Pharmaceutical compositions of non-enveloped viruses

Abstract

The present invention relates to the fields of medicine, gene therapy and medicine, in particular to pharmaceutical compositions of vectors based on recombinant non-enveloped viruses, in particular recombinant adeno-associated viruses (rAVVs), which can be used to treat and prevent various diseases.

Description

[Technical Field]

[0001] The present invention relates to the fields of medicine, gene therapy and medicine, in particular to pharmaceutical compositions of vectors based on recombinant non-enveloped viruses, in particular recombinant adeno-associated viruses (rAAV), which can be used to treat and prevent various diseases. [Background technology]

[0002] Typical representatives of non-enveloped viruses are parvoviruses, noroviruses, as well as rotaviruses and adenoviruses. Simple or non-enveloped viruses consist of nucleic acid and a protein envelope called a capsid. The capsid is made up of repeating morphological subunits called capsomers. The nucleic acid and capsid interact to form a nucleocapsid. Within each virus, oligomerization of capsid proteins during capsid assembly typically results in some type of symmetrical quaternary structure. Most viruses have capsids with helical or icosahedral structures (Lidmar J, Mirny L, Nelson Dr. Virus shapes and buckling transitions in spherical shells. Phys Rev E Stat Nonlin Soft Matter Phys. 2003 Nov;68(5 Pt 1):051910. doi:10.1103 / PhysRevE.68.051910. Epub 2003 Nov 25. PMID:14682823).

[0003] Unlike non-enveloped viruses, the capsid of enveloped viruses is surrounded by a lipid membrane known as the supercapsid. The envelope is derived from the capsid derived from the intracellular membrane of the virus's host, such as the inner nuclear membrane, the Golgi membrane, and the outer membrane of the cell. The presence of a lipid envelope makes the virus less resistant to physical and chemical stress (Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD (1994). Molecular Biology of the Cell (4th ed.). p. 280).

[0004] The capsid of non-enveloped viruses functions to protect the genome during long-term storage and from chemical and physical stresses, such as ultraviolet radiation, extremes in pH or temperature, proteolytic and nucleolytic agents, etc. Non-enveloped viruses can retain these properties for long periods of time in dry conditions on a variety of surfaces, both porous and non-porous (Abad FX, Pinto RM, Bosch A. Survival of enteric viruses on environmental fomites. Appl Environ Microbiol. 1994 Oct;60(10):3704-10. doi: 10.1128 / aem.60.10.3704-3710.1994. PMID: 7986043; PMCID: PMC201876).

[0005] Because viruses transfer their genetic material into the genome of host cells, it has been proposed that the host cell can use the transferred genome as a vector to deliver genetic information to cells. Vectors based on retroviruses, adenoviruses, adeno-associated viruses (AAV), herpes simplex viruses, etc. are used for therapeutic purposes. Among these viruses, adenoviruses and AAV are non-enveloped viruses.

[0006] Recombinant AAV and recombinant adenovirus-based vectors are currently the most widely used and developed gene therapy products, and these vectors are also being used in vaccine development.

[0007] To date, gene therapy has been shown to be a potentially universal approach to treat a wide range of diseases, including infectious diseases, genetic disorders, and malignant neoplasms. Viruses or viral vectors (viral particles) must maintain their structural integrity during production, transportation, storage, and use to exert infectious and biological activity. The structural integrity of viral vectors can be destroyed during production, transportation, storage, and use of vaccines or products containing viral vectors, thus precluding their use as delivery vectors.

[0008] The prior art provides liquid pharmaceutical compositions of AAV (WO2018 / 128689, WO2019 / 094253, WO2020 / 014479, WO2020 / 214929) and liquid pharmaceutical compositions of adenovirus (WO00 / 29024, WO2017 / 013169). Summary of the Invention [Problem to be solved by the invention]

[0009] However, there remains a need for improved, highly stable pharmaceutical compositions comprising recombinant non-enveloped virus-based vectors. [Brief explanation of the drawings]

[0010] [Figure 1] Figure 1 is a graph showing the change in particle size of adeno-associated virus serotype 9-based vectors as a function of time in test formulations under heat stress at 65°C. HisArg, His, Tris, PBS, and PhosCitr are the pharmaceutical compositions HisArg AAV9, His AAV9, Tris AAV9, PBS AAV9, and PhosCitr AAV9, respectively, as shown in Table 9. [Figure 2]Figure 2 is a graph showing the change in particle size of adeno-associated virus serotype 5-based vectors as a function of time in test formulations under heat stress at 40°C. HisArg, His, Tris, PBS, PhosCitr - are the pharmaceutical compositions HisArg AAV5, His AAV5, Tris AAV5, PBS AAV5, and PhosCitr AAV5, respectively, as shown in Table 18. DETAILED DESCRIPTION OF THE INVENTION

[0011] Surprisingly, the present authors have produced a stable pharmaceutical composition containing a vector based on a recombinant non-enveloped virus, particularly a recombinant adeno-associated virus (rAAV). This pharmaceutical composition has increased colloidal stability (higher aggregation temperature), DNA retention stability (higher temperature for DNA extraction), and stability during storage in frozen and liquid form (no loss of titer during storage at (5±3)°C, and reduced loss of titer under heat stress). The developed pharmaceutical composition can be used to treat and prevent various diseases. definition Unless otherwise defined herein, all technical and scientific terms used in connection with the present invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0012] Further, unless otherwise required by context, singular terms shall include plural terms and plural terms shall include the singular term. In this specification and in the claims that follow, unless otherwise dictated by context, the words "include" and "comprise," or variations thereof such as "includes," "including," "comprises," or "comprising," will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0013] The term "pharmaceutical composition" refers to a composition and / or formulation comprising a therapeutically effective amount of a vector based on a recombinant non-enveloped virus and excipients or auxiliary substances (carriers, diluents, fillers, solvents, etc.), the selection and proportions of which depend on the type and route of administration and the dosage.

[0014] As used herein, the term "aqueous composition" refers to a water-based composition, and the water in the composition can be water, water for injection, or saline (0.9% to 1.0% aqueous sodium chloride solution).

[0015] As used herein, the term "lyophilized" refers to a formulation that has undergone a process known in the art as lyophilization, which involves freezing the formulation followed by removal of ice from the frozen contents.

[0016] A pharmaceutical composition is "stable" if the active agent retains its physical and / or chemical stability and / or biological activity for a specified storage period at a storage temperature, e.g., (5±3)°C. Furthermore, the active agent can retain both physical and chemical stability, as well as biological activity. The storage period is adjusted based on stability results under accelerated or natural aging conditions.

[0017] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Also used herein, the term "vector" refers to a recombinant viral particle capable of transporting a nucleic acid.

[0018] The terms "long-term storage" or "long-term stability" should be understood to mean that the pharmaceutical composition can be stored for three months or more, six months or more, one year or more, and the composition can also have a minimum stable shelf-life of at least two years. Generally speaking, the terms "long-term storage" and "long-term stability" further include a stable shelf-life that is at least comparable to or better than the stable shelf-life typically required for currently available commercial formulations, without loss of stability that could render the formulation unsuitable for its intended pharmaceutical application.

[0019] The term "buffering agent" refers to the acid or base component (typically a weak acid or a weak base) of a buffer solution or buffer. Buffering agents help maintain the pH value of a solution at or near a predetermined value, and buffering agents are generally selected to achieve the predetermined value. A buffering agent may be a single compound that produces the desired buffering effect, particularly when the buffering agent is mixed with (and is capable of adequate proton exchange with) an appropriate amount (dependent on the desired predetermined value) of its corresponding "acid / base conjugate."

