Pharmaceutical composition of recombinant adeno-associated virus and use thereof

Abstract

Provided is a pharmaceutical composition, which pharmaceutical composition comprising a recombinant adeno-associated virus (AAV) and a buffer, a stabilizer and a nonionic surfactant. The pharmaceutical composition has good stability, thereby ensuring the stability of the product quality.

Description

A recombinant adeno-associated virus pharmaceutical composition and its use Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a recombinant adeno-associated virus pharmaceutical composition and use thereof. Background Art

[0002] Adeno-associated virus (AAV) belongs to the Parvoviridae family and the Dependovirus genus. Recombinant adeno-associated virus (rAAV) carrying therapeutic genes is widely used in gene transduction, gene therapy, vaccination, and oncolytic therapy. Its advantages include low pathogenicity, wide tissue infection, a broad host cell range (infection and expression in both proliferating and non-proliferating cells), low immunogenicity, prolonged in vivo expression of exogenous genes, and lack of host cell genomic integration, making it widely used in experimental and clinical research.

[0003] During the development of rAAV products, in addition to considering the safety and efficacy of the virus itself, it is also necessary to consider the stability of the product formulation, such as the time and possible environmental changes (temperature, stress, light, etc.) during manufacturing, transportation, storage, and administration. Therefore, to ensure the stability of product quality, formulation development and improvement are required. Summary of the Invention

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

[0005] Specifically, one aspect of the present invention is to provide a pharmaceutical composition comprising a recombinant adeno-associated virus (AAV) and 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, such as a monovalent, divalent, or trivalent salt of sodium citrate, preferably a trivalent salt.

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

[0008] In some embodiments, the stabilizer of the present invention is selected from sucrose, trehalose, mannitol, lactose, galactose, glucose, maltose. In some preferred embodiments, the stabilizer of the present invention is selected from sucrose, trehalose, 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, polyethylene glycol-hydroxystearate (HS15), vitamin E polyethylene glycol succinate (TPGS). In some preferred embodiments, the nonionic surfactant of the present invention is selected from poloxamer 188 (P188), polysorbate 20 (Tween 20), polysorbate 80 (Tween 80).

[0010] In some embodiments, the concentration of the buffer in the pharmaceutical composition of the present invention is 10mM to 200mM; the concentration of the amino acid is 10mM to 200mM; the concentration of the stabilizer is 1% to 20wt%; and the concentration of the nonionic surfactant is 0.001% to 0.1wt%.

[0011] In some embodiments, the concentration of the buffer in the pharmaceutical composition of the present invention is 10mM to 100mM; the concentration of the amino acid is 20mM to 150mM; the concentration of the stabilizer is 2.5% to 10wt%; and the concentration of the nonionic surfactant is 0.001% to 0.05wt%.

[0012] In some embodiments, the concentration of the buffer in the pharmaceutical composition of the present invention is 20mM to 40mM; the concentration of the amino acid is 40mM to 100mM; the concentration of the stabilizer is 3% to 10wt%; and the concentration of the nonionic surfactant is 0.001% to 0.05wt%.

[0013] In some embodiments, the pharmaceutical composition of the present invention comprises 40 mM buffer; 40 mM amino acid; 5% wt of stabilizer; and 0.001% to 0.05 wt% of nonionic surfactant.

[0014] In some embodiments, the pharmaceutical composition of the present invention comprises 40 mM buffer; 100 mM amino acid; 5% wt stabilizer; and 0.001% to 0.05 wt% nonionic surfactant.

[0015] In some embodiments, the pharmaceutical composition of the present invention comprises 40 mM buffer; 60 mM amino acid; 5% wt stabilizer; and 0.001% to 0.05 wt% nonionic surfactant.

[0016] In some embodiments, the pharmaceutical composition of the present invention comprises 20 mM buffer; 100 mM amino acid; 5% wt stabilizer; and 0.001% to 0.05 wt% nonionic surfactant.

[0017] In some embodiments, the pH of the pharmaceutical composition of the present invention is 5.5 to 8.0. In some preferred embodiments, the pH of the pharmaceutical composition of the present invention is 6.0 to 8.0. In some preferred embodiments, the pH of the pharmaceutical composition of the present invention is 5.5 to 7.5. In some preferred embodiments, the pH of the pharmaceutical composition of the present invention is 5.5 to 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 endpoint pH using sodium hydroxide or hydrochloric acid as a pH adjuster.

[0018] In some embodiments, the recombinant adeno-associated virus capsid protein serotype 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 recombinant adeno-associated virus capsid protein serotype of the present invention is AAV8 capsid protein or a modified AAV8 capsid protein.

[0019] In some embodiments, the recombinant adeno-associated virus of the present invention comprises a coding sequence encoding an anti-VEGF protein, wherein the anti-VEGF protein is a fusion protein comprising the amino acid sequence of SEQ ID NO: 1, or comprises a protein having at least 80%, 90%, 95% or 99% homology with the above-mentioned antibody or protein sequence.

