Liquid preparation and application thereof

Abstract

Relates to a liquid preparation and application thereof, in particular to a buffer system used in the liquid preparation, the buffer system comprises soluble salt substances, polyhydric alcohols and a pH buffer solution, the preparation comprises a therapeutically effective amount of virus particles and the buffer system, the buffer system can remarkably improve the stability and dispersity of the virus particles, and the stability and dispersity of the virus particles are improved. The content of aggregates and the content of free nucleic acid are reduced, the signal response strength of the virus particles is not obviously reduced, the virus activity is not obviously reduced, and the dispersion quantity and stability of the virus particles can be obviously improved (such as enhanced).

Description

Liquid preparation and use thereof

[0001] Priority Declaration

[0002] This disclosure claims priority to Chinese patent application No. 2023111231314, filed September 1, 2023. This disclosure incorporates the entirety of the aforementioned Chinese patent application. Technical Field

[0003] The present disclosure relates to the field of biotechnology, and in particular to a liquid preparation and uses thereof. Background Art

[0004] AAV has become a new tool for human gene therapy. Currently, AAV undergoes repeated shaking, refrigerated storage, and even room temperature storage during its manufacturing, storage, transportation, and administration. This situation poses a great challenge to the stability of AAV, resulting in a decrease in transduction efficiency. At the same time, as the application scenarios of AAV continue to increase, it is not only limited to local small-dose administration, so the adverse reactions caused by high-dose systemic administration should receive more attention. In recent years, the viral titer of the products launched on the market is generally maintained at 1×10 13 vg / mL, or even lower, and the solution osmotic pressure is maintained above 350mOsm / kg, or even higher, such as Glybera launched by Uniqure and Roctacian launched by BioMarin. This is not friendly to high-dose systemic administration, will cause unnecessary immune reactions, and will greatly increase the number of administrations, causing pain to patients.

[0005] The clinical research and application of AAV as a gene therapy vector continues to increase. For example, to avoid liver toxicity and liver cancer risks, hemophilia gene therapy must explore safer and more effective AAV vectors and delivery methods. This can alter the tissue distribution of AAV drugs in the human body, making them a gene therapy drug with significant safety advantages. Furthermore, existing AAVs, after delivery into the body, express relatively low levels of FIX, which cannot meet the blood concentrations required for disease treatment. Therefore, the development of AAVs that can meet these blood concentrations is also needed.

[0006] As AAV is increasingly being studied and used clinically as a gene therapy vector, the current challenge is that development and manufacturing, long-term stability, and routes of administration significantly limit the selection of excipients and buffers. Therefore, there is an urgent need to develop an AAV formulation that balances stability, is suitable for different routes of administration and high-concentration doses, and ensures low osmotic pressure.

[0007] Summary of the Invention

[0008] The purpose of the present disclosure is to provide an AAV formulation that has both stability and suitability for different administration routes and high concentration doses while ensuring low osmotic pressure.

[0009] A first aspect of the present disclosure provides a buffer system, which includes a soluble salt substance, a polyol, and a pH buffer solution.

[0010] In another optional example, the soluble salt substance includes a soluble monovalent metal salt.

[0011] In another alternative, the soluble monovalent metal salt comprises a soluble sodium salt.

[0012] In another optional example, the soluble salt substance includes a soluble divalent metal salt.

[0013] In another optional example, the soluble divalent metal salt includes one or more of a soluble calcium salt, a soluble magnesium salt, a soluble zinc salt, a soluble copper salt, a soluble manganese salt, and a soluble chromium salt.

[0014] In another optional example, the polyol includes sucrose, mannitol, and glycerol.

[0015] In another alternative, the pH value of the buffer system is maintained at 7.0-8.0 (eg, 7.0-7.6, or 7.0-7.4).

[0016] In another optional example, the pH buffer is selected from one or more of the following buffers: PB buffer, HEPES buffer.

[0017] In another alternative, the polyol is sucrose.

[0018] In another alternative, the polyol is mannitol.

[0019] In another alternative, the polyol is glycerol.

[0020] In another alternative, the divalent metal ions in the soluble divalent metal salt include Ca 2+ Mg 2+ 、Zn 2+ 、Cu 2+ 、Mn 2+ Cr 2+ .

[0021] In another alternative, the monovalent metal ions in the soluble monovalent metal salt include Na + .

[0022] In another alternative, the Na + Derived from NaCl.

[0023] In another alternative, the Ca2 Derived from CaCl2 and CH3COOCa.

[0024] In another optional example, the Mg 2+ Derived from MgCl2 and Mg2SO4.

[0025] In another optional example, the buffer system further includes a surfactant.

[0026] In another optional example, the surfactant is selected from the following group: F68, Tween 20, or a combination thereof.

[0027] In another optional example, the concentration of the soluble monovalent metal salt in the soluble salt substance is 50-250 mM, such as 80-200 mM, or 100-190 mM, or 100-160 mM.

[0028] In another optional example, the concentration of the polyol (w / v) is 0.5% to 20%, such as 0.8% to 10%, or 0.9% to 5%, based on the total weight volume percentage of the buffer system.

[0029] In another optional example, the concentration of the surfactant (w / v) is 0.0005% to 0.002%, such as 0.0008% to 0.002%, based on the total weight volume percentage of the buffer system.