[0020] The term "buffer" or "buffer solution" refers to an aqueous solution containing a mixture of an acid (typically a weak acid, e.g., acetic acid, citric acid, etc.) and its conjugate base (e.g., acetate or citrate salts, e.g., sodium acetate, sodium citrate, etc., and hydrates of said salts, e.g., sodium acetate trihydrate), or a mixture of a base (typically a weak base, e.g., histidine) and its conjugate acid (e.g., histidine hydrochloride or histidine hydrochloride monohydrate or L-histidine hydrochloride (h / c) monohydrate (m / h) or L-histidine h / cm / h or histidine h / cm / h). The pH value of a "buffer solution" can be slightly changed by adding small amounts of a strong base or strong acid to it, as well as by diluting or concentrating it due to the "buffering effect" provided by the "buffering agent."

[0021] Typically, amino acid is L-amino acid.For example, when histidine and histidine hydrochloride monohydrate are used, they are typically L-histidine and L-histidine hydrochloride monohydrate.For example, when arginine is used, they are typically L-arginine.Amino acid equivalents, such as pharmaceutically acceptable proline salts (for example, proline hydrochloride), can also be used. Abbreviation AAV - adeno-associated virus BHQ-1 - a fluorescence extinguisher used in PCR Ct - cycle threshold FAM - Carboxyfluorescein dye GFP - Green Fluorescent Protein GOI - Gene of Interest MOI - Multiplicity of infection (number of virus particles per cell) rAAV - recombinant adeno-associated virus TU - Transducing Unit Vg - viral genome PCR - polymerase chain reaction ELISA - Enzyme-Linked Immunosorbent Assay FA - Functional Activity SE HPLC - Size Exclusion High Performance Liquid Chromatography The present invention discloses stable pharmaceutical compositions of vectors based on recombinant non-enveloped viruses, in particular recombinant adeno-associated viruses (rAAV), which can be used to treat and prevent a variety of diseases.

[0022] In one aspect, the invention provides a pharmaceutical composition of a recombinant non-enveloped virus-based vector, comprising: (i) a recombinant non-enveloped virus-based vector; (ii) histidine, and (iii) arginine, (iv) sodium chloride; (v) magnesium chloride; (vi) a surfactant; and (vii) Water for injection; The present invention relates to a composition comprising:

[0023] In some embodiments of the invention, histidine is present at a concentration of 2.0 to 3.58 mg / ml, or 2.3 to 3.2 mg / ml, or 2.6 to 3.0 mg / ml, or 2.7 to 2.8 mg / ml, or 2.79 mg / ml.

[0024] In some embodiments of the invention, arginine is present at a concentration of 0.248 to 0.448 mg / ml, or 0.28 to 0.416 mg / ml, or 0.3 to 0.396 mg / ml, or 0.340 to 0.350 mg / ml, or 0.348 mg / ml.

[0025] In some embodiments of the invention, sodium chloride is present in a concentration of 8.0 to 15.0 mg / ml, or 9.5 to 13.5 mg / ml, or 10.5 to 12.5 mg / ml, or 11.0 to 12.0 mg / ml, or 11.7 mg / ml.

[0026] Magnesium chloride can be used in both the form of anhydrous magnesium chloride and its hydrate form. In some aspects of the present invention, the magnesium chloride is magnesium chloride hexahydrate.

[0027] In some embodiments of the invention, magnesium chloride is present in a concentration of 0.15-0.50 mg / ml, or 0.16-0.35 mg / ml, or 0.18-0.25 mg / ml, 0.200-0.210 mg / ml, or 0.203 mg / ml.

[0028] Any pharmaceutically acceptable surfactant can be used as the surfactant. Surfactants are well known in the art and include, but are not limited to, polysorbate 20, polysorbate 80, various poloxamers and Pluronics®, and mixtures thereof.

[0029] In some embodiments of the invention, the surfactant is present in a concentration of 0.01 to 1.0 mg / ml, or 0.01 to 0.5 mg / ml, or 0.01 to 0.2 mg / ml, or 0.03 to 0.15 mg / ml, or 0.05 mg / ml, or 0.1 mg / ml.

[0030] In some aspects of the invention, the pharmaceutical composition comprises: (i) a recombinant non-enveloped virus-based vector; (ii) histidine 2.0 to 3.58 mg / ml; (iii) arginine 0.248 to 0.448 mg / ml; (iv) sodium chloride 8.0 to 15.0 mg / ml; (v) magnesium chloride 0.15 to 0.50 mg / ml; (vi) a surfactant of 0.01 to 1.0 mg / ml; (vii) Water for injection, quantified to 1.0 ml; Includes:

[0031] In some aspects of the invention, the pharmaceutical composition comprises: (i) a recombinant non-enveloped virus-based vector; (ii) histidine 2.3 to 3.2 mg / ml; (iii) arginine 0.28 to 0.416 mg / ml; (iv) sodium chloride 9.5 to 13.5 mg / ml; (v) magnesium chloride 0.16 to 0.35 mg / ml; (vi) a surfactant of 0.01 to 0.2 mg / ml; (vii) Water for injection, quantified to 1.0 ml; Includes:

[0032] In some aspects of the invention, the pharmaceutical composition comprises: (i) a recombinant non-enveloped virus-based vector; (ii) histidine 2.6 to 3.0 mg / ml; (iii) arginine 0.3 to 0.396 mg / ml; (iv) sodium chloride 10.5 to 12.5 mg / ml; (v) magnesium chloride 0.18 to 0.25 mg / ml; (vi) a surfactant of 0.03 to 0.15 mg / ml; (vii) Water for injection, quantified to 1.0 ml; Includes:

[0033] In some aspects of the invention, the pharmaceutical composition comprises: (i) a recombinant non-enveloped virus-based vector; (ii) histidine 2.7 to 2.8 mg / ml; (iii) arginine 0.340 to 0.350 mg / ml; (iv) sodium chloride 11.0 to 12.0 mg / ml; (v) magnesium chloride 0.200 to 0.210 mg / ml; (vi) a surfactant of 0.03 to 0.15 mg / ml; (vii) Water for injection, quantified to 1.0 ml; Includes:

[0034] In some aspects of the invention, the pharmaceutical composition comprises: (i) a recombinant non-enveloped virus-based vector; (ii) histidine 2.79 mg / ml; (iii) arginine 0.348 mg / ml; (iv) sodium chloride 11.7 mg / ml; (v) magnesium chloride 0.203 mg / ml; (vi) 0.05 mg / ml of a surfactant; (vii) Water for injection, quantified to 1.0 ml; Includes:

[0035] In some aspects of the invention, the pharmaceutical composition comprises: (i) a recombinant non-enveloped virus-based vector; (ii) histidine 2.79 mg / ml; (iii) arginine 0.348 mg / ml; (iv) sodium chloride 11.7 mg / ml; (v) magnesium chloride 0.203 mg / ml; (vi) 0.1 mg / ml of a surfactant; (vii) Water for injection, quantified to 1.0 ml; Includes:

[0036] In some aspects of the invention, the magnesium chloride is magnesium chloride hexahydrate. In some embodiments of the present invention, the surfactant is poloxamer 188. In some aspects of the invention, the pharmaceutical composition comprises: (i) a recombinant non-enveloped virus-based vector; (ii) histidine 2.0 to 3.58 mg / ml; (iii) arginine 0.248 to 0.448 mg / ml; (iv) sodium chloride 8.0 to 15.0 mg / ml; (v) magnesium chloride hexahydrate 0.15 to 0.50 mg / ml; (vi) 0.01 to 1.0 mg / ml of a surfactant that is poloxamer 188; (vii) Water for injection, quantified to 1.0 ml; Includes:

