Pharmaceutical compositions comprising aav and methods of making the same

Abstract

The present invention relates to the field of pharmaceuticals, in particular to pharmaceutical compositions comprising AAV and methods of making the same. Furthermore, the present invention relates to the use of making these pharmaceutical compositions. The pharmaceutical compositions of the present invention have significantly improved properties, such as improved stability, solubility, bioavailability and / or potency, etc.

Description

Technical Field

[0001] This invention relates to the field of pharmaceuticals, and more particularly to pharmaceutical compositions comprising AAV. Furthermore, this invention relates to methods for preparing these pharmaceutical compositions and the use of the pharmaceutical compositions of this invention. Background Technology

[0002] Adeno-associated virus (AAV) is a member of the Parvovirus family. It is a small, non-enveloped virus with a single-stranded linear DNA genome of 4.7 kb to 6 kb. The AAV life cycle includes a latent period (after infection, the AAV genome is specifically integrated into the host chromosome at a specific site) and an infective period (after infection with adenovirus or herpes simplex virus, the integrated genome is then rescued, replicated, and packaged into the infecting virus). Its non-pathogenicity, broad host range (including non-dividing cells) infectivity, and potential site-specific chromosomal integration make AAV an attractive tool for gene transfer (see CN 106906241 A).

[0003] The potential of adeno-associated virus (AAV)-based gene delivery vectors for several disease targets has been demonstrated in preclinical disease models and, more recently, in human clinical trials. AAV-based vectors are highly safe because wild-type AAV is non-pathogenic and etiologically unrelated to any known diseases. Furthermore, AAV delivers genes efficiently and maintains transgene expression in many tissues, including the liver, muscle, lungs, retina, and brain.

[0004] The promising applications of adeno-associated viruses (AAVs) necessitate the preparation of suitable AAV formulations. However, the solubility of purified AAV particles, such as AAV2, is limited. Furthermore, AAV2 particles are prone to aggregation. Aggregation can lead to various adverse consequences, such as losses during purification, inconsistencies in purified carrier formulation testing, impaired biodistribution after in vivo administration, and adverse immune responses to the carrier after administration. Existing techniques, such as CN 101018858A, have investigated the inhibition of AAV particle aggregation. However, existing formulations still have various limitations, such as the need for excessively high ionic strength, and the need for further improvement in the solubility and titer of AAVs.

[0005] Therefore, there remains a demand for improved AAV formulations. Summary of the Invention

[0006] Through research, the inventors unexpectedly discovered that MgCl2 can bind to residues on the surface of AAV capsid proteins, preventing the formation of aggregates between AAVs at low concentrations, thereby stabilizing AAVs. This can significantly improve the properties of formulations containing AAVs, such as increasing AAV solubility, titer, and / or formulation stability and potency. For example, for the AAV2 serotype with low solubility, the addition of MgCl2 significantly increased the AAV virus titer (up to 1.6E+13VG / ml), had a significant inhibitory effect on AAV virus aggregation, and improved the stability of the formulation (see Tables 14-19 for example). Based on this, the present invention provides a pharmaceutical composition with improved properties compared to existing pharmaceutical compositions.

[0007] On one hand, the present invention provides a pharmaceutical composition comprising:

[0008] (i)AAV;

[0009] (ii) A buffer of approximately 1 mM to approximately 50 mM;

[0010] (iii) A first pharmaceutically acceptable salt of about 0.1 mM to about 10 mM, wherein the pharmaceutically acceptable salt is MgCl2;

[0011] (iv) Another pharmaceutically acceptable salt of about 50 mM to about 250 mM;

[0012] (v) about 0.0002% (w / v) to about 0.01% (w / v) of nonionic surfactants; and

[0013] (vi) Sugars of about 0.2% (w / v) to about 10% (w / v).

[0014] In some embodiments, the buffer is selected from histidine, glutamate, phosphate, acetate, citrate and tris(hydroxymethyl)aminomethane, preferably histidine, more preferably L-histidine.

[0015] The study in CN 101018858A showed that histidine had no inhibitory effect on AAV virus aggregation (see Table 1 on page 8 of the CN101018858A specification). However, the inventors discovered that histidine, due to its imidazole group, can play a good pH buffering role in the pH range of 5 to 8, significantly improving the problem of easy aggregation and adsorption of high-concentration AAV, and significantly increasing the AAV titer.

[0016] In some embodiments, the additional pharmaceutically acceptable salt is selected from pharmaceutically acceptable sodium, ammonium, or potassium salts, preferably sodium chloride.

[0017] In some embodiments, the nonionic surfactant is poloxamer, preferably poloxamer-188.