[0030] In another optional example, the concentration of the pH buffer is 2-50 mM, such as 5-30 mM, or 8-20 mM.

[0031] In another optional example, the concentration of the soluble divalent metal salt in the soluble salt substance is 0.2-8 mM, such as 0.5-5 mM, or 0.8-2 mM.

[0032] In another optional example, the concentration of sucrose (w / v) is 0.5% to 20%, such as 0.8% to 10%, or 0.9% to 5%, based on the total weight volume percentage of the buffer system.

[0033] In another optional example, the concentration of mannitol (w / v) is 0.5% to 20%, such as 1% to 10%, or 2% to 5%, based on the total weight volume percentage of the buffer system.

[0034] In another optional example, the concentration of glycerol (w / v) is 0.5% to 20%, such as 1% to 10%, or 2% to 5%, based on the total weight volume percentage of the buffer system.

[0035] In another optional example, the osmotic pressure of the buffer system is 280 to 330 mosm / kg.

[0036] In another optional example, the solvent of the buffer system is an organic solvent or an inorganic solvent, and optionally the solvent is an inorganic solvent, for example, the solvent of the buffer system is water.

[0037] In another alternative, the buffer system is further used for one or more purposes selected from the following group:

[0038] (a) Prevent the virus from being damaged by capsid protein fragmentation, aggregation and packaging integrity caused by exposure to low temperature, refrigeration, room temperature and repeated freezing and thawing;

[0039] (b) disperse a greater number of virus particles;

[0040] (c) Ensuring isotonic pressure can realize more application scenarios and avoid adverse reactions caused by high osmotic pressure.

[0041] The second aspect of the present disclosure provides a use of the buffer system described in the first aspect of the present disclosure for preparing a reagent or a kit for improving the stability and dispersibility of virus particles.

[0042] In another alternative embodiment, the reagent or kit is further used for one or more purposes selected from the following group:

[0043] (a) Prevent the virus from being damaged by capsid protein fragmentation, aggregation and packaging integrity caused by exposure to low temperature, refrigeration, room temperature and repeated freezing and thawing;

[0044] (b) disperse a greater number of virus particles;

[0045] (c) Ensuring isotonic pressure can realize more application scenarios and avoid adverse reactions caused by high osmotic pressure.

[0046] A third aspect of the present disclosure provides a kit, comprising:

[0047] i) one or more containers containing the same or different soluble salt substances;

[0048] ii) one or more containers containing the same or different polyols;

[0049] iii) one or more containers containing the same or different pH buffers;

[0050] iv) optionally one or more containers containing the same or different surfactants.

[0051] In another optional example, the soluble salt substance includes a soluble monovalent metal salt.

[0052] In another alternative, the soluble monovalent metal salt comprises a soluble sodium salt.

[0053] In another optional example, the soluble salt substance includes a soluble divalent metal salt.

[0054] In another optional example, the soluble divalent metal salt includes one or more of a soluble calcium salt, a soluble magnesium salt, a soluble zinc salt, a soluble copper salt, a soluble manganese salt, and a soluble chromium salt.

[0055] In another optional example, the polyol includes sucrose, mannitol, and glycerol.

[0056] In another optional example, the surfactant is selected from the following group: F68, Tween 20, or a combination thereof.

[0057] In another alternative embodiment, the kit is further used for one or more purposes selected from the following group:

[0058] (a) Prevent the virus from being damaged by capsid protein fragmentation, aggregation and packaging integrity caused by exposure to low temperature, refrigeration, room temperature and repeated freezing and thawing;

[0059] (b) disperse a greater number of virus particles;

[0060] (c) Ensuring isotonic pressure can realize more application scenarios and avoid adverse reactions caused by high osmotic pressure.

[0061] In another optional example, any two, three, or four (or all) of the above-mentioned containers may be the same (or identical) or different containers.

[0062] In another optional example, any two or three (or all) of the above-mentioned containers can be the same (or identical) or different containers.

[0063] A fourth aspect of the present disclosure provides a method for improving the stability and dispersibility of virus particles, comprising:

[0064] The buffer system described in the first aspect of the present disclosure is provided, and virus particles are placed in the buffer system to improve the stability and dispersibility of the virus particles.

[0065] In another alternative embodiment, the viral particles include AAV viral particles, lentiviral particles, or a combination thereof.

[0066] A fifth aspect of the present disclosure provides a liquid preparation comprising:

[0067] (i) a therapeutically effective amount of viral particles;

[0068] (ii) soluble salts;

[0069] (iii) polyols;

[0070] (iv) pH buffer,

[0071] The pH of the preparation is 7.0 to 8.0 (eg, 7.0 to 7.6, or 7.0 to 7.4).

[0072] In another optional example, the liquid preparation further comprises a surfactant.

[0073] In another optional example, the soluble salt substance includes a soluble monovalent metal salt.

[0074] In another alternative, the soluble monovalent metal salt comprises a soluble sodium salt.

[0075] In another optional example, the soluble salt substance includes a soluble divalent metal salt.

[0076] In another optional example, the soluble divalent metal salt includes one or more of a soluble calcium salt, a soluble magnesium salt, a soluble zinc salt, a soluble copper salt, a soluble manganese salt, and a soluble chromium salt.

[0077] In another optional example, the polyol includes sucrose, mannitol, and glycerol.

