined pH range, one or more salts, one or more sugars or sugar alcohols, one or more bulking agents, and one or more surfactants. The concentrations discussed below or herein in connection with various excipients refer to liquid pharmaceutical formulations, formulations that are subsequently subjected to lyophilization, or reconstituted compositions prepared by reconstitution of a lyophilized composition. The various excipients included in the compositions of the present disclosure include, among others, salts, sugars, and bulking agents. Salts may contribute to the stabilization of AAV particles by mitigating the formation of aggregates, but may significantly reduce the collapse temperature. Sugars can act as cryoprotectants during lyophilization, but can also inhibit the crystallization of bulking agents. Failure to adequately crystallize bulking agents can adversely affect the glass transition temperature and impair storage stability. Therefore, careful selection of excipients and concentrations is necessary to produce stable AAV formulations and lyophilized compositions.

[0069] A. Buffers and pH

[0070] The liquid pharmaceutical formulation, lyophilized pharmaceutical composition, and reconstituted pharmaceutical product of the present disclosure comprise a pharma- ceutically acceptable buffer. In some cases, the buffer includes, but is not limited to, phosphate buffer, histidine buffer, sodium citrate buffer, HEPES buffer, Tris buffer, bicine buffer, glycine buffer, N-glycylglycine buffer, sodium acetate buffer, sodium carbonate buffer, glycylglycine buffer, lysine buffer, arginine buffer, sodium phosphate buffer, and / or mixtures thereof. In some embodiments, the buffer is a phosphate buffer (e.g., sodium phosphate buffer). In some embodiments, the buffer is a Tris buffer.

[0071] In exemplary embodiments, the pharmaceutical compositions of the present disclosure comprise about 1 to about 30, about 1 to about 20 mM, about 5 to about 25 mM, about 5 to about 15, about 7 to about 13 mM, or about 8 to about 12 mM of a buffering agent. In exemplary aspects, the pharmaceutical compositions comprise about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, or about 25 mM of a buffering agent.

[0072] In exemplary embodiments, the pharmaceutical composition of the present disclosure comprises about 1 mM to about 20 mM, about 5 mM to about 15 mM, about 8 mM to about 12 mM, or 10 mM ± 1 mM Tris buffer. In exemplary embodiments, the pharmaceutical composition of the present disclosure comprises about 1 mM to about 20 mM, about 5 mM to about 15 mM, about 8 mM to about 12 mM, or 10 mM ± 1 mM sodium phosphate buffer. In various embodiments, the pharmaceutical composition comprises about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, or about 15 mM Tris buffer. In various embodiments, the pharmaceutical composition comprises about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, or about 15 mM sodium phosphate buffer. In certain embodiments, the pharmaceutical composition comprises 10 mM ± 2 mM Tris buffer. In certain embodiments, the pharmaceutical composition comprises 10 mM ± 2 mM sodium phosphate buffer.

[0073] In exemplary embodiments, the pharmaceutical compositions of the present disclosure have a physiologically compatible pH. In certain embodiments, AAV formulations are provided that contain a buffer suitable for maintaining the formulation at or near a neutral pH.

[0074] In exemplary embodiments, the pH of the pharmaceutical composition is about 6.5 to about 9.0, about 6.5 to about 8.0, about 6.9 to about 7.7, or about 7.0 to about 7.5. In certain embodiments, the pH of the formulation is about 6.5 or about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.0. In exemplary embodiments, the pH of the pharmaceutical composition is about 7.2, about 7.3, or about 7.4. In certain embodiments, the pH of the pharmaceutical composition is 7.3±0.1. In certain embodiments, the pH of the pharmaceutical composition is 7.3±0.05.

[0075] B. Salt

[0076] The liquid pharmaceutical formulations, lyophilized pharmaceutical compositions, and reconstituted pharmaceutical products of the present disclosure comprise one or more pharma- ceutically acceptable salts.

[0077] In various embodiments, pharma- ceutically acceptable salts include, but are not limited to, metal salts such as sodium, potassium, and cesium salts; alkaline earth metal salts such as calcium and magnesium salts; organic amine salts such as triethylamine, guanidine, and N-substituted guanidine salts, acetamidine, and N-substituted acetamidine, pyridine, picoline, ethanolamine, triethanolamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine salts.Pharmaceutically acceptable (basic nitrogen center) salts include, but are not limited to, inorganic acid salts such as hydrochloride, hydrobromide, sulfate, and phosphate; organic acid salts such as trifluoroacetate and maleate; sulfonate salts such as methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, and naphthalenesulfonate; amino acid salts such as alginate, alanine, aspartate, and glutamate; and carbohydrate salts such as gluconate and galacturonate. Non-limiting examples of pharmaceutically acceptable salts include, but are not limited to, sodium, ammonium, potassium, calcium, magnesium salts (e.g., sodium chloride, ammonium chloride, potassium chloride, calcium chloride, magnesium chloride; sodium acetate, ammonium acetate, potassium acetate, calcium acetate, and magnesium acetate; sodium citrate, ammonium citrate, potassium citrate, calcium citrate, and magnesium citrate; sodium phosphate, ammonium phosphate, potassium phosphate, calcium phosphate, and magnesium phosphate; sodium fluoride, ammonium fluoride, potassium fluoride, calcium fluoride, and magnesium fluoride; sodium bromide, ammonium bromide, potassium bromide, calcium bromide, and magnesium bromide; and sodium iodide, ammonium iodide, potassium iodide, calcium iodide, and magnesium iodide). In some cases, the pharmaceutically acceptable salt is sodium chloride. In some cases, the pharmaceutically acceptable salt is arginine hydrochloride (L-arginine hydrochloride).

[0078] In exemplary embodiments, the pharmaceutical compositions of the present disclosure comprise a concentration of from about 0 mM to about 150 mM, from about 5 mM to about 150 mM, from about 5 mM to about 100 mM, from about 5 mM to about 90 mM, from about 5 mM to about 80 mM, from about 5 mM to about 70 mM, from about 5 mM to about 60 mM, from about 5 mM to about 50 mM, from about 5 mM to about 40 mM, from 5 mM to about 30 mM, from about 10 mM to about 50 mM, about 15 mM to about 45 mM, about 20 mM to about 40 mM, about 25 mM to about 35 mM, about 30 mM to about 130 mM, about 40 mM to about 120 mM, about 50 mM to about 110 mM, about 60 mM to about 100 mM, about 70 mM to about 90 mM, or about 75 mM to about 85 mM of a pharma- ceutically acceptable salt (as defined above, e.g., a first salt, a second salt, or both the first salt and the second salt). In exemplary embodiments, the pharmaceutical composition comprises about 0 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 105 mM, about 110 mM, about 115 mM, about 120 mM, about 125 mM, about 130 mM, about 135 mM, about 140 mM, about 145 mM, or about 150 mM of a pharma- ceutically acceptable salt (e.g., a first salt, a second salt, or both the first salt and the second salt).In some embodiments, the pharmaceutical composition comprises about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, , about 31mM, about 32mM, about 33mM, about 34mM, about 35mM, about 36mM, about 37mM, about 38mM, about 39mM, about 40mM, about 41mM, about 42mM, about 43mM, about 44mM , about 45mM, about 46mM, about 47mM, about 48mM, about 49mM, about 50mM, about 51mM, about 52mM, about 53mM, about 54mM, about 55mM, about 56mM, about 57mM, about 58mM, About 59mM, about 60mM, about 61mM, about 62mM, about 63mM, about 64mM, about 65mM, about 66mM, about 67mM, about 68mM, about 69mM, about 70mM, about 71mM, about 72mM, Approximately 73mM, approximately 74mM, approximately 75mM, approximately 76mM, approximately 77mM, approximately 78mM, approximately 79mM, approximately 80mM, approximately 81mM, approximately 82mM, approximately 83mM, approximately 84mM, approximately 85mM, approximately 86mM, approximately 87 mM, about 88 mM, about 89 mM, about 90 mM, about 91 mM, about 92 mM, about 93 mM, about 94 mM, about 95 mM, about 96 mM, about 97 mM, about 98 mM, about 99 mM, about 100 mM, about 101 mM, about 102 mM, about 103 mM, about 104 mM, or about 105 mM of a pharma- ceutically acceptable salt (e.g., a first salt, a second salt, or both the first salt and the second salt).

[0079] In certain embodiments, the pharmaceutical compositions of the present disclosure comprise a concentration of about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, , about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM, or about 50 mM sodium chloride. In some embodiments, the composition comprises 30 mM ± 5 mM sodium chloride. In some embodiments, the composition comprises 30 mM ± 3 mM sodium chloride. In some embodiments, the composition comprises 30 mM ± 1 mM sodium chloride. In certain embodiments, the pharmaceutical compositions of the present disclosure comprise at least one of the following: about 60 mM, about 61 mM, about 62 mM, about 63 mM, about 64 mM, about 65 mM, about 66 mM, about 67 mM, about 68 mM, about 69 mM, about 70 mM, about 71 mM, about 72 mM, about 73 mM, about 74 mM, about 75 mM, about 76 mM, about 77 mM, about 78 mM, about 79 mM, The composition comprises about 80 mM, about 81 mM, about 82 mM, about 83 mM, about 84 mM, about 85 mM, about 86 mM, about 87 mM, about 88 mM, about 89 mM, about 90 mM, about 91 mM, about 92 mM, about 93 mM, about 94 mM, about 95 mM, about 96 mM, about 97 mM, about 98 mM, about 99 mM, or about 100 mM arginine hydrochloride. In some embodiments, the composition comprises 80 mM ± 10 mM arginine hydrochloride. In some embodiments, the composition comprises 80 mM ± 5 mM arginine hydrochloride. In some embodiments, the composition comprises 80 mM ± 3 mM arginine hydrochloride.

[0080] C. Surfactants

[0081] The liquid pharmaceutical formulations, lyophilized pharmaceutical compositions, and reconstituted pharmaceutical products of the present disclosure comprise one or more surfactants.

[0082] As discussed herein, non-ionic surfactants substantially reduce the loss of AAV on the surface and prevent the formation of visible particles in the pharmaceutical composition of the present disclosure.Therefore, in certain embodiments, the pharmaceutical composition contains a stabilizing concentration of non-ionic surfactant.The pharmaceutically acceptable non-ionic surfactants that can be used in the compositions discussed herein include, but are not limited to, polysorbate 80 (Tween 80; PS80), polysorbate 20 (Tween 20; PS20), and various poloxamers (e.g., poloxamer 188), or mixtures thereof. In some cases, specific non-ionic surfactants that may be included in the formulations and compositions of the present disclosure include, for example, polysorbates such as polysorbate 20, polysorbate 28, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 81, and polysorbate 85; poloxamers such as poloxamer 181, poloxamer 188, poloxamer 407; or polyethylene glycol (PEG). In some embodiments, the non-ionic surfactant used in the composition is polysorbate 80. In some embodiments, the non-ionic surfactant used in the composition is poloxamer 188.

[0083] In certain embodiments, the pharmaceutical compositions of the present disclosure comprise from about 0.001% (w / v) to about 0.1% (w / v) of a non-ionic surfactant. In various embodiments, the compositions comprise from about 0.001% (w / v), about 0.0015% (w / v), about 0.002% (w / v), about 0.0025% (w / v), about 0.003% (w / v), about 0.0035% (w / v), about 0.004% (w / v), about 0.0045% (w / v), about 0.0 ... 5% (w / v), about 0.006% (w / v), about 0.0065% (w / v), about 0.007% (w / v), about 0.0075% (w / v), about 0.008% (w / v), about 0.0085% (w / v), about 0.009% (w / v), about 0.0095% (w / v), or about 0.01% (w / v) of a non-ionic surfactant. In various embodiments, the composition comprises about 0.01% (w / v), about 0.015% (w / v), about 0.02% (w / v), about 0.025% (w / v), about 0.03% (w / v), about 0.035% (w / v), about 0.04% (w / v), about 0.045% (w / v), about 0.05% (w / v), about 0.055% (w / v), about 0.065% (w / v), about 0.075% (w / v), about 0.085% (w / v), about 0.090% (w / v), about 0.100% (w / v), about 0.110% (w / v), about 0.125% (w / v), about 0.130% (w / v), about 0.140% (w / v), about 0.150% (w / v), about 0.160% (w / v), about 0.170% (w / v), about 0.180% (w / v), about 0.190% (w / v), about 0.200% (w / v), about 0.210% (w / v), about 0.220% (w / v), about 0.230% (w / v), about 0.240% (w / v), about 0.250% (w / v), about 0.260% (w / v), about 0.270% (w / v), about 0.280% (w / v), about 0.290% (w / v), about 0.30% (w / v), about 0.310% (w / v), about 0.320% (w / v), about 0.330% (w / v), about 0.340% (w / v), about 0 % (w / v), about 0.06% (w / v), about 0.065% (w / v), about 0.07% (w / v), about 0.075% (w / v), about 0.08% (w / v), about 0.085% (w / v), about 0.09% (w / v), about 0.095% (w / v), or about 0.1% (w / v) of a non-ionic surfactant. In certain embodiments, the pharmaceutical compositions of the present disclosure comprise about 0.005% w / v ± about 0.001% w / v of a non-ionic surfactant.