[0037] In some aspects of the invention, the pharmaceutical composition comprises: (i) a recombinant non-enveloped virus-based vector; (ii) histidine 2.3 to 3.2 mg / ml; (iii) arginine 0.28 to 0.416 mg / ml; (iv) sodium chloride 9.5 to 13.5 mg / ml; (v) magnesium chloride hexahydrate 0.16 to 0.35 mg / ml; (vi) 0.01 to 0.2 mg / ml of a surfactant that is poloxamer 188; (vii) Water for injection, quantified to 1.0 ml; Includes:

[0038] In some aspects of the invention, the pharmaceutical composition comprises: (i) a recombinant non-enveloped virus-based vector; (ii) histidine 2.6 to 3.0 mg / ml; (iii) arginine 0.3 to 0.396 mg / ml; (iv) sodium chloride 10.5 to 12.5 mg / ml; (v) magnesium chloride hexahydrate 0.18 to 0.25 mg / ml; (vi) 0.03 to 0.15 mg / ml of a surfactant that is poloxamer 188; (vii) Water for injection, quantified to 1.0 ml; Includes:

[0039] In some aspects of the invention, the pharmaceutical composition comprises: (i) a recombinant non-enveloped virus-based vector; (ii) histidine 2.7 to 2.8 mg / ml; (iii) arginine 0.340 to 0.350 mg / ml; (iv) sodium chloride 11.0 to 12.0 mg / ml; (v) magnesium chloride hexahydrate 0.200 to 0.210 mg / ml; (vi) 0.03 to 0.15 mg / ml of a surfactant that is poloxamer 188; (vii) Water for injection, quantified to 1.0 ml; Includes:

[0040] In some aspects of the invention, the pharmaceutical composition comprises: (i) a recombinant non-enveloped virus-based vector; (ii) histidine 2.79 mg / ml; (iii) arginine 0.348 mg / ml; (iv) sodium chloride 11.7 mg / ml; (v) magnesium chloride hexahydrate 0.203 mg / ml; (vi) 0.05 mg / ml of a surfactant which is poloxamer 188; (vii) Water for injection, quantified to 1.0 ml; Includes:

[0041] In some aspects of the invention, the pharmaceutical composition comprises: (i) a recombinant non-enveloped virus-based vector; (ii) histidine 2.79 mg / ml; (iii) arginine 0.348 mg / ml; (iv) sodium chloride 11.7 mg / ml; (v) magnesium chloride hexahydrate 0.203 mg / ml; (vi) 0.1 mg / ml of a surfactant which is poloxamer 188; (vii) Water for injection, quantified to 1.0 ml; Includes:

[0042] In some aspects of the invention, the histidine is L-histidine. In some aspects of the invention, the arginine is L-arginine. In some embodiments of the invention, the pharmaceutical composition has a pH of 7.0 to 9.0, 7.5 to 8.5, or 7.7 to 8.7, or 8.0, or 8.2.

[0043] In some aspects of the present invention, the pharmaceutical compositions described above are suitable for lyophilization, i.e., the pharmaceutical compositions can act as a pre-lyophilization solution. In one aspect, the present invention relates to pharmaceutical compositions of recombinant non-enveloped virus-based vectors, which are provided in a dry (i.e., powder or granular) form that is dissolved in a suitable solvent (e.g., water) prior to administration. Such pharmaceutical compositions can be prepared, for example, by a process known in the art as lyophilization, i.e., freeze-drying, which involves freezing the product followed by removal of the solvent from the frozen contents.

[0044] In one aspect, the present invention relates to a lyophilized pharmaceutical composition of a recombinant non-enveloped virus-based vector, which is prepared by lyophilizing any of the pharmaceutical compositions described above. Thus, the pharmaceutical composition of the present invention can be an aqueous pharmaceutical composition or a lyophilized pharmaceutical composition (lyophilizate).

[0045] The pharmaceutical compositions described are suitable for delivering therapeutic agents to subjects to treat or prevent various diseases or disorders.These compositions can be used in gene therapy to treat diseases such as hemophilia A, hemophilia B, malignant neoplasms, spinal muscular atrophy, and in vaccine development, for example, to prevent infectious diseases.

[0046] The pharmaceutical composition of the present invention can contain vectors based on recombinant non-enveloped viruses at various concentrations. The concentration of the viral vector depends, for example, on the disease that the pharmaceutical composition will be used to prevent or treat, as well as the age, weight, and health condition of the patient, and therefore may vary from patient to patient. The appropriate dose can be adjusted according to the doctor's decision, so that the patient can be administered one or multiple injections.

[0047] In some aspects of the invention, the recombinant non-enveloped virus-based vector is 1.0 x 10 5 ~1.0×10 14 viral genomes / ml, or 1.0 × 10 9 ~1.0×10 14 viral genomes / ml, or 1.0 × 10 9 ~5.0×10 13 viral genomes / ml, or 1.0 × 10 9 ~1.0×10 13 viral genomes / ml, or (1.0 ± 0.3) × 10 5 viral genomes / ml, or (2.0 ± 0.6) × 10 5 Viral genomes / ml, (5.0±1.5)×10 5 viral genomes / ml, or (1.0 ± 0.3) × 10 6viral genomes / ml, or (2.0 ± 0.6) × 10 6 Viral genomes / ml, (5.0±1.5)×10 6 viral genomes / ml, or (1.0 ± 0.3) × 10 7 viral genomes / ml, or (2.0 ± 0.6) × 10 7 Viral genomes / ml, (5.0±1.5)×10 7 viral genomes / ml, or (1.0 ± 0.3) × 10 8 viral genomes / ml, or (2.0 ± 0.6) × 10 8 Viral genomes / ml, (5.0±1.5)×10 8 viral genomes / ml, or (1.0 ± 0.3) × 10 9 viral genomes / ml, or (2.0 ± 0.6) × 10 9 Viral genomes / ml, (5.0±1.5)×10 9 viral genomes / ml, or (1.0 ± 0.3) × 10 10 viral genomes / ml, or (2.0 ± 0.6) × 10 10 Viral genomes / ml, (5.0±1.5)×10 10 viral genomes / ml, or (1.0 ± 0.3) × 10 11 viral genomes / ml, or (2.0 ± 0.6) × 10 11 Viral genomes / ml, (5.0±1.5)×10 11 viral genomes / ml, or (1.0 ± 0.3) × 10 12 viral genomes / ml, or (2.0 ± 0.6) × 10 12 Viral genomes / ml, (5.0±1.5)×10 12 viral genomes / ml, or (1.0 ± 0.3) × 10 13 viral genomes / ml, or (2.0 ± 0.6) × 10 13 Viral genomes / ml, (5.0±1.5)×10 13 viral genomes / ml, or (1.0 ± 0.3) × 10 14 Present at a concentration of viral genomes / ml.

[0048] The recombinant non-enveloped virus in the pharmaceutical composition may be rAAV, recombinant adenovirus and other types of recombinant non-enveloped viruses. Recombinant adenovirus-based vectors can be produced using any species, strain, serotype, or any combination of species, strains, or serotypes of adenovirus or chimeric adenovirus. Human adenovirus serotypes include any one of serotypes 2, 4, 5, 7, 11, 26, 34, 35, 36, 48, 49, or 50, or combinations, derivatives, variants, or pseudotypes thereof.

[0049] In some embodiments of the present invention, the vector based on a recombinant non-enveloped virus is a rAAV-based vector. This vector can be produced using any strain, AAV serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16) or any combination of strains, serotypes (e.g., rAAV-based vectors containing two or more serotypes), and can contain any serotype of AAV capsid protein (capsid) (AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16), or a combination, derivative, variant or pseudotype thereof.

[0050] In some embodiments of the invention, the rAAV-based vector comprises an AAV5 capsid, or an AAV6 capsid, or an AAV9 capsid. In some aspects of the invention, the AAV5 capsid or AAV6 capsid or AAV9 capsid can be a modified capsid.