[0018] In some embodiments, the sugar is selected from sucrose, trehalose, or a combination thereof; sucrose is preferred.

[0019] The study in CN 101018858A showed that sucrose had no inhibitory effect on the aggregation of AAV virus (see Table 1 on page 8 of the CN101018858A specification). However, the inventors discovered that sugars (especially sucrose) can interact with the capsid proteins of AAV, inhibiting the free energy exchange between AAV and the solvent and reducing AAV aggregation; moreover, sugars do not easily crystallize during cryopreservation, thus sugar excipients can stabilize the conformation of AAV.

[0020] In some implementations, the AAV is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10, with AAV2 being preferred.

[0021] In some embodiments, the concentration of the buffer in the pharmaceutical composition is from about 1 mM to about 50 mM, from about 5 mM to about 25 mM, from about 6 mM to about 20 mM, or from about 8 mM to about 15 mM, for example about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mM, preferably about 10 mM.

[0022] In some embodiments, the concentration of the first pharmaceutically acceptable salt in the pharmaceutical composition is from about 0.1 mM to about 10 mM, from about 0.2 mM to about 5 mM, from about 0.5 mM to about 2 mM; for example, from about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mM, preferably from about 1 mM.

[0023] In some embodiments, the concentration of the additional pharmaceutically acceptable salt in the pharmaceutical composition is about 50 mM to about 250 mM, about 100 mM to about 200 mM, about 120 mM to about 180 mM; for example, about 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or 210 mM, preferably about 150 mM.

[0024] In some embodiments, the concentration of the nonionic surfactant in the pharmaceutical composition is from about 0.0002% (w / v) to about 0.01% (w / v), from about 0.0005% (w / v) to about 0.005% (w / v), from about 0.001% (w / v) to about 0.003% (w / v); for example, about 0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, or 0.01% (w / v), preferably about 0.002% (w / v).

[0025] In some embodiments, the sugar concentration in the pharmaceutical composition is from about 0.2% (w / v) to about 10% (w / v), from about 0.5% (w / v) to about 8% (w / v), from about 1% (w / v) to about 5% (w / v); for example, about 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% (w / v), preferably about 2% (w / v).

[0026] In some embodiments, the pharmaceutical composition has a pH of about 6.0-8.0, such as about 6.5-7.8, or about 7.0-7.5, such as about 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, or 7.8.

[0027] In some embodiments, the pharmaceutical composition comprises AAV particles ranging from 1.0E+10 (VG / ml) to 1.0E+15 (VG / ml), such as 1.0E+11 (VG / ml) to 1.0E+13 (VG / ml), 1.0E+12 (VG / ml) to 1.0E+14 (VG / ml), such as 1.0E+10 (VG / ml), 1.0E+11 (VG / ml), 1.0E+12 (VG / ml), 1.0E+13 (VG / ml), 1.0E+14 (VG / ml), and 1.0E+15 (VG / ml).

[0028] In some implementations, the content of the AAV particles is a ddPCR or qPCR titer.

[0029] In some embodiments, the pharmaceutical composition is a liquid formulation, and preferably also contains a solvent, such as an injectable solvent, including water, preferably water for injection.

[0030] In some embodiments, the pharmaceutical composition is an injectable formulation.

[0031] In some embodiments, the pharmaceutical composition comprises or consists of the following components:

[0032] (i)AAV;

[0033] (ii) Approximately 10 mM of histidine;

[0034] (iii) Approximately 1 mM MgCl2;

[0035] (iv) Approximately 150 mM NaCl;

[0036] (v) Approximately 0.002% (w / v) of poloxamer 188;

[0037] (vi) Approximately 2% (w / v) sucrose; and

[0038] (vii) Water.

[0039] In some embodiments, the pharmaceutical compositions of the present invention have an osmotic pressure of about 200 to about 400 mOsm / kg, about 250 to about 380 mOsm / kg, or about 290 to about 350 mOsm / kg. In some embodiments, the pharmaceutical compositions of the present invention will have an osmotic pressure of, for example, about 200 mOsm / kg, about 210 mOsm / kg, about 220 mOsm / kg, about 230 mOsm / kg, about 240 mOsm / kg, about 250 mOsm / kg, about 260 mOsm / kg, about 270 mOsm / kg, about 280 mOsm / kg, or about 290 mOsm / kg. 0 mOsm / Kg, approximately 300 mOsm / Kg, approximately 310 mOsm / Kg, approximately 320 mOsm / Kg, approximately 330 mOsm / Kg, approximately 340 mOsm / Kg, approximately 350 mOsm / Kg, approximately 360 mOsm / Kg, approximately 370 mOsm / Kg, approximately 380 mOsm / Kg, approximately 390 mOsm / Kg, or approximately 400 mOsm / Kg.