[0078] In another optional example, the pH buffer is selected from one or more of the following buffers: PB buffer, HEPES buffer.

[0079] In another optional example, the surfactant is selected from the following group: F68, Tween 20, or a combination thereof.

[0080] In another optional example, the viral particles include AAV virus and lentiviral particles.

[0081] In another alternative embodiment, the viral particle is AAV virus.

[0082] In another optional example, the viral particle is a lentiviral particle.

[0083] In another optional example, the viral particles are packaged by an adeno-associated viral vector system.

[0084] In another optional example, the structure of the adeno-associated virus vector system is as described in patent application number 202211254858.1 / 202310394646.1.

[0085] In another alternative embodiment, the concentration of the virus particles is 5×10 11 ~5×10 14 vg / mL, for example 2×10 12 ~2×10 14 vg / mL, or 1×10 13 ~1×10 14 vg / mL.

[0086] In another alternative embodiment, the preparation has an osmotic pressure of 280 to 330 mosm / kg.

[0087] In another alternative, the formulation has one or more characteristics selected from the group consisting of:

[0088] (a) the aggregate content of the preparation is <1.5%, such as <1.0%, or <0.8%, or <0.7%;

[0089] (b) the free nucleic acid content of the preparation is <15 ppm, such as <10 ppm, or <8 ppm;

[0090] (c) After a 7-day accelerated destruction test at 37°C, the viral particle signal response intensity of the preparation did not significantly decrease, and the viral activity did not significantly decrease;

[0091] (d) The aggregate content of the preparation after a 7-day accelerated test is less than 1.5%, such as less than 1.0%, or less than 0.8%.

[0092] In another alternative, the aggregate content of the preparation is 0.3% to 1.5%, such as 0.3% to 1%, or 0.3% to 0.7%.

[0093] In another alternative example, the content of free nucleic acid in the preparation after a 7-day accelerated test is 25 ppm to 35 ppm.

[0094] In another alternative embodiment, the preparation undergoes a 7-day accelerated test, and the aggregate content is 0.3% to 0.7%. In another alternative embodiment, the preparation comprises:

[0095] (a)5×10 11 ~5×10 14 A therapeutically effective amount of viral particles at a concentration of vg / mL;

[0096] (b) 50-250 mM soluble salts;

[0097] (c) 0.5% to 20% (w / v) polyol;

[0098] (d) 2-50 mM pH buffer;

[0099] And the pH of the preparation is 7.0-8.0.

[0100] In another alternative, the formulation includes 0.0005% to 0.002% (w / v) of a surfactant.

[0101] In another alternative, the formulation comprises:

[0102] (a)2×10 12 ~2×10 14 A therapeutically effective amount of viral particles at a concentration of vg / mL;

[0103] (b) 80-200 mM soluble salts;

[0104] (c) 0.8% to 10% (w / v) polyol;

[0105] (d) 5-30 mM pH buffer;

[0106] And the pH of the preparation is 7.0-7.6.

[0107] In another alternative, the formulation includes 0.0005% to 0.002% (w / v) of a surfactant.

[0108] In another alternative, the formulation comprises:

[0109] (a)1×10 13 ~1×10 14 A therapeutically effective amount of viral particles at a concentration of vg / mL;

[0110] (b) 100-190 mM soluble salts;

[0111] (c) 0.9% to 5% (w / v) polyol;

[0112] (d) 8-15 mM pH buffer;

[0113] And the pH of the preparation is 7.0-7.4.

[0114] In another alternative, the formulation includes 0.0008% to 0.002% (w / v) of a surfactant.

[0115] The sixth aspect of the present disclosure provides the use of the preparation described in the fifth aspect of the present disclosure for preparing (i) a drug for preventing and / or treating hereditary coagulation factor deficiency; and or (ii) a drug for preventing and / or treating hyperlipidemia-related diseases.

[0116] In another alternative embodiment, the hereditary coagulation factor deficiency disease is hemophilia A, B or C.

[0117] In another optional example, the hereditary coagulation factor deficiency disease is a mammalian hereditary coagulation factor deficiency disease; optionally, the hereditary coagulation factor deficiency disease is a human hereditary coagulation factor deficiency disease.

[0118] In another alternative, the hyperlipidemia-related disease includes cardiovascular disease.

[0119] In another alternative embodiment, the cardiovascular disease includes atherosclerotic cardiovascular disease and familial hypercholesterolemia.

[0120] The seventh aspect of the present disclosure provides a medicine kit, which contains a container and the preparation according to the fifth aspect of the present disclosure located in the container.

[0121] An eighth aspect of the present disclosure provides a method for preparing a stable liquid formulation for improving the stability and dispersibility of viral particles, comprising the steps of:

[0122] (a) mixing a therapeutically effective amount of viral particles, a soluble salt substance, a polyol, and a pH buffer;

[0123] (b) adjusting the pH of the mixture to a pH of 7.0 to 8.0 (e.g., 7.0 to 7.6, or 7.0 to 7.4);

[0124] Thus, a stable liquid preparation with improved stability and dispersibility of virus particles is obtained.