[0020] In some embodiments, the recombinant adeno-associated virus of the present invention comprises:

[0021] (i) rAAV capsid protein, wherein the capsid protein is AAV8 capsid protein or a modified AAV8 capsid protein; and

[0022] (ii) a polynucleotide expression cassette comprising, from 5' to 3' sequence:

[0023] (a) AAV 5′ITR;

[0024] (b) CBA promoter;

[0025] (c)Chi intron;

[0026] (d) Kozak sequence;

[0027] (e) a nucleic acid encoding an anti-VEGF protein, wherein the nucleic acid has a nucleotide sequence as set forth in SEQ ID NO: 2;

[0028] (f) rabbit-globin polyadenylation signal;

[0029] (g) AAV 3'ITR.

[0030] In some specific embodiments, the polynucleotide expression cassette of the present invention comprises the nucleotide sequence of SEQ ID NO: 3 from 5' to 3'.

[0031] In some specific embodiments, the capsid protein of the present invention is a modified AAV8 capsid protein, wherein the amino acids 588 to 592 of the parent AAV8 are replaced by the polypeptide sequence as described in SEQ ID NO: 4.

[0032] In some embodiments, the pharmaceutical composition of the present invention comprises a recombinant adeno-associated virus (AAV) genome at a concentration of about 1*10 7 vg / ml to 1*10 14 In some preferred embodiments, the genome 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.

[0033] In some preferred embodiments, the genome concentration of the recombinant adeno-associated virus (AAV) of the present invention is about 1×10 7 , 1.5x10 7 , 2x10 7 , 2.5x10 7 , 3x10 7 , 3.5x10 7 , 4x10 7 , 4.5x10 7 , 5x10 7 , 5.5x10 7 , 6x10 7 , 6.5x10 7 , 7x10 7 , 7.5x10 7 , 8x10 7 , 8.5x10 7 , 9x10 7 , 9.5x10 7 , 1x10 8 , 1.5x10 8 , 2x10 8 , 2.5x10 8 , 3x10 8 , 3.5x10 8、4x10 8 、4.5x10 8 、5x10 8 、5.5x10 8 、6x10 8 、6.5x10 8 、7x10 8 、7.5x10 8 、8x10 8 、8.5x10 8 、9x10 8 、9.5x10 8 、1x10 9 、1.5x10 9 、2x10 9 、2.5x10 9 、3x10 9 、3.5x10 9 、4x10 9 、4.5x10 9 、5x10 9 、5.5x10 9 、6x10 9 、6.5x10 9 、7x10 9 、7.5x10 9 、8x10 9 、8.5x10 9 、9x10 9 、9.5x10 9 、1x10 10 、1.5x10 10 、2x10 10 、2.5x10 10 、3x10 10 、3.5x10 10 、4x10 10 、4.5x10 10 、5x10 10 、5.5x10 10 、6x10 10 、6.5x10 10 、7x10 10 、7.5x10 10 、8x10 10 、8.5x10 10 、9x10 10 、9.5x10 10 、1x10 11 、1.5x10 11 、2x10 11 、2.5x10 11 、3x1011 , 3.5x10 11 , 4x10 11 , 4.5x10 11 , 5x10 11 , 5.5x10 11 , 6x10 11 , 6.5x10 11 , 7x10 11 , 7.5x10 11 , 8x10 11 , 8.5x10 11 , 9x10 11 , 9.5x10 11 , 1x10 12 , 1.5x10 12 , 2x10 12 , 2.5x10 12 , 3x10 12 , 3.5x10 12 , 4x10 12 , 4.5x10 12 , 5x10 12 , 5.5x10 12 , 6x10 12 , 6.5x10 12 , 7x10 12 , 7.5x10 12 , 8x10 12 , 8.5x10 12 , 9x10 12 , 9.5x10 12 , 1x10 13 , 1.5x10 13 , 2x10 13 , 2.5x10 13 , 3x10 13 , 3.5x10 13 , 4x10 13 , 4.5x10 13 , 5x10 13 , 5.5x10 13 , 6x10 13 , 6.5x10 13 , 7x10 13 , 7.5x10 8 , 8x10 13 , 8.5x10 13 , 9x10 13 , 9.5x10 13 , 1x10 14 (All units are vg / ml).

[0034] In some embodiments, the pharmaceutical composition of the present invention is an intravitreal injection, a subretinal injection preparation, an intrachoroidal injection preparation, an intravenous injection preparation, an intratumoral injection preparation, or an intramuscular injection preparation. In some preferred embodiments, the pharmaceutical composition is an intravitreal injection, a subretinal injection preparation, or an intrachoroidal injection preparation.