[0040] In some embodiments, the pharmaceutical composition is a solid formulation obtained by lyophilizing the liquid formulation.

[0041] Before use, the solid formulation can be reconstituted in a suitable solvent to form the liquid formulation of the present invention.

[0042] In some embodiments, the pharmaceutical compositions of the present invention are stored at temperatures ranging from about -80°C to about 45°C, from about -20°C to about 25°C, from about 20°C to about 40°C, for example, from about -30°C, about -20°C, about 0°C, about 5°C, about 25°C, about 35°C, about 37°C, about 42°C, or about 45°C for at least 5 days, at least 7 days, at least 14 days, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, or at least 10 months. After at least 11 months, at least 12 months, at least 18 months, at least 2 years, 3 years, 4 years or longer, the genomic titer decreases by no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20% or 30%, and / or AAV activity remains at 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%.

[0043] In some embodiments, the pharmaceutical composition of the present invention remains stable after multiple freeze-thaw cycles, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40 cycles, for example, with a genomic titer reduction of no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, or 30%, and / or AAV activity remains at 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%.

[0044] On the other hand, the present invention provides a method for preparing the above-mentioned pharmaceutical composition, which includes the following steps:

[0045] (i) Provide AAV samples;

[0046] (ii) The AAV sample is replaced with an ultrafiltration buffer containing the components of the above-mentioned pharmaceutical composition other than AAV; and

[0047] (iii) The product obtained by centrifugation concentration step (ii) provides the pharmaceutical composition.

[0048] On the other hand, the present invention provides the use of the pharmaceutical composition of the present invention in the preparation of medicaments for the prevention or treatment of ophthalmic diseases, central nervous system diseases, and blood diseases.

[0049] In some implementations, the ophthalmic disease is a gene mutation-related retinal degeneration.

[0050] On the other hand, the present invention provides a method for preventing or treating ophthalmic diseases in subjects in need, the method comprising administering the pharmaceutical composition of the present invention to the subjects.

[0051] In some implementations, the ophthalmic disease is a gene mutation-related retinal degeneration.

[0052] In some implementations, the application is subretinal.

[0053] In some embodiments, the pharmaceutical composition of the invention is administered in an effective amount, such as a therapeutically effective amount or a preventatively effective amount.

[0054] definition:

[0055] The terms used in this invention have the definitions listed below. Unless otherwise defined herein, the terms used in this invention have the meanings commonly understood in the art.

[0056] To explain this specification, the following definitions will be used, and terms used in the singular may also include plural forms, where appropriate. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive.

[0057] As used herein, the term “and / or” means any one of the options or two or more of the options.

[0058] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover situations where the composition consists of the stated elements, integers, or steps. For example, a pharmaceutical composition “comprising” ingredients A, B, and C is also intended to cover a pharmaceutical composition consisting of ingredients A, B, and C.

[0059] "AAV" refers to adeno-associated virus, which can refer to the virus itself or its variants and derivatives, and can be naturally occurring or recombinant. The term "AAV" includes AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), AAV type 9 (AAV-9), canine AAV, sheep AAV, bovine AAV, equine AAV, and primate AAV, as well as their variants and derivatives. Adeno-associated viruses are known in the art, and may also be found, for example, G. Gao et al., Proc Natl Acad Sci USA, 100(10):6081-6086 (May 13, 2003); US-2003-0138772-A1 (July 24, 2003), International Patent Publication No. WO 2005 / 033321, and preferred Chinese Patent Application 201910736573.3, all of which are incorporated herein by reference in their entirety. Preferably, the AAV is AAV-2. Typically, the pharmaceutical compositions of the present invention comprise AAV particles ranging from 1.0E+10 (VG / ml) to 1.0E+15 (VG / ml), such as 1.0E+11 (VG / ml) to 1.0E+13 (VG / ml), 1.0E+12 (VG / ml) to 1.0E+14 (VG / ml), such as 1.0E+10 (VG / ml), 1.0E+11 (VG / ml), 1.0E+12 (VG / ml), 1.0E+13 (VG / ml), 1.0E+14 (VG / ml), and 1.0E+15 (VG / ml).

[0060] The term "pharmaceutically acceptable salt" refers to a salt that can be used for pharmaceutical purposes, is physiologically tolerable when administered to subjects, and generally does not produce adverse effects. The term "pharmaceutically acceptable" includes, but is not limited to, metal salts such as sodium, potassium, and cesium salts; alkaline earth metal salts such as calcium and magnesium salts; and organic amine salts such as triethylamine, trimethylamine, ethanolamine, triethanolamine, guanidine, diisopropylethylamine, and N,N'-dibenzylethylenediamine. The selection and use of pharmaceutically acceptable salts are well known in the art.

