Improved pharmaceutical compositions comprising adeno-associated viral vectors - Patents.com

Abstract

The present invention provides compositions comprising a recombinant AAV and one or more pharma- ceutically acceptable excipients, the compositions having improved stability and half-life compared to other AAV compositions.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from U.S. Application No. 63 / 127,826, filed December 18, 2020, which is incorporated by reference in its entirety.

[0002] Sequence Listing This application has been submitted electronically in ASCII format and contains a Sequence Listing, which is incorporated herein by reference in its entirety. The ASCII copy was created on December 1, 2021, is named 025297_WO029_SL.txt, and is 19,967 bytes in size. [Background technology]

[0003] Gene therapy is a promising method for treating genetic diseases. It introduces healthy copies of defective genes to the patient or inactivates abnormally functioning mutant genes. A genetic disease of particular interest for gene therapy research is hemophilia A. Hemophilia A, also called traditional hemophilia, is an X-linked genetic disease in which the blood clotting process becomes abnormal due to a missing or defective gene that encodes factor VIII. Hemophilia A patients can bleed internally (e.g., within joints and muscles) or externally (e.g., from minor cuts, trauma, or dental procedures). Normal plasma levels of factor VIII range from 50% to 100%. Mild hemophilia A is characterized by levels of 6% to 49% of factor VIII in the blood; patients typically bleed only after severe injury, trauma, or surgery. Moderate hemophilia A is characterized by levels of 1% to 5% of factor VIII in the blood; patients exhibit bleeding episodes after injury. Severe hemophilia A is characterized by levels of less than 1% factor VIII in the blood; patients experience bleeding after injury and often also have frequent spontaneous bleeding in joints and muscles. See also the World Federation of Hemophilia website. Hemophilia A is generally treated with replacement factor VIII either on an as-needed basis (in response to bleeding) or prophylactically. Some patients develop alloantibodies (also known as inhibitors) against the replacement factor, which makes the treatment ineffective. Current therapies are burdensome, requiring frequent intravenous injections, and are of limited supply in developing countries. Thus, gene therapy offers a promising approach to the treatment of hemophilia A.

[0004] Recombinant adeno-associated virus (rAAV) has been investigated as a platform for gene delivery in gene therapy. Adeno-associated virus (AAV) is a small non-enveloped virus belonging to the genus Dependoparvovirus in the family Parvoviridae. The virus is composed of a single-stranded DNA genome packaged in a capsid constructed from three capsid proteins - viral protein (VP) 1, VP2, and VP3. Formulating rAAV preparations into pharmaceutical compositions for clinical use has been a challenge. Commonly used rAAV preparations have been observed to form visible precipitates over the years or during laboratory-generated stress tests. These precipitates, which have remained unresolved, represent a risk to patient safety. Physical stability problems observed with some rAAV formulations can also negatively impact the efficacy of the product in terms of the half-life required for storage, shipping, and administration to patients (see, e.g., Wright et al., Molecular Therapy (2005) 12(1):171-8; Croyle et al., Gene Therapy (2001) 8:1281-90). Thus, there is a need to develop improved formulations for rAAV vectors such that the therapeutic potential of gene therapy can be fully realized. Summary of the Invention

[0005] The present disclosure provides stable rAAV vector formulations suitable for clinical administration. In one embodiment, the present disclosure provides a pharmaceutical composition comprising an rAAV vector, sodium chloride (NaCl), potassium chloride (KCl), disodium phosphate (Na2HPO4), monopotassium phosphate (KH2PO4), magnesium chloride (MgCl2), a polyol (e.g., sucrose), and a poloxamer (e.g., poloxamer 188), optionally comprising about 0.1 mM or less calcium chloride and having a pH of about 7.1 to about 7.5.

[0006] In certain embodiments, the composition comprises about 0.1 to about 2.0 mM magnesium chloride (eg, about 0.5 mM or more, about 1.3 mM or more, or about 1.4 mM).

[0007] In certain embodiments, the composition comprises about 150 to about 200 mM, optionally about 172 mM, sodium chloride.

[0008] In certain embodiments, the composition comprises about 2.5 to about 3.0 mM potassium chloride, optionally about 2.7 mM.

[0009] In certain embodiments, the composition comprises about 5 to about 10 mM, optionally about 8 mM, disodium phosphate.

[0010] In certain embodiments, the composition comprises about 1.0 to about 2.0 mM, optionally about 1.5 mM, monopotassium phosphate.

[0011] In certain embodiments, the composition comprises about 0.5% to about 2% (w / v), optionally about 1% (w / v) sucrose.

[0012] In certain embodiments, the composition comprises about 0.01% to about 0.1% (w / v), optionally about 0.05% (w / v) poloxamer 188.

[0013] In certain embodiments, the disclosure provides a pharmaceutical composition comprising an rAAV vector, about 171.81 mM sodium chloride, about 2.68 mM potassium chloride, about 8.10 mM disodium phosphate, about 1.47 mM potassium phosphate monobasic, about 1.40 mM magnesium chloride, about 1.00% (w / v) sucrose, and about 0.05% (w / v) poloxamer 188, optionally containing less than about 0.1 mM calcium chloride, and having a pH of about 7.1 to about 7.5.

[0014] In other certain embodiments, the disclosure provides a pharmaceutical composition comprising an rAAV vector, about 172 mM sodium chloride, about 2.68 mM potassium chloride, about 8.10 mM disodium phosphate, about 1.47 mM potassium phosphate, about 0.49 mM magnesium chloride, about 1.00% (w / v) sucrose, and about 0.05% (w / v) poloxamer 188, optionally containing less than about 0.1 mM calcium chloride, and having a pH of about 7.1 to about 7.5.

[0015] In certain embodiments, the rAAV in the compositions of the invention includes a genome that includes an expression cassette for a therapeutic protein, e.g., a human Factor VIII polypeptide (e.g., SEQ ID NO: 1). In certain embodiments, the AAV genome includes SEQ ID NO: 2 or nucleotides 131 to 5,024 of SEQ ID NO: 2.

[0016] In certain embodiments, the composition contains rAAV at about 1.0E+12 to about 1.0E+14 vector genomes (vg) per mL, optionally at about 1.0E+13 to about 5.0E+13 vg per mL (e.g., about 1.0E+13 vg per mL).

[0017] In certain embodiments, the rAAV comprises an AAV6 capsid protein (e.g., has an AAV6 capsid). In certain embodiments, the AAV genome comprises inverted terminal repeats (ITRs) derived from AAV2.

[0018] In another aspect, the present disclosure provides a vial containing 5-10 mL, optionally 6.4 mL, of a composition of the invention, which may be constructed, for example, from a cycloolefin copolymer and / or may have a thermoplastic elastomer stopper in place.

[0019] In another aspect, the disclosure provides a method of treating a patient in need of a therapeutic protein, comprising administering to the patient a composition of the present invention. In one embodiment, the disclosure provides a method of increasing serum levels of factor VIII in a human subject in need of such an increase (e.g., a human subject suffering from hemophilia A), comprising intravenously administering to the human subject a therapeutic composition in which the rAAV is a human factor VIII polypeptide. Pharmaceutical compositions for use in such therapeutic methods and the use of the compositions for the manufacture of medicaments for use in such methods are also provided.

[0020] Other features, objects, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description, while illustrating embodiments and aspects of the present invention, is given by way of illustration and not by way of limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the detailed description. [Brief description of the drawings]