[0125] In another alternative embodiment, the stable liquid formulation for improving the stability and dispersibility of viral particles has one or more characteristics selected from the following group:

[0126] (a) the aggregate content of the preparation is less than 1.5%, such as less than 1.0%, or less than 0.8%;

[0127] (b) the free nucleic acid content of the preparation is <15 ppm, such as <10 ppm, or <8 ppm;

[0128] (c) After a 7-day accelerated destruction test at 37°C, the viral particle signal response intensity of the preparation did not significantly decrease, and the viral activity did not significantly decrease;

[0129] (d) The aggregate content of the preparation after a 7-day accelerated test is less than 1.5%, such as less than 1.0%, or less than 0.8%.

[0130] In another alternative, the method further comprises the step of mixing a therapeutically effective amount of viral particles, soluble salts, polyols, and pH buffer with a surfactant.

[0131] It should be understood that within the scope of the present disclosure, the above-mentioned technical features of the present disclosure and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or optional technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0132] Figure 1. Schematic diagram of thermal stability results of different formulations in Example 1;

[0133] Figure 2. Aggregation of different formulations in Example 1 after 10 freeze-thaw cycles;

[0134] Figure 3. Free nucleic acid in different formulations after 10 freeze-thaw cycles in Example 1;

[0135] Figure 4. Percentage of fully loaded AAV after 10 freeze-thaw cycles for different formulations in Example 1;

[0136] Figure 5. Virus aggregation in accelerated tests with different formulations in Example 1;

[0137] Figure 6. Free nucleic acid status of different formulations in accelerated testing in Example 1;

[0138] Figure 7. Percentage of full shells in accelerated tests of different formulations in Example 1;

[0139] Figure 8. Thermal stability of AAV with different formulations in Example 2;

[0140] Figure 9. Signal response intensity of monomeric virus particles in 10 freeze-thaw experiments in Example 2;

[0141] Figure 10. Aggregate ratios in 10 freeze-thaw experiments in Example 2;

[0142] Figure 11. Free nucleic acid in 10 freeze-thaw experiments in Example 2;

[0143] Figure 12. Virus particle loading in 10 freeze-thaw experiments in Example 2;

[0144] Figure 13. Response intensity of accelerated viral particles in Example 2 after 7 days;

[0145] Figure 14. Accelerated viral particle aggregation over 7 days in Example 2;

[0146] Figure 15. Accelerated free nucleic acid status over 7 days in Example 2;

[0147] Figure 16 shows the signal response intensity of monomeric virus particles in the freeze-thaw test of Example 3;

[0148] Figure 17. Effect of freeze-thaw on the purity of AAV in different formulations in Example 3;

[0149] FIG18 shows the response strength of AAV particles with different formulations in the accelerated test of Example 3;

[0150] Figure 19. Effect of different formulations on AAV purity in the accelerated test of Example 3;

[0151] Figure 20. Effects of freeze-thaw on free nucleic acids in different formulations in Example 3;

[0152] Figure 21. Effect of the accelerated test of Example 3 on free nucleic acids;

[0153] Figure 22. Comparative results of clinical experiments in Example 4. DETAILED DESCRIPTION

[0154] After extensive and in-depth research, the inventors of the present disclosure have discovered that a buffer system containing soluble salts, polyols, and a pH buffer can maintain the pH of the solution at 7.0 to 8.0 (e.g., 7.0 to 7.6, or 7.0 to 7.4), including but not limited to 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0, and can significantly improve the stability and / or dispersibility of viral particles. Furthermore, liquid formulations containing soluble salts, polyols, and a pH buffer can also significantly improve the stability and / or dispersibility of viral particles, reduce the aggregate content and the content of free nucleic acid, without significantly reducing the intensity of the viral particle signal response and the viral activity, and can significantly improve (e.g., enhance) the dispersed number and stability of viral particles. The inventors have completed the present disclosure on this basis.

[0155] As used herein, the term "liquid formulation" refers to a preparation in a form that allows the biological activity of the active ingredient to be effective and does not contain other components that are unacceptably toxic to the subject to which the formulation is administered. The subject includes a mammal, which may be a human.

[0156] As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount that is effective in preventing or treating a disease in a pharmacological sense, within the scope of the present disclosure. The antibody is effective for treating the disease. "Treatment" refers to both therapeutic treatment and preventive or defensive measures. Subjects in need of treatment include those already suffering from the relevant condition, as well as those in need of prevention of the relevant disease.

[0157] As used herein, a "pH buffer" refers to a solution prepared by a buffer pair consisting of a salt of a weak acid and its conjugate base, or a salt of a weak base and its conjugate acid, that is capable of slowing down pH changes when a certain amount of other substances is added. The pH buffer disclosed herein maintains the pH value of the solution between 7.0 and 8.0 (e.g., 7.0 to 7.6, or 7.0 to 7.4). An optional buffer herein is PB buffer.

[0158] As used herein, "divalent metal ion" refers to a stable structure with a positive charge resulting from the loss of two electrons from an atom. Common divalent metal ions include Ca 2+ Mg 2+ 、Zn 2+ 、Cu 2+ 、Mn 2+ Cr 2+ The optional divalent metal ion herein is Ca 2+ Mg2+ .

[0159] One object of the present disclosure is to provide an AAV formulation that is stable, suitable for different administration routes, capable of high-concentration dose administration, and ensures low osmotic pressure.

[0160] In one aspect, the present disclosure provides a buffer system comprising a soluble salt substance, a polyol, and a pH buffer solution.