[0061] Non-limiting examples of pharmaceutically acceptable salts include, but are not limited to: sodium salts, ammonium salts, and potassium salts (e.g., sodium chloride, ammonium chloride, and potassium chloride; sodium acetate, ammonium acetate, and potassium acetate; sodium citrate, ammonium citrate, and potassium citrate; sodium phosphate, ammonium phosphate, and potassium phosphate; sodium fluoride, ammonium fluoride, and potassium fluoride; sodium bromide, ammonium bromide, and potassium bromide; and sodium iodide, ammonium iodide, and potassium iodide).

[0062] The term "another pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt other than magnesium chloride. The concentration of the other pharmaceutically acceptable salt in the pharmaceutical composition is about 50 mM to about 250 mM, about 100 mM to about 200 mM, about 120 mM to about 180 mM; for example, about 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or 210 mM.

[0063] The term "treatment" refers to improving a disease or condition (i.e., stopping or slowing the progression of the disease or at least one of its clinical symptoms). Additionally, "treatment" can refer to improving at least one bodily parameter, which may not be perceptible to the patient. Furthermore, "treatment" can also refer to physical (e.g., stabilizing perceptible symptoms) or physiological (e.g., stabilizing bodily parameters) regulation of a disease or condition, or both.

[0064] The term "prevention" refers to the suppression of the occurrence or development of a disease or condition, or symptoms of a particular disease or condition. In some implementations, subjects with a family history of the disease are candidates for preventative programs. Generally, the term "prevention" refers to the administration of a drug before the onset of symptoms or signs, particularly in at-risk subjects.

[0065] The term "effective amount" refers to an amount or dose of the pharmaceutical composition of the present invention that, when administered to a patient in a single or multiple doses, produces the intended effect in the treated patient. The effective amount can be readily determined by a physician skilled in the art by considering a variety of factors, such as: the species of the mammal; its size, age, and general health; the specific disease involved; the degree or severity of the disease; the individual patient's response; the specific active ingredient administered; the mode of administration; the bioavailability characteristics of the administered formulation; the chosen dosing regimen; and the use of any concomitant therapies.

[0066] The term "therapeutic effective amount" refers to the amount that, at the required dose and for the required duration, effectively achieves the desired therapeutic outcome. The therapeutic effective amount of the pharmaceutical composition of the present invention can vary depending on various factors such as disease state, individual age, sex, weight, and the specific active ingredient. A therapeutic effective amount is also a quantity in which the beneficial therapeutic effect of the pharmaceutical composition outweighs any toxic or harmful effects.

[0067] The term "preventive effective dose" refers to the amount of prophylactic dose that, at the required dose and for the required duration, effectively achieves the desired preventive outcome. Typically, because prophylactic doses are administered in subjects before or at an early stage of the disease, the preventive effective dose will usually be less than the therapeutic effective dose.

[0068] The term "subject" refers to a mammal that has or is susceptible to a disease or condition, including but not limited to domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Preferably, the subject is a human.

[0069] The term "buffer" refers to a substance suitable for administration to a subject that maintains a stable pH value. Preferably, the buffer is selected from histidine, glutamate, phosphate, acetate, citrate, and tris(hydroxymethyl)aminomethane. The concentration of the buffer in the pharmaceutical composition of the present invention is from about 1 mM to about 50 mM, from about 5 mM to about 25 mM, from about 6 mM to about 20 mM, or from about 8 mM to about 150 mM, for example, from about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mM.

[0070] As described in the text, a "nonionic surfactant" refers to a substance that, when added in small amounts, can cause a significant change in the interfacial state of a solution system, and that is nonionic. Preferably, the nonionic surfactant is poloxamer, and more preferably poloxamer-188, etc. The concentration of the nonionic surfactant in the pharmaceutical composition is, for example, from about 0.0002% (w / v) to about 0.01% (w / v), from about 0.0005% (w / v) to about 0.005% (w / v), from about 0.001% (w / v) to about 0.003% (w / v); for example, about 0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, or 0.01% (w / v).

[0071] As used herein, the term "solvent" refers to a liquid used to dissolve or suspend the active ingredient (e.g., AAV) and inactive ingredients to form a liquid formulation. The term "injectable solvent" refers to a solvent suitable for injection or infusion into a subject. Solvents that can be used in this invention include, but are not limited to, water (e.g., water for injection), and injectable organic solvents (including, but not limited to, oils for injection), ethanol, propylene glycol, etc., or combinations thereof.

