Adeno-associated virus pharmaceutical composition and its use

Abstract

Provided is a pharmaceutical composition comprising a recombinant adeno-associated virus (AAV), a buffer, an amino acid, a stabilizer, and a nonionic surfactant. The pharmaceutical composition has good stability.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to adeno-associated virus pharmaceutical compositions and their use.

Background Art

[0002] Adeno-associated virus (AAV) belongs to the family Parvoviridae and the genus Dependovirus. Recombinant adeno-associated virus (rAAV) containing a therapeutic gene is widely used in many fields such as gene transfer, gene therapy, vaccination, and oncolytic therapy. It has low pathogenicity, a wide range of infected tissues, a wide range of host cells (infection and expression are possible in both proliferating and non-proliferating cells), low immunogenicity, a long expression time of foreign genes in vivo, and no integration into the host cell genome. Therefore, it is widely used in experimental and clinical research.

[0003] In the development process of rAAV products, in addition to considering the safety and effectiveness of the virus itself, it is also necessary to consider the formulation stability of the product, such as the time and environmental changes (temperature, stress, light, etc.) during production, transportation, storage, and use. Therefore, in order to stably ensure the quality of the product, the development and improvement of the formulation are required.

Summary of the Invention

Problems to be Solved by the Invention

[0004] An object of the present invention is to provide a stable pharmaceutical composition containing recombinant adeno-associated virus (AAV).

Means for Solving the Problems

[0005] Specifically, in one aspect of the present invention, there is provided a pharmaceutical composition comprising recombinant adeno-associated virus (AAV), a buffer, an amino acid, a stabilizer, and a nonionic surfactant.

[0006] In some embodiments, the buffer of the present invention is a citrate buffer. In some preferred embodiments, the citrate is a sodium citrate salt, for example, a monovalent, divalent, or trivalent salt of sodium citrate, preferably the trivalent salt.

[0007] In some embodiments, the amino acid of the present invention is selected from aspartic acid, arginine, glycine, histidine, and proline. More preferably, the amino acid is aspartic acid, proline, or arginine.

[0008] In some embodiments, the stabilizer of the present invention is selected from sucrose, trehalose, mannitol, lactose, galactose, glucose, and maltose. In some preferred embodiments, the stabilizer of the present invention is selected from sucrose, trehalose, and mannitol. In some more preferred embodiments, the stabilizer of the present invention is selected from sucrose or trehalose.

[0009] In some embodiments, the nonionic surfactant of the present invention is selected from poloxamer 188, polysorbate 20, polysorbate 80, polyethylene glycol-hydroxystearate (HS15), and vitamin E polyethylene glycol succinate (TPGS). In some preferred embodiments, the nonionic surfactant of the present invention is selected from poloxamer 188.

[0010] In some embodiments, in the pharmaceutical composition of the present invention, the concentration of the buffer is 1 - 200 mM, the concentration of the amino acid is 5 - 200 mM, the concentration of the stabilizer is 1 - 20 wt%, and the concentration of the nonionic surfactant is 0.001 - 0.1 wt%.

[0011] In some embodiments, in the pharmaceutical composition of the present invention, the concentration of the buffer is 1 - 100 mM, the concentration of the amino acid is 10 - 100 mM, the concentration of the stabilizer is 1 - 20 wt%, and the concentration of the nonionic surfactant is 0.001 - 0.1 wt%.

[0012] In some embodiments, in the pharmaceutical composition of the present invention, the concentration of the buffer solution is 10-100 mM, the concentration of the amino acid is 20-150 mM, the concentration of the stabilizer is 2.5-10 wt%, and the concentration of the non-ionic surfactant is 0.001-0.05 wt%.

[0013] In some embodiments, in the pharmaceutical composition of the present invention, the concentration of the buffer solution is 10-80 mM, the concentration of the amino acid is 10-50 mM, the concentration of the stabilizer is 2.5-10 wt%, and the concentration of the non-ionic surfactant is 0.001-0.05 wt%.

[0014] In some embodiments, in the pharmaceutical composition of the present invention, the concentration of the buffer solution is 20-100 mM, the concentration of the amino acid is 40-150 mM, the concentration of the stabilizer is 3-10 wt%, and the concentration of the non-ionic surfactant is 0.001-0.05 wt%.

[0015] In some specific embodiments, the pharmaceutical composition of the present invention comprises 40 mM of a buffer, 40 mM of an amino acid, 5 wt% of a stabilizer, and 0.001 wt% of a non-ionic surfactant.

[0016] In some specific embodiments, the pharmaceutical composition of the present invention comprises 40 mM of sodium citrate and 40 mM of aspartic acid.

[0017] In some specific embodiments, the pharmaceutical composition comprises 40 mM of sodium citrate, 5 wt% of sucrose, 0.001 wt% of P188, and 40 mM of aspartic acid.

[0018] In some specific embodiments, the pharmaceutical composition comprises 40 mM of sodium citrate, 5 wt% of sucrose, 0.001 wt% of P188, and 40 mM of arginine.

[0019] In some specific embodiments, the pharmaceutical composition comprises 40 mM sodium citrate, 5 wt% sucrose, 0.001 wt% P188, and 40 mM proline.

[0020] In some specific embodiments, the pharmaceutical composition comprises 20 mM sodium citrate, 10 wt% trehalose, 0.001 wt% P188, and 40 mM aspartic acid.

[0021] In some specific embodiments, the pharmaceutical composition comprises 100 mM sodium citrate, 3 wt% sucrose, 0.001 wt% P188, and 80 mM aspartic acid.

[0022] In some specific embodiments, the pharmaceutical composition comprises 40 mM sodium citrate, 5 wt% sucrose, 0.001 wt% P188, and 150 mM aspartic acid.

[0023] In some embodiments, the pharmaceutical composition of the present invention further comprises MgCl₂. The concentration of the magnesium chloride is 0.1 - 10 mM, preferably 1 - 5 mM.