[0035] In some embodiments, the pharmaceutical composition of the present invention is a liquid composition or a pharmaceutical composition obtained by lyophilizing a liquid composition.

[0036] In some embodiments, the pharmaceutical compositions of the present invention are stored in unit dose containers. In some specific embodiments, the unit dose container is a vial or a syringe. In some preferred embodiments, the vial is a glass vial; in other preferred embodiments, the syringe is a prefilled syringe.

[0037] Another aspect of the present invention is to provide use of the pharmaceutical composition according to any one of the preceding claims in the preparation of a medicament for treating diseases associated with VEGF.

[0038] In some embodiments, the VEGF-related disease is an ocular neovascular disease.

[0039] In some preferred embodiments, the eye disease of the present 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, wet age-related macular degeneration with very low vision, choroidal neovascularization secondary to pathological myopia, neovascular glaucoma, iris neovascular disease, and retinopathy of prematurity.

[0040] Another aspect of the present invention is to provide a method for treating a disease, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising the recombinant adeno-associated virus of the present invention to a patient / subject in need thereof.

[0041] In some embodiments, the pharmaceutical composition of the present 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 present invention is administered by suprachoroidal injection or subretinal injection.

[0042] In some embodiments, the VEGF-related disease is an ocular disease.

[0043] In some preferred embodiments, the eye disease of the present 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, wet age-related macular degeneration with very low vision, choroidal neovascularization secondary to pathological myopia, neovascular glaucoma, iris neovascular disease, and retinopathy of prematurity.

[0044] definition

[0045] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise specifically defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art.

[0046] A "pharmaceutical composition" refers to a composition containing one or more recombinant adeno-associated viruses (rAAVs) described herein, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, thereby facilitating the absorption and / or expression of the active ingredient and thereby exerting its biological activity. In this disclosure, the terms "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0047] As used herein, the term "AAV" refers to naturally occurring adeno-associated viruses and recombinant forms of adeno-associated viruses (rAAV), and includes mutant 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 certain embodiments, the AAV is AAV8.

[0048] 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, etc.; organic acid salts, such as citrate, acetate, succinate, etc.; acidic buffers, such as acetic acid, phosphoric acid, hydrochloric acid, carbonic acid, succinic acid, citric acid, histidine hydrochloride, malic acid, etc.; alkaline buffers, such as sodium hydroxide, Tris, HEPES, etc.

[0049] A "citrate" buffer is a buffer that includes citrate ions. Examples of citrate buffers include sodium citrate, potassium citrate, calcium citrate, magnesium citrate, and the like. A preferred citrate buffer is sodium citrate.

[0050] Amino acids are well known in the art and include natural amino acids and unnatural amino acids (synthetic amino acids), exemplified by 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), and glutamic acid (Glu).

[0051] It is found that adding one or more sugars and / or sugar alcohols of appropriate levels (e.g., between about 1% and about 10%) helps the stability of liquid preparations and / or lyophilized preparations as stabilizers. Any sugar can be used as the stabilizer used in pharmaceutical composition of the present invention, and non-limiting examples include: monosaccharide, disaccharide or polysaccharide or water-soluble glucan, including, for example, fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, glucan, trehalose, amylopectin, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch and carboxymethyl cellulose. Sugar alcohol is defined as a hydrocarbon with about 4 to about 8 carbon atoms and a hydroxyl group. Non-limiting examples of sugar alcohols that can be used for pharmaceutical composition provided by the invention include: mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol and arabitol.

[0052] The pharmaceutical compositions of the present invention can be used primarily as nonionic surfactants, which are known in the pharmaceutical field and include, but are not limited to, polysorbate 80 (Tween 80; PS80), polysorbate 20 (Tween 20; PS20) and various poloxamers or pluronics, including Pluronic F-68 and BRIJ 35, or mixtures thereof.

[0053] "Lyophilized formulation" refers to a pharmaceutical composition in liquid or solution form or a formulation or pharmaceutical composition obtained after a liquid or solution formulation has been subjected to a vacuum freeze-drying step.

[0054] In representative embodiments, the pharmaceutical compositions of the present invention have a physiologically compatible pH. In representative aspects, the pH of the pharmaceutical composition is about 5.0 to about 8.0, about 5.5 to about 8.0, about 6.0 to about 8.0, about 5.5 to about 7.5, about 6.0 to about 7.5, about 6.5 to about 7.5. In certain 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. In representative aspects, the pH of the pharmaceutical composition is about 6.0 or about 6.5. In certain embodiments, the pH of the pharmaceutical composition is about 6.0 ± 0.5. In certain embodiments, the pH of the pharmaceutical composition is about 6.0 ± 0.2.

[0055] As used herein, the term "about" refers to an approximate range of plus or minus 10% from a specified value. For example, the expression "about 20%" includes a range of 18-22%. As used herein, "about" also includes this exact amount. Thus, "about 20%" means "about 20%" and "20%."