[0051] rAAV-based vectors can be genetically and / or chemically modified. rAAV-based vectors can be genetically modified to produce rAAV-based vectors with altered receptor utilization, antigenicity, transduction efficiency, and / or tissue tropism, as well as to insert peptide ligands, antibodies, antibody fragments, MHC (major histocompatibility complex) and / or receptors into the viral capsid. For example, rAAV-based vectors can be genetically modified by introducing one or more amino acid mutations, such as point mutations.

[0052] The phrase "more point mutations" refers to 2, 3, 4, 5, 6, 7, 8, 9, or 10 point mutations. Particularly preferred embodiments include naturally occurring conservative substitutions (mutations), i.e., substitutions occurring within a family of amino acids joined at the side chain of an amino acid. In particular, amino acids are typically divided into four families: (1) acidic amino acids are aspartic acid and glutamic acid; (2) basic amino acids are lysine, arginine, and histidine; (3) nonpolar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, it is reasonably predictable that the single substitution of leucine for isoleucine or valine, aspartic acid for glutamic acid, and threonine for serine, or similar conservative substitutions of amino acids for structurally related amino acids, will not significantly affect biological activity. For example, a polypeptide of interest may contain up to about 5-10 conservative or non-conservative amino acid substitutions, as long as the desired function of the molecule remains intact.

[0053] Point mutation variants in the sequence of AAV protein VP1, VP2, or VP3 that use amino acid substitutions are substitutions of at least one amino acid residue in AAV protein VP1, VP2, or VP3 with another amino acid residue.

[0054] Conservative substitutions are shown in Table A under "preferred substitutions."

[0055] [Table 1]

[0056] For example, International Application WO2012 / 145601 describes adeno-associated virus (AAV) particles having a variant capsid protein, wherein the AAV viral particles exhibit higher infectivity of retinal cells compared to wild-type AAV when administered by intravitreal injection. International Application WO2013 / 158879 describes an adeno-associated virus (AAV) vector for delivering heterologous nucleic acid sequences to a subject, comprising a capsid protein VP1 containing one or more lysine substitutions, wherein one lysine substitution is K137R, and the lysine substitution is effective in inhibiting the ubiquitination of the capsid protein, thereby increasing the transduction efficiency of the AAV vector in target cells.

[0057] rAAV-based vectors can be chemically modified to alter tissue tropism. Chemically modified rAAV-based vectors can also exhibit altered receptor utilization, antigenicity, transduction efficiency, and / or tissue tropism. Chemically modified rAAV-based vectors can be created, for example, by using chemoselective reactions that can target specific amino acid side chains and can be used to alter the charge, polarity, hydrophobicity, and hydrogen-bonding potential of the interior of the receptor-binding domain on the AAV capsid.

[0058] In some aspects of the invention, the pharmaceutical compositions of the recombinant non-enveloped virus-based vectors of the invention are intended for parenteral administration. In some aspects of the invention, the pharmaceutical compositions of the recombinant non-enveloped virus-based vectors of the invention are intended for intraocular administration, including intravitreal, subretinal or suprachoroidal administration, as well as intramuscular, intravenous or subcutaneous administration.

[0059] In some aspects of the invention, the pharmaceutical compositions of recombinant non-enveloped virus-based vectors of the invention can be administered intravenously as an infusion. The pharmaceutical composition of the recombinant non-enveloped virus-based vector of the present invention can be used after dilution. To this end, the required amount of composition is transferred from the vial to an infusion container containing sterile 0.9% sodium chloride solution, sterile 5% dextrose solution, or other infusion fluid. The resulting solution is stirred by gently rotating the infusion container.

[0060] In some aspects of the invention, the subject of treatment, or patient, is a mammal, preferably a human subject. The subject can be male or female and of any age. The pharmaceutical composition of the present invention can be stored in any suitable container, such as a glass or plastic container of the desired volume, a vial, an ampoule, a syringe, a cartridge, or a bottle. The container can be equipped with an additional means for administration, such as a dropper, an auto-injector, etc.

[0061] The pharmaceutical compositions of the present invention can be prepared, packaged, or sold in the form of a single unit dose or multiple single unit doses in the form of a ready-to-use formulation. As used herein, the term "single unit dose" refers to a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of the active ingredient is typically equal to the dose of the active ingredient to be administered to a subject, or an appropriate fraction of such a dose, such as, for example, one-half or one-third of such a dose.

[0062] The pharmaceutical composition can be administered as a single therapeutic agent or in combination with additional therapeutic agents as needed.Therefore, in one embodiment, the present method for treatment and / or prevention is used in combination with the administration of a therapeutically effective amount of another active agent.The other active agent can be administered before, during, or after the administration of the pharmaceutical composition of the present invention.The other active agent can be administered as part of the present composition or as a separate formulation. [Example]

[0063] The following examples are provided for a better understanding of the present invention. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

[0064] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to those skilled in the art in light of the teachings of the invention that certain changes and modifications can be made thereto without departing from the spirit or scope of the accompanying embodiments. method 1. Determination of the concentration of the gene of interest (GOI) in the AAV product by PCR.

[0065] The concentration of the GOI in the AAV product was determined by quantitative polymerase chain reaction using forward and reverse primers and a probe conjugated to FAM dye and BHQ-1 quencher. The primers matched the region of the gene encoding the GOI protein. During sample preparation, the samples were first treated with DNase to remove residual plasmid DNA and then with proteinase to disrupt the capsid. A calibration curve was plotted using linearized plasmid (of the corresponding GOI). A 10-fold reduction in concentration of a standard standard was used to plot a calibration curve of log concentration as a function of cycle threshold (Ct). The concentration of viral genomes / ml (vg / ml) in the product was determined from the calibration curve of log concentration as a function of Ct. 2. Determination of viral particle concentration by enzyme-linked immunosorbent assay.

[0066] The concentration of viral vector particles (adeno-associated virus (AAV) capsids) was measured by enzyme-linked immunosorbent assay using the following commercially available kits: PROGEN AAV5 titration ELISA, PROGEN AAV6 titration ELISA, and PROGEN AAV9 titration ELISA.

[0067] High-binding 96-well plates are coated with anti-AAV capsid-binding monoclonal mouse antibodies that recognize conformational epitopes of specific serotypes. AAV capsid proteins bind to the antibodies under temperature incubation, and unbound components of the sample are removed by washing the plate multiple times.

[0068] A biotinylated antibody that binds to AAV capsid proteins under temperature incubation of the plate was used as the detection antibody. Unbound components are removed by washing the plate multiple times. Streptavidin-horseradish peroxidase complex was applied to the plate wells and color was detected. Streptavidin binds to biotin, and unbound components were removed by washing the plate multiple times.

[0069] Tetramethylbenzidine acts as a chromogen. The interaction of horseradish peroxidase, hydrogen peroxide, and tetramethylbenzidine chromogen produces a color reaction. The ELISA reaction was stopped by adding a stop solution (1 normal H2SO4), and the optical density of the solution in the well was measured at a wavelength of 450 nm. The concentration of viral vector particles in the product was determined by a calibration graph plotted using the optical density data of standard solutions with known concentrations of AAV particles. 3. Determination of the functional activity of products based on recombinant adeno-associated viral vectors containing the GFP gene.

[0070] The transduction activity of the product based on a recombinant adeno-associated virus vector containing the GFP gene was measured by GFP expression in CHO-K1 cells induced by the product. For transduction, on the day of analysis, CHO-K1 cells were transfected with 1000 cells / cm 2 1 x 10 per 4 The cells were seeded into 24-well plates in growth medium containing antibiotics (medium for MQD-quantitation assay) at a rate of 19,000 cells per well and incubated for 4-8 hours at 37°C in a 5% CO2 atmosphere to allow the cells to adhere to the plastic surface. Three doses of test sample, with three replicates per dose, were then introduced into the wells containing the cells. After transduction, the product dosage (MOI) for each AAV serotype was pre-determined to produce approximately 5%, 10%, and 20% GFP-positive cells, respectively. The amount of product required for transduction of a single well was calculated using the following formula: V=MOI×S×KC where V is the amount of product required per well (ml), S is the area of ​​a single well (cm), MOI, which is the multiplicity of infection (vg / cl), is the required dose of product, K is the cell seeding density in the well (cells / cm), and C is the concentration of genome-containing capsids in the product (vg / ml).