[0072] As used herein, the term "sugar" includes, but is not limited to, sucrose, trehalose, or combinations thereof. The concentration of sugar in the pharmaceutical compositions of the present invention is from about 0.2% (w / v) to about 10% (w / v), from about 0.5% (w / v) to about 8% (w / v), from about 1% (w / v) to about 5% (w / v); for example, about 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% (w / v).

[0073] The term "genomic titer" refers to, for example, ddPCR or qPCR titers.

[0074] The term “about” when used in conjunction with a numeric value means to cover a range of numeric values ​​that have a lower limit of 10% less than the specified numeric value and an upper limit of 10% greater than the specified numeric value.

[0075] As used in the text, "w / v" refers to "weight / volume". For example, "1% w / v" means 1g / 100ml = 0.01g / ml = 10mg / ml.

[0076] Benefits of the present invention:

[0077] The pharmaceutical compositions of the present invention have significantly improved properties compared to existing pharmaceutical compositions containing AAV, such as improved stability, solubility, titer, bioavailability and / or potency. Attached Figure Description

[0078] Figure 1 Prescription 9 Screening Test Results - Light Intensity and Mass Distribution Map.

[0079] Figure 2 Prescription 10 Screening Test Results - Light Intensity and Mass Distribution Map.

[0080] Figure 3 Prescription 11 Screening Test Results - Light Intensity and Mass Distribution Map.

[0081] Figure 4 Prescription 12 Screening Test Results - Light Intensity and Mass Distribution Map.

[0082] Figure 5 Prescription 13 Screening Test Results - Light Intensity and Mass Distribution Map.

[0083] Figure 6 Prescription 14 Screening Test Results - Light Intensity and Mass Distribution Map. Detailed Implementation

[0084] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments, but the scope of protection of the present invention is not limited to these embodiments. All changes or equivalent substitutions that do not depart from the concept of the present invention are included within the scope of protection of the present invention.

[0085] Unless otherwise specified, the experimental materials and reagents used in the following examples can be obtained from commercial sources, prepared according to existing methods, or prepared according to methods similar to those disclosed in this application.

[0086] It should be understood that, unless otherwise defined or described, the abbreviations used herein have the meanings commonly understood by those skilled in the art. The abbreviations used in the embodiments preferably apply the following meanings.

[0087] Unless otherwise specified or contradicted in the context, the solvent used in the examples is water for injection.

[0088] Example

[0089] Example 1: First round of AAV formulation screening

[0090] (1) Prescription information

[0091] Prescription information is shown in Table 1.

[0092] Table 1 Prescription Information Table

[0093] prescription pH Buffer composition Poloxamer -188 (%) 1 7.4 10mM PB, 180mM NaCl 0.001 2 7.4 10mM PB, 200mM NaCl 0.003 3 7.4 10mM PB, 220mM NaCl 0.003 4 7.4 20mM PB, 360mM NaCl 0.002

[0094] Note: PB = Phosphate Buffer

[0095] (2) Experiment 1 (concentrate first, then change the solution)

[0096] Concentration: Take two ultrafiltration tubes and add 3.5 ml of AAV2 sample (purity 100%, genomic titer: 6.6E+11VG / ml, viral particle count: 5.0E+12VP / ml, the same below; its preparation can be found in Example 1 of Chinese Patent Application 201910736573.3) to each tube. Centrifuge to 0.8 ml.

[0097] Change the solution: Add 1 ml of the solution from prescription 2 and prescription 3 respectively, mix with a pipette, centrifuge to 0.8 ml in the tube, and repeat the operation 5 times.

[0098] (3) Experiment 2 (replace the liquid first, then concentrate)

[0099] Change the medium: Take two ultrafiltration tubes, add 2 ml of AAV2 sample to each, then add 3 ml of Formula 1 and Formula 4 solutions respectively, mix well with a pipette, centrifuge to 2 ml, and repeat the operation 5 times.

[0100] Concentration: Take 2 ml of the sample after changing the liquid and centrifuge to concentrate it to 0.5 ml.

[0101] (4) Results of the first round of prescription screening

[0102] The results of the first round of formulation screening, conducted using dynamic light scattering (DLS), are shown in Table 2. The results indicate that, using the concentration-then-liquid replacement method (Experiment 1), no aggregation was observed in the concentrated samples of Formulations 2 and 3; after the first liquid replacement, no aggregation occurred in either formulation. However, Formulation 2 showed significant aggregation after the second liquid replacement, and Formulation 3 showed the same phenomenon after the third liquid replacement. Using the liquid replacement-then-concentration method (Experiment 2), no aggregation was observed in the samples of Formulations 1 and 4 after the first liquid replacement, but significant aggregation occurred after the second liquid replacement. Considering the convenience of actual production, the concentration-then-liquid replacement method will be adopted subsequently. In summary, Formulations 1–4 are not suitable for this sample and further formulation screening is required.