[0056] The preparation or pharmaceutical composition of the present invention comprises other pharmaceutically acceptable ingredients. In representative aspects, the preparation or pharmaceutical composition comprises any one or a combination of the following: an acidifier, an anticoagulant, an antimicrobial preservative, an antioxidant, a preservative, a base, an inorganic salt, etc.

[0057] An "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also means an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular subject or veterinary subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[0058] The terms "patient" and "subject" are used interchangeably and in their conventional sense to refer to an organism, including humans and non-human animals, suffering from or susceptible to a condition that can be prevented or treated by administering the compositions of the present invention. Examples of subjects include, but are not limited to, humans, chimpanzees, other apes, and monkey species; farm animals such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals, including rodents such as mice, rats, and guinea pigs; and birds, including poultry, wild birds, and game birds such as chickens, turkeys, and other quail, ducks, geese, and the like. The terms do not denote a particular age. Thus, adults, adolescents, and newborn individuals are all subjects.

[0059] "Stable" or "substantially stable" may refer to a pharmaceutical composition that maintains its properties (e.g., pH, osmotic pressure, genome titer, capsid protein purity, rAAV purity, activity, insoluble particles, relative protein expression, etc.) over a long storage period. For example, a storage-stable composition does not have significant impurities due to degradation of the composition over a long period of time, and maintains a high purity, for example, having less than 10% impurities, or less than 9%, or less than 8%, or less than 7%, or less than 6%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1% of degradation products over a long period of time. In some cases, the storage-stable composition has little or very limited insoluble particulates ≥10 μm, or ≥25 μm, or ≥50 μm, or ≥100 μm, or ≥150 μm, or ≥200 μm, or ≥250 μm, or ≥300 μm, or ≥350 μm, or ≥400 μm, or ≥450 μm, or ≥500 μm, or even larger, over an extended period of time. In some cases, the storage-stable composition substantially retains its activity over an extended period of time, for example, the composition retains 100%, or more than 99%, or more than 98%, or more than 97%, or more than 96%, or more than 95%, or more than 94%, or more than 93%, or more than 92%, or more than 91%, or more than 90%, or more than 85%, or more than 80%, or more than 75% of its activity over an extended period of time. An extended period of time is a period of time, for example, more than 1 week, or more than 2 weeks, or more than 3 weeks, or more than 1 month, or more than 2 months, or more than 3 months, or more than 4 months, or more than 6 months, or more than 9 months, or more than 1 year, or more than 1.5 years (e.g., 18 months), or more than 2 years, or more than 2.5 years (e.g., 30 months), or more than 3 years, or more than 3.5 years (e.g., 42 months), or more than 4 years, or more than 4.5 years (e.g., 54 months), or more than 5 years. In some embodiments, the storage-stable pharmaceutical composition is substantially stable at ambient temperature for an extended period of time, for example, at a temperature of 20-40°C, or 25-35°C, or 25-30°C. In some cases, the storage-stable composition is substantially stable at a temperature below ambient temperature for an extended period of time, for example, at a temperature of 0-20°C, or 0-15°C, or 0-10°C, or 2 to 8°C.

[0060] In some cases, "stable" or "substantially stable" can also refer to the thermal stability of AAV in a pharmaceutical composition to withstand high temperatures or temperature changes. Thermal denaturation of AAV involves two pathways: genome ejection and capsid disruption. Genome ejection of the AAV capsid occurs at relatively low temperatures, and DNA begins to escape from the intact capsid. AAV capsid disruption occurs when protein unfolding causes the viral capsid to lose structural integrity and break. In order to monitor or evaluate the thermal stability of AAV, it can be verified by conventional experimental methods in the art such as ELISA, qPCR, AAV-ID, and high-throughput thermal stability analysis. In some embodiments, the present invention utilizes the Uncle (Unchained Labs) multifunctional protein stability analysis system to monitor the genome ejection and capsid disruption of AAV to evaluate the thermal stability of the protein. Genome ejection is tracked using DNA-binding fluorescent dyes (such as SYBR Gold) (nucleic acid dyes can bind to nucleic acids ejected from AAV and emit fluorescence. The more nucleic acids ejected, the stronger the fluorescence signal after binding). The melting temperature (Tm) is determined based on DNA release. A higher Tm temperature indicates better 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, or at least 1°C, or at least 1.5°C, or at least 2°C, or at least 2.5°C, or at least 3°C, or at least 3.5°C, or at least 4°C, or at least 4.5°C, or at least 5°C or more relative to other compositions. AAV capsid disruption can be studied by monitoring the intrinsic protein fluorescence of the capsid protein on Uncle in the absence of a dye to determine its disruption (when the protein is heated to a certain degree, the protein unfolds, the hydrophobic region is exposed, and the protein autofluorescence changes. Therefore, changes in the protein autofluorescence can be used to determine whether the protein structure has changed). The AAV capsid protein in the pharmaceutical composition of the present invention undergoes substantially no structural changes. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] FIG1 is the isothermal stability test result of the sample in Example 2.