[0071] Plates containing transduced cells were incubated in a CO2 incubator (5% CO2, 37°C) for 2–3 days. After the incubation period, the culture medium was removed from the wells, the cells were washed with Hanks' solution, and the cells were removed from the plastic using TrypLE solution (150 μl / well). After detaching the cells from the plastic, Hanks' solution (1:1 with TrypLE) was added to the wells, the cells were collected in a 1.5 ml microtube, and centrifuged at 300 g for 5 min. The cell pellet was resuspended in 150 μl of Hanks' solution. Fifty ml of a working dye solution for determining viable cells in Hanks' solution was added to each sample, except for one isotype control well, and the cells were incubated at room temperature in the dark for 20 min. The stained cells were centrifuged at 300 g for 5 min, the supernatant was carefully selected, and 100 μl of BD Cytofix fixing buffer was added to each sample. The cells were resuspended, and the entire volume of the resulting cell suspension was transferred to a 96-well V-bottom plate for flow cytometry. The plate containing the cells was incubated at room temperature in the dark for 20 minutes. After the incubation period, the plate was centrifuged at 1200 rpm for 5 minutes, the fixation buffer was removed, and the cells were washed with 100 μl / well of stain buffer (PBS containing 0.1% sodium azide and 0.5% BSA). The plate was centrifuged again, the buffer was removed, and then 150 μl / well of stain buffer was introduced, and the cells were carefully resuspended.

[0072] The cells were analyzed by flow cytometer. Cell viability and GFP expression were assessed. For each product dose, the value of FA (functional activity) or TU (transducing units) / ml was calculated using the following formula: TU=P×NV×D where P is the number of GFP-positive viable cells as a percentage, N is the initial number of cells plated (N = S × K), V is the amount of product introduced per well (ml), and D is the product predilution factor.

[0073] The average TU value of the three sample doses was considered as the final result. 4. Determination of viral particle concentration by size exclusion high performance liquid chromatography. The concentration of viral particles was determined by measuring the peak area of ​​the test and standard substances on a high performance liquid chromatograph equipped with an Agilent UV detector, detector wavelengths: 260 nm and 280 nm.

[0074] Column: TSK-gel G3000SWXL 7.8 × 300 mm, 5 μm, 300 Å. Phase A: 0.1 M Na2SO4 in 0.1 M phosphate buffer, pH = 6.7.

[0075] Injection volume: 30μL. Flow rate: 1ml / min. Column temperature: 35°C.

[0076] Detector wavelength: 260nm (4nm), reference wavelength (360nm, bw 100nm); 280nm (4nm), reference wavelength (360nm, bw 100nm). Elution mode: isocratic.

[0077] Chromatography time: 15 min. Peaks were identified in the chromatograms obtained at wavelengths of 280 nm and 260 nm, and their areas were determined. Based on the peak data obtained in the chromatograms of the calibration solutions, linear regression equations were generated for each wavelength. The concentration of virus particles was calculated using the linear regression equation at a wavelength of 280 nm.

[0078] Size-exclusion high performance liquid chromatography was used as an orthogonal method to determine the concentration of viral particles. 5. Preparation of the composition.

[0079] Samples with a target viral genome content were prepared in a Stirred Cell (Millipore) concentration cell under pressure. For this purpose, an initial solution formulation containing viral particles was placed in a diafiltration container, and then at least 10 times the volume of an aqueous solution containing the target formulation, including a buffer, an osmotic agent, and, if necessary, additional water-soluble stabilizers, was introduced into the cell. After the diafiltration process, the solution was concentrated to an optical density above the target density, removed from the device, and the viral genome concentration was determined by PCR. The corresponding excipient solution was then added to the sample to produce a solution containing the target level of viral genomes.

[0080] Samples of solutions containing viral particles were also prepared in Pellicon (Millipore) cassettes in tangential flow mode. For this purpose, the initial solution formulation was placed in a diafiltration tank, and then at least 10 times the volume of a solution containing the target formulation, including buffer, osmotic agent, and, if necessary, additional water-soluble stabilizer, was added to the system. Concentrations of osmotic agent and water-soluble stabilizer can also be added after diafiltration. After the diafiltration process, the solution was concentrated to an optical density exceeding the target density and removed from the system, and the exact values ​​of the optical density and concentration of viral genomes were determined. The corresponding excipient solution was then added to the sample to produce a solution containing the target level of viral genomes.

[0081] Before being aseptically filled into the final container (eg glass / plastic container, vial or syringe), the solution was filtered under sterile conditions using a 0.22 μm membrane. 6. Stability studies.

[0082] The test sample was divided into multiple aliquots and placed into different sterile glass vials: one vial for each reference point was placed in a thermostat and incubated at 25°C for 4 weeks and (5±3)°C for 6 months, with periodic sampling at the reference points according to a schedule; at the time of sampling and after storage, the vials were removed from the thermostat or refrigerator and transferred for analysis. 7. Heat stress.

[0083] The test sample was divided into three portions and placed in the following glass vials: one vial of each formulation was stored in a refrigerator at (5±3)°C, and the remaining vials were placed in a thermostat and incubated at the given temperature for the given period of time. At the time of sampling at the reference points or after heating, the vials were removed from the thermostat, held at room temperature for approximately 15 minutes, and transferred for analysis. 8. Shake.

[0084] The test samples were divided into two portions and placed in the following glass vials: one vial per formulation was stored in a refrigerator at (5±3)°C, and the remaining vials were placed in a thermoshaker and shaken at 800 rpm for a given period at (5±3)°C. At the time of sampling at the reference points or after stress, the vials were removed from the thermoshaker and transferred for analysis. 9. Freeze-thaw.

[0085] The test sample was divided into two aliquots and added to the following glass vials: The vials were placed in a freezer and stored at or below -20°C. Most gene therapy products are stored at temperatures below -70°C, and temperatures below -20°C were chosen as a "worst case" because this method of storage places the sample above its glass transition temperature, thereby placing additional stress on the sample similar to that experienced under freezing. After the sample was completely frozen (8-16 hours), the vial was removed from the freezer and held at room temperature until the contents were completely thawed, with repeated freeze-thaw cycles performed as needed. After a given number of freeze-thaw cycles, the sample was transferred for analysis. 10.Isothermal studies by dynamic light scattering.

[0086] The colloidal stability of the test samples was analyzed using a DynaPro Plate Reader II instrument. 35 μl of each test sample was added to the wells of a 384-well black polymer plate with an optically clear bottom, and then the plate was sealed with a heat-resistant adhesive film. The sealed plate was centrifuged at 3000 rpm and a temperature of 20°C or higher for 3 minutes. Measurements were performed for a given time and at a given temperature. Measurement settings: · Scattered light intensity at θ (angle of scattered light measurement) = 158°. Number of measurements per replicate – 3. Time per measurement - 5s.

[0087] Time trends were analyzed using Dynamics software (Wyatt, USA). 11. Determination of aggregation temperature.

[0088] The aggregation temperature was determined using a DynaPro Plate Reader II. 35 μl of each test sample was added to the wells of a 384-well black polymer plate with an optically clear bottom, and the plate was then sealed with heat-resistant adhesive film. The sealed plate was centrifuged at 3000 rpm at a temperature of 20°C or higher for 3 minutes. Measurement settings: Initial measurement temperature - 25°C. · Scattered light intensity at θ = 158°. Number of measurements per replicate – 3. Time per measurement - 5s. ·Heating rate - 0.15℃ / min. ·Final temperature - 80℃.