[0024] In some embodiments, the pH of the pharmaceutical composition of the present invention is 5.5 - 8.0. In some preferred embodiments, the pH of the pharmaceutical composition of the present invention is 6.0 - 8.0. In some preferred embodiments, the pH of the pharmaceutical composition of the present invention is 5.5 - 7.5. In some preferred embodiments, the pH of the pharmaceutical composition of the present invention is 5.5 - 6.5. In some more preferred embodiments, the pH of the pharmaceutical composition of the present invention is 6.0 ± 0.2. The pharmaceutical composition can be adjusted to the desired final pH using sodium hydroxide or hydrochloric acid as a pH adjuster.

[0025] In some embodiments, the capsid protein serotype of the recombinant adeno-associated virus of the present invention is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or variants thereof. In some preferred embodiments, the capsid protein serotype of the recombinant adeno-associated virus of the present invention is selected from AAV8 or variants thereof.

[0026] In some embodiments, the recombinant adeno-associated virus of the present invention comprises a coding sequence encoding a VEGF antagonist, and the VEGF antagonist is a) a fusion protein comprising the amino acid sequence shown in SEQ ID NO:1, b) a fusion protein comprising the amino acid sequence shown in SEQ ID NO:2, c) one having the antibody heavy chain variable region sequence shown in SEQ ID NO:3 and the antibody light chain variable region sequence shown in SEQ ID NO:4, d) one having the antibody heavy chain variable region sequence shown in SEQ ID NO:5 and the antibody light chain variable region sequence shown in SEQ ID NO:6, preferably a single-chain antibody comprising the amino acid sequence shown in SEQ ID NO:7, or comprises at least one selected from the group consisting of an antibody or a protein having at least 80% homology with the above antibody or protein sequence.

[0027] In some embodiments, the recombinant adeno-associated virus of the present invention is (i) an rAAV capsid protein that is an AAV8 capsid protein or a variant thereof, and (ii) from 5' to 3', (a) an AAV 5' ITR, (b) a CBA promoter, (c) a Kozak sequence, (d) a nucleic acid encoding an anti-VEGF antagonist and having the nucleotide sequence shown in SEQ ID NO:8, (e) a rabbit globin polyadenylation signal, (f) a polynucleotide expression cassette comprising the AAV 3’ ITR in this order.

[0028] In some specific embodiments, the polynucleotide expression cassette of the present invention has the nucleotide sequence shown in SEQ ID NO:9.

[0029] In some embodiments, the genomic concentration of the recombinant adeno-associated virus (AAV) contained in the pharmaceutical composition of the present invention is about 1×10 7 vg / ml to 1×10 14 vg / ml. In some preferred embodiments, the genomic concentration of the recombinant adeno-associated virus (AAV) of the present invention is about 1×10 9 vg / ml to 1×10 13 vg / ml, preferably 1×10 9 vg / ml to 1×10 12 vg / ml, preferably 1×10 10 vg / ml to 1×10 13 vg / ml.

[0030] In some preferred specific embodiments, the genomic concentration of the recombinant adeno-associated virus (AAV) of the present invention is about 1×10 7 , 1.5×10 7 , 2×10 7 , 2.5×10 7 , 3×10 7 , 3.5×10 7 , 4×10 7 , 4.5×10 7 , 5×10 7 , 5.5×10 7 , 6×10 7 , 6.5×10 7 , 7×10 7 , 7.5×10 7 , 8×10 7 , 8.5×10 7 , 9×10 7 , 9.5×10 7 , 1×10 8 , 1.5×10 8 , 2×10 8 , 2.5×10 8 , 3×108 、3.5×10 8 、4×10 8 、4.5×10 8 、5×10 8 、5.5×10 8 、6×10 8 、6.5×10 8 、7×10 8 、7.5×10 8 、8×10 8 、8.5×10 8 、9×10 8 、9.5×10 8 、1×10 9 、1.5×10 9 、2×10 9 、2.5×10 9 、3×10 9 、3.5×10 9 、4×10 9 、4.5×10 9 、5×10 9 、5.5×10 9 、6×10 9 、6.5×10 9 、7×10 9 、7.5×10 9 、8×10 9 、8.5×10 9 、9×10 9 、9.5×10 9 、1×10 10 、1.5×10 10 、2×10 10 、2.5×10 10 、3×10 10 、3.5×10 10 、4×10 10 、4.5×10 10 、5×10 10 、5.5×10 10 、6×10 10 、6.5×10 10 、7×10 10 、7.5×10 10 、8×10 10 、8.5×10 10 、9×10 10 、9.5×10 10 、1×10 11 、1.5×10 11 、2×10 11、2.5×10 11 、3×10 11 、3.5×10 11 、4×10 11 、4.5×10 11 、5×10 11 、5.5×10 11 、6×10 11 、6.5×10 11 、7×10 11 、7.5×10 11 、8×10 11 、8.5×10 11 、9×10 11 、9.5×10 11 、1×10 12 、1.5×10 12 、2×10 12 、2.5×10 12 、3×10 12 、3.5×10 12 、4×10 12 、4.5×10 12 、5×10 12 、5.5×10 12 、6×10 12 、6.5×10 12 、7×10 12 、7.5×10 12 、8×10 12 、8.5×10 12 、9×10 12 、9.5×10 12 、1×10 13 、1.5×10 13 、2×10 13 、2.5×10 13 、3×10 13 、3.5×10 13 、4×10 13 、4.5×10 13 、5×10 13 、5.5×10 13 、6×10 13 、6.5×10 13 、7×10 13 、7.5×10 13 、8×10 13 、8.5×10 13 、9×10 13 、9.5×10 13 、1×10 14(All units are vg / ml).

[0031] In some embodiments, the pharmaceutical composition of the present invention is an intravitreal injection, a subretinal injection, an intrachoroidal injection, an intravenous injection, an intratumoral injection, or an intramuscular injection. In some preferred embodiments, the pharmaceutical composition is an intravitreal injection, a subretinal injection, or an intrachoroidal injection.