[0021] [Figure 1A] FIG. 1A summarizes the characterization of particulate matter found in samples of formulations containing SB-525. [Figure 1B] FIG. 1B summarizes the characterization of particulate matter found in samples of formulations containing SB-525. [Figure 1C] FIG. 1C summarizes the characterization of particulate matter found in samples of formulations containing SB-525. [Figure 1D] FIG. 1D summarizes the characterization of particulate matter found in samples of formulations containing SB-525. [Figure 1E] FIG. 1E summarizes the characterization of particulate matter found in samples of formulations containing SB-525. [Figure 1F] FIG. IF summarizes the characterization of particulate matter found in samples of formulations containing SB-525. [Diagram 2]FIG. 2 shows a scheme of buffer exchange by tangential flow filtration (TFF). [Diagram 3] FIG. 3 shows the formulation, filling, and finishing steps of the AAV6 vector formulation. [Figure 4A] Figure 4A shows vector genome (vg) titers and infectious titers of AAV6 vector formulations after multiple freeze / thaw cycles. Error bars shown are ± percentage RSD from N > 3 sample measurements of particle concentration by MADLS or ± allowed test variation for vg titers, mean tissue culture infectious dose (TCID50), ELISA, and SE-LC. RSD: relative standard deviation. MADLS: multi-angle dynamic light scattering. SE-LC: size exclusion liquid chromatography. [Figure 4B] Figure 4B shows capsid titers and particle concentrations of AAV6 vector formulations after multiple freeze / thaw cycles. Error bars shown are ± percentage RSD from N > 3 sample measurements for particle concentration by MADLS or ± allowed test variation for vg titer, mean tissue culture infectious dose (TCID50), ELISA, and SE-LC. [Figure 4C] FIG. 4C is a table showing the results of typical purity quality attributes of frozen / thawed AAV6 vector formulation samples. [Figure 4D] FIG. 4D is a table showing the strength quality attribute results of frozen / thawed AAV6 vector formulation samples. [Figure 5A] Figure 5A shows vg and infectious titers over a 6 month period in ambient (25°C / 60% RH) conditions. RH: relative humidity. [Figure 5B] FIG. 5B shows capsid titers and particle concentrations over a 6 month period under ambient (25° C. / 60% RH) conditions. [Figure 5C] Figure 5C is a table showing the results of general purity quality attributes for samples incubated under ambient (25°C / 60%RH) conditions. ND: No data. NAA: No analysis available. [Figure 5D] FIG. 5D is a table showing the results of strength quality properties for samples incubated under ambient (25° C. / 60% RH) conditions. [Figure 6A] FIG. 6A shows vg and infectious titers over a 3-month period under stress (40° C. / 75% RH) conditions. [Figure 6B] FIG. 6B shows capsid titers and particle concentrations over a 7-month period under stressed (40° C. / 75% RH) conditions. [Figure 6C-1] FIG. 6C is a table showing the results of general purity quality attributes of samples incubated under stress (40° C. / 75% RH) conditions. [Figure 6C-2] FIG. 6C is a table showing the results of general purity quality attributes of samples incubated under stress (40° C. / 75% RH) conditions. [Figure 6D-1] FIG. 6D is a table showing the results of strength quality properties of samples incubated under stress (40° C. / 75% RH) conditions. [Figure 6D-2] FIG. 6D is a table showing the results of strength quality properties of samples incubated under stress (40° C. / 75% RH) conditions. [Figure 7] FIG. 7 is a table showing the quality attributes and failure criteria associated with the stabilized samples. [Figure 8] FIG. 8 is a table showing the inter-sample formulation evaluation summary. [Figure 9] FIG. 9 is a table showing test endpoint trend lines for common purity quality attributes and formulation evaluation. [Figure 10-1] FIG. 10 is a table showing the long-term (24 months) stability of SB-525 formulations at the intended storage temperature (−70° C.). [Figure 10-2] FIG. 10 is a table showing the long-term (24 months) stability of SB-525 formulations at the intended storage temperature (−70° C.). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Detailed Description of the Invention The present disclosure provides a pharmaceutical composition comprising an AAV vector and one or more pharma- ceutically acceptable excipients. The AAV vector composition of the present invention may comprise a rAAV whose genome carries an expression cassette for a protein of interest (e.g., a therapeutic protein). The inventors have unexpectedly found that an AAV vector formulation that is substantially free of calcium (e.g., does not contain calcium added in the formulation) exhibits improved stability and half-life compared to conventional compositions. Calcium has typically been included in previous formulations due to the conventional belief that it improves the stability of AAV compositions (see, e.g., Turnbull et al., Hum Gene Ther. (2000) 11(4):629-35; Cotmore et al., J Virol. (2010) 84(4):1945-56). The inventors have found that the AAV vector compositions of the invention have improved appearance (e.g., clarity and colorlessness), more stable pH, and less aggregation (as determined by product quality attributes under freeze / thaw cycles and accelerated stability conditions). Calcium ions are not required for product function or stability, as AAV vector products formulated without calcium exhibit good product stability.

[0023] I. Preparation of recombinant AAV The virus preparations described herein may be obtained by any known production system, such as mammalian cell AAV production systems (e.g., 293T or HEK293 cell-based systems) and insect cell AAV production systems (e.g., sf9 insect cell-based systems and / or systems using baculovirus helper vectors). The virus preparations may be purified from cell cultures using well-known techniques, such as discontinuous cesium chloride density gradients (see, e.g., Grieger, Mol Ther Methods Clin Dev. (2016) 3:16002).

[0024] The compositions of the invention may include AAV of various AAV serotypes, such as AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV8.2, AAV9, AAVrhlO, AAV10, and AAV11, as well as any or combination of mutant, hybrid, chimeric, or pseudotyped AAVs thereof. "Pseudotyped" or "cross-packaged" rAAV refers to recombinant AAV in which the capsid has been replaced with that of another AAV serotype, e.g., to alter the transduction efficiency or tropism profile of the virus (see, e.g., Balaji et al., J Surg Res. (2013) 184(1):691-8). "Chimeric" or "hybrid" rAAV refers to a recombinant AAV whose capsid is composed of capsid proteins from different serotypes and / or whose capsid proteins are chimeric proteins having sequences from different serotypes (e.g., serotypes 1 and 2; see, e.g., Hauck et al., Mol Ther. (2003) 7(3):419-25. For example, compositions of the invention may include a recombinant AAV whose genome, including the ITRs, is from one serotype, such as AAV2, but whose capsid is from another serotype (e.g., AAV2 / 8, AAV2 / 5, AAV2 / 6, AAV2 / 9, or AAV2 / 6 / 9). See, e.g., U.S. Patent Nos. 7,198,951 and 9,585,971.

[0025] II. Preparation of recombinant AAV Once purified, the AAV preparation can be formulated as described herein, for example, by tangential flow filtration, normal flow filtration (using a stirred cell), gel filtration, dialysis, column chromatography, and / or buffer exchange through a desalting column to obtain a composition containing the desired components. As an example, the purified virus preparation can be first concentrated by ultrafiltration (UF) followed by diafiltration (DF) to 10 or more equivalent volumes of the desired formulated aqueous solution. See also the Examples below.

[0026] The solution of the formulation may contain isotonicity agents, stabilizers, surfactants, and buffers.The buffers may include, for example, acetate, succinate (e.g., disodium succinate hexahydrate), succinic acid, gluconic acid, citrate, histidine, acetate, phosphate, phosphoric acid, ascorbic acid, ascorbate, tartaric acid, maleate, maleic acid, glycine, lactate, lactate, bicarbonate, carboxylic acid, sodium benzoate, benzoic acid, edetate, imidazole, tris, and mixtures thereof. In certain embodiments, the formulated solution contains sodium chloride and / or potassium chloride, e.g., about 150-200 mM, respectively (e.g., about 150 mM, about 155 mM, about 160 mM, about 165 mM, about 168 mM, about 170 mM, about 171 mM, about 171.1 mM, about 171.2 mM, about 171.3 mM, about 171.4 mM, about 171.5 mM, about 171.6 mM, about 171. 7 mM, about 171.8 mM, about 171.9 mM, or about 172 mM) and about 2.5-3.0 mM (e.g., about 2.5 mM, about 2.6 mM, about 2.61 mM, about 2.61 mM, about 2.63 mM, about 2.64 mM, about 2.65 mM, about 2.66 mM, about 2.67 mM, about 2.68 mM, about 2.69 mM, or about 2.7 mM, about 2.8 mM, about 2.9 mM, about 3.0 mM).

[0027] The formulation solution may be phosphate buffered, for example, with disodium phosphate and / or monopotassium phosphate. In some embodiments, the total phosphate ion concentration in the formulation solution is about 8 to 12 mM (e.g., about 9.6 mM or about 9.57 mM). In some embodiments, the formulation solution contains about 5-10 mM (e.g., about 5 mM, about 6 mM, about 7 mM, about 7.9 mM, about 8 mM or about 8.1 mM, about 8.2 mM, about 8.5 mM, about 9 mM, or about 10 mM) of disodium phosphate and about 1-2 mM (e.g., about 1 mM, about 1.2 mM, about 1.3 mM, about 1.45 mM, about 1.47 mM, about 1.48 mM, about 1.49 mM, about 1.5, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM, or about 2 mM) of monopotassium phosphate. The formulation may have a pH of about 6.5-8.0 (e.g., about 7.1-7.5, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5).

[0028] The formulation solution may contain magnesium but does not contain added calcium. In some embodiments, the formulation solution contains about 0.1 to 2.0 mM (e.g., about 0.5 to about 1.4 mM, about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.42 mM, about 0.44 mM, about 0.45 mM, about 0.46 mM, about 0.47 mM, about 0.48 mM, about 0.49 mM, about 0.5, about 0.55 mM, or about 0.6 mM) magnesium chloride. The formulation solution does not contain added calcium and the pharmaceutical composition is prepared from an AAV preparation, but the formulation solution may have trace amounts of calcium carried over from the AAV manufacturing and purification process. For example, the pharmaceutical composition may contain less than about 0.10 mM calcium (e.g., less than about 0.09, 0.07, 0.05, 0.03, or 0.01 mM) as measured by colorimetric assay. In some embodiments, the pharmaceutical composition contains undetectable calcium as measured by colorimetric assay. In some embodiments, the pharmaceutical composition does not contain calcium (i.e., 0 mM calcium).

[0029] The formulation solution may contain a polyol, such as mannitol, trehalose, sorbitol, erythritol, isomalt, lactitol, maltitol, xylitol, glycerol, lactitol, ethylene glycol, propylene glycol, polyethylene glycol, inositol, fructose, glycol, mannose, sucrose, sorbose, xylose, lactose, maltose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch, water-soluble glucan, or mixtures thereof. In some embodiments, the formulation solution contains about 0.5% to 2% (e.g., about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, or about 1%) (w / v) sucrose.

[0030] The formulation solution may contain a non-ionic or ionic hydrophilic surfactant. Examples of surfactants include polysorbates, poloxamers, tritons, sodium dodecyl sulfate, sodium lauryl sulfate, sodium octyl glucoside, lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine, stearyl sulfobetaine, lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, stearyl sarcosine, linoleyl betaine, myristyl betaine, cetyl betaine, lauramidopropyl betaine, cocamidopropyl betaine, linoleyl propyl betaine, myristamidopropyl betaine, palmidopropyl betaine, isostearamidopropyl betaine, myristamidopropyl dimethylamine, palmidopropyl dimethylamine, isostearamidopropyl dimethylamine, sodium cocoyl methyl taurate, disodium oleoyl methyl taurate, dihydroxypropyl PEG-5 linoleic ammonium chloride, polyethylene glycol, polypropylene glycol, sorbitan monostearate (e.g., Spans), acid esters of glycerol, and mixtures thereof. In some embodiments, the surfactant may be polysorbate (PS) 20, PS-21, PS-40, PS-60, PS-61, PS-65, PS-80, PS-81, PS-85, PEG-3350, poloxamer 188, and mixtures thereof. In some embodiments, the formulation solution comprises about 0.01% to 0.1% (e.g., about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, or about 0.1%) (w / v) of poloxamer 188.