[0089] The temperature trend and aggregation point were determined using Dynamics software (Wyatt, USA). 12. Determining the Melting Temperature of Capsid Proteins The melting temperature of recombinant adeno-associated virus capsid proteins was determined by differential scanning fluorimetry. Five microliters of diluted SyproOrange fluorescent dye (Thermo Fisher Scientific, USA), which specifically binds to the hydrophobic region of protein molecules, was added to 45 μl of test sample, resulting in a final dilution of 1000x the dye. The resulting mixture was transferred to a 96-well PCR plate, sealed with heat-resistant adhesive film, centrifuged at 3000 rpm for 3 minutes, and placed in an amplifier equipped with a fluorescence detector. Heating was performed from 25 to 85°C in 0.15°C increments, and fluorescence was detected during heating according to the manufacturer's instructions. Results were processed using CFX Manager (Bio-Rad, USA) software. 13. Determining the temperature for DNA extraction.

[0090] The temperature for DNA extraction from recombinant adeno-associated virus-based vector capsids was determined by differential scanning fluorimetry. Five microliters of diluted SybrGOLD fluorescent dye (Thermo Fisher Scientific, USA), which specifically binds nucleic acids, was added to 45 μl of test sample, resulting in a final dilution of 2000x. The resulting mixture was transferred to a 96-well PCR plate, sealed with heat-resistant adhesive film, centrifuged at 3000 rpm for 3 minutes, and placed in an amplifier equipped with a fluorescence detector. Heating was performed from 25 to 85°C in 0.15°C increments, and fluorescence was detected during heating according to the manufacturer's instructions. Results were processed using CFX Manager (Bio-Rad, USA) software. 14. Determination of sample homogeneity by dynamic light scattering.

[0091] Sample homogeneity was determined by dynamic light scattering using a DynaPro Plate Reader II. 35 μl of each test sample was added to the wells of a 384-well black polymer plate with an optically clear bottom, and the plate was then sealed with heat-resistant adhesive film. The sealed plate was centrifuged at 3000 rpm at a temperature of 20°C or higher for 3 minutes. Measurement settings: ·Measurement temperature: 25℃. Hold at this temperature for 1 minute before starting the measurement. · Scattered light intensity at θ = 158°. Number of measurements per replicate – 10. Time per measurement - 5s.

[0092] The size and polydispersity of the samples were determined using Dynamics software (Wyatt, USA). Example 1. Selection of excipient formulation The study aims to evaluate the possibility of storing recombinant adeno-associated virus vector products in liquid form at a temperature of (5±3) degrees, as well as to select the optimal excipient formulation to further study the stability of products with various serotypes.

[0093] The excipient solution must provide stability for the viral vector under the expected storage conditions. The following excipients were screened: tris(hydroxymethyl)aminomethane (buffering agent, maintaining required pH level), sodium chloride (stabilizer, osmolality adjuster), magnesium chloride hexahydrate (stabilizer), trehalose dihydrate (stabilizer, osmolality adjuster, cryoprotectant), poloxamer p188 (solubilizer), L-histidine (buffering agent, maintaining required pH level, stabilizer), L-arginine (buffering agent, maintaining required pH level, stabilizer), and L-proline (stabilizer).

[0094] The excipient formulations for the test pharmaceutical compositions are shown in Table 1.

[0095] [Table 2]

[0096] Approximately 1 x 10 rAAV serotype 5-based vectors carrying the GOI 10Studies were conducted on samples containing 0.1 mg / mL of PEG / 2000 vg / mL. Samples of various excipient formulations were prepared by diafiltration as per Method 5. To determine the effect of various excipients on vector stability, samples in the test formulations were stressed as per Methods 7, 8, and 9. Samples were analyzed before and after stress as per Methods 1, 2, 3, and 4. Results are shown in Tables 2-5.

[0097] [Table 3]

[0098] [Table 4]

[0099] [Table 5]

[0100] [Table 6]

[0101] The stress panel surprisingly revealed a favorable effect of using a histidine-arginine buffer on AAV stability. Formulations containing a histidine-arginine buffer were surprisingly found to exhibit high thermal stability. Pharmaceutical compositions based on a given buffer system showed minimal reduction in vector particle concentration after heat stress at 50°C, as analyzed by ELISA and PCR.

[0102] An accelerated stability study was also conducted on Formulation 2 shown in Table 1, per Method 6. Samples at benchmark points were analyzed per Methods 1, 2, and 3. The results of the analysis are shown in Tables 6-8.

[0103] [Table 7]

[0104] [Table 8]

[0105] [Table 9]

[0106] As shown by the data from the stability results for 6 months at (5±3)°C and 1 month at (25±2)°C, Formulation 2 showed good stability throughout the storage period. Example 2. Study of the pharmaceutical composition of recombinant adeno-associated virus serotype 9-based vectors.

[0107] The study used five pharmaceutical compositions, shown in Table 9, containing recombinant adeno-associated virus serotype 9-based vectors consisting primarily of empty capsids, at two concentrations: 1 x 10 12 and 1 × 10 13 particles / ml. Test samples were prepared by diafiltration as in Method 5.

[0108] [Table 10]

[0109] Colloidal stability studies at elevated temperatures. To measure colloidal stability, we determined the aggregation temperature of recombinant adeno-associated virus particle-based vectors in five formulations as per Method 11. The results are shown in Table 10.

[0110] [Table 11]

[0111] We also studied the thermal stability due to heat stress, as per Method 7. Sample homogeneity was analyzed by dynamic light scattering, as per Method 14, before and after exposure to a temperature of 65°C for 96 hours. The results are shown in Table 11.

[0112] [Table 12]

[0113] Additionally, samples were controlled by dynamic light scattering during 120 minutes at elevated temperature of 65° C. as per method 10. The data are shown in FIG. The pharmaceutical composition HisArg AAV9 exhibited the best properties in terms of colloidal stability at elevated temperatures: the given pharmaceutical composition exhibited the highest aggregation temperature of 76.0°C and the smallest mean particle radius during and after thermal stress. Study of colloidal stability under multiple freeze-thaw cycles The effect of multiple freeze-thaw cycles on the colloidal stability of recombinant adeno-associated virus particle-based vectors was studied at two test sample concentrations as per Method 9. Before and after stress, the homogeneity of the samples was analyzed by dynamic light scattering as per Method 14. The results are shown in Table 12.

[0114] [Table 13]

[0115] The pharmaceutical composition HisArg AAV9 has the smallest radius and polydispersity level and exhibits reduced levels of aggregation under freezing compared to other solutions. Example 3. Study of the pharmaceutical composition of recombinant adeno-associated virus serotype 6-based vectors.

[0116] The study used five pharmaceutical compositions containing rAAV serotype 6-based vector material bearing a GOI. The pharmaceutical compositions tested are listed in Table 13. Test samples were prepared by diafiltration as per Method 5.

[0117] [Table 14]

[0118] Colloidal stability studies at elevated temperatures. To measure colloidal stability, we determined the aggregation temperature of recombinant adeno-associated virus particle-based vectors in five formulations as per Method 11. We measured the aggregation temperature of 1 x 10 12 Pharmaceutical compositions containing viral vectors at concentrations of 0.05 mg / ml were used. The results are shown in Table 14. The results were analyzed using the heat map tool in Microsoft Excel.

[0119] [Table 15]

[0120] The pharmaceutical composition HisArg AAV6 exhibited the best colloidal stability properties at elevated temperatures, with the given pharmaceutical composition exhibiting the highest aggregation temperature of 72.9°C. Pharmaceutical composition: Study of the conformational and chemical stability of HisArg AAV6.