[0032] In some embodiments, the pharmaceutical composition of the present invention is a liquid composition or a pharmaceutical composition obtained by lyophilizing the liquid composition.

[0033] In some embodiments, the pharmaceutical composition of the present invention is stored in a unit dose container. In some specific embodiments, the unit dose container is a vial or a syringe. In some preferred embodiments, the vial is a glass vial, and in some other preferred embodiments, the syringe is a prefilled syringe.

[0034] Another aspect of the present invention provides the use of the pharmaceutical composition according to any one of the above claims in the manufacture of a drug for treating a disease related to VEGF.

[0035] In some embodiments, the disease related to VEGF is an ocular angiogenesis disease.

[0036] In some preferred embodiments, the ocular disease of the present invention is selected from age-related macular degeneration, retinal angiogenesis, choroidal angiogenesis, diabetic retinopathy, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy and diabetic retinal edema, macular edema secondary to retinal vein occlusion, polypoidal choroidal vasculopathy, exudative age-related macular degeneration with extremely low vision, choroidal angiogenesis secondary to pathologic myopia, neovascular glaucoma, iris angiogenesis disease, retinopathy of prematurity.

[0037] In another aspect of the invention, there is provided a method of treating a disease, comprising administering to a patient / subject in need thereof a therapeutically effective amount of the pharmaceutical composition comprising the recombinant adeno-associated virus of the invention.

[0038] In some embodiments, the pharmaceutical composition of the invention is administered by subcutaneous injection, intramuscular injection, intravenous injection, intratumoral injection, intravitreal injection, suprachoroidal injection, or subretinal injection. In some preferred embodiments, the pharmaceutical composition of the invention is administered by suprachoroidal injection or subretinal injection.

[0039] In some embodiments, the disease associated with VEGF is an ocular disease.

[0040] In some preferred embodiments, the eye disease of the invention is selected from age-related macular degeneration, retinal neovascularization, choroidal neovascularization, diabetic retinopathy, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy and diabetic retinal edema, macular edema secondary to retinal vein occlusion, polypoidal choroidal vasculopathy, exudative age-related macular degeneration with extremely low vision, choroidal neovascularization secondary to pathologic myopia, neovascular glaucoma, iris neovascularization disease, retinopathy of prematurity. Definitions

[0041] To facilitate understanding of the present invention, specific technical terms and scientific terms are specifically defined below. Unless otherwise explicitly defined herein, all other technical terms and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0042] "Pharmaceutical composition" means comprising one or more recombinant adeno-associated viruses (rAAVs) described herein and other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to a living being and to facilitate absorption and / or expression of the active ingredient for exerting biological activity. In the present disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0043] As used herein, the term "AAV" refers to naturally occurring adeno-associated viruses and recombinant forms of adeno-associated virus (rAAV), and includes variant forms of AAV. The term AAV further includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3, AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, primate AAV, and non-primate AAV. In some embodiments, AAV is AAV8.

[0044] Pharmaceutically acceptable buffers are well known in the art and include, but are not limited to, inorganic acid salts such as phosphates (sodium or potassium), bicarbonates, organic acid salts such as citrates, acetates, succinates, acidic buffers such as acetic acid, phosphoric acid, hydrochloric acid, carbonic acid, succinic acid, citric acid, histidine hydrochloride, malic acid, and alkaline buffers such as sodium hydroxide, Tris, HEPES, etc.

[0045] A "citrate" buffer is a buffer containing citrate ions. Examples of citrate buffers include sodium citrate, potassium citrate, calcium citrate, magnesium citrate, etc. A preferred citrate buffer is sodium citrate.

[0046] Amino acids are well known in the art and include natural amino acids and non-natural amino acids (synthetic amino acids). Examples include alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), tryptophan (Trp), methionine (Met), glycine (Gly), serine (Ser), threonine (Thr), cysteine (Cys), tyrosine (Tyr), asparagine (Asn), glutamine (Gln), selenocysteine (Sec), pyrrolysine (Pyl), lysine (Lys), arginine (Arg), histidine (His), aspartic acid (Asp), glutamic acid (Glu), etc.

[0047] It has been found that adding one or more sugars and / or sugar alcohols as stabilizers at appropriate levels (e.g., about 1% to about 10%) is advantageous for improving the stability of liquid formulations and / or lyophilized formulations. Any sugar can be used as a stabilizer for the pharmaceutical compositions of the present invention, and non-limiting examples thereof include monosaccharides, disaccharides or polysaccharides, or water-soluble dextran, such as fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, dextran, trehalose, amylopectin, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch and carboxymethyl cellulose. Sugar alcohols are defined as hydrocarbons having about 4 to about 8 carbon atoms and one hydroxyl group. Non-limiting examples of sugar alcohols that can be used in the pharmaceutical compositions according to the present invention include mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol.

[0048] The main non-ionic surfactants that can be used in the pharmaceutical compositions of the present invention are known in the pharmaceutical field and include various poloxamers or pluronics, including but not limited to polysorbate 80 (Tween 80, PS80), polysorbate 20 (Tween 20, PS20), and pluronic F-68 and BRIJ 35, or mixtures thereof.

[0049] The "lyophilized formulation" means a pharmaceutical composition in the form of a liquid or solution, or a formulation or pharmaceutical composition obtained by vacuum freeze-drying a liquid or solution formulation.

[0050] In representative embodiments, the pharmaceutical composition of the present invention has a physiologically compatible pH. Typically, the pH of the pharmaceutical composition is from about 5.0 to about 9.0, from about 5.5 to about 8.0, from about 6.0 to about 8.0, from about 5.5 to about 7.5, from about 6.0 to about 7.5, from about 6.5 to about 7.5. In some embodiments, the pH of the formulation is about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, 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. Typically, the pH of the pharmaceutical composition is about 7.0 or about 7.5. In some embodiments, the pH of the pharmaceutical composition is about 6.0 ± 0.2. In some embodiments, the pH of the pharmaceutical composition is about 7.0 ± 0.2. In some embodiments, the pH of the pharmaceutical composition is about 8.0 ± 0.2.