[0031] In some embodiments, the pharmaceutical composition comprises an AAV vector in formulation F2 or F3. The components of F2 and F3 are shown in Table A below. Dulbecco's phosphate buffered saline (DPBS) (containing calcium and magnesium) is a commonly used formulation for cell culture. F0 is another conventional calcium-containing formulation. As shown in the examples below, F2 and F3 are superior to DPBS and F0 when formulating AAV. Table A. Formulation Comparison [Table 1]

[0032] As used herein, the concentrations of various components in a formulation may be expressed with a decimal point of 0, 1, or 2. Thus, for example, in F2, the concentrations of NaCl, KCl, Na2HPO4, KH2PO4, MgCl2, and sucrose may be expressed as 171.80 (or 171.8 or 172) mM, 2.68 (or 2.7) mM, 8.10 (or 8.1 or 8) mM, 1.47 (or 1.5) mM, 0.49 (or 0.5) mM, and 1.00% (or 1.0% or 1%) (w / v), respectively. In F3, the concentrations of NaCl, KCl, Na2HPO4, KH2PO4, MgCl2, and sucrose may be expressed as 171.80 (or 171.8 or 172) mM, 2.68 (or 2.7) mM, 8.10 (or 8.1 or 8) mM, 1.47 (or 1.5) mM, 1.40 (or 1.4) mM, and 1.00% (or 1.0% or 1%) (w / v), respectively.

[0033] The pharmaceutical composition may further comprise one or more preservatives, such as ascorbic acid (vitamin C), sulfites, sorbates, benzoates, phenol, m-cresol, benzyl alcohol, benzalkonium chloride, phenoxyethanol, and / or parabens (e.g., methylparaben). In some embodiments, the pharmaceutical composition does not include added preservatives.

[0034] The pharmaceutical composition may contain other agents that enhance the effectiveness of the pharmaceutical composition.The pharmaceutical composition may include a delivery vehicle, such as a liposome, a nanocapsule, a microparticle, a microsphere, a lipid particle, and a vesicle.

[0035] III. A typical recombinant AAV In an exemplary embodiment, the present disclosure provides an improved pharmaceutical composition comprising a rAAV vector for factor VIII gene therapy. The rAAV vector, designated PF-07055480 / SB-525 (or herein "SB-525"), is of the 2 / 6 pseudoserotype and comprises a recombinant genome with an AAV6 capsid and an AAV2 inverted terminal repeat (ITR). The genome of SB-525 carries an expression cassette encoding a B-domain deleted (BDD) form of human factor VIII (FVIII). See, e.g., WO2017 / 074526 (SEQ ID NO: 37). SB-525 is administered as an intravenous (IV) dose and has liver specificity to provide long-term hepatic production of factor VIII protein in hemophilia A patients. See, WO2020 / 028830. The secreted FVIII protein has an amino acid sequence identical to that of the approved recombinant antihemophilic factors (Refacto® and Xyntha®).

[0036] The genome of SB-525 contains an expression cassette for human Factor VIII type BDD, which has the amino acid sequence shown below: [Table 2]

[0037] The signal peptide portion of SEQ ID NO:1 is shown in the box above and is cleaved off when the protein is secreted.

[0038] The SB-525 genome contains the following nucleotide sequence: [Table 3] [Table 4]

[0039] In the above sequence, the left (5') ITR (AAV2 ITR) spans nucleotides 1 to 130, and the right (3') ITR (AAV2 ITR) spans nucleotides 5025 to 5132. Both ITRs are shown as boxes.

[0040] IV. Use of recombinant AAV preparations The pharmaceutical compositions of the present invention may be supplied by an article of manufacture (e.g., a kit) that includes a vial (e.g., a pretreated glass vial or COP vial) and instructions for use. In some embodiments, each vial contains about 1E+11 to 1E+15 vg per mL of AAV in 0.5 to 50 mL (e.g., 1 to 10 mL). In some embodiments, each vial contains 5E+12 to 1E+14 / mL (e.g., 1E+13 vg / mL). In some embodiments, each vial contains 6E+13 vg in 6 mL.

[0041] The composition may be administered to a patient one or more times. For example, the composition may be administered to a patient at intervals of less than one month, three months, six months, nine months, or one year. In some embodiments, the composition may be administered to a patient at intervals of less than two years, five years, seven years, ten years, or fifteen years. The pharmaceutical composition may be provided to a patient in need thereof by a route appropriate for the disease being treated. For example, the composition may be administered by intravenous injection, intraarterial injection, intracerebral injection, intraperitoneal injection, portal vein injection, or intramuscular injection. For example, the SB-525 pharmaceutical composition may be provided intravenously to a hemophilia A patient at 1E+11 to 1E+15 vg / kg, for example, 1E+11 to 1E+14 (e.g., 1E+12 to 1E+14) vg / kg. In certain embodiments, the SB-525 pharmaceutical composition may be provided intravenously to a hemophilia A patient at 5E+11, 6E+11, 7E+11, 8E+11, 9E+11, 1E+12, 2E+12, 3E+12, 4E+12, 5E+12, 6E+12, 7E+12, 8E+12, 9E+12, 1E+13, 2E+13, 3E+13, 4E+13, 5E+13, 6E+13, 7E+13, 8E+13, 9E+13, or 1E+14 vg / kg. In certain embodiments, the SB-525 composition is provided intravenously to a hemophilia A patient at a dose of about 6E+13 vg / kg.

[0042] In some embodiments, the patient has severe or moderate hemophilia A. In some further embodiments, the patient does not have inhibitors (alloantibodies to factor VIII). In some embodiments, the patient does not have neutralizing antibodies to AAV6. The patient may be an adult or adolescent patient (≧12 years old) or a pediatric patient (<12 years old).

[0043] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those skilled in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein may also be used in the practice or testing of this disclosure. In case of conflict, the present specification, including definitions, shall control. In general, the nomenclature and techniques used in connection with cardiology, medicine, medicinal and pharmaceutical chemistry, and cell biology described herein are those well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer's instructions, as commonly practiced in the art, or as described herein. Furthermore, unless otherwise required by context, singular terms shall include the plural, and plural terms shall include the singular. Throughout this specification and the embodiments, the words "have" and "comprise", or variations such as "has", "having", "comprises" or "comprising" are understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of other integers or groups of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although many references are cited herein, this citation does not acknowledge that any of these references form part of the common general knowledge in the art. As used herein, the term "approximately" or "about" as applied to one or more target values ​​means a value similar to the stated reference value. In certain embodiments, the term refers to a range of values ​​that falls within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (upwards or below) of the stated reference value, unless otherwise indicated or clear from the context.

[0044] In order that this invention may be better understood, the following examples are set forth. These examples are for illustrative purposes only and should not be construed as limiting the scope of this invention in any way. EXAMPLES

[0045] The following examples describe studies in which the inventors found that current SB-525 formulations form precipitates upon freeze / thaw cycling, a common test for evaluating half-life and long-term stability of biological products. The inventors also found that precipitates formed only in buffer formulations (i.e., buffer formulations that do not contain active viral vector components). Thus, the inventors have found a novel formulation that solves this problem.

[0046] In this study, SB-525 virus preparations were prepared at approximately 1.0E+13 vg / mL in phosphate buffered saline (PBS) with CaCl2, MgCl2, 35 mM NaCl, 1% sucrose, and 0.05% Kolliphor® P188 (poloxamer 188), sealed in 5 mL 6 mL Aseptic Technologies crystal closed vials, formulated, and stored at ≦−65°C. For stability studies, SB-525 compositions were subjected to stability at −0°C, 5°C, and 25°C for 24 months (Table 5). Five cycles of uncontrolled freeze / thaw (F / T) (Table 1) and 24-hour agitation (AG) (Table 2) were also performed. Formulation (DP) quality attributes were analyzed according to Tables 5-8.

[0047] Example 1: Freeze / thaw cycling and agitation studies Materials and Methods SB-525 was supplied at 1.0E+13 vg / mL (nominal) in formulation buffer containing the following components: 0.90 mM CaCl2, 0.49 mM MgCl2, 2.68 mM KCl, 1.47 mM KH2PO4, 172 mM NaCl, 8.10 mM Na2HPO4, 1% (w / v) sucrose, 0.05% (w / v) poloxamer 188 (pH 7.36). Twenty-three vials were stored at -70°C, 11 at 5°C, and 8 at 25°C.

[0048] In freeze / thaw (F / T) cycling studies, samples were cycled for 3 or 5 cycles of uncontrolled F / T as shown in Table 1. Table 1. DP non-controlled F / T period [Table 5]

[0049] For the stirring test, the samples were stirred at room temperature as shown in Table 2. Table 2.DP stirring settings [Table 6]

[0050] Vials stored at -70°C, 5°C, and 25°C were analyzed for (i) appearance (liquid), pH, dynamic light scattering (DLS), and high accuracy liquid particle counter (HIAC); (ii) vg identity (qPCR), vg titer (qPCR), capsid titer (ELISA), capsid identity (ELISA), and infectious titer (TCID 50 ); (iii) in vitro FVIII activity (bioassay); or (iv) analyzed for UV260 / 280, reduced CE-SDS, SEC titer_260 / 280.