[0084] In some exemplary embodiments, the pharmaceutical compositions of the present disclosure comprise from about 0.001% (w / v) to about 0.01% (w / v) polysorbate 80. In some exemplary embodiments, the pharmaceutical compositions of the present disclosure comprise from about 0.001% (w / v) to about 0.01% (w / v) poloxamer 188. In some embodiments, the pharmaceutical compositions comprise from about 0.001% (w / v), about 0.0015% (w / v), about 0.002% (w / v), about 0.0025% (w / v), about 0.003% (w / v), about 0.0035% (w / v), about 0.004% (w / v), about 0.0045% (w / v), about 0.005% (w / v), about 0.00 55% (w / v), about 0.006% (w / v), about 0.0065% (w / v), about 0.007% (w / v), about 0.0075% (w / v), about 0.008% (w / v), about 0.0085% (w / v), about 0.009% (w / v), about 0.0095% (w / v), or about 0.01% (w / v) polysorbate 80. In some embodiments, the pharmaceutical composition comprises about 0.001% (w / v), about 0.0015% (w / v), about 0.002% (w / v), about 0.0025% (w / v), about 0.003% (w / v), about 0.0035% (w / v), about 0.004% (w / v), about 0.0045% (w / v), about 0.005% (w / v), about 0.00 55% (w / v), about 0.006% (w / v), about 0.0065% (w / v), about 0.007% (w / v), about 0.0075% (w / v), about 0.008% (w / v), about 0.0085% (w / v), about 0.009% (w / v), about 0.0095% (w / v), or about 0.01% (w / v) of poloxamer 188. In certain embodiments, the pharmaceutical composition of the present disclosure comprises about 0.005% w / v ± 0.001% w / v of polysorbate 80. In certain embodiments, the pharmaceutical composition of the present disclosure comprises about 0.005% w / v ± 0.001% w / v of poloxamer 188.

[0085] D. Sugars

[0086] The liquid pharmaceutical formulations, lyophilized pharmaceutical compositions, and reconstituted pharmaceutical products of the present disclosure comprise one or more sugars.

[0087] The inclusion of one or more sugars (e.g., about 1% to about 20%) improves the stability of the liquid and / or lyophilized formulations of the present disclosure. For example, sugars allow for better properties during freeze / thaw cycles. Thus, in certain embodiments, the pharmaceutical compositions of the present disclosure contain about 1% to about 10% of one or more sugars. Any sugar may be used in various embodiments, such as a monosaccharide, disaccharide, or polysaccharide, or a water-soluble glucan, including, for example, fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, dextran, trehalose, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch, and carboxymethyl cellulose. In some embodiments, the sugar is sucrose, trehalose, or a combination thereof. In certain embodiments, the trehalose is trehalose dihydrate. In some embodiments, the sugar is sucrose. In some embodiments, the sugar is trehalose.

[0088] Sugars may be used individually or in combination. In some embodiments, the sugar, or combinations thereof, are present in the formulation or composition at a concentration of about 1% to about 10% (w / v), about 2% to about 8% (w / v), about 2.5% to about 7.5% (w / v), about 3% to about 7% (w / v), or about 4% to about 6% (w / v). In some embodiments, the pharmaceutical compositions of the present disclosure comprise about 1.0% (w / v), about 1.1% (w / v), about 1.2% (w / v), about 1.3% (w / v), about 1.4% (w / v), about 1.5% (w / v), about 1.6% (w / v), about 1.7% (w / v), about 1.8% (w / v), about 1.9% (w / v), about 2.0% (w / v), about 2.1% (w / v), about 2.2% (w / v), about 2.3% (w / v), about 2.4% (w / v), about 2.5% (w / v), about 2.6% (w / v), Approximately 2.7% (w / v), approximately 2.8% (w / v), approximately 2.9% (w / v), approximately 3.0% (w / v), approximately 3.1% (w / v), approximately 3.2% (w / v), approximately 3.3% (w / v), approximately 3.4% (w / v), approximately 3.5% (w / v), approximately 3.6% ( 3.7% (w / v), 3.8% (w / v), 3.9% (w / v), 4.0% (w / v), 4.1% (w / v), 4.2% (w / v), 4.3% (w / v), 4.4% (w / v), 4.5% (w / v), approx. 4.6% (w / v), approximately 4.7% (w / v), approximately 4.8% (w / v), approximately 4.9% (w / v), approximately 5.0% (w / v), approximately 5.1% (w / v), approximately 5.2% (w / v), approximately 5.3% (w / v), approximately 5.4% (w / v), approximately 5.5% (w) / v), approximately 5.6% (w / v), approximately 5.7% (w / v), approximately 5.8% (w / v), approximately 5.9% (w / v), approximately 6.0% (w / v), approximately 6.1% (w / v), approximately 6.2% (w / v), approximately 6.3% (w / v), approximately 6.4% (w / v), approximately 6 .5%(w / v), approximately 6.6%(w / v), approximately 6.7%(w / v), approximately 6.8%(w / v), approximately 6.9%(w / v), approximately 7.0%(w / v), approximately 7.1%(w / v), approximately 7.2%(w / v), approximately 7.3%(w / v), approximately 7.4%(w / v), about 7.5% (w / v), about 7.6% (w / v), about 7.7% (w / v), about 7.8% (w / v), about 7.9% (w / v), about 8.0% (w / v), about 8.1% (w / v), about 8.2% (w / v), about 8.3% (w / v), about 8.4% (w / v), about 8.5% (w / v), about 8.6% (w / v), about 8.7% (w / v), about 8.8% (w / v), about 8.9% (w / v), about 9.0% (w / v), about 9.1% (w / v), about 9.2% (w / v), about 9.3% (w / v), about 9.4% (w / v), about 9.5% (w / v), about 9.6% (w / v), about 9.7% (w / v), about 9.8% (w / v), about 19.9% ​​(w / v), or about 10% (w / v) sugar.

[0089] In various embodiments, the compositions of the present disclosure comprise from about 2.5% w / v to about 7.5% w / v sucrose. In some embodiments, the compositions contain from 4% w / v to 6% w / v sucrose. In some embodiments, the compositions contain 5% w / v ± 0.5% w / v sucrose. In various embodiments, the compositions of the present disclosure comprise from about 2.5% w / v to about 7.5% w / v trehalose. In some embodiments, the compositions contain from 4% w / v to 6% w / v trehalose. In some embodiments, the compositions contain 5% w / v ± 0.5% w / v trehalose.

[0090] E. Bulking Agents

[0091] The liquid pharmaceutical formulations, lyophilized pharmaceutical compositions, and reconstituted pharmaceutical products of the present disclosure comprise one or more bulking agents.

[0092] The inclusion of one or more sugars (e.g., about 1% to about 10%) improves the stability of the liquid and / or lyophilized formulations of the present disclosure. For example, the bulking agents discussed herein allow for the production of better lyophilized compositions with improved stability and reconstitution properties. Thus, in certain embodiments, the pharmaceutical compositions of the present disclosure contain about 1% to about 10% of one or more bulking agents. In various embodiments, the bulking agents included in the pharmaceutical compositions of the present disclosure may include, but are not limited to, polymers such as mannitol, glycine, dextran, polyvinylpyrrolidone, carboxymethylcellulose, lactose, sorbitol, or xylitol. In some embodiments, the bulking agent is mannitol. In some embodiments, the bulking agent is glycine.

[0093] Bulking agents may be used individually or in combination. In some embodiments, the bulking agent, or combinations thereof, is present in the formulation or composition at a concentration of about 1% to about 10% (w / v), about 1% (w / v) to about 6% (w / v), about 2% to about 4% (w / v), or about 3% (w / v) to about 3.5% (w / v). In some embodiments, the pharmaceutical compositions of the present disclosure comprise about 1.0% (w / v), about 1.1% (w / v), about 1.2% (w / v), about 1.3% (w / v), about 1.4% (w / v), about 1.5% (w / v), about 1.6% (w / v), about 1.7% (w / v), about 1.8% (w / v), about 1.9% (w / v), about 2.0% (w / v), about 2.1% (w / v), about 2.2% (w / v), about 2.3% (w / v), about 2.4% (w / v), about 2.5% (w / v), about 2.6% (w / v), about 2.7% (w / v), about 2.8% (w / v), about 2.9% (w / v), about 3.0% (w / v), about 3.1% (w / v), about 3.2% (w / v), about 3.3% (w / v), about 3.4% (w / v), about 3.5% (w / v), about 3.6% (w / v), about 3.7% (w / v), about 3.8% (w / v), about 3.9% (w / v), about 4.0% (w / v), about 4.1% (w / v), about 4.2% (w / v), about 4.3% (w / v), about 4.4% (w / v), about 4.5% (w / v), about 4.6% (w / v), about 4.7% (w / v), about 4.8% (w / v), about 4.9% (w / v), about 5.0% (w / v), about 5.1% (w / v), about 5.2% (w / v), about 5.3% (w / v), about 5.4% (w / v), about 5. .7% (w / v), approximately 2.8% (w / v), approximately 2.9% (w / v), approximately 3.0% (w / v), approximately 3.1% (w / v), approximately 3.2% (w / v), approximately 3.3% (w / v), approximately 3.4% (w / v), approximately 3.5% (w / v), approximately 3.6% (w / v) ), approximately 3.7% (w / v), approximately 3.8% (w / v), approximately 3.9% (w / v), approximately 4.0% (w / v), approximately 4.1% (w / v), approximately 4.2% (w / v), approximately 4.3% (w / v), approximately 4.4% (w / v), approximately 4.5% (w / v), approximately 4.6% ( 4.7% (w / v), 4.8% (w / v), 4.9% (w / v), 5.0% (w / v), 5.1% (w / v), 5.2% (w / v), 5.3% (w / v), 5.4% (w / v), 5.5% (w / v), approx. 5 .6%(w / v), approx. 5.7%(w / v), approx. 5.8%(w / v), approx. 5.9%(w / v), approx. 6.0%(w / v), approx. 6.1%(w / v), approx. 6.2%(w / v), approx. 6.3%(w / v), approx. 6.4%(w / v), approx. 6.5%(w / v) , about 6.6% (w / v), about 6.7% (w / v), about 6.8% (w / v), about 6.9% (w / v), about 7.0% (w / v), about 7.1% (w / v), about 7.2% (w / v), about 7.3% (w / v), about 7.4% (w / v), about 7.5% ( 7.6%(w / v), 7.7%(w / v), 7.8%(w / v), 7.9%(w / v), 8.0%(w / v), 8.1%(w / v), 8.2%(w / v), 8.3%(w / v), 8.4%(w / v), 8.5% (w / v), about 8.6% (w / v), about 8.7% (w / v), about 8.8% (w / v), about 8.9% (w / v), about 9.0% (w / v), about 9.1% (w / v), about 9.2% (w / v), about 9.3% (w / v), about 9.4% (w / v), about 9.5% (w / v), about 9.6% (w / v), about 9.7% (w / v), about 9.8% (w / v), about 19.9% ​​(w / v), or about 10% (w / v) of bulking agent.

[0094] In various embodiments, the compositions of the present disclosure comprise about 2% w / v to about 4.5% w / v mannitol. In some embodiments, the compositions contain 3% w / v to 3.5% w / v mannitol. In some embodiments, the compositions contain 3.3% w / v ± 0.1% w / v mannitol. In various embodiments, the compositions of the present disclosure comprise about 2% w / v to about 4.5% w / v glycine. In some embodiments, the compositions contain 3% w / v to 3.5% w / v glycine. In some embodiments, the compositions contain 3.3% w / v ± 0.1% w / v glycine.

[0095] F. Excipient ratio

[0096] In various embodiments, the liquid pharmaceutical formulations, lyophilized pharmaceutical compositions, and reconstituted pharmaceutical products of the present disclosure contain defined ratios of specific excipients to enhance the stability of the composition. In some embodiments, the compositions contain a specific ratio of bulking agent and sugar (bulking agent:sugar). In some embodiments, the compositions contain a specific ratio of two salts (first salt:second salt).

[0097] In some cases, the ratio of bulking agent:sugar is about 1:1 to 1:10. In various embodiments, the ratio of bulking agent:sugar is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 2:3, about 2:5, about 2:7, about 2:9, about 3:4, about 3:5, about 3:7, about 3:8, about 3:10, about 4:5, about 4:7, about 4:9, about 5:6, about 5:7, about 5:8, about 5:9, about 6:7, about 7:8, about 7:9, about 7:10, or about 8:9. In some embodiments, the ratio of bulking agent:sugar is about 3.1:5, about 3.2:5, about 3.3:5, about 3.4:5, or about 3.5:5. In some cases, the ratio of bulking agent:sugar is 3.3:5. In some cases, the bulking agent is mannitol and the sugar is sucrose, and the ratio is about 1:1 to 1:10. In various embodiments, the ratio of mannitol:sucrose is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 2:3, about 2:5, about 2:7, about 2:9, about 3:4, about 3:5, about 3:7, about 3:8, about 3:10, about 4:5, about 4:7, about 4:9, about 5:6, about 5:7, about 5:8, about 5:9, about 6:7, about 7:8, about 7:9, about 7:10, or about 8:9. In some embodiments, the ratio of mannitol:sucrose is about 2.8:5, about 2.9:5, about 3.0:5, about 3.1:5, about 3.2:5, about 3.3:5, about 3.4:5, about 3.5:5, about 3.6:5, about 3.7:5, or about 3.8:5. In some cases, the ratio of mannitol:sucrose is 3.3:5.

[0098] In some cases, the ratio of the first salt:second salt is about 1:1 to 1:10. In various embodiments, the ratio of the first salt:second salt is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 2:3, about 2:5, about 2:7, about 2:9, about 3:4, about 3:5, about 3:7, about 3:8, about 3:10, about 4:5, about 4:7, about 4:9, about 5:6, about 5:7, about 5:8, about 5:9, about 6:7, about 7:8, about 7:9, about 7:10, or about 8:9. In some embodiments, the ratio of the first salt:second salt is about 2.5:8, about 2.6:8, about 2.7:8, about 2.8:8, about 2.9:8, about 3.0:8, about 3.1:8, about 3.2:8, about 3.3:8, about 3.4:8, or about 3.5:8. In some cases, the ratio of the first salt:second salt is 3:8. In some cases, the first salt is sodium chloride and the second salt is L-arginine hydrochloride, and the ratio is about 1:1 to 1:10. In various embodiments, the ratio of NaCl:L-arginine hydrochloride is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 2:3, about 2:5, about 2:7, about 2:9, about 3:4, about 3:5, about 3:7, about 3:8, about 3:10, about 4:5, about 4:7, about 4:9, about 5:6, about 5:7, about 5:8, about 5:9, about 6:7, about 7:8, about 7:9, about 7:10, or about 8:9. In some embodiments, the ratio of NaCl:L-arginine hydrochloride is about 2.5:8, about 2.6:8, about 2.7:8, about 2.8:8, about 2.9:8, about 3.0:8, about 3.1:8, about 3.2:8, about 3.3:8, about 3.4:8, or about 3.5:8. In some cases, the ratio of NaCl:L-arginine hydrochloride is 3:8.