[0161] In an optional example, the buffer system includes a first buffer system; the first buffer system includes soluble salt substances, polyols and pH buffer, and the osmotic pressure of the first buffer system is 280 to 330 mosm / kg.

[0162] In another optional example, in the first buffer system, the soluble salt substances include one or more soluble monovalent metal salts and / or one or more soluble divalent metal salts; the polyol includes one or more of sucrose, mannitol and glycerol; and the pH buffer is selected from PB buffer and / or HEPES buffer.

[0163] In another alternative, in the first buffer system, the soluble monovalent metal salt includes a sodium salt, such as sodium chloride; the soluble divalent metal salt includes Ca 2+ Mg 2+ 、Zn 2+ 、Cu 2+ 、Mn 2+ and Cr 2+ One or more of 2+ and Mg 2+ .

[0164] In another optional example, in the first buffer system, the soluble salt substance includes a soluble monovalent metal salt.

[0165] In another alternative, in the first buffer system, the soluble monovalent metal salt includes a soluble sodium salt.

[0166] In another optional example, in the first buffer system, the soluble salt substance includes a soluble divalent metal salt.

[0167] In another optional example, in the first buffer system, the soluble divalent metal salt includes one or more of a soluble calcium salt, a soluble magnesium salt, a soluble zinc salt, a soluble copper salt, a soluble manganese salt, and a soluble chromium salt.

[0168] In another optional example, in the first buffer system, the soluble divalent metal salt is a soluble calcium salt and / or a soluble magnesium salt.

[0169] In another optional example, in the first buffer system, the soluble monovalent metal salt is a sodium salt, and the soluble divalent metal salt is a soluble calcium salt and a soluble magnesium salt.

[0170] In another optional example, in the first buffer system, the polyol includes one or more of sucrose, mannitol or glycerol.

[0171] In another optional example, in the first buffer system, the polyol is sucrose and / or glycerol;

[0172] In another optional example, in the first buffer system, the polyol is sucrose.

[0173] In another optional example, in the first buffer system, the soluble monovalent metal salt is a sodium salt, the soluble divalent metal salt is a soluble calcium salt and a soluble magnesium salt, and the polyol is sucrose.

[0174] In another optional example, in the first buffer system, the pH buffer is selected from one or any several buffers of the following group: PB buffer and / or HEPES buffer.

[0175] In another optional example, in the first buffer system, the pH buffer is a PB buffer.

[0176] In another optional example, in the first buffer system, the soluble monovalent metal salt is a sodium salt, the soluble divalent metal salt is a soluble calcium salt and a soluble magnesium salt, the polyol is sucrose, and the pH buffer is a PB buffer.

[0177] In another optional example, in the first buffer system, the Na + Derived from NaCl.

[0178] In another alternative, in the first buffer system, the Ca 2 Derived from CaCl2 and CH3COOCa.

[0179] In another optional example, in the first buffer system, the Mg 2+ Derived from MgCl2 and Mg2SO4.

[0180] In another optional example, in the first buffer system, the concentration of the soluble monovalent metal salt in the soluble salt substance is 50-250 mM, such as 80-200 mM, or 100-190 mM, or 100-160 mM.

[0181] In another optional example, in the first buffer system, the concentration of the soluble divalent metal salt is 0.2-8 mM, such as 0.5-5 mM, or 0.8-2 mM.

[0182] In another optional example, in the first buffer system, the concentration of the soluble monovalent metal salt is 50-250 mM, and the concentration of the soluble divalent metal salt is 0.2-8 mM.

[0183] In another optional example, in the first buffer system, the concentration of the soluble monovalent metal salt is 100-160 mM, and the concentration of the soluble divalent metal salt is 0.8-2 mM.

[0184] In another optional example, in the first buffer system, the concentration of the soluble monovalent metal salt is 80-200 mM, and the concentration of the soluble divalent metal salt is 0.5-5 mM.

[0185] In another optional example, in the first buffer system, the concentration (w / v) of the polyol is 0.5% to 20%, such as 0.8% to 10%, or 0.9% to 5%, based on the total weight volume percentage of the buffer system.

[0186] In another optional example, in the first buffer system, the concentration of sucrose (w / v) is 0.5% to 20%, such as 0.8% to 10%, or 0.9% to 5%, calculated as a percentage by weight of the total volume of the buffer system.

[0187] In another optional example, in the first buffer system, the concentration of the soluble monovalent metal salt is 50-250 mM, the concentration of the soluble divalent metal salt is 0.2-8 mM, and the concentration (w / v) of sucrose is 0.5%-20%.

[0188] In another optional example, in the first buffer system, the concentration of the soluble monovalent metal salt is 80-200 mM, the concentration of the soluble divalent metal salt is 0.5-5 mM, and the concentration (w / v) of sucrose is 0.8%-10%.

[0189] In another optional example, in the first buffer system, the concentration of the soluble monovalent metal salt is 100-160 mM, the concentration of the soluble divalent metal salt is 0.8-2 mM, and the concentration (w / v) of sucrose is 0.9%-5%.

[0190] In another optional example, in the first buffer system, the concentration of mannitol (w / v) is 0.5% to 20%, such as 1% to 10%, or 2% to 5%, based on the total weight volume percentage of the buffer system.