[0051] Samples subjected to the F / T cycle were analyzed for (i) appearance (liquid), pH, osmolality, DLS, HIAC; (ii) vg titer (qPCR), capsid titer and identity (ELISA), reducing SDS-PAGE, and infectious titer (TCID 50 ); or (iii) analyzed for UV260 / 280, SEC titer_260 / 280, RP-HPLC, in vitro FVIII activity (bioassay), and reduced CE-SDS.

[0052] Samples subjected to agitation (AG) for 24 hours were analyzed for (i) appearance (liquid), pH, osmolality, DLS, and HIAC; (ii) vg titer (qPCR), capsid titer and identity (ELISA), reducing SDS-PAGE, infectious titer (TCID 50 ); or (iii) analyzed for UV260 / 280, SEC titer_260 / 280, RP-HPLC, in vitro FVIII activity (bioassay), and reduced CE-SDS.

[0053] result The DP stability of SB-525, the results from 5 cycles of uncontrolled F / T, and the 24 hour agitation group are described below.

[0054] As shown in Table 3 below, there was little change in color, clarity, pH, hydrodynamic radius (measured by DLS), vg titer, capsid titer, capsid purity (measured by SDS-PAGE), infectious titer, UV260 / 280 (a surrogate measure of empty:total capsid ratio), and biological activity (relative % potency) when samples were stored at the test conditions for up to one month. There was a change in visible particles at 5°C and 25°C. All samples were frozen and stored at T ゼロ At (T0), it appeared white and opaque. Table 3. Stability results [Table 7] [Table 8]

[0055] As shown in Table 4, there was no significant change in capsid purity after one month under all storage conditions. Table 4. Capsid purity stability results [Table 9]

[0056] As shown in Table 5, there was little (NMT: less than; LOQ: limit of quantification) aggregation or formation of high molecular weight species (HMWS) after one month in all storage conditions. Table 5. SEC titer stability results after 1 month Objective, accelerated, and stress storage conditions [Table 10]

[0057] As shown in Table 6, there was no significant increase in subvisible particles (by HIAC) after one month for all storage conditions. At all time points, for the ≥10 μm and ≥25 μm ranges (acceptance criteria are <6,000 particles >10 μm per container and 600 particles >25 μm per container), the USP <787> It showed substandard particles. Table 6. HIAC stability results [Table 11]

[0058] As shown in Table 7, F / T / AG stress did not result in significant changes in color, clarity, pH, hydrodynamic radius (measured by DLS), vg titer, capsid titer, infectious titer, UV260 / 280 (a surrogate measure of empty:total capsid ratio), capsid purity (measured by RP-HPLC), and biological activity (relative % potency). There was a change in visible particles in F / T. Table 7. Cycle Uncontrolled F / T and 24-hour AG Results [Table 12]

[0059] As shown in Table 8, there was little change in reduced CE-SDS due to F / T / AG stress. Table 8. CE-SDS reduction results for 5 cycles uncontrolled F / T and 24 h AG [Table 13]

[0060] As shown in Table 9, there was little change in the stirred / HMWS formulation due to F / T / AG stress. Table 9. SEC titer results for 5 cycle uncontrolled F / T and 24-hour AG [Table 14]

[0061] As shown in Table 10, F / T significantly increased the number of sub-visible particles (by HIAC). The particles by F / T were greater than the USP 100 for the ≥ 10 μm and ≥ 25 μm ranges. <787> Although the particles by AG were above the USP standard for the ranges of ≥ 10 μm and ≥ 25 μm, <787> It was below standard. Table 10. HIAC Results for 5 Cycle Uncontrolled F / T and 24-Hour AG [Table 15]

[0062] The results above indicate that SB-525 samples showed no significant changes in color, clarity, pH, hydrodynamic radius (measured by dynamic light scattering), vg titer, capsid titer, infectious titer, empty:total capsid ratio (measured by UV260 / 280), biological activity (relative % potency), capsid purity (measured by SDS-PAGE, reduced CE-SDS and RP-HPLC), or viral particle titer when stored at -70°C, 5°C, or 25°C for up to 1 month. Additionally, either F / T or AG stress did not significantly alter color, clarity, pH, hydrodynamic radius (measured by dynamic light scattering), vg titer, capsid titer, infectious titer, empty:total capsid ratio (measured by UV260 / 280), biological activity (relative % potency), capsid purity (measured by SDS-PAGE, reduced CE-SDS, RP-HPLC), or viral particle titer. However, F / T caused a significant increase in subvisible particles (by HIAC) and an increase in visible particles after 1 month of storage at 5°C and 25°C. The study was terminated at 1 month due to these results.

[0063] Example 2: Characterization of particulate matter in DP vials after F / T Samples from the above stability studies of SB-525 formulations, in which particulate matter was observed, were further analyzed for isolation and identification.

[0064] A formulation sample that had undergone 5 freeze / thaw cycles ("DP after 5F / T") and a sample that had been stored at 25°C for 1 month (DP after 1M at 25°C) were analyzed. Particulate matter was isolated on a 0.8 μm gold filter. It was then imaged on the filter under partial ring illumination using a Keyence VHX6000 digital microscope at 150x magnification. The filter was then transferred to a Fourier transform infrared (FTIR) microscope and a spectrum was obtained for the material. The FTIR spectrum was compared to the KnowItAll database of known spectra. A portion of the DP sample after 1M at 25°C was also scraped off the filter onto a glass slide and imaged under linear and cross polarized light using a Nikon Eclipse ME600 polarizing microscope. A portion of the gold filter was cut and analyzed on a JEOL6000SEM equipped with an EDS module. The sample was also analyzed by Raman microscopy.

[0065] Digital imaging of all samples showed a white solid that appeared semi-crystalline. Under crossed polars, the "DP after 5F / T" sample showed no birefringence, indicating either amorphous material or isotropic crystals. SEM / EDS analysis showed mostly oxygen, phosphorus and calcium. FTIR analysis identified the material as a salt, but further identification was required. Raman microscopy was able to detect peaks characteristic of calcium phosphate, indicating that the white particulate material in all samples was calcium phosphate. Figure 1A-F summarizes the particle characterization studies.

[0066] Example 3: Reformulation of SB-525 This example describes experiments testing new SB-525 formulations that can avoid the precipitation problems seen above. These experiments evaluated the short-term stability of SB-525DP when reformulated by (1) removing calcium, (2) removing calcium and magnesium, or (3) increasing sucrose to 8.5%. These changes are described to prevent particle generation by (1) removing the source of particles, (2) removing both divalent cations due to MgCl2 solubility concerns, or (3) increasing the stability of the formulation to freeze / thaw stress, respectively. Two pilot batches were buffer exchanged and filled at 2.5 mL into 6 mL AT vials to track worst case conditions for surface area to volume (SA / V) filling. After filling / finalization, the vials were subjected to 5 uncontrolled freeze / thaw cycles (≦−65° C. relative to ambient) followed by stability at ≦−65° C. (intended storage), 2-8° C. (stress; liquid storage), 25° C. (accelerated; liquid storage), and 40° C. (aggressive / forced degradation conditions; liquid storage). These conditions were chosen as they were expected to reveal differences between the formulations.

[0067] SB-525 virus was purified from the clarified bulk harvest, formulated at approximately 1.0E+13 vg / mL in phosphate buffered saline (PBS) (containing CaCl2, MgCl2, 35 mM NaCl, 1% sucrose, and 0.05% Kolliphor® P188 (poloxamer 188)), filled into vials, and stored at ≦−65° C.

[0068] material and method SB-525 formulated bulk drug substance contained SB-525 (1.0E+13 vg / mL (nominal)) in 0.90 mM CaCl2, 0.49 mM MgCl2, 2.68 mM KCl, 1.47 mM KH2PO4, 172 mM NaCl, 8.10 mM Na2HPO4, 1% (w / v) sucrose, 0.05% (w / v) poloxamer 188 (pH 7.0-7.6), was sterile filtered, and stored at ≤-65°C in 125 mL HDPE bottles. Na2HPO4 (sodium phosphate, dibasic, anhydrous), KH2PO4 (potassium phosphate dihydrogen), CaCl2 (calcium chloride dihydrate), and MgCl2 (magnesium chloride hexahydrate) were supplied by JT Baker or Fischer. Poloxamer 188 was supplied by BASF. Storage vials were 6mL Aseptic Technologies (AT) Closed Crystal Vials (Aseptic Technologies; cat# VIA-060000), vials with primary packaging stoppers, cycloolefin copolymer (COC) thermoplastic elastomer stoppers in place, and yellow caps. Prior to DP filling, the formulated bulk drug substance (FBDS) was filled into 125mL high density polyethylene (HDPE) bottles and frozen at ≦−65°C. It was then thawed at ambient bottle temperature, buffer exchanged, filtered, and DP filled into 6mL AT vials using an Aseptic Technologies M1 unit. One pilot batch material was buffer exchanged by TFF at approximately 40 psi pressure using a 50 kDa filter unit for a total of 10 exchange volumes. The next pilot batch was buffer exchanged at approximately 40 psi using an Amicon stirred cell on a 50 kDa NMW PES filter for a total of 10 exchange volumes. These two exchange methods have been used previously for AAV without significant material loss or absorption.