[0103] Table 2 Summary of the first round of prescription screening results

[0104]

[0105] Note: - indicates that this operation was not performed, and the same applies below. The reason for not performing this operation is that aggregation occurred during the liquid change and concentration process.

[0106] Example 2. Second round of AAV formulation screening

[0107] (1) Prescription information

[0108] The molar concentrations of buffer salt and NaCl were adjusted, and the concentration of surfactant Poloxamer-188 was increased. At the same time, the type of buffer salt was changed, i.e., histidine buffer system was added, to further investigate the effect of different formulations on the product. Detailed formulation information is shown in Table 3.

[0109] Table 3 Prescription Information Table

[0110] prescription pH Buffer composition Poloxamer -188 (%) 5 7.5 10mM PB, 200mM NaCl 0.003 6 7.5 10mM PB, 220mM NaCl 0.003 7 7.5 20mM PB, 360mM NaCl 0.002 8 7.5 10mM His, 200mM NaCl 0.005

[0111] (2) Concentration and liquid replacement

[0112] Concentration: Take 4 ultrafiltration tubes, add 5ml of AAV2 sample to each tube, centrifuge for about 12 minutes to reduce to 1ml, which is a 5-fold concentration.

[0113] Change the solution: Add 1 ml of the solution from the above prescriptions (5-8) respectively, mix with a pipette, centrifuge to 0.8 ml, repeat the operation 5 times, that is, change the solution 5 times.

[0114] (3) Results of the second round of prescription screening

[0115] qPCR method for determining genomic titers: Linearized plasmids were prepared to achieve a standard curve concentration range of 0.001 pg / μl to 1000 pg / μl, i.e., a titer range of 2.82E2 VG / μl to 2.82E8 VG / μl. The test samples were diluted to within this range. Based on the direct correlation between fluorescence signal intensity and sample titer, the titer of the test sample was calculated using the standard curve. Reference: Practical utilization of recombinant AAV vector reference standards: focus on vector genome titration by free ITR qPCR. Methods & Clinical Development(2016)5,16019

[0116] ddPCR method for determining genomic titers: A method for absolute quantification of nucleic acid molecules. It uses water-in-oil droplets to diffuse into 20,000 tiny reaction systems, each undergoing independent PCR amplification. Finally, the endpoint fluorescence signal of each droplet is read, and the initial concentration of the target gene is absolutely quantified using Poisson distribution correction. Reference: Evaluation of a Droplet Digital Polymerase Chain Reaction Format for DNA CopyNumber Quantification. Anal. Chem. 2012, 84, 1003-1011.

[0117] The results of the second round of formulation screening are shown in Tables 4 and 5. The results indicate that the particle size and monomer mass percentage of formulations 5-8 did not change significantly before and after concentration and medium change, but formulations 6 and 7 showed some degree of aggregation during the medium change process. Based on the qPCR and ddPCR genomic titer results after concentration and five medium changes, it can be concluded that the loss of genomic titer after five concentration and medium changes for formulation 8 was significantly lower than that of other formulations. In summary, formulation 8 is significantly superior to other formulations, and further screening will be conducted using formulation 8.

[0118] Table 4 Summary of the Second Round of Prescription Screening Results - 1

[0119]

[0120] Table 5 Summary of the Second Round of Prescription Screening Results - 2

[0121]

[0122] Example 3. Third round of AAV formulation screening

[0123] (1) Prescription information

[0124] The effects of the concentration of surfactant P-188, the types of protective agents (sucrose and trehalose), and the presence or absence of metal ions (MgCl2) on product stability were further investigated. The formulation information is shown in Table 6.

[0125] Table 6 Prescription Information Table

[0126]

[0127] (2) Concentration and liquid replacement

[0128] Concentration: Take 3 ultrafiltration tubes, add 7ml of AAV2 sample to each tube, and centrifuge to 1ml;

[0129] Change the buffer solution: Add 1 ml of the above-mentioned prescription buffer solution, mix with a pipette, centrifuge to 1 ml, and repeat the operation 5 times.

[0130] (3) Results of the third round of prescription screening

[0131] Detection was performed using dynamic light scattering. The results of the third round of prescription screening are shown in Tables 7-9. Figure 1-3 The results showed that the particle size and monomer mass percentage of formulations 9-11 did not change significantly before and after concentration and medium replacement. After concentration and five medium replacements, the genomic titers of formulations 9-11 decreased by 18%, 24%, and 16%, respectively (it should be noted that unless otherwise specified or contradicted by the context, the genomic titers in the examples refer to ddPCR titers). Simultaneously, combined with the light intensity distribution results, it can be concluded that the proportion of aggregates in formulation 11 after five concentration and medium replacements was lower than that of other formulations, indicating that sucrose has a better protective effect than trehalose, and the addition of MgCl2 can effectively prevent the formation of aggregates. In summary, formulation 11 is significantly superior to other formulations, but its osmotic pressure is relatively high; therefore, further optimization of salt ion and sucrose concentrations is necessary.