[0062] FIG2 is the isothermal stability test result of the sample in Example 2 after 5 freeze-thaw cycles.

[0063] FIG3 is the isothermal stability test result of the sample in Example 3.

[0064] FIG4 shows the isothermal stability test results of the sample in Example 3 after 5 freeze-thaw cycles.

[0065] FIG5 is the isothermal stability test result of the sample in Example 5. DETAILED DESCRIPTION

[0066] The present invention will be further described below with reference to specific examples. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0067] Example 1 Preparation of recombinant adeno-associated virus vector (rAAV) stock solution

[0068] Conventional DNA recombination and cloning techniques are used to construct and clone a recombinant plasmid containing the target protein gene of the present invention in Escherichia coli for subsequent preparation of recombinant AAV virus.

[0069] First, a polynucleotide expression cassette and recombinant plasmid encoding the gene of interest are constructed. Exemplarily, the 5' to 3' sequence of the expression cassette or plasmid includes: AAV 5' ITR, CBA promoter, Chi intron, Kozak sequence, nucleic acid encoding an anti-VEGF protein, rabbit-globin polyadenylation signal, and AAV 3' ITR. The anti-VEGF protein encoding sequence in the polynucleotide expression cassette and recombinant plasmid encodes an aflibercept fusion protein. Aflibercept has the amino acid sequence described in SEQ ID NO: 1. A recombinant adeno-associated viral vector (rAAV) is prepared using a three-plasmid co-transfection process known in the art. The AAV capsid used in this example is a modified AAV8 capsid protein, which includes a polypeptide sequence described in SEQ ID NO: 4 (RGNQQNTARQ) replacing amino acids Q588 to A592 (QQNTA) of the parent AAV8. The construction of the plasmid containing the capsid protein is described in detail in the related patent PCT / CN2022 / 142185 (Invention Title: Modified AAV Capsid Protein and Its Use), which is introduced here.

[0070] AAV packaging is generated by co-transfecting cells with three plasmids: a first plasmid containing the ITRs flanked by the anti-VEGF protein coding sequence, constructed as described above; a second plasmid encoding the Rep / Cap genes, as described above; and a third plasmid containing adenoviral helper genes. After transfection, cells are harvested, lysed, and the virus is released. Viral titer is determined by PCR and used for future use.

[0071] Example 2

[0072] The rAAV prepared in Example 1 was used to prepare a sample according to Table 1, wherein the concentration of rAAV was 1×10 12 vg / mL.

[0073] Table 1

[0074] The thermal stability of rAAV in the above formulations was verified by isothermal stability experiments. In this experiment, the sample was maintained at a specific temperature for a period of time (e.g., 18 hours, 24 hours), and structural changes in the rAAV capsid protein (e.g., aggregation / denaturation) were monitored in real time using fluorescence, static light scattering (SLS), and dynamic light scattering (DLS). The specific procedure was as follows: 9 μl of the above formulation sample was added to a Uni tube and analyzed using the "Isothermal Toolbox" program in the high-throughput multiparameter protein temperature analysis system (UNcle, UNCHAINED LABS). The parameters of the high-throughput multiparameter protein temperature analysis system were: temperature 30°C, constant temperature time: 5 minutes, observation time: 20 hours, UV 266 filter 1 (0.50), blue laser filter 3 (0.25). The thermal stability of the protein samples in different formulations was observed over time (abscissa) using the relative BCM (300-430 nm) response (ordinate). The results are shown in Figure 1. By superimposing the time-varying curves of the samples to be compared and observing the early and rapid rise of the curves, the early and rapid aggregation / denaturation of the samples can be determined, thereby evaluating the sample stability. As can be seen from the thermal stability curves in Figure 1, the curves of the citric acid buffer system formulations (Formulations 6-10) are more gradual and have relatively better thermal stability than those of the phosphate buffer system formulations (Formulations 1-3) and the HPEPS buffer system formulations (Formulations 4-6). Furthermore, in the citric acid buffer system formulations, the addition of arginine (Formulations 9-10) is more helpful in improving the stability of the formulations than proline (Formulation 7).

[0075] At the same time, samples of the formulations were subjected to five freeze-thaw cycles, and the isothermal thermal stability of the samples was again assessed using a high-throughput multi-parameter protein temperature analysis system, using the same conditions as the unfrozen-thawed samples. The results are shown in Figure 2. Figure 2 also shows that, consistent with pre-freeze-thaw conditions, after freeze-thaw cycles, the curves of the citric acid buffer formulations (formulations 6-10) were smoother and more stable than those of the phosphate buffer formulations (formulations 1-3) and HPEPS buffer formulations (formulations 4-6), with formulations 9-10 exhibiting the best stability.