[0191] In another optional example, in the first buffer system, the concentration of the glycerol (w / v) is 0.5% to 20%, such as 1% to 10%, or 2% to 5%, based on the total weight volume percentage of the buffer system.

[0192] In another optional example, in the first buffer system, the concentration of the soluble monovalent metal salt is 50-250 mM, the concentration of the soluble divalent metal salt is 0.2-8 mM, and the concentration (w / v) of the glycerol is 0.5%-20%.

[0193] In another optional example, in the first buffer system, the concentration of the soluble monovalent metal salt is 80-200 mM, the concentration of the soluble divalent metal salt is 0.5-5 mM, and the concentration (w / v) of the glycerol is 1%-10%.

[0194] In another optional example, in the first buffer system, the concentration of the soluble monovalent metal salt is 100-160 mM, the concentration of the soluble divalent metal salt is 0.8-2 mM, and the concentration (w / v) of the glycerol is 2%-5%.

[0195] In another optional example, in the first buffer system, the concentration of the pH buffer is 2-50 mM, such as 5-30 mM, or 8-20 mM.

[0196] In another optional example, in the first buffer system, the pH value of the buffer system is maintained at 7.0-8.0 (eg, 7.0-7.6, or 7.0-7.4).

[0197] In another optional example, in the first buffer system, the buffer system further includes a surfactant.

[0198] In another optional example, in the first buffer system, the surfactant is selected from one or more of polyoxyethylene polyoxypropylene ether block copolymer (Poloxamer 188, F68), polysorbate (Tween), polyethylene glycol (PEG), polyethylene glycol p-isooctylphenyl ether (Triton), fatty acid sorbitan (Span), sucrose fatty acid ester (SE), polyoxyethylene fatty alcohol ether (Brij) and polyoxyethylene fatty acid ester (Myeij).

[0199] In another optional example, in the first buffer system, the surfactant is selected from the following group: F68, Tween, or a combination thereof.

[0200] In another optional example, in the first buffer system, the Tween includes Tween20.

[0201] In another optional example, in the first buffer system, the concentration (w / v) of the surfactant is 0.0005% to 0.002%, for example, 0.0008% to 0.002%, based on the total weight volume percentage of the buffer system.

[0202] In another optional example, in the first buffer system, the solvent of the buffer system is an organic solvent or an inorganic solvent, and optionally the solvent is an inorganic solvent, for example, the solvent of the buffer system is water.

[0203] In another optional example, the buffer system includes a second buffer system, and the second buffer system includes a soluble monovalent metal salt, a soluble divalent metal salt with a complexing effect, a polyol and a pH buffer.

[0204] In another alternative, the second buffer system comprises a sodium salt such as sodium chloride, and a divalent metal salt such as CaCl. 2+ Mg 2+ 、Zn 2+ 、Cu 2+ 、Mn 2+ and Cr 2+ One or more of 2+ and Mg 2+ ; The polyol includes one or more of sucrose, mannitol and glycerol; the pH buffer is selected from PB buffer and / or HEPES buffer.

[0205] In another optional example, in the second buffer system, the soluble monovalent metal salt is a sodium salt, and the soluble divalent metal salt is a soluble calcium salt and a soluble magnesium salt.

[0206] In another optional example, in the second buffer system, the soluble monovalent metal salt is a sodium salt, and the soluble divalent metal salt is a soluble calcium salt and a soluble magnesium salt.

[0207] In another optional example, in the second buffer system, the soluble monovalent metal salt is a sodium salt, the soluble divalent metal salt is a soluble calcium salt and a soluble magnesium salt, and the polyol is sucrose.

[0208] In another optional example, in the second buffer system, the soluble monovalent metal salt is a sodium salt, the soluble divalent metal salt is a soluble calcium salt and a soluble magnesium salt, the polyol is sucrose, and the pH buffer is a PB buffer.

[0209] In another optional example, in the second buffer system, the concentration of the soluble monovalent metal salt is 50-250 mM, for example, 80-200 mM, or 100-190 mM, or 100-160 mM; the concentration of the soluble divalent metal salt is 0.2-8 mM, for example, 0.5-5 mM, or 0.8-2 mM; based on the total weight volume percentage of the buffer system, the concentration of the polyol is 0.5%-20%, for example, 0.8%-10%, or 0.9%-5%; the concentration of the pH buffer solution is 2-50 mM, for example, 5-30 mM, or 8-20 mM; optionally, the pH value of the buffer system is 7.0-8.0, for example, 7.0-7.6, or 7.0-7.4.

[0210] In another optional example, in the second buffer system, the soluble monovalent metal salt is a sodium salt, the soluble divalent metal salt is a soluble calcium salt and a soluble magnesium salt, the concentration of the soluble monovalent metal salt is 50 to 250 mM, and the concentration of the soluble divalent metal salt is 0.2 to 8 mM.

[0211] In another optional example, in the second buffer system, the soluble monovalent metal salt is a sodium salt, the soluble divalent metal salt is a soluble calcium salt and a soluble magnesium salt, the concentration of the soluble monovalent metal salt is 100-160 mM, and the concentration of the soluble divalent metal salt is 0.8-2 mM.