[0069] The AT vials were filled at 2.5 mL with a target of approximately 1.0E+13 vg / mL, freeze / thawed five times from ≦−65° C. to ambient conditions at an uncontrolled rate, and then subjected to stability in a non-GMP storage unit. The vials were subjected to the stability pull schedule outlined in Table 11 and tested by the methods described in Table 12. The formulations tested are outlined in Table 13. Table 11. Capsid purity stability schedule [Table 16] Table 12. Analytical tests [Table 17] Table 13. Formulations [Table 18]

[0070] result Formulation characterization To ensure that the correct formulation was achieved, the SB-525DP formulations were tested to determine osmolality, P188 concentration, sucrose concentration, and capsid ratio and purity (by reduced CGE). The results of these tests for M05-M08 are shown in Table 14 below. Capsid purity and ratios, as measured by rCGE, showed little difference. Osmolality is within the expected range depending on the sucrose level. The M05-M08 samples showed the correct concentration of P188. Additionally, calcium and magnesium concentrations were confirmed by CEDEX to be within the expected range for all formulations. Table 14. Formulation characterization [Table 19]

[0071] Vector genome titer (vg / mL) The vector genome titer results for M05-M08 are shown in Table 15 below. At ≦-65°C and 2-8°C conditions, little change was observed after 4 weeks of storage. After 3 days at 40°C, the Ca / Mg-free formulation showed a trend towards a decrease in vector genome titer. This result is considered within the experimental variability, but is consistent with the vp titer and UV260 / 280 results for this formulation. Table 15. Genomic titers (vg / mL) [Table 20]

[0072] Visual appearance All formulations exhibited color and clarity standards equivalent to or less than B9 color and Opalescence Reference 1, respectively, used for visual inspection against a black and white background for all time points and conditions (see, e.g., Ph. Eur. 7.0, 20201, 20202 (01 / 2008); Millipore Sigma Color Reference Solution B). Visual appearance results show that only the "control" formulation (M05) exhibited too many to count (TMTC) white flaky particles when placed at 25°C for one week or more, or at 40°C for three or more days. M06 and M07 formulations exhibited few TMTC visible particles; however, the particle state of all of these samples was fibrous particles. Although these particles were not identified in this study, the fibrous particles are more characteristic of exogenous particles (e.g., filter particles) versus flaky particles characteristic of calcium phosphate. Also, the appearance of fibrous / exogenous particles is negligible in laboratory development tests where the DP is not generated under strictly controlled environmental conditions.

[0073] Cumulative invisible particles Subvisible cumulative particle analysis by HIAC showed that the only formulation with significant subvisible particle counts was the "Control" formulation, which tended to increase under aggressive stability conditions (higher temperatures and longer periods). The other formulations (M06-M08) showed only negligible particle counts regardless of time or conditions.

[0074] Functional biological assays (potency) The functional FVIII bioassay results are shown in Table 16 below. The results showed that there was little change in potency when samples were maintained at either ≦-65° C. or 2-8° C. At 25° C., there was a trend towards decreased potency for all formulations (M05-M08) after 4 weeks. At 40° C., there was a significant decrease in potency for the Ca / Mg-free formulation after 3 days. Table 16. Functional biological assays (relative potency) [Table 21]

[0075] SEC The SEC results are shown below. Percent aggregation results are shown in Table 17 below. No significant trend in aggregation was observed at stability conditions ≦-65°C, 2-8°C, or 25°C. At 40°C, there was a trend towards increased aggregation for the Ca / Mg-free formulation with less than 7% HMWS after 1 month. The "calcium-free" formulation showed approximately 3% HMWS after 1 month at 40°C. Other formulations showed no significant increase in aggregation when placed at ≦-65°C or 2-8°C for 5 weeks, 25°C for 4 weeks, and 40°C for up to 1 week. Table 17. Concentration of aggregates by SEC (%HMWS) [Table 22]

[0076] The viral particle results are shown in Table 18 below. All formulations maintained at ≦-65°C, 2-8°C, or 25°C showed no significant change in viral particle titer. However, at 40°C, there was a significant decrease in particle titer for formulations without Ca / Mg after 3 days and 1 week. Other formulations (M05-M06, M08) showed no significant decrease in viral particle titer when maintained at ≦-65°C or 2-8°C for 5 weeks, at 25°C for 4 weeks, and at 40°C for up to 1 week. Table 18. Viral particle titers by SEC (vp / mL) [Table 23]

[0077] The results of the UV260 / 280 ratio are shown in Table 19 below. There was a decreasing trend in the UV260 / 280 ratio for the Ca / Mg-free formulations when maintained at 40° C. UV260 / 280 is considered a surrogate measure of the empty / total particle ratio. The other formulations (M05, M06, and M08) did not show any change in the UV260 / 280 ratio at any condition or time point, as shown. Table 19. UV260 / 280 ratio by SEC [Table 24]

[0078] pH As shown in Table 20 below, there was little change in pH for all formulations at any condition and time point. Table 20. pH [Table 25]

[0079] Mass spectrometry characterization (deamidation) The results of deamidation as determined by mass spectrometry are shown in Table 21. Only samples stored at 40°C were used in this study since samples stored at lower temperatures were not expected to show significant differences between formulations over the 5 week study period.

[0080] Results were presented using the TO of the control formulation as TO for all formulations. For all deamidation sites, the N57G and N94H hot spots in AAV VP1 were the two that showed the most significant increase in deamidation at 3 weeks at 40°C. Other sites also showed increased deamidation, but to a much lesser extent (not shown). These two hot spots are of particular importance as they are predicted to affect transduction efficiency. The Ca / Mg-free formulation showed the most significant increase in deamidation. All other formulations (M05, M06, and M08) showed similar levels of increased deamidation after 3 days and 3 weeks at 40°C. Table 21. Deamidation by mass spectrometry (% at 40°C) [Table 26]

[0081] conclusion Three possible variations on the current SB-525 formulation were evaluated: a calcium-free formulation, a calcium- and magnesium-free formulation, and a formulation with high sucrose concentrations. Examples 1 and 2 show the random occurrence of numerous white flaky particles in both the buffer and the formulation, particularly after freeze / thaw cycling followed by stability at either 25° C. for one week or more or at 40° C. for three or more days. The particles were identified as calcium phosphate.

[0082] The results herein show that calcium chloride is not an essential excipient for formulation stability under intended or stressed storage conditions. Also, no significant differences were observed between the "calcium-free" and "high sucrose" formulations at 40°C for up to 2 weeks, at 25°C for up to 4 weeks, and at 5°C and -70°C for up to 5 weeks. However, it was observed that the removal of both calcium and magnesium from the formulations caused changes in formulation quality attributes, including a greater reduction in vg and vp titers and a higher degree of deamidation and aggregation, especially when maintained at 40°C. Furthermore, it was found that only the conventional (control) formulation continued to show white flaky particles throughout the 5 weeks.

[0083] Example 4: Further reformulation of SB-525 To prevent particle generation, three types of changes to the formulation were tested in this example: (1) increasing the sucrose concentration from 1% to 5-10% to increase stability against FT stress, (2) removing divalent cations to remove a source of particles and prevent possible solubility issues, and (3) increasing NaCl to change the ionic strength of the formulation.

[0084] More specifically, the experiments in this example evaluated the stability of SB-525 buffer when reformulated with one of several changes compared to the conventional (control) formulation; i.e., 172 mM NaCl, 8.10 mM Na2HPO4, 2.68 mM KCl, 1.47 mM KH2PO4, 0.90 mM CaCl2, 0.49 mM MgCl2, 1% (w / v) sucrose, and 0.05% (w / v) poloxamer 188, pH 7.0-7.6. The formulations were filled at 5 mL into 6 mL AT vials and subjected to 5 uncontrolled F / T cycles (≦-65°C relative to ambient) followed by thermal stability at 2-8°C (accelerated; liquid storage) and 25°C (stress; liquid storage) to evaluate the various buffers on particle formation.

[0085] Test Design The formulations tested were formulated by adding sodium and potassium chloride and phosphate to water, followed by sucrose, as shown in Table 22. If necessary, magnesium chloride was prepared in a separate 50 mL solution, followed by calcium chloride, and transferred to the larger solution. Poloxamer 188 was then added to the solution. The formulations were then made to volume, pH tested, and filtered through a 0.22 μm PES filter. Table 22. Formulations [Table 27]

[0086] These formulations were filled into AT vials at 5 mL, subjected to 5 uncontrolled freeze / thaw cycles, and subjected to stability at either 5° C. or 25° C. The stability run schedule is outlined in Table 23, where X indicates appearance (liquid); A indicates osmolality (freezing point depression), conductivity, viscosity, and density; B indicates light obscuration by HIAC; C indicates P188 concentration; and D indicates pH. Table 23. Stability Schedule 0139-M03~-M08 [Table 28]

[0087] Formulation properties SB-525 buffer formulations were tested to determine conductivity, osmolality, density, and viscosity. P188 concentration was also measured at 2.5 weeks of 5° C. storage and 5 days at 25° C. The results of these tests are shown in Table 24 below. The conductivity, osmolality, density, and viscosity results for M03-M09 were all as expected - higher amounts of sucrose resulted in higher osmolality, density, and viscosity; more NaCl resulted in higher osmolality and conductivity. The P188 concentration results were within the method variability for each set of results, confirming that the correct P188 concentration was achieved during formulation. Table 24. Formulation characterization [Table 29]

[0088] pH As shown in Table 25 below, no significant changes in pH were observed for any of the formulations during FT cycling. Table 25. pH results [Table 30]

[0089] Visual appearance All samples showed B9 color and opalescence reference 1 at each condition and time point tested, or less. Visual appearance results consistently showed too many (TMTC) white flaky particles to count at 5°C and 25°C from 5 days to 8 weeks for the control formulation only (M09). The 5% sucrose formulation showed random occurrence of TMTC visible particles. Many samples also showed 1-5 fibrous particles. Although these particles were not identified in this study, the fibrous particles are more characteristic of exogenous particles (e.g., filter particles) versus flaky particles characteristic of calcium phosphate. Also, the appearance of fibrous / exogenous particles is negligible in laboratory development studies where DP is not generated under strictly controlled environmental conditions.