[0099] G. Additional Optional Ingredients

[0100] The liquid pharmaceutical formulations, lyophilized pharmaceutical compositions, and reconstituted pharmaceutical products of the present disclosure may also optionally include additional ingredients. In exemplary embodiments, the formulation or pharmaceutical composition includes any one or combination of the following: acidifying agents, additives, adsorbents, air displacing agents, alkalizing agents, anti-caking agents, anticoagulants, antimicrobial preservatives, antioxidants, preservatives, bases, binders, buffers, chelating agents, coating agents, colorants, drying agents, detergents, diluents, disinfectants, disintegrants, dispersants, dissolution enhancers, dyes, emollients, emulsifiers, emulsions, and the like. A cation stabilizer, a filler, a film-forming agent, a flavor enhancer, a flavoring agent, a glidant, a gelling agent, a granulating agent, a humectant, a lubricant, a mucoadhesive agent, an ointment base, an ointment, an oily vehicle, an organic base, a tablet base, a pigment, a plasticizer, an abrasive, a preservative, a sequestering agent, a solubilizing agent, a solvent, a stabilizer, a surface active agent, a surfactant, a suspending agent, a sweetening agent, a therapeutic agent, a thickening agent, an isotonicity agent, a viscosity increasing agent, a water absorbing agent, a water miscible co-solvent, a water softener, or a humectant.

[0101] Lyophilization and Composition The lyophilized composition is prepared by lyophilization of the pharmaceutical formulation described above or discussed herein.In various aspects, the present disclosure includes such lyophilized compositions and methods for producing stable lyophilized pharmaceutical compositions.The embodiment of the method for producing the lyophilized AAV composition discussed herein can also include producing an aqueous formulation, which is then lyophilized.

[0102] The freeze-drying process generally includes (i) freezing, (ii) annealing, (iii) primary drying, and (iv) secondary drying. In some cases, freezing is performed at a temperature of -45°C ± 5°C for 60 minutes ± 10 minutes. In some cases, freezing is performed both before and after annealing. In some cases, annealing is performed at a temperature of -20°C ± 5°C for 6 hours ± 30 minutes. In some cases, primary drying is performed at a temperature of -26°C ± 5°C for 50 hours ± 10 hours. In some cases, secondary drying is performed at a temperature of 35°C ± 5°C for 5 to 20 hours, or 15 hours ± 5 hours.

[0103] In one embodiment, the rAAV sample is filled into a glass vial and placed in a lyophilization chamber. The sample may be first incubated at about 5° C. for about 60 minutes, cooled to about 5° C. to about −45° C. at a ramp rate of about 0.5° C. / min, followed by an isothermal hold for about 60 minutes. Annealing may be performed at about −20° C. for about 6 hours at a ramp rate of about 0.5° C. / min, cooled to about −45° C. at a ramp rate of about 0.5° C. / min, followed by a hold for about 60 minutes. Primary drying may be performed at about −26° C. under a chamber pressure of about 50 mTorr for about 50 hours, followed by secondary drying at about 35° C. under about 100 mTorr for about 10 or 15 hours, with a ramp rate of about 0.3° C. / min for both primary and secondary drying. At the end of the lyophilization cycle, the sample may be backfilled with nitrogen gas at about 80% of atmospheric pressure, capped, and sealed.

[0104] In one embodiment, the rAAV sample is filled into a glass vial and placed in a lyophilization chamber. The sample may first be incubated at a temperature of about 1° C. to about 10° C. for about 30 minutes to about 90 minutes, cooled from the incubation temperature to a freezing temperature of about −30° C. to about −60° C. at a ramp rate of about 0.1° C. / min to about 1° C. / min, followed by an isothermal hold for about 30 minutes to about 90 minutes. Annealing may be performed at a temperature of about −10° C. to about −30° C. for a time of about 4 hours to about 8 hours at a ramp rate of about 0.1° C. / min to about 1° C. / min, and then the sample may be cooled to a temperature of about −30° C. to about −60° C. at a ramp rate of about 0.1° C. / min to about 1° C. / min, followed by a hold for about 30 minutes to about 90 minutes. With a ramp rate of about 0.1° C. / min to about 1° C. / min for both primary and secondary drying, primary drying may be carried out at a temperature of about −20° C. to about −30° C. and under a chamber pressure of about 40 mTorr to about 60 mTorr for about 40 hours to about 60 hours, followed by secondary drying at a temperature of about 25° C. to about 45° C. and under a pressure of about 75 mTorr to about 125 mTorr for about 5 hours to about 20 hours. At the end of the freeze-drying cycle, the samples may be back-filled with nitrogen gas at about 70% to 90% of atmospheric pressure, capped and sealed.

[0105] In some embodiments, the rAAV sample is first incubated at a temperature of about 10° C., about 9° C., about 8° C., about 7° C., about 6° C., about 5° C., about 4° C., about 3° C., about 2° C., about 1° C., or about 0° C. This incubation may be maintained for a period of about 10 minutes to about 120 minutes or more. In some embodiments, the rAAV sample is first incubated for a period of 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, or 120 minutes or more.

[0106] During lyophilization, the temperature of the composition (e.g., the rAAV sample) may be reduced, for example, to a temperature below the freezing point of water in the composition. For example, the temperature of the composition may be reduced to 0° C. or below, or -5° C. or below, or -10° C. or below, or -15° C. or below, or -20° C. or below, or -25° C. or below, or -30° C. or below, or -35° C. or below, or -40° C. or below, or -45° C. or below, or -50° C. or below, or -55° C. or below, or -60° C. or below, or -65° C. or below, or -70° C. or below during freezing as part of the lyophilization process. In various embodiments, the rAAV sample is held at a freezing temperature (e.g., -45°C) for about 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, or more.

[0107] In some embodiments, annealing is performed at a temperature of about −10° C. to about −30° C. In some cases, annealing is performed at a temperature of about −10° C., about −11° C., about −12° C., about −13° C., about −14° C., about −15° C., about −16° C., about −17° C., about −18° C., about −19° C., about −20° C., about −21° C., about −22° C., about −23° C., about −24° C., about −25° C., about −26° C., about −27° C., about −28° C., about −29° C., or about −30° C. In various embodiments, the sample is maintained at the annealing temperature for a period of about 3 hours to about 9 hours or more. In some cases, the sample is maintained at the annealing temperature for about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, about 5.5 hours, about 6 hours, about 6.5 hours, about 7 hours, about 7.5 hours, about 8 hours, about 8.5 hours, or about 9 hours or more.

[0108] In some embodiments, additional freezing is performed after annealing. In various embodiments, this additional freezing can be performed at a temperature and for a duration as described above or herein. For example, the rAAV sample can be reduced to about -45°C for about 60 minutes.

[0109] Primary drying may be carried out as part of a freeze-drying process at a temperature of about -10°C to about -50°C, at a pressure of about 1 mTorr to about 10 Torr, and for a period of about 10 hours to about 200 hours or more.

[0110] In some embodiments, primary drying is carried out at a temperature of about -20°C to about -30°C. In various embodiments, primary drying is carried out at a temperature of about -10°C, about -11°C, about -12°C, about -13°C, about -14°C, about -15°C, about -16°C, about -17°C, about -18°C, about -19°C, about -20°C, about -21°C, about -22°C, about -23°C, about -24°C, about -25°C, about -26°C, about -27°C, about -28°C, about -29°C, or about -30°C. In some cases, primary drying is carried out at a temperature of about -25°C. In some cases, primary drying is carried out at a temperature of about -26°C. In some cases, primary drying is carried out at a temperature of about -27°C.

[0111] In some embodiments, the pressure surrounding the composition during primary drying is reduced to standard atmospheric pressure or less. For example, the pressure surrounding the composition can be reduced to 100 Torr or less, or 10 Torr or less, or 1 Torr or less, or 500 mTorr or less, or 400 mTorr or less, or 300 mTorr or less, or 200 mTorr or less, or 100 mTorr or less, or 90 mTorr or less, or 80 mTorr or less, or 70 mTorr or less, or 60 mTorr or less, or 50 mTorr or less, or 40 mTorr or less, or 30 mTorr or less, or 20 mTorr or less, or 10 mTorr or less, or 5 mTorr or less, or 1 mTorr or less. In some cases, the pressure surrounding the composition is reduced to about 40 mTorr to about 60 mTorr or less. In some cases, the pressure surrounding the composition is reduced to about 45 mTorr to about 55 mTorr or less. In some cases, the pressure surrounding the composition is reduced to about 50 mTorr.

[0112] In various embodiments, primary drying is carried out for about 10 hours to about 200 hours or more. In some cases, primary drying is carried out for about 10 hours, about 15 hours, about 20 hours, about 25 hours, about 30 hours, about 35 hours, about 40 hours, about 45 hours, about 50 hours, about 55 hours, about 60 hours, about 65 hours, about 70 hours, about 75 hours, about 80 hours, about 85 hours, about 90 hours, about 95 hours, about 100 hours, about 120 hours, about 140 hours, about 160 hours, about 180 hours, or about 200 hours or more. In some cases, primary drying is carried out for about 40 hours to about 60 hours. In some cases, primary drying is carried out for about 45 hours to about 55 hours. In some cases, primary drying is carried out for about 50 hours.

[0113] Secondary drying may be carried out as part of a freeze-drying process at a temperature of about 5° C. to about 65° C., at a pressure of about 1 mTorr to about 10 Torr, and for a period of about 1 hour to about 30 hours or more.

[0114] In some embodiments, secondary drying is carried out at a temperature of about 20° C. to about −50° C. In various embodiments, secondary drying is carried out at a temperature of about 10° C., about 11° C., about 12° C., about 13° C., about 14° C., about 15° C., about 16° C., about 17° C., about 18° C., about 19° C., about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., about 25° C., about 26° C., about 27° C., about 28° C., about 29° C., about 30° C., about 31° C., about 32° C., about 33° C., about 34° C., about 35° C., about 36° C., about 37° C., about 38° C., about 39° C., about 40° C., about 41° C., about 42° C., about 43° C., about 44° C., about 45° C., about 46° C., about 47° C., about 48° C., about 49° C., or about 50° C. In some cases, the secondary drying is carried out at a temperature of about 30° C. to about 40° C. In some cases, the secondary drying is carried out at a temperature of about 35° C.

[0115] In some embodiments, the pressure surrounding the composition during secondary drying is reduced to less than standard atmospheric pressure. For example, the pressure surrounding the composition can be reduced to 100 Torr or less, or 10 Torr or less, or 1 Torr or less, or 500 mTorr or less, or 400 mTorr or less, or 300 mTorr or less, or 200 mTorr or less, or 100 mTorr or less, or 90 mTorr or less, or 80 mTorr or less, or 70 mTorr or less, or 60 mTorr or less, or 50 mTorr or less, or 40 mTorr or less, or 30 mTorr or less, or 20 mTorr or less, or 10 mTorr or less, or 5 mTorr or less, or 1 mTorr or less. In some cases, the pressure surrounding the composition is reduced to about 150 mTorr to about 50 mTorr or less. In some cases, the pressure surrounding the composition is reduced to about 125 mTorr to about 75 mTorr or less. In some cases, the pressure surrounding the composition is reduced to about 100 mTorr.

[0116] In various embodiments, the secondary drying is carried out for about 1 hour to about 30 hours or more. In some cases, the primary drying is carried out for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, or about 30 hours or more. In some cases, the primary drying is carried out for about 5 hours to about 20 hours. In some cases, the primary drying is carried out for about 10 hours. In some cases, the primary drying is carried out for about 15 hours.

[0117] When changing from one temperature to another during the freeze-drying process, the ramp rate may be about 0.5° C. / min. In various embodiments, the ramp rate may be about 0.1° C. / min, about 0.2° C. / min, about 0.3° C. / min, about 0.4° C. / min, about 0.5° C. / min, about 0.6° C. / min, about 0.7° C. / min, about 0.8° C. / min, about 0.9° C. / min, or about 1° C. / min. In some cases, the ramp rate may be about 2° C. / min, about 3° C. / min, about 4° C. / min, or about 5° C. / min.

[0118] In various embodiments, the lyophilization process produces a lyophilized composition with a low moisture content (e.g., less than 3% moisture). In various embodiments, the lyophilized composition has a moisture content of 3.0% or less, 2.9% or less, 2.8% or less, 2.7% or less, 2.6% or less, 2.5% or less, 2.4% or less, 2.3% or less, 2.2% or less, 2.1% or less, 2.0% or less, 1.9% or less, 1.8% or less, 1.7% or less, 1.6% or less, 1.5% or less, 1.4% or less, 1.3% or less, 1.2% or less, 1.1% or less, 1.0% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less as measured by Karl Fischer (KF) titration or another method (e.g., as described below). In some embodiments, the lyophilized composition has a water content of about 0.5% to about 0.8% as measured by Karl Fischer titration or another method (e.g., as described below). In some embodiments, the lyophilized composition has a water content of 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, or 0.8% as measured by Karl Fischer titration or another method (e.g., as described below). In certain embodiments, the lyophilized composition has 3% or less water as measured by Karl Fischer titration. In certain embodiments, the lyophilized composition has 2% or less water as measured by Karl Fischer titration. In certain embodiments, the lyophilized composition has 1.5% or less water as measured by Karl Fischer titration. In certain embodiments, the lyophilized composition has 1% or less water as measured by Karl Fischer titration. In certain embodiments, the lyophilized composition has 0.9% or less water as measured by Karl Fischer titration. In certain embodiments, the lyophilized composition has 0.8% or less water as measured by Karl Fischer titration. In certain embodiments, the lyophilized composition has 0.7% or less water as measured by Karl Fischer titration. In certain embodiments, the lyophilized composition has 0.6% or less water as measured by Karl Fischer titration.In certain embodiments, the lyophilized composition has 0.5% or less water as measured by Karl Fischer titration. In certain embodiments, the lyophilized composition has 0.4% or less water as measured by Karl Fischer titration. In certain embodiments, the lyophilized composition has 0.3% or less water as measured by Karl Fischer titration. In certain embodiments, the lyophilized composition has 0.2% or less water as measured by Karl Fischer titration. Other methods for determining the moisture content of a composition include, but are not limited to, loss on drying (LOD) (measuring the amount of water and volatiles in a sample when the sample is dried under certain conditions), electrolyte sensors (e.g., using a P2O5 sensor), piezoelectric adsorption, oxide sensors, aluminum oxide sensors, absorption spectroscopy, and near infrared (NIR).