[0076] Example 3

[0077] The rAAV prepared in Example 1 was used to prepare a sample according to Table 2, wherein the concentration of rAAV was 1×10 12 vg / mL.

[0078] Table 2

[0079] The thermal stability of rAAV in the above formulations was verified by isothermal stability experiments. In this isothermal stability experiment, the sample was maintained at a specific temperature for a period of time (e.g., 18 hours, 24 hours), and structural changes in the rAAV capsid protein (e.g., aggregation / denaturation) were monitored in real time using fluorescence, static light scattering (SLS), and dynamic light scattering (DLS). The specific procedure was as follows: 9 μl of the above formulation sample was added to a Uni tube and analyzed using the "Isothermal Toolbox" program in the high-throughput multi-parameter protein temperature analysis system (UNcle, UNCHAINED LABS). The parameters of the high-throughput multi-parameter protein temperature analysis system were: temperature 30°C, constant temperature time: 5 minutes, observation time: 18 hours, UV 266 filter 1 (0.50), Blue Laser Filter 3 (0.25). The thermal stability of the protein samples in different formulations was observed over time (abscissa) using the relative BCM (300-430 nm) response (ordinate). The results are shown in Figure 3. By superimposing the time-dependent curves of the samples to be compared and observing the early and rapid rise of the curves, the early and rapid rise of the sample aggregation / denaturation can be determined, thereby evaluating the sample stability. As shown in Figure 3, among the citrate buffer system formulations, the curves of the formulations with arginine (Formulations 11, 14-15) are more gradual than those with proline and aspartic acid (Formulations 12-13), indicating relatively better thermal stability.

[0080] At the same time, the formulation samples were subjected to five freeze-thaw cycles. The isothermal thermal stability of the samples was again assessed using a high-throughput multi-parameter protein temperature analysis system, using the same conditions as the unfrozen-thawed samples. The results are shown in Figure 4. Consistent with the pre-freeze-thaw conditions, the curves for the formulations supplemented with arginine (Formulations 11, 14-15) after freeze-thaw cycles showed a more gradual change than those supplemented with proline and aspartic acid (Formulations 12-13), indicating relatively better thermal stability.

[0081] Example 4

[0082] The rAAV prepared in Example 1 was used to prepare a sample according to Table 3, wherein the concentration of rAAV was 1×10 12 vg / mL.

[0083] Table 3

[0084] Sample testing: Uncle equipment was used to test the Tm value of the capsid protein in each preparation, as well as the Tm value of gene expression after the addition of nucleic acid dyes. Simultaneously, each sample was freeze-thawed five times (≤-65°C for ≥2 hours, then room temperature for ≥0.5 hours, with each cycle consisting of one cycle). The post-freeze-thaw Tm value was then tested with the addition of nucleic acid dyes.

[0085] To determine the Tm value of the capsid protein in the formulation, 9 μl of the formulation sample was added to a Uni tube. Real-time analysis was performed using the "Tm, Tagg & Optional DLS" program in a high-throughput multi-parameter protein temperature analysis system (UNcle, UNCHAINED LABS). The system settings were: start temperature 15°C, incubation time 180 s, ramp rate 0.5°C / min, end temperature 95°C, UV 266 Opaque (0.00), and Blue Laser open (1.00).

[0086] The specific operation of testing the Tm value of gene ejection after adding nucleic acid dye is as follows: first, treat the rAAV sample with nucleic acid dye, and then use fluorescence, static light scattering (SLS), dynamic light scattering (DLS) and other methods to detect the gene ejection of the sample in a certain temperature range (25℃~95℃) in real time. TM GLOD nucleic acid dye, 9 μl, was added to a Uni tube. Real-time detection was then performed using the "Tm, Tagg & Optional DLS" program in a high-throughput multi-parameter protein temperature analysis system (UNcle, UNCHAINED LABS). The system settings were: start temperature 15°C, incubation time 180 s, ramp rate 0.5°C / min, end temperature 95°C, UV 266 Opaque (0.00), and Blue Laser open (1.00).

[0087] The results are shown in Table 4. It can be seen that the rAAV preparation at pH 6 has better stability than that at pH 7 and pH 8.

[0088] Table 4

[0089] Example 5

[0090] The rAAV prepared in Example 1 was used to prepare a sample according to Table 5, wherein the concentration of rAAV was 1×10 13 vg / mL.