[0212] In another optional example, in the second buffer system, the soluble monovalent metal salt is a sodium salt, the soluble divalent metal salt is a soluble calcium salt and a soluble magnesium salt, the concentration of the soluble monovalent metal salt is 80-200 mM, and the concentration of the soluble divalent metal salt is 0.5-5 mM.

[0213] In another optional example, in the second buffer system, the soluble monovalent metal salt is a sodium salt, the soluble divalent metal salt is a soluble calcium salt and a soluble magnesium salt, the polyol is sucrose, the concentration of the soluble monovalent metal salt is 50 to 250 mM, the concentration of the soluble divalent metal salt is 0.2 to 8 mM, and the concentration of the polyol is 0.5% to 20%.

[0214] In another optional example, in the second buffer system, the soluble monovalent metal salt is a sodium salt, the soluble divalent metal salt is a soluble calcium salt and a soluble magnesium salt, the polyol is sucrose, the concentration of the soluble monovalent metal salt is 100-160 mM, the concentration of the soluble divalent metal salt is 0.8-2 mM, and the concentration of the polyol is 0.8%-10%.

[0215] In another optional example, the soluble monovalent metal salt is a sodium salt, the soluble divalent metal salt is a soluble calcium salt and a soluble magnesium salt, the polyol is sucrose, the concentration of the soluble monovalent metal salt is 80-200 mM, the concentration of the soluble divalent metal salt is 0.5-5 mM, and the concentration of the polyol is 0.9%-5%.

[0216] In another optional example, the second buffer system also includes a surfactant, which includes one or more of polyoxyethylene polyoxypropylene ether block copolymer (Poloxamer 188, F68), polysorbate (Tween), polyethylene glycol (PEG), polyethylene glycol p-isooctylphenyl ether (Triton), fatty acid sorbitan (Span), sucrose fatty acid ester (SE), polyoxyethylene fatty alcohol ether (Brij) and polyoxyethylene fatty acid ester (Myeij), such as F68.

[0217] In another optional example, in the second buffer system, the concentration of the surfactant is 0.0005% to 0.002% (w / v).

[0218] In another aspect, the present disclosure provides use of the aforementioned first buffer system or second buffer system for preparing a reagent or kit for improving the stability and dispersibility of virus particles.

[0219] In another alternative embodiment, the improving the stability and dispersibility of the virus particles includes one or more of the following:

[0220] (i) Prevent the virus from breaking, aggregating, or damaging its capsid protein or packaging integrity during storage at low temperatures, room temperature, or repeated freezing and thawing;

[0221] (ii) disperse a greater number of virus particles;

[0222] (iii) It has isotonic pressure. Within the lower isotonic pressure range, adverse reactions caused by high osmotic pressure can be avoided, which is conducive to the use of the product in more application scenarios.

[0223] In another optional example, the virus includes AAV virus, lentivirus or recombinant AAV virus; the serotype of the AAV virus or recombinant AAV virus includes AAV2, AAV4, AAV5, AAV6, AAV6.2FF, AAV8, AAV9, AAVPHP.eB, AAVPHP.S or AAVPHP.B.

[0224] In another optional example, the target gene carried by the recombinant AAV virus is selected from one or more of the following: tyrosine hydroxylase (TH) gene, GTP-cyclohydrolase I (GCH1) gene, aromatic amino acid dopa decarboxylase (AADC) gene, coagulation factor VIII gene, coagulation factor IX gene, anti-AIDS neutralizing antibody coding sequence, CD4 receptor neutralizing antibody coding sequence, anti-VEGF or VEGFR antibody coding sequence, anti-IgE antibody coding sequence and / or anti-PCSK9 antibody or its antigen coding sequence antibody sequence related to hyperlipidemia, such as Alirocumab antibody or Evolocumab antibody.

[0225] In another optional example, one or more of the soluble salt substances are packaged in the same or different containers, one or more of the polyols are packaged in the same or different containers, and one or more of the pH buffers are packaged in the same or different containers.

[0226] Another aspect of the present disclosure provides a kit comprising:

[0227] (i) one or more containers containing the same or different soluble salt substances;

[0228] (ii) one or more containers containing the same or different polyols;

[0229] (iii) one or more containers containing the same or different pH buffers;

[0230] (iv) optionally one or more containers containing the same or different surfactants.

[0231] Optionally, the combination of the soluble salt substance, the polyol, the pH buffer and the surfactant is selected from the aforementioned first buffer system or the second buffer system.

[0232] In another alternative embodiment, the kit is further used for one or more purposes selected from the following group:

[0233] (a) Prevent the virus from breaking, aggregating, or damaging its capsid protein or packaging integrity during storage at low temperatures, room temperature, or repeated freezing and thawing;

[0234] (b) disperse a greater number of virus particles;

[0235] (c) It has isotonic pressure. Within the lower isotonic pressure range, adverse reactions caused by high osmotic pressure can be avoided, which is conducive to the use of the product in more application scenarios.

[0236] In another optional example, any two, three, or four (or all) of the above-mentioned containers may be the same (or identical) or different containers.

[0237] In another optional example, any two or three (or all) of the above-mentioned containers can be the same (or identical) or different containers.

[0238] Another aspect of the present disclosure provides a method for improving the stability and / or dispersibility of viral particles, comprising:

[0239] The buffer system (the first buffer system or the second buffer system) described in the aforementioned aspect of the present disclosure is provided, and virus particles are placed in the buffer system to improve the stability and / or dispersibility of the virus particles.