[0119] Because the moisture content of the above-mentioned freeze-dried composition is low, the freeze-dried composition may be in the form of a solid.In some cases, the solid freeze-dried composition is a powder.In some cases, the freeze-dried composition may facilitate the storage of the composition for a long period of time (e.g., compared with the liquid formulation of the same composition).For example, the freeze-dried composition may be a storage-stable composition, as discussed above or herein, in which the composition is substantially stable for a long period of time.

[0120] Reconstituted compositions (e.g., reconstituted pharmaceutical products) prepared by reconstitution of lyophilized compositions are also encompassed within the scope of the present disclosure. Such reconstituted compositions may be administered to a subject, for example, by injection or intravenous administration. In some embodiments, prior to administration of the reconstituted composition to a subject, for example, the solid lyophilized composition described above may be combined with a liquid to provide a liquid composition suitable for administration, for example, by injection or intravenous administration. In some cases, prior to administration of the composition to a subject, the solid lyophilized composition may be combined with water (e.g., water for injection, WFI) or a buffer / buffer (e.g., as described above) to provide an aqueous composition suitable for administration, for example, by injection or intravenous administration. For example, the lyophilized composition may be reconstituted with water (e.g., water for injection, WFI) or a buffer / buffer (e.g., as described above) to generate a reconstituted dosage unit suitable for administration, for example, by injection or intravenous administration to a subject.

[0121] In various embodiments, the reconstituted dosage unit has a pH that is compatible with physiological conditions. In some cases, the pH of the reconstituted dosage unit is in the range of 6-8. In some cases, the pH of the reconstituted dosage unit is in the range of 6.5-7.5. For example, the pH of the reconstituted dosage unit can be in the range of 7-7.5. In some cases, the pH of the reconstituted dosage unit is 7.0. In some cases, the pH of the reconstituted dosage unit is 7.1. In some cases, the pH of the reconstituted dosage unit is 7.2. In some cases, the pH of the reconstituted dosage unit is 7.3. In some cases, the pH of the reconstituted dosage unit is 7.4. In some cases, the pH of the reconstituted dosage unit is 7.5.

[0122] The reconstituted dosage unit may contain a predetermined amount of rAAV particles of the present disclosure, calculated in an amount sufficient to produce a desired therapeutic effect in a subject. The amount of rAAV particles in the reconstituted dosage unit administered to a subject may depend on the subject being treated, the severity of the disease, and the method of administration. For example, the reconstituted dosage unit may contain the amount of rAAV particles administered as disclosed herein in a therapeutically effective amount.

[0123] Reconstitution of the freeze-dried composition discussed herein can be accomplished within 1 minute. In some cases, reconstitution of the freeze-dried composition is accomplished within 30 seconds or less. In some cases, reconstitution of the freeze-dried composition is accomplished within 20 seconds or less. In some cases, reconstitution of the freeze-dried composition is accomplished within 15 seconds or less. In various embodiments, reconstitution of the freeze-dried composition is accomplished within 60 seconds or less, 55 seconds or less, 50 seconds or less, 45 seconds or less, 40 seconds or less, 35 seconds or less, 30 seconds or less, 25 seconds or less, 20 seconds or less, 15 seconds or less, 10 seconds or less, or 5 seconds or less. In exemplary aspects, the reconstituted composition is free or substantially free of visible particles.

[0124] Stability of formulations and compositions The pharmaceutical preparations and compositions of the present invention exhibit a high level of stability.The term "stable" as used herein in relation to pharmaceutical preparations means that the AAV particles in the pharmaceutical preparation retain an acceptable degree of structure and / or function and / or biological activity after storage for a specified amount of time.A preparation can be stable even if the AAV particles contained therein do not retain 100% of their pre-storage structure and / or function and / or biological activity after storage for a specified amount of time.In certain circumstances, the retention of about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of the structure and / or function and / or biological activity of the AAV particles after storage for a specified amount of time can be considered "stable".

[0125] Reference to the stability of a pharmaceutical formulation or composition "after" a specified period is intended to mean that a measurement of a stability parameter (e.g., % intact capsid, vector genome titer, or average particle size) is made at or near the end of the specified period, and is not intended to mean that the pharmaceutical formulation or composition will necessarily maintain the same degree of stability for the parameter measured thereafter. For example, reference to a particular stability after 6 months means that the stability measurement is made at or near 6 months after the start of the study. Additional methods for assessing the stability of AAV particles in formulations, including lyophilized compositions, are demonstrated in the examples provided below.

[0126] The storage stability of a lyophilized composition can be assessed with a reconstituted composition prepared from the lyophilized composition after storage for a specified period of time.

[0127] In some cases, the lyophilized composition that is stable upon reconstitution (e.g., as a reconstituted pharmaceutical product) comprises an average particle size of about 20 nm to about 40 nm, as determined by dynamic light scattering. In some cases, the average particle size is about 25 nm to about 35 nm, as determined by dynamic light scattering. In various embodiments, the average particle size is about 20 nm, about 21 nm, about 22 nm, about 23 nm, about 24 nm, about 25 nm, about 26 nm, about 27 nm, about 28 nm, about 29 nm, about 30 nm, about 31 nm, about 32 nm, about 33 nm, about 34 nm, about 35 nm, about 36 nm, about 37 nm, about 38 nm, about 39 nm, or about 40 nm, or less, as determined by dynamic light scattering.

[0128] In some cases, a stable lyophilized composition comprises an average particle size that upon reconstitution (e.g., as a reconstituted pharmaceutical product) does not change by more than about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% after storage for a specified period of time compared to the average particle size in the composition before lyophilization, as determined by dynamic light scattering.

[0129] In some cases, the stable lyophilized composition, upon reconstitution (e.g., as a reconstituted pharmaceutical product), comprises a population of rAAV particles that comprises at least 80% complete capsids as determined by anion exchange chromatography (e.g., AEX-UPLC). In some cases, the stable lyophilized composition, upon reconstitution, comprises a population of rAAV particles that comprises at least 95% complete capsids as determined by AEX-UPLC. In some cases, the population of rAAV particles comprises complete capsids or comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complete capsids as determined by AEX-UPLC.

[0130] In some cases, the stable lyophilized composition, upon reconstitution (e.g., as a reconstituted pharmaceutical product), comprises a population of rAAV particles that comprises a percentage of complete capsids as determined by AEX-UPLC, and the percentage of complete capsids in the reconstituted composition is within 10% of the percentage of complete capsids in the pharmaceutical formulation prior to lyophilization. In some cases, the percentage of complete capsids in the reconstituted pharmaceutical product is within 2% of the percentage of complete capsids in the pharmaceutical formulation prior to lyophilization. In some cases, the percentage of complete capsids in the reconstituted pharmaceutical product is within 1% of the percentage of complete capsids in the pharmaceutical formulation prior to lyophilization. In some cases, the percentage of complete capsids in the reconstituted pharmaceutical product is within 0.5% of the percentage of complete capsids in the pharmaceutical formulation prior to lyophilization. In various embodiments, the percentage of complete capsids in the reconstituted pharmaceutical product is within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4.5%, within 4%, within 3.5%, within 3%, within 2.5%, within 2%, within 1.5%, within 1%, within 0.75%, within 0.5%, or within 0.25% of the percentage of complete capsids in the pre-lyophilized pharmaceutical formulation as determined by AEX-UPLC.

[0131] In some cases, the stable lyophilized composition upon reconstitution (e.g., as a reconstituted pharmaceutical product) comprises a vector genome titer determined by droplet digital polymerase chain reaction (ddPCR) that is within 10% of the vector genome titer present in the pharmaceutical formulation prior to lyophilization. In some cases, the vector genome titer is within 7% of the vector genome titer present in the formulation prior to lyophilization. In some cases, the vector genome titer is within 5% of the vector genome titer present in the formulation prior to lyophilization. In various embodiments, the stable lyophilized composition upon reconstitution (e.g., as a reconstituted pharmaceutical product) comprises a vector genome titer determined by ddPCR that is within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the vector genome titer present in the pharmaceutical formulation prior to lyophilization.

[0132] In any of the various embodiments discussed above or herein, the storage stability of the lyophilized composition, as assessed by a reconstituted composition prepared from the lyophilized composition, may be measured after storage at a particular temperature for a defined period of time. In some cases, the period is 1 month. In some cases, the period is 3 months. In some cases, the period is 6 months. In various embodiments, the period is 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, 21 months, 24 months, 30 months, 36 months, 42 months, 48 ​​months, or 5 years or more. In various embodiments, the stability parameters may be measured after a period of storage (as described above) at temperatures of -30°C, -20°C, -10°C, -5°C, 0°C, 2-8°C, 5°C, 10°C, 15°C, 20°C, or 25°C. In some cases, the storage period and temperature is 1 month at a temperature between 2-8° C. In some cases, the storage period and temperature is 3 months at a temperature between 2-8° C. In some cases, the storage period and temperature is 6 months at a temperature between 2-8° C.

[0133] In some cases, a stable lyophilized composition, upon reconstitution (e.g., as a reconstituted pharmaceutical product), has storage parameters, as discussed above or herein, that remain the same or substantially the same as the parameters measured in the formulation prior to lyophilization.

[0134] Exemplary Formulations and Compositions According to one embodiment of the invention, a pharmaceutical formulation comprises (i) recombinant adeno-associated virus (rAAV) particles, (ii) a buffering agent at a concentration of about 1 mM to about 20 mM, (iii) a first salt at a concentration of about 10 mM to about 50 mM, (iv) a second salt at a concentration of about 20 mM to 100 mM, (v) a sugar at a concentration of about 1% w / v to about 10% w / v, (vi) a bulking agent at a concentration of about 1% w / v to about 6% w / v, and (vii) a surfactant at a concentration of about 0.001% w / v to about 0.1% w / v, wherein the formulation has a pH of about 6.8 to about 7.8.

[0135] In some cases, the pharmaceutical formulation comprises (i) recombinant adeno-associated virus (rAAV) particles, (ii) Tris buffer at a concentration of 5 mM to 15 mM, (iii) sodium chloride at a concentration of 20 mM to 40 mM, (iv) L-arginine hydrochloride at a concentration of 30 mM to 90 mM, (v) sucrose at a concentration of 3% w / v to 7% w / v, (vi) mannitol at a concentration of 2% w / v to 5% w / v, (vii) polysorbate 80 or poloxamer 188 at a concentration of 0.005% w / v to 0.015% w / v, and the formulation has a pH of 7.1 to 7.5.

[0136] In some cases, the pharmaceutical formulation comprises (i) recombinant adeno-associated virus (rAAV) particles selected from the group consisting of AAV1 serotype, AAV2 serotype, AAV3 serotype, AAV4 serotype, AAV5 serotype, AAV6 serotype, AAV7 serotype, AAV8 serotype, AAV9 serotype, and AAV10 serotype; (ii) Tris buffer at a concentration of 5 mM to 15 mM; (iii) sodium chloride at a concentration of 20 mM to 40 mM; (iv) L-arginine hydrochloride at a concentration of 30 mM to 90 mM; (v) sucrose at a concentration of 3% w / v to 7% w / v; (vi) mannitol at a concentration of 2% w / v to 5% w / v; (vii) polysorbate 80 or poloxamer 188 at a concentration of 0.005% w / v to 0.015% w / v; and the formulation has a pH of 7.1 to 7.5.

[0137] In some cases, the pharmaceutical formulation comprises (i) recombinant adeno-associated virus (rAAV) particles of serotype AAV1, (ii) Tris buffer at a concentration of 5 mM to 15 mM, (iii) sodium chloride at a concentration of 20 mM to 40 mM, (iv) L-arginine hydrochloride at a concentration of 30 mM to 90 mM, (v) sucrose at a concentration of 3% w / v to 7% w / v, (vi) mannitol at a concentration of 2% w / v to 5% w / v, (vii) polysorbate 80 or poloxamer 188 at a concentration of 0.005% w / v to 0.015% w / v, and the formulation has a pH of 7.1 to 7.5.

[0138] In some cases, the pharmaceutical formulation comprises (i) recombinant adeno-associated virus (rAAV) particles of serotype AAV5, (ii) Tris buffer at a concentration of 5 mM to 15 mM, (iii) sodium chloride at a concentration of 20 mM to 40 mM, (iv) L-arginine hydrochloride at a concentration of 30 mM to 90 mM, (v) sucrose at a concentration of 3% w / v to 7% w / v, (vi) mannitol at a concentration of 2% w / v to 5% w / v, (vii) polysorbate 80 or poloxamer 188 at a concentration of 0.005% w / v to 0.015% w / v, and the formulation has a pH of 7.1 to 7.5.