[0051] As used herein, the term "about" means an approximate range of plus or minus 10% from a specific numerical value. For example, the expression "about 20%" includes the range from 18% to 22%. As used herein, "about" also includes the exact amount. Thus, "about 20%" means both "about 20%" and "20%".

[0052] The formulation or pharmaceutical composition of the present invention includes other pharmaceutically acceptable components. In representative embodiments, the formulation or pharmaceutical composition includes any one or a combination of an acidifying agent, an anticoagulant, an antibacterial preservative, an antioxidant, a preservative, an alkali, an inorganic salt, etc.

[0053] "Effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or disorder of a medical condition. An effective amount also means an amount sufficient to enable or facilitate a diagnosis. The effective amount for a particular subject or veterinary subject can vary depending on factors such as the disorder being treated, the general health of the subject, the method, route and dosage of administration, and the severity of side effects. The effective amount can be the maximum dosage or dosing schedule that avoids significant side effects or toxic effects.

[0054] The terms "patient" and "subject" are used interchangeably and in their conventional sense refer to organisms, including humans and non-human animals, that are suffering from, or are susceptible to, a disorder that can be prevented or treated by administration of the compositions of the present invention. Examples of subjects include humans, chimpanzees, other apes, monkey species, livestock such as cattle, sheep, pigs, goats, horses, domesticated mammalian pets such as dogs and cats, laboratory animals including rodents such as mice, rats, guinea pigs, poultry such as chickens, turkeys, other fowl, ducks, geese, wild birds, and game birds, but are not limited thereto. The term does not denote a particular age. Thus, adults, adolescents, and newly born individuals are all included.

[0055] "Stable" or "substantially stable" can refer to a pharmaceutical composition that maintains its properties (e.g., pH, osmotic pressure, genomic titer, purity of capsid protein, purity of rAAV, activity, stabilization of insoluble microparticles) over a long period of storage. For example, a composition with storage stability does not contain obvious impurities due to the decomposition of the composition over a long period, maintains a high purity, e.g., the impurities are 10% or less, or the decomposition products are 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less over a long period. In some cases, a composition with storage stability contains few, if any, insoluble microparticles of 10 μm or more, 25 μm or more, 50 μm or more, 100 μm or more, 150 μm or more, 200 μm or more, 250 μm or more, 300 μm or more, 350 μm or more, 400 μm or more, 450 μm or more, or 500 μm or more, and contains them in very small amounts over a long period. In some cases, a composition with storage stability substantially retains its activity over a long period, e.g., the composition maintains 100%, 99% or more, 98% or more, 97% or more, 96% or more, 95% or more, 94% or more, 93% or more, 92% or more, 91% or more, 90% or more, 85% or more, 80% or more, or 75% or more of its activity over a long period. "Long period" means 1 week or more, 2 weeks or more, 3 weeks or more, 1 month or more, 2 months or more, 3 months or more, 4 months or more, 6 months or more, 9 months or more, 1 year or more, 1.5 years (e.g., 18 months) or more, 2 years or more, 2.5 years (e.g., 30 months) or more, 3 years or more, 3.5 years (e.g., 42 months) or more, 4 years or more, 4.5 years (e.g., 54 months) or more, or 5 years or more. In some embodiments, a pharmaceutical composition with storage stability is substantially stable over a long period at ambient temperature, e.g., at a temperature of 20 - 40°C, or 25 - 35°C, or 25 - 30°C. In some cases, a composition with storage stability is substantially stable over a long period at a temperature lower than ambient temperature, e.g., at a temperature of 0 - 20°C, or 0 - 15°C, or 0 - 10°C, or 2 - 8°C.

[0056] In some cases, "stable" or "substantially stable" may refer to the thermal stability of AAV in a pharmaceutical composition that withstands high temperatures or temperature changes. Two pathways are involved in the thermal denaturation of AAV: genome ejection and capsid disruption. Genome ejection from the AAV capsid occurs at relatively low temperatures, and the DNA begins to escape from the intact capsid. AAV capsid disruption occurs when the viral capsid loses its structural integrity due to protein unfolding and is disrupted. To monitor or evaluate the thermal stability of AAV, it can be verified through conventional experimental methods in this field, such as Elisa, qPCR, AAV-ID, and high-throughput thermal stability analysis. In some embodiments, the present invention uses the Uncle (Unchained Labs) multifunctional protein stability analysis system to monitor genome ejection and capsid disruption of AAV and evaluate the thermal stability of the protein. A DNA-binding fluorescent dye (e.g., SYBR Gold) is used to track genome ejection (the nucleic acid dye can bind to the nucleic acid ejected from AAV and emit fluorescence. The more nucleic acid is ejected, the stronger the fluorescence signal after binding), and the melting temperature (Tm) is determined based on DNA release. The higher the Tm temperature, the higher the thermal stability of AAV. In some cases, the Tm of AAV in the pharmaceutical composition of the present invention is increased by at least 0.5 °C, at least 1 °C, at least 1.5 °C, at least 2 °C, at least 2.5 °C, at least 3 °C, at least 3.5 °C, at least 4 °C, at least 4.5 °C, at least 5 °C or more compared to other compositions. The capsid disruption of AAV can be studied with Uncle in the absence of dye by monitoring the intrinsic protein fluorescence of the capsid protein to determine its disruption (since the protein is heated to a certain extent, the protein unfolds, hydrophobic regions are exposed, and changes occur in the autofluorescence of the protein, so it is possible to determine whether the structure of the protein has changed based on the change in the autofluorescence of the protein). The AAV capsid protein in the pharmaceutical composition of the present invention undergoes almost no structural change.