[0091] Table 5

[0092] The thermal stability of rAAV in the above formulations was verified by isothermal stability experiments. In this isothermal stability experiment, the sample was maintained at a specific temperature for a period of time (e.g., 18 hours, 24 hours), and structural changes in the rAAV capsid protein (e.g., aggregation / denaturation) were monitored in real time using fluorescence, static light scattering (SLS), and dynamic light scattering (DLS). The specific procedure was as follows: 9 μl of the above formulation sample was added to a Uni tube and the "Isothermal" program was selected from the "Isothermal Toolbox" in the high-throughput multi-parameter protein temperature analysis system (UNcle, UNCHAINED LABS). The parameters of the high-throughput multi-parameter protein temperature analysis system were: temperature 30°C, constant temperature time: 5 minutes, observation time: 18 hours, UV 266 filter 1 (0.50), blue laser filter 3 (0.25). The thermal stability changes of the protein samples in different formulations over time (abscissa) were observed by the relative BCM (300-430 nm) response value (ordinate). The results are shown in Figure 5. By superimposing the time-dependent curves of the samples to be compared and observing the early and rapid rise of the curves, we can determine the early and rapid rate of sample aggregation / denaturation, and thus evaluate the sample stability. As can be seen from the thermal stability curves in Figure 5, Formulation 15 exhibits a more gradual rise and relatively better thermal stability than Formulations 19-21.

[0093] After the formulation in Table 5 was placed at 25°C for 5 days, the protein expression level was tested. The method included taking a HEK293 cell suspension (density 1×10 5 100 μl / well of the solution was added to a 96-well plate and cultured overnight in a 37°C, 5% carbon dioxide incubator. 10 100 μl / well of a 500 μg / ml sample solution of the preparation was added and incubated in a 37°C, 5% CO2 incubator for 24 hours. The 96-well plate was then removed and the supernatant discarded. 100 μl each of DMEM complete medium and Luciferase colorimetric solution, restored to room temperature, was added to each well and incubated in the dark at room temperature for 5 minutes. Finally, the samples were read using a microplate reader for Luciferase colorimetric readings. Samples from each group stored in a refrigerator ≤-65°C were simultaneously tested, and the results were used as the control sample data. The ratio of the test results of the samples after 5 days at 25°C to the control sample was the relative protein expression level. The results are shown in Table 6.

[0094] Table 6

[0095] Example 6

[0096] The rAAV prepared in Example 1 was used to prepare a sample according to Table 7, wherein the concentration of rAAV was 1×10 13 vg / mL.

[0097] Table 7

[0098] After the formulation in Table 7 was placed at 25°C for 5 days, the protein expression was tested. The method included taking a HEK293 cell suspension (density 1×10 5 100 μl / well of the solution was added to a 96-well plate and cultured overnight in a 37°C, 5% carbon dioxide incubator. 10 100 μl / well of the sample solution (vg / ml) was added and incubated in a 37°C, 5% CO2 incubator for 24 hours. The 96-well plate was then removed and the supernatant discarded. 100 μl each of DMEM complete medium and Luciferase colorimetric solution, restored to room temperature, was added to each well and incubated in the dark at room temperature for 5 minutes. Finally, the samples were read using a microplate reader for Luciferase colorimetric readings. Samples from each group stored in a refrigerator ≤-65°C were simultaneously tested, and the results were used as the control sample data. The ratio of the test results of the samples after 5 days at 25°C to the control sample was the relative protein expression level. The results are shown in Table 8.

[0099] Table 8

[0100] Example 7

[0101] For the preparation of 1.0×10 13 Long-term stability (≤-65°C, 12 months and freeze-thaw cycle, 3 months) of formulation sample 15 at a concentration of vg / mL was evaluated. Evaluation criteria included genomic titer, relative protein expression after infection, and protein biological activity after infection, as shown in Table 9.

[0102] Table 9

Claims

1. A pharmaceutical composition comprising a recombinant adeno-associated virus, characterized in that: The pharmaceutical composition further comprises a buffer, an amino acid, a stabilizer, and a nonionic surfactant, wherein the buffer is a citrate buffer, preferably, the citrate is sodium citrate; the amino acid is selected from aspartic acid, arginine, glycine, histidine, and proline, preferably, the amino acid is arginine; the stabilizer is selected from sucrose, trehalose, mannitol, lactose, galactose, glucose, and maltose, preferably sucrose or trehalose; the nonionic surfactant is selected from poloxamer 188, polysorbate 20, polysorbate 80, HS15, and TPGS, preferably poloxamer 188, polysorbate 20, and polysorbate 80.

2. A pharmaceutical composition according to any one of the preceding claims, characterized in that The concentration of the buffer in the pharmaceutical composition is 10mM to 200mM, the concentration of the amino acid is 10mM to 200mM, the concentration of the stabilizer is 1% to 20wt%, and the concentration of the non-ionic surfactant is 0.001% to 0.1wt%; preferably, the concentration of the buffer in the pharmaceutical composition is 10mM to 100mM, the concentration of the amino acid is 20mM to 150mM, the concentration of the stabilizer is 2.5% to 10wt%, and the concentration of the non-ionic surfactant is 0.001% to 0.05wt%; more preferably, the concentration of the buffer in the pharmaceutical composition is 20mM to 40mM, the concentration of the amino acid is 40mM to 100mM, the concentration of the stabilizer is 3% to 10wt%, and the concentration of the non-ionic surfactant is 0.001% to 0.05wt%.