[0139] In some cases, the pharmaceutical formulation comprises (i) recombinant adeno-associated virus (rAAV) particles of serotype AAV8, (ii) Tris buffer at a concentration of 5 mM to 15 mM, (iii) sodium chloride at a concentration of 20 mM to 40 mM, (iv) L-arginine hydrochloride at a concentration of 30 mM to 90 mM, (v) sucrose at a concentration of 3% w / v to 7% w / v, (vi) mannitol at a concentration of 2% w / v to 5% w / v, (vii) polysorbate 80 or poloxamer 188 at a concentration of 0.005% w / v to 0.015% w / v, and the formulation has a pH of 7.1 to 7.5.

[0140] In some cases, the pharmaceutical formulation comprises (i) recombinant adeno-associated virus (rAAV) particles of serotype AAV9, (ii) Tris buffer at a concentration of 5 mM to 15 mM, (iii) sodium chloride at a concentration of 20 mM to 40 mM, (iv) L-arginine hydrochloride at a concentration of 30 mM to 90 mM, (v) sucrose at a concentration of 3% w / v to 7% w / v, (vi) mannitol at a concentration of 2% w / v to 5% w / v, (vii) polysorbate 80 or poloxamer 188 at a concentration of 0.005% w / v to 0.015% w / v, and the formulation has a pH of 7.1 to 7.5.

[0141] In some cases, the pharmaceutical formulation comprises (i) recombinant adeno-associated virus (rAAV) particles, (ii) Tris buffer at a concentration of 10 mM ± 1 mM, (iii) sodium chloride at a concentration of 30 mM ± 3 mM, (iv) L-arginine hydrochloride at a concentration of 40 mM ± 5 mM or 80 mM ± 5 mM, (v) sucrose at a concentration of 5% w / v ± 1% w / v, (vi) mannitol at a concentration of 3.3% w / v ± 0.5% w / v, (vii) polysorbate 80 or poloxamer 188 at a concentration of 0.01% w / v ± 0.002% w / v, and the formulation has a pH of 7.3 ± 0.1.

[0142] In some instances, the pharmaceutical formulation comprises: (i) recombinant adeno-associated virus (rAAV) particles selected from the group consisting of AAV1 serotype, AAV2 serotype, AAV3 serotype, AAV4 serotype, AAV5 serotype, AAV6 serotype, AAV7 serotype, AAV8 serotype, AAV9 serotype, and AAV10 serotype; (ii) Tris buffer at a concentration of 10 mM ± 1 mM; (iii) 30 mM ± 3 mM (iv) L-arginine hydrochloride at a concentration of 40 mM ± 5 mM or 80 mM ± 5 mM; (v) sucrose at a concentration of 5% w / v ± 1% w / v; (vi) mannitol at a concentration of 3.3% w / v ± 0.5% w / v; and (vii) polysorbate 80 or poloxamer 188 at a concentration of 0.01% w / v ± 0.002% w / v, wherein the formulation has a pH of 7.3 ± 0.1.

[0143] In some cases, the pharmaceutical formulation comprises (i) recombinant adeno-associated virus (rAAV) particles of serotype AAV1, (ii) Tris buffer at a concentration of 10 mM ± 1 mM, (iii) sodium chloride at a concentration of 30 mM ± 3 mM, (iv) L-arginine hydrochloride at a concentration of 40 mM ± 5 mM or 80 mM ± 5 mM, (v) sucrose at a concentration of 5% w / v ± 1% w / v, (vi) mannitol at a concentration of 3.3% w / v ± 0.5% w / v, (vii) polysorbate 80 or poloxamer 188 at a concentration of 0.01% w / v ± 0.002% w / v, and the formulation has a pH of 7.3 ± 0.1.

[0144] In some cases, the pharmaceutical formulation comprises (i) recombinant adeno-associated virus (rAAV) particles of serotype AAV5, (ii) Tris buffer at a concentration of 10 mM ± 1 mM, (iii) sodium chloride at a concentration of 30 mM ± 3 mM, (iv) L-arginine hydrochloride at a concentration of 40 mM ± 5 mM or 80 mM ± 5 mM, (v) sucrose at a concentration of 5% w / v ± 1% w / v, (vi) mannitol at a concentration of 3.3% w / v ± 0.5% w / v, (vii) polysorbate 80 or poloxamer 188 at a concentration of 0.01% w / v ± 0.002% w / v, and the formulation has a pH of 7.3 ± 0.1.

[0145] In some cases, the pharmaceutical formulation comprises (i) recombinant adeno-associated virus (rAAV) particles of serotype AAV8, (ii) Tris buffer at a concentration of 10 mM ± 1 mM, (iii) sodium chloride at a concentration of 30 mM ± 3 mM, (iv) L-arginine hydrochloride at a concentration of 40 mM ± 5 mM or 80 mM ± 5 mM, (v) sucrose at a concentration of 5% w / v ± 1% w / v, (vi) mannitol at a concentration of 3.3% w / v ± 0.5% w / v, (vii) polysorbate 80 or poloxamer 188 at a concentration of 0.01% w / v ± 0.002% w / v, and the formulation has a pH of 7.3 ± 0.1.

[0146] In some cases, the pharmaceutical formulation comprises (i) recombinant adeno-associated virus (rAAV) particles of serotype AAV9, (ii) Tris buffer at a concentration of 10 mM ± 1 mM, (iii) sodium chloride at a concentration of 30 mM ± 3 mM, (iv) L-arginine hydrochloride at a concentration of 40 mM ± 5 mM or 80 mM ± 5 mM, (v) sucrose at a concentration of 5% w / v ± 1% w / v, (vi) mannitol at a concentration of 3.3% w / v ± 0.5% w / v, (vii) polysorbate 80 or poloxamer 188 at a concentration of 0.01% w / v ± 0.002% w / v, and the formulation has a pH of 7.3 ± 0.1.

[0147] In some cases, the pharmaceutical formulation comprises (i) recombinant adeno-associated virus (rAAV) particles, (ii) phosphate buffer at a concentration between 5 mM and 15 mM, (iii) sodium chloride at a concentration between 20 mM and 40 mM, (iv) L-arginine hydrochloride at a concentration between 30 mM and 90 mM, (v) sucrose at a concentration between 3% w / v and 7% w / v, (vi) mannitol at a concentration between 2% w / v and 5% w / v, (vii) polysorbate 80 or poloxamer 188 at a concentration between 0.005% w / v and 0.015% w / v, and the formulation has a pH of 7.1 to 7.5.

[0148] In some cases, the pharmaceutical formulation comprises (i) recombinant adeno-associated virus (rAAV) particles selected from the group consisting of AAV1 serotype, AAV2 serotype, AAV3 serotype, AAV4 serotype, AAV5 serotype, AAV6 serotype, AAV7 serotype, AAV8 serotype, AAV9 serotype, and AAV10 serotype; (ii) phosphate buffer at a concentration of 5 mM to 15 mM; (iii) sodium chloride at a concentration of 20 mM to 40 mM; (iv) L-arginine hydrochloride at a concentration of 30 mM to 90 mM; (v) sucrose at a concentration of 3% w / v to 7% w / v; (vi) mannitol at a concentration of 2% w / v to 5% w / v; (vii) polysorbate 80 or poloxamer 188 at a concentration of 0.005% w / v to 0.015% w / v; and the formulation has a pH of 7.1 to 7.5.

[0149] In some cases, the pharmaceutical formulation comprises (i) recombinant adeno-associated virus (rAAV) particles of serotype AAV1, (ii) phosphate buffer at a concentration of 5 mM to 15 mM, (iii) sodium chloride at a concentration of 20 mM to 40 mM, (iv) L-arginine hydrochloride at a concentration of 30 mM to 90 mM, (v) sucrose at a concentration of 3% w / v to 7% w / v, (vi) mannitol at a concentration of 2% w / v to 5% w / v, (vii) polysorbate 80 or poloxamer 188 at a concentration of 0.005% w / v to 0.015% w / v, and the formulation has a pH of 7.1 to 7.5.

[0150] In some cases, the pharmaceutical formulation comprises (i) recombinant adeno-associated virus (rAAV) particles of serotype AAV5, (ii) phosphate buffer at a concentration of 5 mM to 15 mM, (iii) sodium chloride at a concentration of 20 mM to 40 mM, (iv) L-arginine hydrochloride at a concentration of 30 mM to 90 mM, (v) sucrose at a concentration of 3% w / v to 7% w / v, (vi) mannitol at a concentration of 2% w / v to 5% w / v, (vii) polysorbate 80 or poloxamer 188 at a concentration of 0.005% w / v to 0.015% w / v, and the formulation has a pH of 7.1 to 7.5.

[0151] In some cases, the pharmaceutical formulation comprises (i) recombinant adeno-associated virus (rAAV) particles of serotype AAV8, (ii) phosphate buffer at a concentration of 5 mM to 15 mM, (iii) sodium chloride at a concentration of 20 mM to 40 mM, (iv) L-arginine hydrochloride at a concentration of 30 mM to 90 mM, (v) sucrose at a concentration of 3% w / v to 7% w / v, (vi) mannitol at a concentration of 2% w / v to 5% w / v, (vii) polysorbate 80 or poloxamer 188 at a concentration of 0.005% w / v to 0.015% w / v, and the formulation has a pH of 7.1 to 7.5.

[0152] In some cases, the pharmaceutical formulation comprises (i) recombinant adeno-associated virus (rAAV) particles of serotype AAV9, (ii) phosphate buffer at a concentration of 5 mM to 15 mM, (iii) sodium chloride at a concentration of 20 mM to 40 mM, (iv) L-arginine hydrochloride at a concentration of 30 mM to 90 mM, (v) sucrose at a concentration of 3% w / v to 7% w / v, (vi) mannitol at a concentration of 2% w / v to 5% w / v, (vii) polysorbate 80 or poloxamer 188 at a concentration of 0.005% w / v to 0.015% w / v, and the formulation has a pH of 7.1 to 7.5.

[0153] In some cases, the pharmaceutical formulation comprises (i) recombinant adeno-associated virus (rAAV) particles, (ii) phosphate buffer at a concentration of 10 mM ± 1 mM, (iii) sodium chloride at a concentration of 30 mM ± 3 mM, (iv) L-arginine hydrochloride at a concentration of 40 mM ± 5 mM or 80 mM ± 5 mM, (v) sucrose at a concentration of 5% w / v ± 1% w / v, (vi) mannitol at a concentration of 3.3% w / v ± 0.5% w / v, (vii) polysorbate 80 or poloxamer 188 at a concentration of 0.01% w / v ± 0.002% w / v, and the formulation has a pH of 7.3 ± 0.1.

[0154] In some instances, the pharmaceutical formulation comprises: (i) recombinant adeno-associated virus (rAAV) particles selected from the group consisting of AAV1 serotype, AAV2 serotype, AAV3 serotype, AAV4 serotype, AAV5 serotype, AAV6 serotype, AAV7 serotype, AAV8 serotype, AAV9 serotype, and AAV10 serotype; (ii) a phosphate buffer at a concentration of 10 mM ± 1 mM; (iii) a phosphate buffer solution at a concentration of 30 mM ± 3 mM; (iv) L-arginine hydrochloride at a concentration of 40 mM ± 5 mM or 80 mM ± 5 mM; (v) sucrose at a concentration of 5% w / v ± 1% w / v; (vi) mannitol at a concentration of 3.3% w / v ± 0.5% w / v; and (vii) polysorbate 80 or poloxamer 188 at a concentration of 0.01% w / v ± 0.002% w / v, wherein the formulation has a pH of 7.3 ± 0.1.

[0155] In some cases, the pharmaceutical formulation comprises (i) recombinant adeno-associated virus (rAAV) particles of serotype AAV1, (ii) phosphate buffer at a concentration of 10 mM ± 1 mM, (iii) sodium chloride at a concentration of 30 mM ± 3 mM, (iv) L-arginine hydrochloride at a concentration of 40 mM ± 5 mM or 80 mM ± 5 mM, (v) sucrose at a concentration of 5% w / v ± 1% w / v, (vi) mannitol at a concentration of 3.3% w / v ± 0.5% w / v, (vii) polysorbate 80 or poloxamer 188 at a concentration of 0.01% w / v ± 0.002% w / v, and the formulation has a pH of 7.3 ± 0.1.

[0156] In some cases, the pharmaceutical formulation comprises (i) recombinant adeno-associated virus (rAAV) particles of serotype AAV5, (ii) phosphate buffer at a concentration of 10 mM ± 1 mM, (iii) sodium chloride at a concentration of 30 mM ± 3 mM, (iv) L-arginine hydrochloride at a concentration of 40 mM ± 5 mM or 80 mM ± 5 mM, (v) sucrose at a concentration of 5% w / v ± 1% w / v, (vi) mannitol at a concentration of 3.3% w / v ± 0.5% w / v, (vii) polysorbate 80 or poloxamer 188 at a concentration of 0.01% w / v ± 0.002% w / v, and the formulation has a pH of 7.3 ± 0.1.

[0157] In some cases, the pharmaceutical formulation comprises (i) recombinant adeno-associated virus (rAAV) particles of serotype AAV8, (ii) phosphate buffer at a concentration of 10 mM ± 1 mM, (iii) sodium chloride at a concentration of 30 mM ± 3 mM, (iv) L-arginine hydrochloride at a concentration of 40 mM ± 5 mM or 80 mM ± 5 mM, (v) sucrose at a concentration of 5% w / v ± 1% w / v, (vi) mannitol at a concentration of 3.3% w / v ± 0.5% w / v, (vii) polysorbate 80 or poloxamer 188 at a concentration of 0.01% w / v ± 0.002% w / v, and the formulation has a pH of 7.3 ± 0.1.

[0158] In some cases, the pharmaceutical formulation comprises (i) recombinant adeno-associated virus (rAAV) particles of serotype AAV9, (ii) phosphate buffer at a concentration of 10 mM ± 1 mM, (iii) sodium chloride at a concentration of 30 mM ± 3 mM, (iv) L-arginine hydrochloride at a concentration of 40 mM ± 5 mM or 80 mM ± 5 mM, (v) sucrose at a concentration of 5% w / v ± 1% w / v, (vi) mannitol at a concentration of 3.3% w / v ± 0.5% w / v, (vii) polysorbate 80 or poloxamer 188 at a concentration of 0.01% w / v ± 0.002% w / v, and the formulation has a pH of 7.3 ± 0.1.