[0090] Invisible particle count Subvisible particle analysis by HIAC showed that the only formulation that showed significant and consistent subvisible particle counts was the control formulation (M09), which worsened with continued storage at higher stability conditions (25°C). The other formulations (M03-M08) showed less significant particle counts regardless of time or condition, and no overall trend was observed.

[0091] conclusion This example evaluated the effect of freeze / thaw stress and thermal stability on the differentiation of several formulations from a conventional (control) SB-525 formulation. Seven different formulations were filled into AT crystal closed vials, subjected to freeze / thaw cycles, and stored at 5° C. and 25° C. for 8 weeks. Throughout all test conditions, only the control formulation consistently exhibited white flaky particles.

[0092] After the FT cycle, none of the formulations showed significant visible particle formation or pH change, therefore, all formulations were subjected to short-term stability at 5°C and 25°C for 8 weeks. Only the control formulation (M09) was shown to consistently generate significant visible particles upon stability. The "200mM NaCl" (M03), "5% sucrose" (M04), and "10% sucrose" (M06) formulations were not selected for subsequent reformulation studies, as the particle results were comparable (i.e., low visible particle generation). Notably, the "5% sucrose" formulation (M04) showed some formation of white flaky particles, indicating that this sucrose concentration may not be sufficient to prevent particle generation due to FT stress, and "10% sucrose" (M06) introduces manufacturing challenges due to its high viscosity. Thus, based on these results, the "8.5% sucrose" (M05), "calcium-free" (M07), and "calcium- and magnesium-free" (M08) formulations may be promising.

[0093] Example 5: Final formulation of SB-525 The composition and description of the SB-525 (PF-07055480) drug substance (DS) are shown in Table 26. The formulation had a final pH of 7.3 ± 0.3. Excipient concentrations were achieved in whole or in part by the addition of base buffer (phosphate buffered saline). The DS concentration target was 1.0E+13 vg / mL (0.5E+13 to 2.5E+13 vg / mL) with a DS range of 50-250% of the DS target. Table 26. Description of SB-525 Drug Substance Formulation [Table 31]

[0094] The composition and description of the SB-525 (PF-07055480) formulation are shown in Table 27. The DP was at a final pH of 7.3 ± 0.3. Excipient concentrations were achieved in whole or in part by the addition of base buffer (phosphate buffered saline). The DP concentration target was 1.0E+13 vg / mL (0.3E+13 to 3.0E+13 vg / mL) with a DP range of 30-300% of the DP target. Table 27. Description of SB-525 Drug Substance Formulation [Table 32]

[0095] Container and filling volume The filtered drug substance was packaged in sterile high density polyethylene (HDPE) bottles. The DP was filled into AT10 mL cycloolefin copolymer (COC) vials (Aseptic Technologies VIA-101800) with thermoplastic elastomer stoppers in place. The DS and DP were stored at -60°C to -90°C. Each vial contained 6.4 mL of DP (desired extractable volume of 6 mL) with 6.4E+13 (nominal 6.0E+13) vg of SB-525AAV.

[0096] Characterization of DP The viscosity, osmolality, density, and conductivity of SB-525DP were measured. The density was 1.0106 g / mL (20° C.). The viscosity was 1.112 cP (20° C.). The osmolality was 374 mOsm / kg. The conductivity was 17.80 mS / cm (20° C.).

[0097] Example 6: AAV6 viral vector drug substance formulation Examples 6-8 below describe further reformulation and accelerated stability studies performed to evaluate AAV2 / 6 viral vector formulations with various concentrations of divalent cation salts. As in the previous examples, the viral vector has an AAV6 capsid and a recombinant genome (including AAV2 ITRs). The viral vector here carries a transgene encoding alpha-L-iduronidase (IDUA). This transgene helps patients with a deletion of IDUA, such as those with mucopolysaccharidosis type I (MPS-I), also known as Hurler syndrome (lysosomal storage disease). The AAV genome is shown as SEQ ID NO: 28 in Table 5 of US2020 / 0246486. Because the viral genome is located within the AAV capsid and is not exposed to the formulation, it is expected that the observations in Examples 6-8 will be applicable to AAV6 vectors carrying other transgenes, as in the previous examples.

[0098] This study was also conducted to identify new formulations that do not form precipitates after freeze / thaw cycles.The stability of four AAV6 viral vector drug substance formulations containing various concentrations of divalent cation salts was evaluated.

[0099] The formulations tested contained various concentrations of divalent cation salts (Ca 2+ and Mg 2+ ) was included. As described above, the conventional AAV6 formulation as well as the formulation buffer (without AAV6) all showed the formation of small amounts of visible precipitates after multiple freeze / thaw cycles. The precipitated particles were determined to be composed of calcium phosphate salts. As the formulation undergoes freeze / thaw cycles during typical use, the potential for salt precipitation could pose a risk to patient safety and to formulation quality. The studies shown in Examples 6-8 below were aimed at finding improved formulations that would not have such precipitation issues.

[0100] In this study, samples of AAV6 viral vector formulations were prepared as different drug substance formulations by tangential flow filtration and the samples were spiked with a high concentration "spiking buffer". The reformulated samples were then incubated under various accelerated stability conditions and the quality attributes of the samples were measured after incubation. The quality attribute results were evaluated taking into account known method variability where appropriate. Outliers and trend results over the study period were identified for each of the four formulations. A pass, neutral, or fail score was assigned for each quality attribute and formulation based on the results at each study endpoint. Finally, the overall quality attribute stability scores of each of the four formulations were compared to each other. The study is described in detail below.

[0101] Sample preparation The AAV6 formulations prepared herein contained approximately 1.0E+13 vg / mL of AAV6 donor vector suspended in formulation buffer F0, as described below in Table 1. Buffer F0 was formulated in 1% w / v sucrose and 0.05% w / v poloxamer 188 based on the method of Dulbecco's phosphate buffered saline with the divalent cations calcium (as CaCl2) and magnesium (as MgCl2), an additional amount of approximately 35 mM sodium chloride.

[0102] To transfer the AAV6 material to another buffer formulation, a buffer exchange by tangential flow filtration was performed as shown in Figure 2. Briefly, several vials of AAV6 formulation formulated in Buffer F0 were thawed and transferred to a single ultrafiltration / diafiltration (UF / DF) reservoir. Ultrafiltration was first performed to concentrate the material approximately 2x. The concentrated material was then diafiltered with at least 10x an equivalent volume of formulation buffer F1. The recovered intermediate material, labeled "A" in Figure 2, was suspended in formulation buffer F1 and should contain approximately 2.0E+13 vg copies / mL.

[0103] The intermediate UF / DF product "A" was then split into three portions and formulated into three different buffers. Three additional formulation buffers (F1, F2, and F3; Table 28) were prepared from Dulbecco's Phosphate Buffered Saline with the addition of sodium chloride, sucrose, and poloxamer 188 (e.g., Pluronic® F-68). These three buffers did not contain calcium, but instead contained different amounts of magnesium. Buffer F1 contained no calcium or magnesium components, buffer F2 contained magnesium at a molar equivalent to F0, and buffer F3 contained an additional amount of magnesium to account for the molar proportion of calcium removed from buffer F0. Table 28. Formulation buffer composition [Table 33]

[0104] To simplify sample preparation and allow for the preparation of different buffers, "spike buffers" were prepared that were 6 times more concentrated than buffers F2 and F3. These F2 and F3 "6x concentration" buffers were mixed with F1 in a 1:5 ratio to generate the desired compositions of buffers F2 and F3, respectively, as shown in Figure 3.

[0105] To prepare these formulation buffers, each dry component was weighed using a microbalance. The components were mixed and dissolved in deionized water for injection. These buffers were then titrated to the desired pH range with 0.1M NaOH or 0.1M HCI, if necessary. Finally, these buffers were formulated with sucrose and poloxamer 188 at the desired concentrations.

[0106] The composition of the prepared drug substance formulation was determined by several semi-quantitative measurements for confirmation, and the results are listed in Table 29. Ca was measured using a commercially available colorimetric assay. 2+ (Bio Vision Cat#K380-250) and Mg 2+The concentrations of calcium ions (Bio Vision Cat#K385-l00) were measured. In both colorimetric assays, the sample concentrations were within the dynamic linear range of measurement, and the measured signals and calculated concentrations trended in line with the theoretical sample composition. Critically, the calcium signals of formulations F1, F2, and F3 all matched the assay negative control. Thus, these samples were calcium-free, and the TFF sample preparation process was effective in removing calcium present in the F0 starting material. Magnesium measurements also confirmed Mg 2+ It was also confirmed that β-lactam α-amyloid was removed by TFF and added at appropriate concentrations in F2 and F3. Table 29. Laboratory measured parameters of formulated drug substance [Table 34]

[0107] qPCR was also performed to measure vg titer and multi-angle dynamic light scattering (MADLS) was performed to measure particle concentration. Again, these results were within the range expected from the sample preparation, indicating that the buffer exchange and formulation steps were performed as intended.