[0121] Conformational and chemical stability studies were performed to determine the denaturation temperature of capsid proteins as per Method 12 and the temperature of DNA extraction from capsids as per Method 13. 12 The experiments were carried out using recombinant adeno-associated virus serotype 6 vector material at a concentration of 10 ...

[0122] [Table 16]

[0123] The pharmaceutical composition HisArg AAV6 exhibited high conformational and chemical stability at high temperatures for DNA extraction and high denaturation temperatures of the capsid protein. Example 4. Study of the pharmaceutical composition of recombinant adeno-associated virus serotype 5-based vectors.

[0124] The study used five pharmaceutical compositions containing rAAV serotype 5-based vector material bearing a GOI. The pharmaceutical compositions tested are listed in Table 18. Test samples were prepared by diafiltration as per Method 5.

[0125] [Table 17]

[0126] Colloidal stability studies at elevated temperatures. To measure colloidal stability, we determined the aggregation temperature of recombinant adeno-associated virus particles in five formulations as per method 11. 12 The pharmaceutical compositions containing the viral vectors at a concentration of 1000 mg / ml were used. The results are shown in Table 19.

[0127] [Table 18]

[0128] We also studied the thermal stability due to heat stress as per Method 7. Sample homogeneity was analyzed by dynamic light scattering as per Method 14 before and after exposure to a temperature of 40°C for 96 hours. The results are shown in Table 20.

[0129] [Table 19]

[0130] Additionally, samples were controlled by dynamic light scattering during 200 minutes at elevated temperature of 40° C. as per method 10. The data are shown in FIG. The pharmaceutical composition HisArg AAV5 exhibited the best colloidal stability properties at elevated temperatures: the given composition exhibited the highest aggregation temperature of 59.3°C, the smallest mean particle radius during elevated temperature exposure, and the smallest mean radius and polydispersity after thermal stress. Study of colloidal stability under multiple freeze-thaw cycles.

[0131] The effect of multiple freeze-thaw cycles on the colloidal stability of recombinant adeno-associated virus particles was studied as per Method 9. Before and after stress, the homogeneity of the samples was analyzed by dynamic light scattering as per Method 14. The results are shown in Table 21.

[0132] [Table 20]

[0133] The pharmaceutical composition HisArg AAV5 exhibited the smallest size and polydispersity of the samples tested. Conformational and chemical stability studies.

[0134] Conformational and chemical stability studies were performed to determine the denaturation temperature of capsid proteins as per Method 12 and the temperature of DNA extraction from capsids as per Method 13. 13 The experiments were carried out using recombinant adeno-associated virus serotype 5 material at a concentration of 10 ...

[0135] [Table 21]

[0136] The HisArg AAV5 pharmaceutical composition exhibited the highest DNA extraction temperature, indicating its high stabilization properties. The HisArg AAV5 pharmaceutical composition also exhibited a high capsid protein denaturation temperature of 87.0°C.

[0137] Example 5. Study of the effect of excipient formulation on the DNA extraction propensity of recombinant adeno-associated virus-based vectors. To study the effect of pharmaceutical composition on the tendency to extract DNA from capsids, we used two pharmaceutical compositions shown in Table 23. A given formulation contains 1 x 10 13 The best results were obtained in studies of recombinant adeno-associated serotype 5 particles at a concentration of vg / ml.

[0138] [Table 22]

[0139] Research, 1x10 12 Assays were performed using vector material based on rAAV serotypes 2, 5, 6, 8, and 9 carrying the GOI at concentrations of 0.05 mg / ml. Test samples were prepared by diafiltration as in Method 5. Analysis was performed as in Method 13.

[0140] [Table 23]

[0141] For all test vectors based on adeno-associated virus particles of various serotypes, the following: L-histidine 2.79mg / ml L-arginine 0.348mg / ml Sodium chloride 11.7mg / ml Magnesium chloride 0.203mg / ml Poloxamer 188 0.1mg / ml The pharmaceutical composition allowed for higher temperatures for DNA extraction compared to alternative pharmaceutical compositions based on histidine buffer systems, demonstrating better DNA retention stability inside the capsids of test articles in a given formulation.

[0142] The developed compositions can be applied to adeno-associated viruses and other types of non-enveloped viruses.

Claims

1. 1. A pharmaceutical composition of a recombinant non-enveloped virus-based vector, comprising: (i) a recombinant non-enveloped virus-based vector; (ii) histidine, and (iii) arginine, and (iv) sodium chloride; (v) magnesium chloride; (vi) a surfactant; and (vii) water for injection; A pharmaceutical composition comprising:

2. 2. The pharmaceutical composition of claim 1, wherein the histidine is present in a concentration of 2.0 to 3.58 mg / ml.

3. histidine is present at a concentration of 2.3 to 3.2 mg / ml, or histidine is present at a concentration of 2.6 to 3.0 mg / ml, or 2. The pharmaceutical composition of claim 1, wherein the histidine is present in a concentration of 2.7 to 2.8 mg / ml.

4. 10. The pharmaceutical composition of claim 1, wherein the arginine is present in a concentration of 0.248 to 0.448 mg / ml.

5. arginine is present at a concentration of 0.28 to 0.416 mg / ml, or arginine is present at a concentration of 0.3 to 0.396 mg / ml, or 10. The pharmaceutical composition of claim 1, wherein the arginine is present in a concentration of 0.340 to 0.350 mg / ml.

6. 10. The pharmaceutical composition of claim 1, wherein the sodium chloride is present in a concentration of 8.0 to 15.0 mg / ml.

7. sodium chloride is present at a concentration of 9.5 to 13.5 mg / ml, or sodium chloride is present at a concentration of 10.5 to 12.5 mg / ml, or 10. The pharmaceutical composition of claim 1, wherein the sodium chloride is present in a concentration of 11.0 to 12.0 mg / ml.

8. 2. The pharmaceutical composition of claim 1, wherein the magnesium chloride is present in a concentration of 0.15 to 0.50 mg / ml.

9. Magnesium chloride is present in a concentration of 0.16 to 0.35 mg / ml, or Magnesium chloride is present in a concentration of 0.18 to 0.25 mg / ml, or 10. The pharmaceutical composition of claim 1, wherein the magnesium chloride is present in a concentration of 0.200 to 0.210 mg / ml.

10. 10. The pharmaceutical composition of claim 1, wherein the surfactant is present in a concentration of 0.01 to 1.0 mg / ml.

11. the surfactant is present in a concentration of 0.01 to 0.5 mg / ml; or the surfactant is present in a concentration of 0.01 to 0.2 mg / ml; or 2. The pharmaceutical composition of claim 1, wherein the surfactant is present in a concentration of 0.03 to 0.15 mg / ml.