[0159] In any of the various pharmaceutical formulations described above, the surfactant is polysorbate 80. In any of the various pharmaceutical formulations described above, the surfactant is poloxamer 188.

[0160] In various embodiments, the present disclosure provides a lyophilized composition prepared by lyophilization of any one of the above-mentioned pharmaceutical formulations, or a reconstituted composition (or pharmaceutical product) prepared by reconstituting any such lyophilized composition.

[0161] Containers, kits and unit dosage forms The present disclosure includes a container and a kit that contains or includes the formulation or composition discussed herein.In some cases, the container contains a pre-lyophilized pharmaceutical composition, a lyophilized composition, or a reconstituted composition or pharmaceutical product.In some cases, the container is a glass vial.The present disclosure also includes a kit that contains the container and instructions for use (e.g., in therapeutic applications discussed below).

[0162] In exemplary aspects, the composition comprising the AAV particles is contained in a glass or plastic container, e.g., a glass vial, or a plastic tube, as described herein. In exemplary embodiments, the glass or plastic container is any one of those known in the art or commercially available. In some cases, the disclosure includes disposing about 0.1 ml to about 25 ml (e.g., about 1 ml to about 10 ml) of the composition comprising the AAV particles in the glass or plastic container.

[0163] Providing the composition in a container may facilitate maintaining the composition as a sterile composition. For example, the container may be configured to maintain the composition enclosed within the container in a sterile environment. Thus, the container may be a sealed container, for example, the container may include a seal, such as a water-tight and / or air-tight seal. The seal may be removable from the container to allow a user to access the contents of the container. In some instances, the seal may be a frangible seal, or in other instances, the seal may be configured to allow a needle, cannula, or syringe to be inserted into the interior of the container without removing the seal from the container. In some cases, a seal configured to allow access to the interior of the container without removing the seal from the container may facilitate maintaining the contents of the container (e.g., the composition within the container) in a sterile environment prior to administration of the composition to a subject. Suitable materials for the seal include, but are not limited to, rubber or polymer seals, such as, for example, silicone rubber, natural rubber, styrene butadiene rubber, ethylene-propylene copolymer, polychloroprene, polyacrylate, polybutadiene, polyurethane, styrene butadiene, and the like, and combinations thereof. For example, in certain embodiments, the seal is a septum that can be punctured by a needle, syringe, or cannula. The seal can also provide convenient access to the sample in the container as well as a protective barrier covering the opening of the container. In some instances, the seal is a removable seal, such as a screw-on or snap-on cap, or other suitable sealing element, that can be applied to the opening of the container. For example, a screw-on cap can be screwed onto the opening before or after the sample is added to the container.

[0164] In some cases, the container is a unit dose container. A unit dose container refers to a container that contains one or more unit doses for administration to a subject. In some embodiments, the unit dose container contains a predetermined amount of the subject composition calculated in an amount sufficient to produce a desired effect in the subject. Certain embodiments of the composition may be provided in a unit dose container suitable for individual administration of a precise dose. The amount of active composition administered to a subject may depend on the subject being treated, the severity of the disease, and the method of administration. For example, the unit dose container may contain an amount of the composition administered as disclosed herein in an amount effective to achieve a desired effect in the subject being treated. In certain instances, the unit dose container contains a composition having a therapeutically effective amount of AAV. In certain embodiments, the unit dose container is a vial. In some cases, the vial is a sealed vial (e.g., as described above for a sealed container).

[0165] The container may be constructed of any convenient material that is compatible with the AAV and other components of the composition. For example, the container can be a solid-compatible container configured to contain a solid (e.g., a lyophilized composition). In some instances, the container is a liquid-compatible container configured to contain a liquid. The container may also be solid- and liquid-compatible, where the container is configured to contain a solid and a liquid. In some cases, the liquid in the container may be an aqueous liquid, and in these cases, the container may be compatible with an aqueous composition. By "compatible" it is meant that the container is substantially inert (e.g., does not react significantly) with the liquid and / or composition, or other components that come into contact with the container. Examples of suitable container materials include, but are not limited to, glass and plastic. For example, the container may be constructed of glass, such as, but not limited to, silicate glass, borosilicate glass, sodium borosilicate glass, fused quartz glass, fused silica glass, and the like. Other examples of suitable container materials include, but are not limited to, plastics such as polypropylene, polymethylpentene, polytetrafluoroethylene (PTFE), perfluoroether (PFE), fluorinated ethylene propylene (FEP), perfluoroalkoxyalkane (PFA), polyethylene terephthalate (PET), polyethylene (PE), polyether ether ketone (PEEK), polystyrene, etc. In a particular example, as described above, the container is a vial and thus may be a glass vial. As described above, the container may be a sealed container and thus may be a sealed glass vial.

[0166] In exemplary embodiments, the volume of a composition comprising an AAV disposed in a glass or plastic container is about 0.1 to about 25 ml, about 0.1 ml to about 10 ml, about 0.1 ml to about 5 ml, about 0.2 ml to about 6 ml, about 0.2 ml to about 5 ml, about 0.25 ml to about 5 ml, about 2 ml to about 5 ml, about 2.5 ml to about 5 ml, about 3 ml to about 5 ml, about 4 ml to about 5 ml, about 5 ml to about 10 ml, about 6 ml, about 9 ml, or about 7 ml to about 8 ml. In some cases, the volume is about 0.1 ml, about 0.2 ml, about 0.25 ml, about 0.3 ml, about 0.4, about 0.5 ml, about 0.6 ml, about 0.7 ml, about 0.75 ml, about 0.8 ml, about 0.9 ml, about 1 ml, about 2 ml, about 2.5 ml, about 3 ml, about 3.5 ml, about 4 ml, about 4.5 ml, about 5 ml, about 5.5 ml, about 6 ml, about 6.5 ml, about 7 ml, about 7.5 ml, about 8 ml, about 8.5 ml, about 9 ml, about 9.5 ml, or about 10 ml.

[0167] Therapeutic Use The pharmaceutical formulations and compositions of the present invention are useful, inter alia, for the treatment, prevention, and / or amelioration of any disease or disorder treatable by gene therapy. In exemplary embodiments, the pharmaceutical compositions discussed herein can be administered to a subject in an amount effective to treat the disorder.

[0168] The compositions disclosed herein can be formulated for administration via known methods, such as intravenous administration (e.g., as a bolus or by continuous infusion over a period of time) or administration by injection.In certain embodiments, the AAV formulations provided herein can be administered systemically or locally.Systemic administration includes, but is not limited to, intravenous administration.Local administration includes, but is not limited to, subcutaneous and intramuscular injection administration.

[0169] When administered to a subject, the liquid or reconstituted dosage unit may contain a therapeutically effective amount of AAV such that the reconstituted dosage unit delivers between 1E10 viral genomes (or vector genomes) per milliliter (vg / ml) and 5E15 vg / ml.

[0170] In certain embodiments, the administration of the liquid or reconstituted dosage unit to the subject is carried out according to a treatment regimen. For example, in some cases, the subject to be treated may be prescribed a treatment regimen by a medical professional. In some cases, the treatment regimen includes a single administration of the dosage unit. Generally, the subject administered the AAV pharmaceutical product generates anti-drug antibodies against the serotype of AAV administered to the subject, thereby preventing further administration of the pharmaceutical product to produce a therapeutic effect. This does not exclude the possibility that further administration of the same gene of interest contained in the rAAV pharmaceutical product can be administered using a different AAV serotype.

[0171] In certain embodiments, the reconstituted dosage unit of the present disclosure can be administered before, simultaneously, or after other active agents for treating related or unrelated conditions, for example, in combination therapy. Examples of such additional therapies include radiation therapy, surgery, and chemotherapy. When provided simultaneously with other active agents, the reconstituted dosage unit of the present disclosure can be provided in the same formulation or in different formulations. For example, simultaneous therapy can be achieved by administering a pharmaceutical composition having a reconstituted dosage unit and at least one other active agent, such as a chemotherapeutic agent, which can be combined to provide a therapeutically effective dose according to a specific treatment regimen. The administration of the separate pharmaceutical compositions can be performed at the same time or at different times (e.g., sequentially, in any order, on the same day, or on different days), as long as the therapeutically effective effect of the combination of these substances is caused in the subject being treated.

[0172] Thus, aspects of the present disclosure further include combination therapy. Combination therapy means that the AAV composition (e.g., as described herein) can be used in combination with another therapeutic agent to treat a single disease or condition. In certain embodiments, the AAV formulation or composition of the present disclosure can be administered simultaneously with the administration of another therapeutic agent. In certain embodiments, the AAV formulation or composition of the present disclosure can be administered before or after the administration of another therapeutic agent. In certain embodiments, the combination therapy includes two or more AAV compositions that contain different serotypes and / or different genes of interest. EXAMPLES

[0173] The following examples are provided to provide those skilled in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventor regards as his invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is degrees Celsius, and pressure is at or near atmospheric pressure.

[0174] Example 1: Pre-lyophilized formulation screening study This study was designed to screen for formulations that were suitable for lyophilization and potentially had liquid stability at 25° C. for up to 2 days.

[0175] Approximately 3 mL of each formulation shown in Table 1 was prepared. In a laminar flow hood, each formulation was filter sterilized using a 0.2 μm filter and dispensed into 2R RTU glass vials for a target fill volume of 0.7 mL. All vials were sealed with rubber stoppers. At least duplicate vials were prepared for each formulation.

[0176] [Table 1]

[0177] Particle size distribution by dynamic light scattering (DLS) was performed on a Zetasizer Ultra system (Malvern, Westborough, MA) using 90 degree side scattering with a ZEN2112 low volume quartz cuvette. Five replicates were collected per sample, with 10 acquisitions of 10 seconds each for each replicate.

[0178] The data demonstrated that sucrose and mannitol were compatible with AAV8 and did not induce the formation of AAV8 aggregates. Formulations F1, F4, and F7 showed comparable dynamic light scattering (DLS) results (approximately 25n) at both t=0 and 2 days at 25°C and 60% relative humidity (other samples showed possible reversible aggregation). DLS assays showed that all formulations were stable after 2 days of exposure to 25°C / 60% RH. Furthermore, no obvious recovery loss (by ddPCR) or purity change (empty vs. intact capsid ratio by AEX-UPLC) was observed for formulation F7 after 2 days of incubation at 25°C / 60% RH.

[0179] Example 2: Freeze-drying feasibility study This study was designed to evaluate the lyophilization process feasibility of seven formulations proposed for AAV8 lyophilization process development.

[0180] Sucrose was used as the cryoprotectant and mannitol (3.3% w / v) was used as the bulking agent. The lyophilization designs were evaluated using formulations with varying concentrations of L-arginine HCl and NaCl (0 mM, 30 mM, 50 mM, and 80 mM), two concentrations of sucrose (1.2% w / v and 5% w / v), and a formulation with 0.005% w / v P188. The seven formulations evaluated in this study are shown in Table 2.

[0181] [Table 2]

[0182] In a laminar flow hood, each formulation was filter sterilized using a 0.2 μm filter and dispensed into 2 mL glass vials for a target fill volume of 0.4 mL. All vials were sealed with rubber stoppers. At least 10 vials of each formulation were prepared. The Tc decay temperature or Teu eutectic temperature of each formulation was measured by freeze-drying microscopy.

[0183] The load for each lyophilization cycle contained 70 2 mL glass vials, 10 vials for each formulation F1-F7. Each formulation was grouped together. The vials were evaluated for moisture content, cake appearance, and Tg (glass transition) analysis. Dynamic light scattering was used to detect aggregation, which is the major degradation pathway for AAV products.

[0184] The lyophilization cycle is shown in Table 3 below.

[0185] [Table 3]

[0186] Freeze-drying experiments were performed using an FTS LyoStar™ 3 freeze-dryer (SP Scientific, Stone Ridge, NY). Ready-to-use (RTU) type I glass vials (SCHOTT, Mülheim, Germany) were placed in the freeze-drying shelf as described above. A two-wire thermocouple (Omega, Norwalk, CT) was used to monitor the temperature profile of the placebo product throughout the freeze-drying process. Samples were first incubated at 5°C for 60 min, cooled from 5°C to -45°C at a ramp rate of 0.5°C / min, followed by an isothermal hold for 60 min. Annealing was performed at -20°C for 6 h at a ramp rate of 0.5°C / min, cooled to -45°C at a ramp rate of 0.5°C / min, followed by a hold for 60 min. Primary drying was at -26°C for 50 hours under 100 mTorr chamber pressure, followed by secondary drying at 35°C for 10 hours under 100 mTorr, with a heating ramp rate of 0.3°C / min for both primary and secondary drying. At the end of the lyophilization cycle, samples were back-filled with 80% atmospheric nitrogen gas, capped with 13 mm Flurotec® chlorobutyl stoppers, and sealed with 13 mm aluminum flip-off seals (West, Jersey Shore, PA).

[0187] The color and cake appearance of the lyophilized products were assessed by visual inspection.

[0188] The glass transition temperatures Tg' of the most freeze-concentrated solutions of the liquid formulations and Tg of the solid freeze-dried formulations were determined on a DSC2500 (TA Instruments, New Castle, DE). To determine Tg', 40 uL of sample solution was pipetted into a Tzero pan and tightly sealed with a Tzero sealing lid. To determine Tg, the freeze-dried samples were ground and prepared in a humidity-controlled room to minimize moisture contamination of the samples during sample preparation. The sample powder was then transferred to a Tzero pan and tightly sealed with a Tzero sealing lid. The samples were equilibrated at -80°C and held isothermal for 5 minutes, then ramped to 25°C at 2°C / min, adjusting by 0.5°C every 60 seconds. Data analysis was performed on TRIOS.