3. A pharmaceutical composition according to any one of the preceding claims, characterized in that The pharmaceutical composition comprises 40mM buffer, 40mM amino acid, 5%wt stabilizer, and 0.001% to 0.05wt% non-ionic surfactant; or the pharmaceutical composition comprises 40mM buffer, 100mM amino acid, 5%wt stabilizer, and 0.001% to 0.05wt% non-ionic surfactant; or the pharmaceutical composition comprises 40mM buffer, 60mM amino acid, 5%wt stabilizer, and 0.001% to 0.05wt% non-ionic surfactant; or the pharmaceutical composition comprises 20mM buffer, 100mM amino acid, 5%wt stabilizer, and 0.001% to 0.05wt% non-ionic surfactant.

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

2.

5. A pharmaceutical composition according to any one of the preceding claims, characterized in that The recombinant adeno-associated virus capsid protein serotype is AAV8 capsid protein or a modified AAV8 capsid protein.

6. A pharmaceutical composition according to any preceding claim, characterized in that The recombinant adeno-associated virus comprises a coding sequence encoding an anti-VEGF protein, and the anti-VEGF protein is a fusion protein comprising the amino acid sequence of SEQ ID NO: 1; preferably, the recombinant adeno-associated virus comprises: (i) rAAV capsid protein, wherein the capsid protein is AAV8 capsid protein or a modified AAV8 capsid protein; and (ii) a polynucleotide expression cassette comprising, from 5' to 3': (a) AAV 5'ITR; (b) CBA promoter; (c) Chi intron; (d) Kozak sequence; (e) a nucleic acid encoding an anti-VEGF protein, wherein the nucleic acid has a nucleotide sequence as described in SEQ ID NO: 2; (f) rabbit-globin polyadenylation signal; (g) AAV 3'ITR; More preferably, the polynucleotide expression cassette comprises the nucleotide sequence of SEQ ID NO: 3 from 5' to 3'.

7. The pharmaceutical composition according to claim 5, characterized in that The capsid protein is a modified AAV8 capsid protein, and the modified AAV8 capsid protein is replaced by the polypeptide sequence described in SEQ ID NO: 4 at amino acids 588 to 592 of the parent AAV8.

8. A pharmaceutical composition according to any one of the preceding claims, characterized in that The pharmaceutical composition is an intravitreal injection, a subretinal injection preparation, an intrachoroidal injection preparation, an intravenous injection preparation, an intratumoral injection preparation or an intramuscular injection preparation; preferably, the pharmaceutical composition is an intravitreal injection, a subretinal injection preparation, or an intrachoroidal injection preparation.

9. A pharmaceutical composition according to any one of the preceding claims, characterized in that The pharmaceutical composition is a liquid preparation.

10. A pharmaceutical composition according to any one of the preceding claims, characterized in that The pharmaceutical composition is a lyophilized preparation.

11. A pharmaceutical composition according to any one of the preceding claims, characterized in that The pharmaceutical compositions are stored in unit dose containers.

12. The pharmaceutical composition according to claim 11, characterized in that The unit dose container is a vial or a syringe.

13. The pharmaceutical composition according to claim 12, characterized in that The vial is a glass vial.

14. The pharmaceutical composition according to claim 12, characterized in that The syringe is a pre-filled syringe.

15. A pharmaceutical composition according to any preceding claim, characterized in that The genome concentration of the recombinant adeno-associated virus in the pharmaceutical composition is about 1*10 7 vg / ml to 1*10 14 vg / ml; preferably, the genome concentration of the recombinant adeno-associated virus is about 1*10 9 vg / ml to 1*10 13 vg / ml; more preferably, the genome concentration of the recombinant adeno-associated virus is about 1*10 9 vg / ml to 1*10 12 vg / ml.

16. Use of a pharmaceutical composition according to any preceding claim in the preparation of a medicament for treating a disease associated with VEGF.

17. A method for treating a disease associated with VEGF, the method comprising administering to a patient / subject in need thereof a therapeutically effective amount of the pharmaceutical composition of any one of claims 1 to 15.

18. The use according to claim 16 or the method according to claim 17, characterized in that The disease associated with VEGF is an ocular neovascularization disease.

19. The use according to claim 16 or the method according to claim 17, characterized in that The ocular neovascular disease 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, wet age-related macular degeneration with very low vision, choroidal neovascularization secondary to pathological myopia, neovascular glaucoma, iris neovascular disease, and retinopathy of prematurity.

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