[0108] The bulk formulated product was filtered and filled into 2 mL cyclized olefin polymer (COP) vials (West Pharma / Daikyo CZ Cat#19550057) at 0.5 mL / vial.

[0109] Sample Incubation The vials of formulation were incubated under various conditions as described below. Freeze / thaw cycling was performed on the days listed in Table 30. Table 30. Freeze / Thaw Cycling Sample Dates [Table 35]

[0110] Samples were subjected to "accelerated, stress" conditions (40°C / 75% relative humidity (RH)) or "accelerated, ambient" conditions (25°C / 60% RH) on D3, D7, 2 weeks, 1 month, 2 months, 3 months, or 6 months of their execution. Samples were stored at 2-8°C and analyzed within 24 hours of their execution.

[0111] All run samples were aliquoted into polycarbonate flip-top centrifuge tubes for testing of the sample of interest. Aliquots were stored at 2-8°C for less than 2 months between active testing. If not tested within a reasonable time from the run date, the aliquots were stored at ≦-65°C until testing could be performed. All archived samples and remaining material after testing were stored at ≦-65°C for long term storage.

[0112] Example 7: Testing of AAV6 drug substance formulations The samples were tested including the quality attributes listed in Table 31. Abbreviations are as follows: DLS, dynamic light scattering; MADLS, multi-angle dynamic light scattering; HMWS, high molecular weight species; SE-LC, size exclusion liquid chromatography; and AEX-LC, anion exchange liquid chromatography. The appearance of the solutions was evaluated to ensure that they were clear, colorless, and free of particulate matter. Solutions that were turbid, cloudy, or contained visible particles did not meet the evaluation criteria. Table 31. Quality attributes of the formulations evaluated. [Table 36]

[0113] First sample The quality attribute results from all starting formulated product samples are shown in Tables 32 and 33. The results were grouped by the quality attribute categories of total, purity, and strength. Table 32. Total and purity quality attribute results for the starting (t0) sample [Table 37] Table 33. Quality attribute results for concentrations in the starting (t0) sample [Table 38]

[0114] The initial infectious titer results were mostly lower for F3 compared to the other starting samples. It also showed a significantly higher vg / TCID ratio than the other samples. This was mainly due to the TCID 50 This was due to assay variability. None of the other results appeared to be significantly affected.

[0115] Freeze / thaw cycle samples The results of freeze / thaw cycling of the four different formulations are shown in Figures 4A-D. The data show that freeze / thaw cycling did not affect solution pH, monomeric capsid size, sample size distribution, or total capsid content in all four formulations, nor did it result in significant amounts of HMWS or soluble aggregates. However, freeze / thaw cycling caused visual appearance failure in the starting F0 control formulation. After 5xF / T cycles, small white flaky precipitates were observed that had the characteristics of previously observed calcium phosphate precipitates. These precipitates were not detected in formulations F1, F2, or F3. The data also show that freeze / thaw cycling did not significantly affect any of the formulation strength properties, taking into account the variability of the test method.

[0116] Specimens under accelerated temperature of 25℃ / 60% RH (ambient) Results of testing four different formulations incubated at ambient conditions (25°C / 60% RH) are shown in Figure 5A-D. The data show that appearance and pH were not affected by the 25°C conditions. Sample size distributions appeared to broaden, polydispersity index (PDI) increased, and monomer peak average size increased slightly with longer incubation times. High molecular weight species (HMWS) also began to form with incubation time. In general, these HMWS were more readily detected by SE-LC than DLS. Charge separation of total and empty capsids appeared to be lost after 1-2 months of incubation at 25°C, and therefore % total capsids could not be measured by AEX-LC. Capsid titer and monomer concentration were not affected by the 25°C conditions, nor did vg titer appear to be affected. TCID 50 was decreased at 3 and 6 months for F0, F1, and F2, but the same results were not observed for F3.

[0117] Specimen under 40℃ / 75% RH stress The results of stressed condition (40°C / 75% RH) incubation of four different formulations are shown in Figures 6A-D. The data show that 40°C / 75% RH conditions significantly affected several formulation quality attributes. The particle size distribution was significantly broadened and HMWS had formed. Although SE-LC appears to be more sensitive than DLS for detecting smaller amounts of HMWS, if larger amounts of HMWS (possible high molecular weight species) were present, these particles may not have been detected by SE-LC. High molecular weight species may have been filtered or did not pass through the LC column to reach the detector.

[0118] After 3 months at 40°C, all solutions began to appear cloudy, possibly due to the formation of large insoluble aggregates or HMWS. Solution pH did not appear to be affected. TCID 50 was significantly decreased for all samples after 1 month at 40°C / 75% RH, with the least decrease from onset seen for F3.

[0119] Example 8: Study Endpoint Measurements Intra-sample analysis was performed for each formulation. Within each formulation, each study endpoint result for each quality attribute was scored against the acceptance and failure criteria as described in Figure 7. The stability study endpoints of interest were as follows: 10x freeze / thaw cycles at <-65°C for less than 12 hours per cycle and less than 6 hours at ambient temperature, 3 months at 25°C / 60% RH, and 1 month at 40°C / 75% RH. For each study endpoint, the quality attributes described in Figure 7 were determined based on known established method variability as well as an overall evaluation of all data generated in this study.

[0120] To perform a sample-to-sample analysis or relative evaluation of each formulation, each endpoint score for each quality attribute was compared for each formulation. When one quality attribute was considered, an overall Pass, Neutral, or Fail score was assigned for each formulation by comparing the relative endpoint scores across the four considered formulations as a whole. For example, if all endpoint results showed accepted criteria passed for a given formulation, an overall Pass score was assigned for that quality attribute for that formulation. The number of neutral and failed endpoint conditions was taken into account when assigning the overall quality attribute score for that formulation. For certain quality attributes, such as appearance, one failure endpoint score is sufficient to score an overall failure for that formulation. For other quality attributes, such as monomer concentration, one failure endpoint at the 40°C condition was applied to all formulations (except F3); therefore, this failure endpoint was not fully scored. TCID 50 The results contained information only and no scores were assigned.

[0121] These inter-sample analytical scores are shown in Figures 8 and 9. A summary of all overall quality attribute scores for each formulation is shown in Table 34, with formulation F0 used as the control. Table 34. Summary of formulation evaluation between samples [Table 39]

[0122] The overall quality attribute scores during the accelerated stability study show that formulations F3 and F2 are superior to F0 and F1. Formulation F3 appears to be the best, with an overall passing quality attribute score of 8 and 2 intermediate scores. Formulation F2 also performed reasonably well. F0 showed an overall failing score for appearance, confirming the initial objective of improving this formulation. F1 appeared to show the most aggregates and had the most HMWS detected.

[0123] Example 9: Long-term temperature testing The stability of SB-525 (PF-07055480) formulations provided at 1.00E+13 vg / mL in formulation buffer was tested over extended temperature. The formulation buffer contained the following components: 0.49 mM MgCl2, 2.68 mM KCl, 1.47 mM KH2PO4, 172 mM NaCl, 8.10 mM Na2HPO4, 1% (w / v) sucrose, 0.05% (w / v) poloxamer 188 (pH 7.4).

[0124] The vials containing the formulation were incubated at (i) -70°C for 0 days (T0), (ii) -150°C for 3 days (T 3日間 ), and (iii) at −150°C for 14 days (T 14日間 ) and stored. Formulation samples were subsequently analyzed for (i) appearance (liquid), (ii) reduced CE-SDS, (iii) SEC titer_260 / 280, and (iv) in vitro FVIII activity (bioassay). Formulation buffer samples were also analyzed for (i) container integrity.

[0125] As shown below in Table 35, there was no significant change in color, clarity, reduced CE-SDS, SEC potency_260 / 280 (a surrogate measure of empty:total capsid ratio), or biological activity (relative % potency) when samples were stored under the test conditions, including storage at -150°C for up to 14 days. No effect was observed on container integrity after storage at -150°C for 14 days. Table 35. Stability results [Table 40]

[0126] Example 10: Simulated shipping and long-term temperature testing The stability of SB-525 formulations was tested under anticipated shipping conditions including shock, air pressure, drop, and vibration. SB-525 formulations were provided at 1.00E+13vg / mL in formulation buffer containing the following components: 0.49mM MgCl2, 2.68mM KCl, 1.47mM KH2PO4, 172mM NaCl, 8.10mM Na2HPO4, 1% (w / v) sucrose, 0.05% (w / v) poloxamer 188 (pH 7.4).

[0127] Test sample vials containing the formulation were subjected to concurrently applied transport hazards (e.g., shock, air pressure, drop, and vibration) using a worst-case global transport profile. Control sample vials were not subjected to concurrently applied transport hazards. As part of the challenge, the test vials were kept at -35°C for 40 hours and then at -70°C for an additional 40 hours. After challenge, the control and test formulation vials were stored at -70°C and were evaluated at day 0 (T0), 6 months (T 6ヶ月 ), and at 10 months (T 10ヶ月 ). Formulation buffer vials were stored at -70°C and tested at T0. Formulation samples were analyzed for (i) appearance (liquid), (ii) reduced CE-SDS, (iii) SEC titer_260 / 280, and (iv) in vitro FVIII activity (bioassay). Formulation buffer samples were analyzed for (i) container integrity.