Brief Description of the Drawings

[0057]

Figure 1A

Figure 1B

Figure 2

Embodiments for Carrying Out the Invention

[0058] The present invention will be further described below with reference to specific examples. It should be understood that these examples are used only for the purpose of explaining the present invention and do not limit the scope of the present invention. Example 1 Preparation of Recombinant Adeno-Associated Virus Vector (rAAV) Stock Solution

[0059] Using conventional DNA recombination technology and cloning technology, a recombinant plasmid containing the target protein gene of the present invention was constructed and cloned in Escherichia coli, and then used for the preparation of recombinant AAV virus. First, a polynucleotide expression cassette and a recombinant plasmid encoding the target gene were constructed. Exemplarily, the expression cassette or plasmid contains, in order from 5' to 3', AAV 5' ITR, CBA promoter, Kozak sequence, coding sequence nucleic acid encoding the anti-VEGF protein, rabbit globin polyadenylation signal, and AAV 3' ITR. The polynucleotide expression cassette and the recombinant plasmid contain a coding sequence encoding the Conbercept fusion protein (target gene). Here, Conbercept has the amino acid sequence shown in SEQ ID NO:2. The recombinant adeno-associated virus vector (rAAV) is prepared through a three-plasmid co-transfection process known in the art. After transfecting three plasmids (pAAV-Conbercept:pAAV8-RC-Kan:pHelper-Kan) into HEK293 cells, the rAAV genomic titer is detected for later use. Example 2

[0060] Preparation of buffer: Appropriate amounts of sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, and poloxamer 188 were weighed respectively, using water as the solvent, and a buffer solution containing 180 mM sodium chloride, 10 mM disodium hydrogen phosphate - sodium dihydrogen phosphate buffer, and 0.001 wt% poloxamer 188 was prepared, and the pH value was adjusted to about 7 with dilute hydrochloric acid or sodium hydroxide solution. Preparation of sample solution: An appropriate amount of the stock solution in Example 1 was taken and injected into a dialysis card. Dialysis and liquid exchange were carried out 3 times, 2 hours each time, so that the volume ratio of the formulation buffer to the stock solution was 100 times or more, and 3 sets of different formulations were obtained. In the finally obtained sample, the concentration of disodium hydrogen phosphate - sodium dihydrogen phosphate buffer was 10 mM, the concentration of sodium chloride was 180 mM, the concentration of poloxamer 188 was 0.001 wt%, the pH was about 7, and the rAAV concentration was 1×10 11 vg / ml. The individually packaged formulation samples were left at room temperature (for 3 days), and the stability of the samples was examined by SDS-PAGE using the purity of the rAAV capsid protein as an index. The results are shown in Figures 1A and 1B. As a result of observing the samples at room temperature for 3 days (Figure 1B), a distinct band appeared at 70 kDa (lane 4) compared with the initial sample (Figure 1A). Example 3

[0061] Preparation of buffer solution: Appropriate amounts of glycine, sodium citrate, trehalose, and poloxamer 188 were weighed respectively. Using water as the solvent, a buffer solution containing 40 mM glycine, 40 mM sodium citrate buffer, 5 wt% trehalose, and 0.001 wt% poloxamer 188 was prepared, and the pH value was adjusted to about 7.2 with dilute hydrochloric acid or sodium hydroxide solution. Preparation of sample solution: An appropriate amount of the stock solution in Example 1 was taken and injected into a dialysis card. Dialysis and liquid exchange were performed 3 times, 2 hours each time, so that the volume ratio of the formulation buffer to the stock solution was 100 times or more. The concentration of rAAV in the sample solution was 1×10 11 vg / ml. Example 4

[0062] Preparation of buffer solution: Appropriate amounts of aspartic acid, sodium citrate, trehalose, and poloxamer 188 were weighed respectively. Using water as the solvent, a buffer solution containing 40 mM aspartic acid, 40 mM sodium citrate buffer, 5 wt% trehalose, and 0.001 wt% poloxamer 188 was prepared, and the pH value was adjusted to about 7.2 with dilute hydrochloric acid or sodium hydroxide solution. Preparation of sample solution: An appropriate amount of the stock solution in Example 1 was taken and injected into a dialysis card. Dialysis and liquid exchange were performed 3 times, 2 hours each time, so that the volume ratio of the formulation buffer to the stock solution was 100 times or more. The concentration of rAAV in the sample solution was 1×10 11 vg / ml. Example 5

[0063] Preparation of buffer solution: Appropriate amounts of aspartic acid, sodium citrate, sucrose, and poloxamer 188 were weighed respectively. Using water as the solvent, a buffer solution containing 40 mM aspartic acid, 40 mM sodium citrate buffer, 5 wt% sucrose, and 0.001 wt% poloxamer 188 was prepared, and the pH value was adjusted to about 6.0 with dilute hydrochloric acid or sodium hydroxide solution. Preparation of sample solution: An appropriate amount of the stock solution in Example 1 was taken and injected into a dialysis card. Dialysis and liquid exchange were performed three times for 2 hours each so that the volume ratio of the formulation buffer to the stock solution was 100 times or more, and five sets of sample solutions were prepared. The concentrations of rAAV in the sample solutions were 1.0×10 13 vg / mL, 5.0×10 12 vg / mL, 1.0×10 12 vg / mL, 1.0×10 11 vg / mL, and 1.0×10 10 vg / mL, respectively. The long-term stability (≤ -65°C, 9 months) of the prepared sample with a concentration of 1.0×10 13 vg / mL was examined. As shown in Table 1, the indices to be examined included pH, osmotic pressure, genomic titer, purity of capsid protein, purity of rAAV, insoluble fine particles, and the like.

[0064]

Table 1

[0065] The stock solution in Example 1 was taken, and a formulation solution of rAAV was prepared according to the formulations in Table 2-1 and Table 2-2 below. Here, the concentration of AAV was 1.0×10 12 vg / mL. The pH value of the solution was adjusted with dilute hydrochloric acid or sodium hydroxide solution.