[0189] The collapse temperature (Tc) of the formulations was determined with a Lyostat 3 freeze-drying microscope (Linkam Scientific, Waterfield, UK) equipped with a polarizing filter and an Olympus camera. Approximately 5 uL of sample solution was placed on a glass cover slip placed on the cooling stage under the microscope. The sample was first frozen to -45°C and then heated to 0°C at 1°C / min under vacuum (100 mTorr). Images were acquired using Linsys32 software, exported, and used to determine Tc.

[0190] The residual moisture content of the lyophilized samples was determined using a Vapor Pro RX moisture analyzer and dry air generator (Arizona Instrument, Chandler, AZ). The lyophilized cake was broken into small pieces in a sealed glass vial prior to analysis. The airflow rate was set at 75 mL / min and the drying temperature was set at 75° C. To calculate the percentage of residual moisture content, the measured weight of the residual moisture was divided by the weight of the lyophilized cake.

[0191] Particle size distribution by dynamic light scattering (DLS) was performed on a Zetasizer Ultra system (Malvern, Westborough, MA) using 90 degree side scattering with a ZEN2112 low volume quartz cuvette. Five replicates were collected per sample, with 10 acquisitions of 10 seconds each for each replicate. The results of the DLS assay for each of the seven formulations are shown in Figure 2. Of the seven formulations evaluated, F7 was the only formulation that showed minimal changes in size distribution profile compared to the pre-lyophilized sample.

[0192] Example 3: Buffer Screening Study

[0193] The stability of rAAV8 formulations using Tris versus sodium phosphate buffer was evaluated. The mean particle size to identify aggregates was determined both pre- and post-lyophilization, the % complete capsid was determined pre- and post-lyophilization, and the viral genome titer was determined pre- and post-lyophilization. Lyophilization was performed as described above in Example 2.

[0194] The rAAV8 capsid contained a gene of interest (GOI) for therapeutic use. The rAAV8 vector was generated by triple transfection of HEK293 cells at Regeneron Pharmaceuticals (Tarrytown, NY).

[0195] The sodium phosphate formulation contained 1.00E13 vg / mL rAAV8-GOI, 10 mM sodium phosphate at pH 7.3, 0.005% w / v P188, 5% w / v sucrose, 3.3% w / v mannitol, 30 mM NaCl, and 80 mM L-arginine hydrochloride. The Tris formulation contained 1.00E13 vg / mL rAAV8-GOI, 10 mM Tris buffer at pH 7.3, 0.005% w / v P188, 5% w / v sucrose, 3.3% w / v mannitol, 30 mM NaCl, and 80 mM L-arginine hydrochloride.

[0196] Particle size distribution by dynamic light scattering (DLS) was performed on a Zetasizer Ultra system (Malvern, Westborough, MA) using 90 degree side scattering with a ZEN2112 low volume quartz cuvette. Five replicates were collected per sample, with 10 acquisitions of 10 seconds each for each replicate.

[0197] The ratio of empty to full capsids was determined by anion exchange ultra-performance liquid chromatography (AEX-UPLC) using a Waters Acquity UPLC H-Class system equipped with PDA and FRR detectors (Waters, Milford, MA) and a CIMac AAV empty / full 0.1 mL, 1.3 μm analytical column (BIA Separation, Slovenia). Empty and full capsids were eluted at 0.8 mL / min using a linear gradient containing bis-tris propane and tetramethylammonium chloride at pH 8.5. Peak identification and integration were performed using a Waters Empower chromatography data system.

[0198] Vector genome titers were determined using a QX200 Droplet Digital AAV samples were prepared in triplicate and digested with DNase I (Ambion Part No. AM2224) to remove non-encapsulated DNA, followed by digestion with Proteinase K (Invitrogen Part No. 25530049) to disrupt the capsid. A ddPCR master mix containing primers and probe sets was then added and mixed with the reaction mixture. An aliquot of 22 μL was then removed and introduced into the droplet generator for droplet dispensing. After droplet generation, vector genomic DNA contained in the droplets was amplified by PCR and analyzed with QuantaSoft software on the droplet reader.

[0199] The results of the stability evaluation before and after lyophilization are shown in Table 5 below.

[0200] [Table 4]

[0201] The results of the stability evaluation showed excellent results in Tris buffer after lyophilization. The difference in mean particle size is believed to be the result of nanobubbles in the reconstituted composition rather than aggregation of AAV particles, as discussed in more detail in Example 7 below.

[0202] Example 4: Evaluation of sugar and salt concentrations Sucrose concentration, as well as NaCl and arginine HCL concentrations, were evaluated to determine their effect on rAAV8-GOI stability. Eight formulations were evaluated, as shown in Table 6 below. Lyophilization of these eight formulations was performed as described above in Example 2.

[0203] [Table 5]

[0204] Particle size distribution both before and after lyophilization by dynamic light scattering (DLS) was performed on a Zetasizer Ultra system (Malvern, Westborough, MA) using 90 degree side scattering with a ZEN2112 low volume quartz cuvette. Five replicates were collected per sample, with 10 acquisitions of 10 seconds each for each replicate.

[0205] The results are shown in Table 7 below.

[0206] [Table 6]

[0207] Formulation F7, containing 30 mM NaCl, 80 mM L-Arg HCl, and 5% sucrose, showed the least increase in particle size among the formulations tested. This formulation also showed good solution stability for 2 days at 25° C. without signs of aggregation. The observed differences in particle size distribution are believed to be the result of nanobubbles in the reconstituted composition rather than aggregation of AAV particles, as discussed in more detail in Example 7 below.

[0208] The residual moisture content in Formulation F7 after lyophilization was analyzed to be 0.7%. The residual moisture content was determined using a Vapor Pro RX moisture analyzer and dry air generator (Arizona Instrument, Chandler, AZ). The lyophilized cake was broken into small pieces in a sealed glass vial prior to analysis. The airflow rate was set at 75 mL / min and the drying temperature was set at 75° C. To calculate the percentage of residual moisture content, the measured weight of the residual moisture was divided by the weight of the lyophilized cake.

[0209] The Tc and Tg' of formulation (F7) were measured using lyophilization microscopy and DSC, which were determined as -34° C. and -43° C., respectively, and the results are shown in Figures 6A-6B.

[0210] As shown in Figure 9A and Figure 9B, reducing the total ionic strength in the formulation to 80 mM resulted in a significant decrease (about 18%) in vg titer recovery after lyophilization. The addition of 2 mM magnesium chloride helped to improve vg titer recovery by about 6%, but did not show an equivalent stabilizing effect on vg titer recovery after lyophilization compared to that achieved with the control formulation. This observation was unexpected based on the assumption that salts would not be effective stabilizers for proteins in lyophilized pharmaceutical products because they would crystallize during lyophilization and not remain in the amorphous matrix, a prerequisite for stabilizing proteins in the dry form. These results confirmed that salts with a total ionic strength of about 110 mM provide a stabilizing effect for lyophilized AAV8 formulations.

[0211] Example 5: Effect of bulking agent and concentration on freeze-dried products The effect of mannitol on the rAAV8-GOI lyophilized product was evaluated, as well as the concentrations of mannitol, sucrose, and salt.

[0212] The formulations with and without mannitol were lyophilized and evaluated for appearance, reconstitution time, and residual moisture content as described above in Example 2. The two evaluated formulations are shown below in Table 8, along with the results of the evaluation.

[0213] [Table 7]

[0214] The presence of mannitol improved the appearance of the cake and reduced the residual moisture content, as shown in FIG.

[0215] The effect of varying concentrations of mannitol, sucrose, NaCl, arginine-HCl, and MgCl2 on rAAV8-GOI formulations was also evaluated. The six evaluated formulations are shown below in Table 9. All formulations contained 1E13 AAV8-GOI, 0.02% w / v P188, and 10 mM Tris at pH 7.3.

[0216] [Table 8]

[0217] The results are expected to show that varying the concentrations of these components will yield comparable stability results.

[0218] Example 6: Surfactant screening test Stability evaluation of rAAV8 formulations was performed using various concentrations of poloxamer 188 (P188) or polysorbate 80 (PS80). Mean particle size to identify aggregates was determined both pre- and post-lyophilization, % intact capsids were determined pre- and post-lyophilization, and % viral genome recovery was determined pre- and post-lyophilization as described above in Example 3. Lyophilization was performed as described above in Example 2.

[0219] In the first study, three concentrations of P188 were evaluated in a formulation containing 1E13 vg / mL rAAV8-GOI, 10 mM sodium phosphate at pH 7.3, 5% w / v sucrose, 3.3% w / v mannitol, 30 mM NaCl, and 80 mM L-Arg-HCl. The results are shown in Table 10 below.

[0220] [Table 9]

[0221] In a second study, the effect of using PS80 instead of P188 was evaluated. Each of the formulations contained 1E13 vg / mL rAAV8-GOI, 10 mM sodium phosphate at pH 7.3, 5% w / v sucrose, 3.3% w / v mannitol, 30 mM NaCl, and 80 mM L-Arg-HCl, and either 0.005% w / v P188 or 0.005% w / v PS80. The results are shown in Table 11 below.

[0222] [Table 10]

[0223] In the third study, recovery at different concentrations of detergent was evaluated. Each of the formulations contained 1E13 vg / mL rAAV8-GOI, 10 mM sodium phosphate at pH 7.3, 5% w / v sucrose, 3.3% w / v mannitol, 30 mM NaCl, and 80 mM L-Arg-HCl, and various detergents / concentrations. Two different types of non-ionic detergents (PS80 and P188 at 0.005% w / v) and different concentrations of P188 (0.005, 0.01, and 0.02% w / v) were tested. As shown in Figure 4, formulations with both 0.005% PS80 and P188 showed similar levels of recovery loss (91% and 90%, respectively) in the t=0 samples after lyophilization, suggesting that the observed recovery loss was not associated with a specific type of non-ionic detergent. However, increasing the surfactant concentration from 0.005% to 0.01% to 0.02% w / v resulted in increased recovery after lyophilization (90, 95, and 96%, respectively), suggesting that the observed recovery losses are likely due to AAV adsorption to type I glass vials during the lyophilization process. The greatest increase in recovery after lyophilization occurred when the P188 concentration was increased from 0.005% to 0.01%, and further increases in surfactant concentration appeared to have minimal impact. Thus, a surfactant concentration of approximately 0.01% w / v would be sufficient to mitigate AAV adsorption to glass vials within the titer range tested.

[0224] Example 7: Stability of rAAV lyophilized formulations at 2-8°C Six formulations (three placebo and three rAAV8) were prepared to evaluate the stability of the lyophilized pharmaceutical product over time. Lyophilization was performed as described above in Example 2, except the length of secondary drying was extended to 10 hours. The formulations are identified below in Table 12. All six formulations contained 10 mM sodium phosphate buffer at pH 7.3.

[0225] [Table 11]

[0226] The color and cake appearance of the lyophilized products were assessed by visual inspection and the reconstitution times were determined as described above in Example 2. The results are shown in Table 13 below.

[0227] [Table 12]

[0228] The average particle size to characterize the aggregates was determined both before and after lyophilization as described above in Example 3. The results are shown in Table 14 below.

[0229] [Table 13]

[0230] The rAAV-7 formulation that demonstrated the best stability based on aggregation (Table 14) was further evaluated over a 6 month period and monitored for appearance, particle size, % intact capsid, and viral genome titer as described above in Example 3. The results are shown below in Table 15.

[0231] [Table 14]

[0232] rAAV-7 (AAV8 serotype) showed good stability at 5°C for up to six months.

[0233] As discussed above in Examples 3 and 4, the majority of the observed increase in mean particle size can be explained by the presence of nanobubbles in the reconstituted composition. For example, sample rAAV-7 demonstrated good storage stability after lyophilization of the AAV product compared to the formulation prior to lyophilization, however, upon completion of lyophilization, an increase in particle size of approximately 10 nm was detected by DLS in the reconstituted rAAV-7 formulation (post-lyophilization t=0, Table 15), suggesting that larger sized particle species arose during the lyophilization process. To investigate the underlying cause, a placebo sample (formulation without AAV particles) was lyophilized and identified larger particle species with an estimated size of approximately 100-200 nm (Figure 3). This is in the same size range as nanobubbles previously observed in reconstituted lyophilized protein products (data not shown), likely arising from microvoids within the lyophilized cake. As no obvious particle size increase was detected in the rAAV-7 formulation up to 6 months after lyophilization, the larger sized particle species that arose during lyophilization are likely due to nanobubbles rather than AAV aggregation. Furthermore, stability results from the lyophilized rAAV-7 formulations showed no evidence to suggest that the presence of nanobubbles in the lyophilized cake had an obvious adverse effect on the AAV product storage stability.

[0234] Example 8: Short-term stability of rAAV lyophilized formulations at room temperature Room temperature stability of at least 48 hours in the liquid state for lyophilized formulations is desirable to support manufacturing operations such as holding steps before filling and dispensing the product into glass vials prior to lyophilization. To evaluate in-solution stability testing, 0.7 mL of the AAV8 product in formulation F7 was filled into 2 mL type I glass vials and incubated at 25 °C for 2 days. As shown in Figure 7A, Figure 7B, and Figure 7C, formulation F7 demonstrated good in-solution stability with no detectable changes in selected critical quality attributes (CQAs) of AAV, including particle size by DLS to assess aggregation, viral genome (vg) titer by ddPCR to assess recovery, or empty-to-intact capsid ratio by AEX to assess capsid purity.

[0235] 1×1013 Another formulation containing AAV8 at vg / mL, 10 mM Tris, 30 mM sodium chloride, 80 mM L-arginine hydrochloride, 5% w / v sucrose, 3.3% w / v mannitol, and 0.01% w / v P188 at pH 7.3 was prepared and evaluated for stability in solution for 2 days and one month at 25° C. The results are shown in Figures 10A, 10B, and 10C, and Figures 11A, 11B, and 11C, respectively. No significant changes were observed in CQAs including particle size, vg titer, and empty to full capsid ratio. No significant changes were observed in samples using cell-based potency assays (data not shown).