12. (i) a recombinant non-enveloped virus-based vector; (ii) histidine 2.0 to 3.58 mg / ml; (iii) arginine 0.248 to 0.448 mg / ml; (iv) sodium chloride 8.0 to 15.0 mg / ml; (v) magnesium chloride 0.15 to 0.50 mg / ml; (vi) a surfactant in an amount of 0.01 to 1.0 mg / ml; (vii) water for injection to make 1.0 ml; 2. The pharmaceutical composition of claim 1, comprising:

13. (i) a recombinant non-enveloped virus-based vector; (ii) histidine 2.3 to 3.2 mg / ml; (iii) arginine 0.28 to 0.416 mg / ml; (iv) sodium chloride 9.5 to 13.5 mg / ml; (v) magnesium chloride 0.16 to 0.35 mg / ml; (vi) a surfactant in an amount of 0.01 to 0.2 mg / ml; (vii) water for injection to make 1.0 ml; 2. The pharmaceutical composition of claim 1, comprising:

14. (i) a recombinant non-enveloped virus-based vector; (ii) histidine 2.6 to 3.0 mg / ml; (iii) arginine 0.3 to 0.396 mg / ml; (iv) sodium chloride 10.5 to 12.5 mg / ml; (v) magnesium chloride 0.18 to 0.25 mg / ml; (vi) a surfactant of 0.03 to 0.15 mg / ml; (vii) water for injection to make 1.0 ml; 2. The pharmaceutical composition of claim 1, comprising:

15. (i) a recombinant non-enveloped virus-based vector; (ii) histidine 2.7 to 2.8 mg / ml; (iii) arginine 0.340 to 0.350 mg / ml; (iv) sodium chloride 11.0 to 12.0 mg / ml; (v) magnesium chloride 0.200 to 0.210 mg / ml; (vi) a surfactant of 0.03 to 0.15 mg / ml; (vii) water for injection to make 1.0 ml; 2. The pharmaceutical composition of claim 1, comprising:

16. A pharmaceutical composition wherein histidine is present at a concentration of 2.79 mg / ml.

17. 10. The pharmaceutical composition of claim 1, wherein arginine is present at a concentration of 0.348 mg / ml.

18. 10. The pharmaceutical composition of claim 1, wherein the sodium chloride is present in a concentration of 11.7 mg / ml.

19. 10. The pharmaceutical composition of claim 1, wherein the magnesium chloride is present in a concentration of 0.203 mg / ml.

20. 10. The pharmaceutical composition of claim 1, wherein the surfactant is present at a concentration of 0.05 mg / ml or 0.1 mg / ml.

21. (i) a recombinant non-enveloped virus-based vector; (ii) histidine 2.79 mg / ml; (iii) arginine 0.348 mg / ml; (iv) sodium chloride 11.7 mg / ml; (v) magnesium chloride 0.203 mg / ml; (vi) 0.05 mg / ml of a surfactant; (vii) water for injection to make 1.0 ml; Contains, or (i) a recombinant non-enveloped virus-based vector; (ii) histidine 2.79 mg / ml; (iii) arginine 0.348 mg / ml; (iv) sodium chloride 11.7 mg / ml; (v) magnesium chloride 0.203 mg / ml; (vi) 0.1 mg / ml of a surfactant; (vii) water for injection to make 1.0 ml; The pharmaceutical composition of claim 1 comprising:

22. 2. The pharmaceutical composition of claim 1, wherein the magnesium chloride is magnesium chloride hexahydrate.

23. 2. The pharmaceutical composition of claim 1, wherein the surfactant is poloxamer 188.

24. (i) a recombinant non-enveloped virus-based vector; (ii) histidine 2.0 to 3.58 mg / ml; (iii) arginine 0.248 to 0.448 mg / ml; (iv) sodium chloride 8.0 to 15.0 mg / ml; (v) magnesium chloride hexahydrate, 0.15 to 0.50 mg / ml; (vi) 0.01 to 1.0 mg / ml of a surfactant which is poloxamer 188; (vii) water for injection to make 1.0 ml; 2. The pharmaceutical composition of claim 1, comprising:

25. (i) a recombinant non-enveloped virus-based vector; (ii) histidine 2.3 to 3.2 mg / ml; (iii) arginine 0.28 to 0.416 mg / ml; (iv) sodium chloride 9.5 to 13.5 mg / ml; (v) magnesium chloride hexahydrate, 0.16 to 0.35 mg / ml; (vi) 0.01 to 0.2 mg / ml of a surfactant which is poloxamer 188; (vii) water for injection to make 1.0 ml; 2. The pharmaceutical composition of claim 1, comprising:

26. (i) a recombinant non-enveloped virus-based vector; (ii) histidine 2.6 to 3.0 mg / ml; (iii) arginine 0.3 to 0.396 mg / ml; (iv) sodium chloride 10.5 to 12.5 mg / ml; (v) magnesium chloride hexahydrate, 0.18 to 0.25 mg / ml; (vi) 0.03 to 0.15 mg / ml of a surfactant which is poloxamer 188; (vii) water for injection to make 1.0 ml; 2. The pharmaceutical composition of claim 1, comprising:

27. (i) a recombinant non-enveloped virus-based vector; (ii) histidine 2.7 to 2.8 mg / ml; (iii) arginine 0.340 to 0.350 mg / ml; (iv) sodium chloride 11.0 to 12.0 mg / ml; (v) magnesium chloride hexahydrate, 0.200 to 0.210 mg / ml; (vi) 0.03 to 0.15 mg / ml of a surfactant which is poloxamer 188; (vii) water for injection to make 1.0 ml; 2. The pharmaceutical composition of claim 1, comprising:

28. (i) a recombinant non-enveloped virus-based vector; (ii) histidine 2.79 mg / ml; (iii) arginine 0.348 mg / ml; (iv) sodium chloride 11.7 mg / ml; (v) magnesium chloride hexahydrate, 0.203 mg / ml magnesium chloride; (vi) 0.05 mg / ml of a surfactant which is Poloxamer 188; (vii) water for injection to make 1.0 ml; Contains, or (i) a recombinant non-enveloped virus-based vector; (ii) histidine 2.79 mg / ml; (iii) arginine 0.348 mg / ml; (iv) sodium chloride 11.7 mg / ml; (v) magnesium chloride hexahydrate, 0.203 mg / ml magnesium chloride; (vi) 0.1 mg / ml of a surfactant which is Poloxamer 188; (vii) water for injection to make 1.0 ml; 2. The pharmaceutical composition of claim 1, comprising:

29. 2. The pharmaceutical composition of claim 1, wherein the histidine is L-histidine.

30. 2. The pharmaceutical composition of claim 1, wherein the arginine is L-arginine.

31. 10. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition has a pH of 7.0 to 9.

0.

32. the pharmaceutical composition has a pH of 7.7 to 8.7; or 10. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition has a pH of 7.5 to 8.

5.

33. 10. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition has a pH of 8.

2.

34. 1.0 x 10 ９ ~1.0 x 10 １４ 10. The pharmaceutical composition of claim 1, wherein the composition is present at a concentration of viral genomes / ml.

35. The vector based on a recombinant non-enveloped virus is (1.0±0.3)×10 ９ Viral genomes / ml, (2.0±0.6)×10 ９ Viral genomes / ml, (5.0±1.5) × 10 ９ Viral genomes / ml, (1.0±0.3) x 10 １０ Viral genomes / ml, (2.0±0.6)×10 １０ Viral genomes / ml, (5.0±1.5) × 10 １０ Viral genomes / ml, (1.0±0.3) x 10 １１ Viral genomes / ml, (2.0±0.6)×10 １１ Viral genomes / ml, (5.0±1.5) × 10 １１ Viral genomes / ml, (1.0±0.3) x 10 １２ Viral genomes / ml, (2.0±0.6)×10 １２ Viral genomes / ml, (5.0±1.5) × 10 １２ Viral genomes / ml, (1.0±0.3) x 10 １３ Viral genomes / ml, (2.0±0.6)×10 １３ Viral genomes / ml, (5.0±1.5) × 10 １３ viral genomes / ml, or (1.0±0.3) x 10 １４ 10. The pharmaceutical composition of claim 1, wherein the composition is present at a concentration of viral genomes / ml.

36. The pharmaceutical composition of claim 1 , wherein the recombinant non-enveloped viral vector is an rAAV-based vector.

37. 37. The pharmaceutical composition of claim 36, wherein the rAAV-based vector comprises a capsid of an AAV5 serotype, an AAV6 serotype, or an AAV9 serotype.

38. 38. The pharmaceutical composition of claim 37, wherein the capsid of an AAV5 serotype, an AAV6 serotype, or an AAV9 serotype can be a modified capsid.

39. 39. A pharmaceutical composition according to any one of claims 1 to 38, which is suitable for lyophilization.

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