[0066]

Table 2-1

[0067] [[ID=4,0]]

Table 2-2

[0068] The thermal stability of rAAV in the above formulation solution was examined by the protein thermal stability (Tm) experiment. In the Tm experiment, first, a nucleic acid dye was added to the rAAV sample for treatment, and then the gene injection of the sample in a specific temperature range (25°C to 95°C) was detected in real time by fluorescence, static light scattering (SLS), dynamic light scattering (DLS), etc. The specific operation method is as follows. Add 20X SYBRTM GLOD nucleic acid dye to the above formulation sample, collect 9 μl into a Uni-tube, select the "Tm, Tagg&Optional DLS" program on the high-throughput multi-parameter protein temperature analysis system (UNcle, UNCHAINED LABS), and perform real-time detection. The set parameters of the high-throughput multi-parameter protein temperature analysis system are as follows. Starting temperature: 25°C, incubation time: 180 s, heating rate: 0.5°C / min, ending temperature: 95°C, UV 266 Opaque (0.00), Blue Laser open (1.00). The results of the samples are shown in Table 3-1 and Table 3-2.

[0069]

Table 3-1

[0070]

Table 3-2

[0071] Collect the stock solution of Example 1, and prepare a formulation solution of rAAV according to the formulation in Table 4 below, where the concentration of AAV is 1.0×10 12 vg / mL respectively. Adjust the pH value of the solution with dilute hydrochloric acid or sodium hydroxide solution.

[0072]

Table 4

[0073] The Tm test was carried out with reference to the method of Example 6. The results are shown in Table 5.

[0074]

Table 5

[0075] The stock solution of Example 1 was collected, and a formulation solution of rAAV was prepared according to the formulation in Table 6 below. Here, the concentration of AAV was 1.0×10 12 vg / mL each. The pH value of the solution was adjusted with dilute hydrochloric acid or sodium hydroxide solution.

[0076]

Table 6

[0077] The Tm test was performed with reference to the method of Example 6. The results are shown in Table 7.

[0078]

Table 7

[0079] The stock solution of Example 1 was collected, and a formulation solution of rAAV was prepared according to the formulation in Table 8 below. Here, the concentration of AAV was 1.0×10 12 vg / mL each. The pH value of the solution was adjusted with dilute hydrochloric acid or sodium hydroxide solution.

[0080]

Table 8

[0081] The thermal stability of rAAV in the above-mentioned formulation solution was examined by an isothermal stability experiment. The sample was held at a specific temperature for a certain period (e.g., 18 h, 24 h), and structural changes (e.g., aggregation / denaturation) of the rAAV capsid protein in the sample were detected in real time by fluorescence, static light scattering (SLS), dynamic light scattering (DLS), etc. The specific operation method is as follows. 9 μl of the above-mentioned formulation sample was collected into a Uni-tube, and "Isothermal Toolbox" was selected on a high-throughput multi-parameter protein temperature analysis system (UNcle, UNCHAINED LABS), and the program "Isothermal" was selected for detection. The setting parameters of the high-throughput multi-parameter protein temperature analysis system are as follows. Temperature: 42 °C, constant temperature time: 5 min, examination time: 18 h, UV 266 Filter1 (0.50), Blue Laser Filter3 (0.25). Through the relative BCM (300 - 430 nm) response value, changes in the thermal stability of proteins in various formulations were observed. By overlapping the time-course curves of the samples to be compared and observing the rising speed of the curves, the speed of aggregation / denaturation of the samples was grasped, and the stability of the samples was evaluated.

[0082] The results are shown in Figure 2. As can be seen from Figure 2, for the sample of Example 6-1, the change in the curve is gentle within 0 - 18 h, and the relative BCM index fluctuates at 1 ± 0.002. Under the condition of 42 °C, there is almost no aggregation or denaturation during the incubation process of the sample, and the stability is good. For the samples of Examples 9-1, 9-7, and 9-9, the curve changes relatively greatly, and the relative BCM index fluctuates within the range of 1 - 1.007. For the samples of Examples 9-2, 9-4, 9-5, 9-6, and 9-8, the curve changes even more greatly, and the relative BCM index fluctuates in the range of 1 - 1.012. For the sample of Example 9-3, the curve changes the most greatly, and the relative BCM index fluctuates in the range of 1 - 1.016. Example 10

[0083] The stock solution of Example 1 was collected, and a formulation solution of rAAV was prepared according to the formulation in Table 9 below. Here, the concentration of AAV was 1.0×10 12 vg / mL respectively. The pH value of the solution was adjusted with dilute hydrochloric acid or sodium hydroxide solution.

[0084]

Table 9

[0085] As a result of performing the isothermal stability test according to the method of Example 9, the formulation samples of Examples 10-1, 10-2, and 10-3 had a gentle curve change within 0 to 18 h, a relative BCM value of 1±0.002, and maintained good thermal stability. Example 11

[0086] The stock solution of Example 1 was collected, and a formulation solution of rAAV containing 40 mM sodium citrate, 5 wt% sucrose, 0.001 wt% P188, and 40 mM aspartic acid was prepared. Here, the concentration of AAV was 1.0×10 12 vg / mL respectively. The pH value of the solution was adjusted to 6.0±0.2 with dilute hydrochloric acid or sodium hydroxide solution. As a result of performing the isothermal stability test according to the method of Example 9, the above-mentioned rAAV formulation sample had a gentle curve change within 0 to 18 h, a relative BCM value of 1±0.002, and maintained good thermal stability. Example 12

[0087] The stock solution of Example 1 was collected, and a formulation solution of rAAV containing 20 mM sodium citrate, 10 wt% sucrose, 0.001 wt% P188, 40 mM aspartic acid, and 1 mM magnesium chloride hexahydrate was prepared. Here, the concentration of AAV was 1.0×10 12 vg / mL respectively. The pH value of the solution was adjusted to 6.0±0.2 with dilute hydrochloric acid or sodium hydroxide solution. As a result of performing the isothermal stability test according to the method of Example 9, the above rAAV formulation sample had a gentle curve change within 0 to 18 h, a relative BCM value of 1 ± 0.002, and maintained good thermal stability. Example 13