[0132] Table 7. Screening and detection results of prescription 9

[0133]

[0134] Table 8. Screening and detection results of prescription 10

[0135]

[0136] Table 9. Screening and detection results of prescription 11

[0137]

[0138] Example 4. Fourth round of AAV formulation screening

[0139] (1) Prescription information

[0140] The concentrations of salt ions and sucrose were further optimized to maintain the osmotic pressure within the range of 300–400 mOsm / kg. The prescription information is shown in Table 10.

[0141] Table 10 Prescription Information Table

[0142]

[0143] (2) Concentration and liquid replacement

[0144] Concentration: Take two ultrafiltration tubes, add 5 ml of AAV2 sample to each, and centrifuge to 1 ml;

[0145] Change the buffer solution: Add 1 ml of the above-mentioned prescription buffer solution, mix with a pipette, centrifuge to 1 ml, and repeat the operation 5 times.

[0146] (3) Results of the fourth round of prescription screening

[0147] The results of the fourth round of prescription screening are shown in Tables 11 and 12. Figure 4-5 The results showed that the particle size and monomer mass percentage of formulations 12 and 13 did not change significantly before and after concentration and medium replacement. After concentration and five medium replacements, the genomic titers of formulations 12 and 13 decreased by 23% and 11%, respectively. Furthermore, combined with the light intensity distribution results, it can be concluded that the proportion of aggregates in formulation 13 was slightly lower than that in formulation 12 after five concentration and medium replacements, and the osmotic pressure of both formulations was within the range of 300–400 mOsm / Kg. In summary, formulation 13 was selected as the formulation for further optimization.

[0148] Table 11 Screening and detection results for prescription 12

[0149]

[0150] Table 12 Screening and Detection Results of Prescription 13

[0151]

[0152] Example 5. Fifth round of AAV formulation screening

[0153] (1) Prescription information

[0154] Poloxamer-188 plays a role in solubilization and anti-aggregation in AAV virus preparation buffer. Higher concentrations of poloxamer-188 may be unstable. Therefore, the concentration of poloxamer-188 was optimized during this round of formulation screening. Formulation information is shown in Table 13.

[0155] Table 13 Prescription Information Table

[0156]

[0157] (2) Concentration and liquid replacement

[0158] Buffer change: Take one ultrafiltration tube, add 2.0 ml of AAV2 sample, then add 2.0 ml of buffer, mix well, and centrifuge to 2.0 ml. Repeat the above operation, changing the buffer a total of 7 times.

[0159] Concentration: Centrifuge to 1.0 ml and 0.5 ml respectively, which means concentrating by 2 times and 4 times.

[0160] (3) Results of the fifth round of prescription screening

[0161] The results of the fifth round of prescription screening are shown in Table 14 and Figure 6 The results showed that the particle size, monomer mass percentage, and genomic titer did not change significantly after seven liquid changes in Formulation 14. Furthermore, combined with the light intensity distribution results, it can be concluded that no significant aggregation occurred in the samples under this formulation. Concentrations of 2-fold and 4-fold indicate that the product exhibits good stability under this formulation, with an osmotic pressure ranging from 300 to 400 mOsm / kg. In summary, this formulation (10 mM H₂O, 150 mM NaCl, 0.002% poloxamer-188, 2% sucrose, 1 mM MgCl₂, pH 7.5) was selected as the formulation for this AAV2.

[0162] Table 14 Screening and Detection Results of Prescription 14

[0163]

[0164] Example 6: Stability determination of the formulation. Low-titer (1.0E+13VG / ml) and high-titer (1.8E+13VG / ml) AAV2 samples were used to investigate the stability of AAV2 in the formulation (Formulation 14) through accelerated testing at 5±3℃, high-temperature testing at 37±3℃, and repeated freeze-thaw cycles. Specifically, the AAV2 formulation of Formulation 14 with the above-mentioned titers was placed in a constant temperature and humidity chamber protected from light. The chamber temperatures were 5±3℃ for accelerated testing and 37±3℃ for high-temperature testing. The formulation was then placed in a -80℃ freezer, frozen, and then allowed to thaw completely at room temperature. These freeze-thaw cycles were repeated 3 and 6 times.