[0128] As shown below in Table 36, there was no significant change in color, clarity, reduced CE-SDS, SEC titer_260 / 280 (a surrogate measure of empty:total capsid ratio), or biological activity (relative % potency) when samples were stored for up to 10 months under the test conditions. There was no effect on container integrity at T0. Table 36. Stability results [Table 41]

[0129] Example 11: Formulation Stability Data - 24 Months The purpose of this study was to establish the long-term (24 months) stability of the SB-525 formulation at the intended storage temperature (-70°C). In this study, the SB-525 formulation (DP) was purified from Sf9 insect cells and formulated at the intended 1.00E+13 vector vg / mL in 8.10 mM Na2HPO4, 1.47 mM KH2PO4, 0.49 mM MgCl2, 2.68 mM KCl, 172 mM NaCl, 1% (w / v) sucrose, and 0.05% (w / v) poloxamer P188 (pH 7.3±0.3). The DP was then filled at 6.4 mL into 10 mL Aseptic Technologies (AT) crystal closed vials and stored at -60°C to -90°C.

[0130] Results from DP stability studies including 5 cycles of uncontrolled F / T and 24 hours of agitation are shown in Figure 10. The data show that by 24 months at -70°C, 12 months at 5°C, and 3 months at 25°C / 60% RH, there were no clear trends observed in color, clarity, pH, non-visible cumulative particulate matter, capsid purity and VP ratio (by R-CGE), %HMMS and UV260 / 280 (by SEC-HPLC), and capsid titer. The 12-month sample at 5°C recorded "one long fibrous particle" in the solution that appeared to be extraneous in morphology in natural and uncontrolled laboratory conditions (i.e., open lab space) testing; all other samples recorded "essentially no visible particles." No changes in these quality attributes were observed after 5 cycles of uncontrolled F / T or 24 hours of agitation.

[0131] For all time points and conditions tested, the variability of results for genome titers and transmitted virus titers as well as infection ratios was within the expected measurement variability and showed no obvious trends. Results of in vitro relative potency, a method that indicates sensitivity and accuracy of stability, showed a slight trend towards decreased potency at the intended storage conditions (-70°C) after 24 months, but these results are within the clinical stability acceptance criteria. A trend towards decreased stability was also seen in potency at 3 months at 25°C / 60% RH and 12 months at 5°C.

[0132] Container integrity (CCIT) was also tested at 18 months at the intended storage conditions (-70°C) with a validated headspace analyzer and the results were recorded. Six conditioned vials were used for this analysis. P188 concentration was also stable at -70°C for 18 months, at 5°C for 12 months, and at 25°C / 60% RH for up to 3 months. A slight decrease in P188 concentration was observed at 24 months at -70°C.

Claims

1. Recombinant adeno-associated virus (rAAV) vector, Sodium chloride (NaCl), Potassium chloride (KCl), Disodium phosphate (Na ２ HPO ４ ), Monopotassium phosphate (KH ２ PO ４ ), Magnesium chloride (MgCl ２ ), Polyols, and Poloxamer A pharmaceutical composition comprising, wherein the pharmaceutical composition contains about 0.1 mM or less of calcium chloride and has a pH of about 7.1 to about 7.

5.

2. The pharmaceutical composition according to claim 1, wherein the polyol is sucrose.

3. The pharmaceutical composition according to claim 1 or 2, wherein the poloxamer is poloxamer 188.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the pharmaceutical composition contains about 0.1 to about 2.0 mM magnesium chloride.

5. The pharmaceutical composition according to claim 4, wherein the pharmaceutical composition contains about 0.5 mM or more of magnesium chloride.

6. The pharmaceutical composition according to claim 4, wherein the pharmaceutical composition contains about 1.4 mM magnesium chloride.

7. The pharmaceutical composition according to claim 4, wherein the pharmaceutical composition contains about 1.3 mM or more of magnesium chloride.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the pharmaceutical composition contains about 150 to about 200 mM sodium chloride.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the pharmaceutical composition contains about 2.5 to about 3.0 mM potassium chloride.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the pharmaceutical composition contains about 5 to about 10 mM disodium phosphate.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the pharmaceutical composition contains about 1.0 to about 2.0 mM monopotassium phosphate.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the pharmaceutical composition contains about 0.5% to about 2% (w / v) of sucrose.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the pharmaceutical composition contains about 0.01% to about 0.1% (w / v) of poloxamer 188.

14. Recombinant adeno-associated virus (rAAV) vector, Approximately 171.81 mM sodium chloride, Approximately 2.68 mM potassium chloride, Approximately 8.10 mM disodium phosphate, Approximately 1.47 mM monopotassium phosphate, Approximately 1.40 mM magnesium chloride, Approximately 1.00% (w / v) sucrose, and Approximately 0.05% (w / v) of poloxamer 188 A pharmaceutical composition comprising, wherein the pharmaceutical composition contains about 0.1 mM or less of calcium chloride and has a pH of about 7.1 to about 7.

5.

15. Recombinant adeno-associated virus (rAAV) vector, Approximately 172 mM sodium chloride, Approximately 2.68 mM potassium chloride, Approximately 8.10 mM disodium phosphate, Approximately 1.47 mM monopotassium phosphate, Approximately 0.49 mM magnesium chloride, Approximately 1.00% (w / v) sucrose, and Approximately 0.05% (w / v) of poloxamer 188 A pharmaceutical composition comprising, wherein the pharmaceutical composition contains about 0.1 mM or less of calcium chloride and has a pH of about 7.1 to about 7.

5.

16. The pharmaceutical composition according to any one of claims 1 to 15, wherein the rAAV comprises a genome including an expression cassette for a therapeutic protein.

17. The pharmaceutical composition according to claim 16, wherein the therapeutic protein is a human factor VIII polypeptide.

18. A recombinant adeno-associated virus (rAAV) vector comprising a genome containing an expression cassette for the expression of human factor VIII polypeptide, Approximately 171.81 mM sodium chloride, Approximately 2.68 mM potassium chloride, Approximately 8.10 mM disodium phosphate, Approximately 1.47 mM monopotassium phosphate, Approximately 1.40 mM magnesium chloride, Approximately 1.00% (w / v) sucrose, and Approximately 0.05% (w / v) of poloxamer 188 A pharmaceutical composition comprising, wherein the pharmaceutical composition contains about 0.1 mM or less of calcium chloride and has a pH of about 7.1 to about 7.

5.

19. A recombinant adeno-associated virus (rAAV) vector comprising a genome containing an expression cassette for the expression of human factor VIII polypeptide, Approximately 172 mM sodium chloride, Approximately 2.68 mM potassium chloride, Approximately 8.10 mM disodium phosphate, Approximately 1.47 mM monopotassium phosphate, Approximately 0.49 mM magnesium chloride, Approximately 1.00% (w / v) sucrose, and Approximately 0.05% (w / v) of poloxamer 188 A pharmaceutical composition comprising, wherein the pharmaceutical composition contains about 0.1 mM or less of calcium chloride and has a pH of about 7.1 to about 7.

5.

20. The pharmaceutical composition according to any one of claims 17 to 19, wherein the human factor VIII polypeptide comprises SEQ ID NO:

1.

21. The pharmaceutical composition according to any one of claims 17 to 19, wherein the genome of the rAAV comprises SEQ ID NO: 2 or nucleotides 131 to 5024 of SEQ ID NO:

2.

22. The pharmaceutical composition according to any one of claims 1 to 21, wherein the pharmaceutical composition contains the rAAV in an amount of about 1.0 E+12 to about 1.0 E+14 vector genomes (vg) per 1 mL.

23. The pharmaceutical composition according to claim 22, wherein the pharmaceutical composition contains the rAAV at approximately 1.0E+13vg per 1 mL.

24. The pharmaceutical composition according to any one of claims 1 to 23, wherein the rAAV comprises an AAV6 capsid protein.

25. A vial containing 5 to 10 mL of the pharmaceutical composition according to any one of claims 1 to 24.

26. A vial according to claim 25, comprising a cycloolefin copolymer.

27. The vial according to claim 25 or 26, wherein the vial has a thermoplastic elastomer stopper at a predetermined position.

28. The pharmaceutical composition according to claim 8, wherein the pharmaceutical composition contains about 172 mM sodium chloride.

29. The pharmaceutical composition according to claim 9, wherein the pharmaceutical composition contains about 2.7 mM potassium chloride.

30. The pharmaceutical composition according to claim 10, wherein the pharmaceutical composition comprises about 8 mM disodium phosphate.

31. The pharmaceutical composition according to claim 11, wherein the pharmaceutical composition contains about 1.5 mM monopotassium phosphate.

32. The pharmaceutical composition according to claim 12, wherein the pharmaceutical composition contains about 1% (w / v) sucrose.

33. The pharmaceutical composition according to claim 13, wherein the pharmaceutical composition contains about 0.05% (w / v) of poloxamer 188.

34. The pharmaceutical composition according to claim 22, wherein the pharmaceutical composition contains the rAAV in an amount of about 1.0 E+13 to about 5.0 E+13 vector genomes (vg) per 1 mL.

35. The pharmaceutical composition according to claim 24, wherein the rAAV comprises the inverted terminal sequence (ITR) of AAV2.

36. A pharmaceutical composition according to any one of claims 1 to 24 and 28 to 35, for use in the treatment of hemophilia A in human subjects.

Images