[0088] The stock solution of Example 1 was collected, and a formulation solution of rAAV containing 100 mM sodium citrate, 3 wt% sucrose, 0.001 wt% P188, 80 mM aspartic acid, and 1 mM magnesium chloride hexahydrate was prepared. Here, the concentration of AAV was 1.0×10 12 vg / mL. The pH value of the solution was adjusted to 6.0 ± 0.2 with dilute hydrochloric acid or sodium hydroxide solution. As a result of performing the isothermal stability test according to the method of Example 9, the above rAAV formulation sample had a gentle curve change within 0 to 18 h, a relative BCM value of 1 ± 0.002, and maintained good thermal stability. Example 14

[0089] The stock solution of Example 1 was collected, and a formulation solution of rAAV containing 40 mM sodium citrate, 5 wt% sucrose, 0.001 wt% P188, 150 mM aspartic acid, and 1 mM magnesium chloride hexahydrate was prepared. Here, the concentration of AAV was 1.0×10 12 vg / mL. The pH value of the solution was adjusted to 6.0 ± 0.2 with dilute hydrochloric acid or sodium hydroxide solution. As a result of performing the isothermal stability test according to the method of Example 9, the above rAAV formulation sample had a gentle curve change within 0 to 18 h, a relative BCM value of 1 ± 0.002, and maintained good thermal stability. Example 15

[0090] The stock solution of Example 1 was collected, and a formulation solution of rAAV containing 40 mM sodium citrate, 5 wt% sucrose, 0.001 wt% P188, 40 mM aspartic acid, and 1 mM magnesium chloride hexahydrate was prepared. Here, the concentration of AAV was 1.0×10 11It is vg / mL. The pH value of the solution was adjusted to 6.0 ± 0.2 with dilute hydrochloric acid or sodium hydroxide solution. As a result of performing the isothermal stability test according to the method of Example 9, the above rAAV formulation sample had a gentle curve change within 0 to 18 h, a relative BCM value of 1 ± 0.002, and maintained good thermal stability. Example 16

[0091] The stock solution of Example 1 was collected, and an rAAV formulation solution containing 40 mM sodium citrate, 5 wt% sucrose, 0.001 wt% P188, 40 mM aspartic acid, and 1 mM magnesium chloride hexahydrate was prepared. Here, the concentration of AAV was 1.0×10 10 vg / mL, and the pH value of the solution was adjusted to 6.0 ± 0.2 with dilute hydrochloric acid or sodium hydroxide solution. As a result of performing the isothermal stability test according to the method of Example 9, the above rAAV formulation sample had a gentle curve change within 0 to 18 h, a relative BCM value of 1 ± 0.002, and maintained good thermal stability. Example 17

[0092] The stock solution of Example 1 was collected, and an rAAV formulation solution containing 40 mM sodium citrate, 5 wt% sucrose, 0.001 wt% P188, 40 mM aspartic acid, and 1 mM magnesium chloride hexahydrate was prepared. Here, the concentration of AAV was 1.0×10 13 vg / mL, and the pH value of the solution was adjusted to 6.0 ± 0.2 with dilute hydrochloric acid or sodium hydroxide solution. As a result of performing the isothermal stability test according to the method of Example 9, the above rAAV formulation sample had a gentle curve change within 0 to 18 h, a relative BCM value of 1 ± 0.002, and maintained good thermal stability.

Claims

1. A pharmaceutical composition comprising a recombinant adeno-associated virus, a buffer, an amino acid, a stabilizer, and a nonionic surfactant, wherein the buffer is a citrate buffer.

2. The citrate is a sodium citrate salt, and more preferably, the sodium citrate salt is a trivalent sodium citrate salt. The pharmaceutical composition according to claim 1, wherein the sodium citrate salt is a trivalent sodium citrate salt.

3. The amino acid is selected from aspartic acid, arginine, glycine, histidine, and proline, and more preferably, the amino acid is aspartic acid, proline, and arginine. The pharmaceutical composition according to any one of the preceding claims, wherein the amino acid is aspartic acid, proline, and arginine.

4. The stabilizer is selected from sucrose, trehalose, mannitol, lactose, galactose, glucose, and maltose, and preferably, the stabilizer is sucrose or trehalose. The pharmaceutical composition according to any one of the preceding claims, wherein the stabilizer is sucrose or trehalose.

5. The nonionic surfactant is selected from poloxamer 188, polysorbate 20, polysorbate 80, HS15, and TPGS, and preferably, the nonionic surfactant is poloxamer 188. The pharmaceutical composition according to any one of the preceding claims, wherein the nonionic surfactant is poloxamer 188.

6. The concentration of the buffer in the pharmaceutical composition is 1 to 200 mM, the concentration of the amino acid is 5 to 200 mM, the concentration of the stabilizer is 1 to 20 wt%, and the concentration of the nonionic surfactant is 0.001 to 0.1 wt%. The pharmaceutical composition according to any one of the preceding claims, wherein the concentration of the buffer in the pharmaceutical composition is 1 to 200 mM, the concentration of the amino acid is 5 to 200 mM, the concentration of the stabilizer is 1 to 20 wt%, and the concentration of the nonionic surfactant is 0.001 to 0.1 wt%.

7. The concentration of the buffer in the pharmaceutical composition is 10 to 100 mM, the concentration of the amino acid is 20 to 150 mM, the concentration of the stabilizer is 2.5 to 10 wt%, and the concentration of the nonionic surfactant is 0.001 to 0.05 wt%. The pharmaceutical composition according to any one of the preceding claims, wherein the concentration of the buffer in the pharmaceutical composition is 10 to 100 mM, the concentration of the amino acid is 20 to 150 mM, the concentration of the stabilizer is 2.5 to 10 wt%, and the concentration of the nonionic surfactant is 0.001 to 0.05 wt%.