[0236] Example 9: Long-term stability of rAAV lyophilized formulations at 2-8°C The long-term storage stability of the lyophilized AAV8 pharmaceutical product in formulation F7 at 2-8°C was evaluated for cake appearance and reconstitution time (data not shown) at 0, 1, 3, 6, and 12 months, as well as particle size, vg titer, and empty-to-full capsid ratio in the reconstituted AAV8 formulation. The results are shown in Figure 8A, Figure 8B, and Figure 8C. No significant changes were observed for all the above assessments in the post-lyophilization reconstituted formulation, indicating that the lyophilized AAV8 pharmaceutical product in formulation F7 is stable during long-term storage at 2-8°C for up to twelve months.

[0237] The approximately 10 nm increase in particle size by DLS in the 0 hour post-lyophilization sample (FIG. 8A) compared to the 0 hour pre-lyophilization sample can be attributed to nanobubbles, as discussed in Example 7. There is no evidence to suggest that the presence of nanobubbles significantly impacts the quality or stability of the lyophilized protein product, other than contributing to the number of submicron particles detectable in the reconstituted lyophilized protein formulation.

[0238] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to be within the scope of the appended claims. *******

Claims

1. 1. A pharmaceutical formulation comprising: (i) recombinant adeno-associated virus (rAAV) particles; (ii) a buffering agent at a concentration of about 1 mM to about 20 mM; (iii) a first salt at a concentration of about 10 mM to about 50 mM; (iv) a second salt at a concentration of about 60 mM to 100 mM; (v) a sugar at a concentration of about 1% w / v to about 10% w / v; (vi) a bulking agent at a concentration of about 1% w / v to about 6% w / v; and (vii) a surfactant at a concentration of about 0.001% w / v to about 0.1% w / v; wherein the formulation has a pH of about 6.8 to about 7.

8.

2. 2. The pharmaceutical formulation of claim 1, wherein the buffer is a Tris buffer or a sodium phosphate buffer.

3. The pharmaceutical formulation of claim 2 , wherein the buffer is a Tris buffer.

4. The pharmaceutical formulation according to any one of claims 1 to 3, wherein the concentration of the buffering agent is from 5 mM to 15 mM.

5. 5. The pharmaceutical formulation of claim 4, wherein the concentration of buffering agent is 10 mM ± 2 mM.

6. The pharmaceutical formulation of any one of claims 1 to 5, wherein the first salt is sodium chloride, potassium chloride, or magnesium chloride.

7. 7. The pharmaceutical formulation of claim 6, wherein the first salt is sodium chloride.

8. The pharmaceutical formulation of any one of claims 1 to 7, wherein the concentration of the first salt is from 20 mM to 40 mM.

9. 9. The pharmaceutical formulation of claim 8, wherein the concentration of the first salt is 30 mM ± 5 mM.

10. The pharmaceutical formulation of any one of claims 1 to 9, wherein the second salt is arginine hydrochloride.

11. The pharmaceutical formulation of any one of claims 1 to 10, wherein the concentration of the second salt is from 30 mM to 90 mM.

12. 12. The pharmaceutical formulation of claim 11, wherein the concentration of the second salt is 40 mM ± 5 mM or 80 mM ± 5 mM.

13. The pharmaceutical formulation according to any one of claims 1 to 12, wherein the sugar is sucrose or trehalose.

14. 14. The pharmaceutical formulation of claim 13, wherein the sugar is sucrose.

15. A pharmaceutical formulation according to any one of claims 1 to 14, wherein the concentration of sugar is between 3% w / v and 7% w / v.

16. 16. The pharmaceutical formulation of claim 15, wherein the concentration of sugar is 5% w / v ± 1% w / v.

17. The pharmaceutical formulation according to any one of claims 1 to 16, wherein the bulking agent is mannitol or glycine.

18. 18. The pharmaceutical formulation of claim 17, wherein the bulking agent is mannitol.

19. 19. The pharmaceutical formulation of any one of claims 1 to 18, wherein the concentration of the bulking agent is from 2% w / v to 5% w / v.

20. 20. The pharmaceutical formulation of claim 19, wherein the concentration of the bulking agent is 3.3% w / v ± 0.5% w / v.

21. The pharmaceutical formulation according to any one of claims 1 to 20, wherein the surfactant is a polysorbate or a poloxamer.

22. 22. The pharmaceutical formulation of claim 21, wherein the polysorbate is polysorbate 80.

23. 22. The pharmaceutical formulation of claim 21, wherein the poloxamer is poloxamer 188.

24. 24. The pharmaceutical formulation of any one of claims 1 to 23, wherein the concentration of surfactant is from 0.001% w / v to 0.1% w / v.

25. 25. The pharmaceutical formulation of claim 24, wherein the surfactant concentration is 0.01% w / v ± 0.005% w / v.

26. 26. The pharmaceutical formulation of any one of claims 1 to 25, wherein the pH of the formulation is from 7.0 to 7.

6.

27. 27. The pharmaceutical formulation of claim 26, wherein the pH of the formulation is 7.3±0.

1.

28. 77. A pharmaceutical formulation according to any one of claims 1 to 76, wherein the ratio of bulking agent to sugar is from 1:1 to 1:7 or from 1:1 to 1:

3.

29. 29. The pharmaceutical formulation of claim 28, wherein the ratio of mannitol to sucrose is from 1:1 to 1:7 or from 1:1 to 1:

3.

30. 30. The pharmaceutical formulation of any one of claims 1 to 29, wherein the ratio of the first salt to the second salt is from 1:1 to 1:

4.

31. 31. The pharmaceutical formulation of claim 30, wherein the ratio of sodium chloride to arginine hydrochloride is 1:1 to 1:

4.

32. 32. The pharmaceutical formulation of any one of claims 1 to 31, wherein the formulation has a total ionic strength of 70 mM to 120 mM.

33. 33. The pharmaceutical formulation of claim 32, wherein the total ionic strength is 80 mM ± 5 mM or 110 mM ± 10 mM.

34. 34. The pharmaceutical formulation of any one of claims 1 to 33, comprising from 1x10E10 vg / mL to 1x10E15 vg / mL.

35. 35. The pharmaceutical formulation of claim 34, comprising from 1 x 10E12 vg / mL to 1 x 10E14 vg / mL.

36. 36. The pharmaceutical formulation of claim 35, comprising about 1 x 10E13 vg / mL.

37. 1. A pharmaceutical formulation comprising: (i) recombinant adeno-associated virus (rAAV) particles at a concentration of 1×10E10 vg / mL to 1×10E15 vg / mL; (ii) phosphate buffer at a concentration of 10 mM±1 mM; (iii) sodium chloride at a concentration of 30 mM±3 mM; (iv) L-arginine hydrochloride at a concentration of 80 mM±8 mM; (v) sucrose at a concentration of 5% w / v±0.5% w / v; (vi) mannitol at a concentration of 3.3% w / v±0.3% w / v; and (vii) poloxamer 188 at a concentration of 0.005% w / v±0.001% w / v, wherein said formulation has a pH of 7.3±0.

2.

38. 1. A pharmaceutical formulation comprising: (i) recombinant adeno-associated virus (rAAV) particles at a concentration of 1×10E10 vg / mL to 1×10E15 vg / mL; (ii) Tris buffer at a concentration of 10 mM±1 mM; (iii) sodium chloride at a concentration of 30 mM±3 mM; (iv) L-arginine hydrochloride at a concentration of 80 mM±8 mM; (v) sucrose at a concentration of 5% w / v±0.5% w / v; (vi) mannitol at a concentration of 3.3% w / v±0.3% w / v; and (vii) poloxamer 188 at a concentration of 0.01% w / v±0.002% w / v; wherein the formulation has a pH of 7.3±0.

2.

39. 39. The pharmaceutical formulation of claim 37 or 38, wherein the rAAV particles are at a concentration of 1x10E12 vg / mL to 1x10E14 vg / mL.

40. 39. The pharmaceutical formulation of claim 37 or 38, wherein the rAAV particles are at a concentration of about 1 x 10E13 vg / mL.

41. 41. The pharmaceutical formulation of any one of claims 1 to 40, wherein the rAAV particles are of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-DJ, AAV-DJ / 8, AAV-Rh10, AAV-retro, AAV-PHP.B, AAV8-PHP.eB, or AAV-PHP.S.

42. 42. The pharmaceutical formulation of claim 41, wherein the rAAV particles are of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10.

43. 43. The pharmaceutical formulation of claim 42, wherein the rAAV particles are of serotype AAV1.

44. 43. The pharmaceutical formulation of claim 42, wherein the rAAV particles are of serotype AAV5.

45. 43. The pharmaceutical formulation of claim 42, wherein the rAAV particles are of serotype AAV8.

46. 43. The pharmaceutical formulation of claim 42, wherein the rAAV particles are of serotype AAV9.

47. 47. The pharmaceutical formulation of any one of claims 1 to 46, wherein the formulation has an average particle size of about 20 nm to about 40 nm as determined by dynamic light scattering.

48. 48. The pharmaceutical formulation of any one of claims 1 to 47, wherein the formulation contains a population of rAAV particles that contain at least 95% intact capsids as determined by anion exchange chromatography.

49. 49. A stable lyophilized pharmaceutical composition formed by lyophilizing the pharmaceutical formulation of any one of claims 1 to 48.

50. 50. The lyophilized pharmaceutical composition of claim 49, having a moisture content of 1.5% or less.

51. 51. The lyophilized pharmaceutical composition of claim 50, having a moisture content of 1.0% or less.

52. 52. The lyophilized pharmaceutical composition of claim 51, having a moisture content of 0.5% to 0.8%, or 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, or 0.8%.

53. The lyophilized pharmaceutical composition of any one of claims 49 to 52, which can be reconstituted in 30 seconds or less.

54. 54. The lyophilized pharmaceutical composition of claim 53, which can be reconstituted in 20 seconds or less.

55. 55. The lyophilized pharmaceutical composition of claim 54, which can be reconstituted in 15 seconds or less.

56. 56. A reconstituted pharmaceutical product formed by reconstituting the lyophilized pharmaceutical composition of any one of claims 49 to 55.

57. 57. The reconstituted pharmaceutical product of claim 56, comprising an average particle size of about 20 nm to about 40 nm as determined by dynamic light scattering.

58. 58. The reconstituted pharmaceutical product of claim 56 or 57, comprising a population of rAAV particles comprising at least 95% intact capsids as determined by anion exchange chromatography.

59. 59. The reconstituted pharmaceutical product of any one of claims 56-58, comprising a population of rAAV particles comprising a percentage of complete capsids as determined by anion exchange chromatography, wherein the percentage of complete capsids in the reconstituted pharmaceutical product is within 2% of the percentage of complete capsids in the pharmaceutical formulation prior to lyophilization.

60. 60. The reconstituted pharmaceutical product of claim 59, wherein the percentage of complete capsids in the reconstituted pharmaceutical product is within 1% of the percentage of complete capsids in the pharmaceutical formulation prior to lyophilization.

61. 61. The reconstituted pharmaceutical product of claim 60, wherein the percentage of complete capsids in the reconstituted pharmaceutical product is within 0.5% of the percentage of complete capsids in the pharmaceutical formulation prior to lyophilization.

62. 62. The reconstituted pharmaceutical product of any one of claims 56-61, comprising a vector genome titer as determined by droplet digital polymerase chain reaction, wherein the vector genome titer in the reconstituted pharmaceutical product is within 10% of the vector genome titer present in the pharmaceutical formulation prior to lyophilization.

63. 63. The reconstituted pharmaceutical product of claim 62, wherein the vector genome titer in the reconstituted pharmaceutical product is within 7% of the vector genome titer present in the pharmaceutical formulation prior to lyophilization.

64. 64. The reconstituted pharmaceutical product of any one of claims 57-63, formed by reconstituting the lyophilized pharmaceutical composition after storage for one month at a temperature of 2-8°C.

65. 64. The reconstituted pharmaceutical product of any one of claims 57-63, formed by reconstituting the lyophilized pharmaceutical composition after storage for 3 months at a temperature of 2-8°C.

66. 64. The reconstituted pharmaceutical product of any one of claims 57-63, formed by reconstituting the lyophilized pharmaceutical composition after storage for 6 months at a temperature of 2-8°C.

67. A container containing the pharmaceutical formulation according to any one of claims 1 to 48.

68. A container containing the freeze-dried pharmaceutical composition according to any one of claims 49 to 55.

69. 67. A container containing a reconstituted pharmaceutical product according to any one of claims 56 to 66.

70. 70. The container according to any one of claims 67 to 69, which is a glass vial.

71. A kit comprising a container according to any one of claims 67 to 70 and instructions for use.

72. A method for producing a stable lyophilized pharmaceutical composition according to any one of claims 49 to 55, comprising carrying out a lyophilization process on the pharmaceutical formulation according to any one of claims 1 to 48.

73. 73. The method of claim 72, wherein the freeze-drying process comprises: (i) freezing, (ii) annealing, (iii) primary drying, and (iv) secondary drying.

74. 74. The method of claim 73, wherein the freezing is carried out at a temperature of -45°C ± 5°C for 60 minutes ± 10 minutes.

75. 75. The method of claim 73 or 74, wherein the freezing is performed both before and after the annealing.

76. 76. The method of any one of claims 73 to 75, wherein the annealing is carried out at a temperature of -20°C ± 5°C for 6 hours ± 30 minutes.

77. 77. The method of any one of claims 73 to 76, wherein the primary drying is carried out at a temperature of -26°C±5°C for 50 hours±10 hours.

78. 78. The method of any one of claims 73 to 77, wherein the secondary drying is carried out at a temperature of 35°C ± 5°C for a period of 5 to 20 hours, or 15 hours ± 5 hours.

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