[0165] 1. Accelerated (5±3℃) test results

[0166] The accelerated (5±3℃) assay results of the formulations are shown in Tables 15 and 16. The results indicate that after 30 days of storage at (5±3℃), the pH, particle size, monomer mass ratio, and genomic titer of both AAV2 formulations did not change significantly; the infectivity, i.e., the infectivity titer (IU), of both formulations was higher than 1.0E+10. Therefore, the AAV2 formulations exhibit good stability after 30 days of storage at (5±3℃).

[0167] Table 15 Results of accelerated test of low titer (1.0E+13VG / ml) AAV2 (5±3℃)

[0168]

[0169] Table 16 Results of accelerated test (5±3℃) of high titer (1.8E+13VG / ml) AAV2

[0170]

[0171] 2. High temperature (37±3℃) test results

[0172] The results of high-temperature (37±3℃) testing of the formulations are shown in Tables 17 and 18. The results indicate that after 10 days of storage at (37±3℃), the pH, particle size, monomer mass ratio, and genomic titer of both AAV2 formulations did not change significantly; the infectivity, i.e., the infectivity titer (IU), of both formulations was higher than 1.0E+10. Therefore, the AAV2 formulations exhibit good stability after 10 days of storage at (37±3℃).

[0173] Table 17 Results of High-Temperature Test (37±3℃) for Low-Titer (1.0E+13VG / ml) AAV2

[0174]

[0175] Table 18 Results of High-Titer (1.8E+13VG / ml) AAV2 High-Temperature Test (37±3℃)

[0176]

[0177] 3. Results of repeated freeze-thaw tests

[0178] The freeze-thaw test results of the formulations are shown in Table 19. The results indicate that after six freeze-thaw cycles, the pH, particle size, monomer mass ratio, and genomic titer of both AAV2 formulations did not change significantly; the infectivity, i.e., the infectivity titer (IU), of both formulations was higher than 1.0E+10. Therefore, the AAV2 formulations exhibit good stability after six freeze-thaw cycles.

[0179] Table 19. Results of Freeze-Thaw Test

[0180]

[0181] The results showed that the AAV2 formulation of the present invention has high solubility and good stability.

[0182] The foregoing describes exemplary embodiments of the present invention. Those skilled in the art should understand that these disclosures are merely exemplary, and various other substitutions, adaptations, and modifications can be made within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments listed herein.

Claims

1. A pharmaceutical composition comprising or consisting of the following components: (i) AAV, wherein the AAV is AAV2; (ii) Approximately 10 mM of histidine; (iii) Approximately 1 mM MgCl2; (iv) Approximately 150 mM NaCl; (v) Approximately 0.002% poloxamer 188 on a w / v basis; (vi) Approximately 2% sucrose by w / v; and (vii) Water; The term "about" refers to a range of numerical values ​​that have a lower limit that is 10% smaller than the specified numerical value and an upper limit that is 10% larger than the specified numerical value.

2. The pharmaceutical composition of claim 1, having a pH of 6.0-8.

0.

3. The pharmaceutical composition of claim 2, having a pH of 6.5-7.

8.

4. The pharmaceutical composition of claim 3, having a pH of about 7.

5. The term "about" refers to a range of numerical values ​​that have a lower limit that is 10% smaller than the specified numerical value and an upper limit that is 10% larger than the specified numerical value.

5. The pharmaceutical composition according to any one of claims 1-4, comprising AAV particles at a concentration between 1.0E+10 (VG / ml) and 1.0E+15 (VG / ml).

6. The pharmaceutical composition of claim 5, comprising AAV particles at a concentration between 1.0E+12 (VG / ml) and 1.0E+14 (VG / ml).

7. The pharmaceutical composition of claim 5, wherein the content of the AAV particles is determined by ddPCR or qPCR titer.

8. The pharmaceutical composition according to any one of claims 1-4 and 6-7, wherein it is a liquid formulation comprising water for injection.

9. The pharmaceutical composition according to any one of claims 1-4 and 6-7, wherein it is an injectable preparation.

10. The pharmaceutical composition according to any one of claims 1-4 and 6-7, having an osmotic pressure of 200 to 400 mOsm / Kg.

11. The pharmaceutical composition of claim 10, having an osmotic pressure of 290 to 350 mOsm / kg.

12. A solid dosage form obtained by freeze-drying the pharmaceutical composition as described in any one of claims 1-11.

13. A method for preparing a pharmaceutical composition according to any one of claims 1-11, comprising the following steps: (i) Provide AAV samples; (ii) The AAV sample is replaced with an ultrafiltration buffer, the buffer containing the components of the pharmaceutical composition according to any one of claims 1-11 other than AAV; and (iii) The product obtained by centrifugation concentration step (ii) provides a pharmaceutical composition.

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