8. The concentration of the buffer in the pharmaceutical composition is 20 to 100 mM, the concentration of the amino acid is 40 to 150 mM, the concentration of the stabilizer is 3 to 10 wt%, and the concentration of the nonionic surfactant is 0.001 to 0.05 wt%. The pharmaceutical composition according to any one of the preceding claims, wherein the concentration of the buffer in the pharmaceutical composition is 20 to 100 mM, the concentration of the amino acid is 40 to 150 mM, the concentration of the stabilizer is 3 to 10 wt%, and the concentration of the nonionic surfactant is 0.001 to 0.05 wt%.

9. The pharmaceutical composition according to any one of the preceding claims, characterized in that it contains 40 mM of a buffer, 40 mM of an amino acid, 5 wt% of a stabilizer, and 0.001 wt% of a non-ionic surfactant.

10. The pharmaceutical composition according to any one of the preceding claims, characterized in that it contains 40 mM of sodium citrate, 40 mM of aspartic acid, 5 wt% of sucrose, and 0.001 wt% of P188.

11. MgCl ２ The pharmaceutical composition according to any one of the preceding claims, further comprising

12. The pharmaceutical composition according to any one of the preceding claims, characterized in that the pH is 5.5 to 8.0, preferably 6.0 to 8.0, preferably 5.5 to 6.5, preferably 6.0 ± 0.

2.

13. The genomic concentration of the recombinant adeno-associated virus is about 1×10 ７ vg / ml to 1×10 １４ vg / ml, preferably about 1×10 ９ vg / ml to 1×10 １３ vg / ml, more preferably about 1×10 ９ vg / ml to 1×10 １２ vg / ml, and the pharmaceutical composition according to any one of the preceding claims, characterized in that.

14. The pharmaceutical composition according to any one of the preceding claims, characterized in that the capsid protein serotype of the recombinant adeno-associated virus is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or a variant thereof.

15. The pharmaceutical composition according to any one of the preceding claims, characterized in that the capsid protein serotype of the recombinant adeno-associated virus is selected from AAV8 or a variant thereof.

16. The recombinant adeno-associated virus contains a coding sequence encoding a VEGF antagonist, and the VEGF antagonist is a) a fusion protein containing the amino acid sequence shown in SEQ ID NO: 1, b) a fusion protein containing the amino acid sequence shown in SEQ ID NO: 2, c) one having the antibody heavy chain variable region sequence shown in SEQ ID NO: 3 and the antibody light chain variable region sequence shown in SEQ ID NO: 4, d) having the antibody heavy chain variable region sequence shown in SEQ ID NO: 5 and the antibody light chain variable region sequence shown in SEQ ID NO: 6, preferably a single-chain antibody containing the amino acid sequence shown in SEQ ID NO: 7, or at least one selected from the group consisting of an antibody or protein having at least 80% homology with the above antibody or protein sequence. The pharmaceutical composition according to any one of the preceding claims, characterized in that it contains at least one.

17. The recombinant adeno-associated virus is (i) an rAAV capsid protein that is an AAV8 capsid protein or a variant thereof, (ii) from 5' to 3', (a) an AAV 5' ITR, (b) CBA promoter, (c) Kozak sequence, (d) a nucleic acid encoding an anti-VEGF antagonist and having the nucleotide sequence shown in SEQ ID NO: 8, (e) rabbit globin polyadenylation signal, (f) a polynucleotide expression cassette containing an AAV 3' ITR in this order, and the pharmaceutical composition according to any one of the preceding claims, characterized by comprising the same. (Claim 18) The polynucleotide expression cassette has the nucleotide sequence shown in SEQ ID NO: 9, and the pharmaceutical composition according to any one of the preceding claims, characterized by comprising the same. (Claim 19) An intravitreal injection, a subretinal injection, an intrachoroidal injection, an intravenous injection, an intratumoral injection or a intramuscular injection, preferably an intravitreal injection, a subretinal injection, an intrachoroidal injection, and the pharmaceutical composition according to any one of the preceding claims, characterized by comprising the same. (Claim 20) A liquid preparation, and the pharmaceutical composition according to any one of the preceding claims, characterized by comprising the same. (Claim 21) A freeze-dried preparation, and the pharmaceutical composition according to any one of the preceding claims, characterized by comprising the same. (Claim 22) Stored in a unit dose container, and the pharmaceutical composition according to any one of the preceding claims, characterized by comprising the same. (Claim 23) The unit dose container is a vial or a syringe, and the pharmaceutical composition according to claim 22, characterized by comprising the same. (Claim 24) The vial is a glass vial, and the pharmaceutical composition according to claim 22, characterized by comprising the same. (Claim 25) The syringe is a prefilled syringe, and the pharmaceutical composition according to claim 22, characterized by comprising the same. (Claim 26) Use of the pharmaceutical composition according to any one of the preceding claims in the manufacture of a drug for treating a disease related to VEGF. (Claim 27) A method for treating a disease related to VEGF, comprising administering a therapeutically effective amount of the pharmaceutical composition according to any one of claims 1 to 25 to a patient / subject in need thereof. (Claim 28) The disease related to VEGF is an ocular neovascular disease, and the use according to claim 26 or the method according to claim 27, characterized by comprising the same. (Claim 29) The ocular angiogenesis disease is selected from age-related macular degeneration, retinal angiogenesis, choroidal angiogenesis, diabetic retinopathy, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy and diabetic retinal edema, macular edema secondary to retinal vein occlusion, polypoidal choroidal vasculopathy, exudative age-related macular degeneration with extremely reduced vision, choroidal angiogenesis secondary to pathologic myopia, neovascular glaucoma, iris angiogenesis disease, retinopathy of prematurity, the use or method according to claim 28, characterized in that.

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