[0240] In another alternative embodiment, the viral particles include AAV viral particles, lentiviral particles, or a combination thereof.

[0241] A fifth aspect of the present disclosure provides a liquid preparation comprising:

[0242] (i) Virus particles;

[0243] (ii) soluble salts;

[0244] (iii) polyols;

[0245] (iv) pH buffer.

[0246] In another optional example, the liquid preparation includes viral particles and the buffer system (the first buffer system or the second buffer system) described in the above aspects.

[0247] In another alternative, the viral particles include a therapeutically effective amount of viral particles.

[0248] In another optional example, the virus includes AAV virus, lentivirus or recombinant AAV virus; the serotype of the AAV virus or recombinant AAV virus includes AAV2, AAV4, AAV5, AAV6, AAV6.2FF, AAV8, AAV9, AAVPHP.eB, AAVPHP.S or AAVPHP.B.

[0249] In another optional example, the target gene carried by the recombinant AAV virus is selected from one or more of the following: tyrosine hydroxylase (TH) gene, GTP-cyclohydrolase I (GCH1) gene, aromatic amino acid dopa decarboxylase (AADC) gene, coagulation factor VIII gene, coagulation factor IX gene or anti-hyperlipidemia-related antibody sequence, such as Alirocumab antibody or Evolocumab antibody.

[0250] In another optional example, the viral particles are packaged by an adeno-associated viral vector system.

[0251] In another optional example, the structure of the adeno-associated virus vector system is as described in patent application number PCT / CN2022 / 115831 or CN202310394646.1.

[0252] In another alternative embodiment, the concentration of the virus particles is 5×10 11 ~5×10 14 vg / mL, for example 2×10 12 ~2×10 14 vg / mL, or 1×10 13 ~1×10 14 vg / mL.

[0253] In another optional example, the osmotic pressure of the liquid preparation is 280 to 330 mosm / kg.

[0254] In another alternative, the liquid formulation has one or more characteristics selected from the following group:

[0255] (a) The aggregate content of the liquid preparation is less than 1.5% (the aggregate content refers to a form of expression of product purity. In SEC chromatography, three signals can be separated: AAV monomers, AAV aggregates, and free nucleic acids. The percentage of aggregates refers to the percentage of aggregate signals to the total of the above signals), for example, less than 1.0%, or less than 0.8%, or less than 0.7%;

[0256] (b) the free nucleic acid content of the liquid preparation is less than 15 ppm, for example, less than 10 ppm, or less than 8 ppm;

[0257] (c) After a 7-day accelerated destruction test at 37°C, the liquid preparation showed no significant decrease in the intensity of the viral particle signal response and no significant decrease in viral activity;

[0258] (d) The liquid preparation undergoes a 7-day accelerated destruction test at 37° C., and the aggregate content is less than 1.5%, such as less than 1.0%, or less than 0.8%.

[0259] In another alternative, the aggregate content of the liquid preparation is 0.3% to 1.5%, such as 0.3% to 1%, or 0.3% to 0.7%.

[0260] In another optional example, the free nucleic acid content of the liquid preparation after a 7-day 37° C. accelerated destruction test is 25 ppm to 35 ppm.

[0261] In another optional example, the liquid preparation undergoes a 7-day accelerated destruction test at 37° C., and the aggregate content is 0.3% to 0.7%.

[0262] In another alternative, the liquid preparation comprises:

[0263] (a)5×10 11 ~5×10 14 A therapeutically effective amount of viral particles at a concentration of vg / mL;

[0264] (b) 50-250 mM soluble salts;

[0265] (c) 0.5% to 20% (w / v) polyol;

[0266] (d) 2-50 mM pH buffer;

[0267] And the pH of the liquid preparation is 7.0-8.0.

[0268] In another alternative, the liquid preparation includes 0.0005% to 0.002% (w / v) of a surfactant.

[0269] In another alternative, the liquid preparation comprises:

[0270] (a)2×10 12 ~2×10 14 A therapeutically effective amount of viral particles at a concentration of vg / mL;

[0271] (b) 80-200 mM soluble salts;

[0272] (c) 0.8% to 10% (w / v) polyol;

[0273] (d) 5-30 mM pH buffer;

[0274] And the pH of the liquid preparation is 7.0-7.6.

[0275] In another alternative, the liquid preparation includes 0.0005% to 0.002% (w / v) of a surfactant.

[0276] In another alternative, the liquid preparation comprises:

[0277] (a)1×10 13 ~1×10 14 A therapeutically effective amount of viral particles at a concentration of vg / mL;

[0278] (b) 100-190 mM soluble salts;

[0279] (c) 0.9% to 5% (w / v) polyol;

[0280] (d) 8-15 mM pH buffer;

[0281] And the pH of the liquid preparation is 7.0-7.4.

[0282] In another alternative, the liquid preparation includes 0.0008% to 0.002% (w / v) of a surfactant.

[0283] Another aspect of the present disclosure provides the use of the liquid preparation described in the aforementioned aspects of the present disclosure for preparing (i) a drug for preventing and / or treating hereditary coagulation factor deficiency; and / or (ii) a drug for preventing and / or treating hyperlipidemia-related diseases.

[0284] Another aspect of the present disclosure provides a method for preventing and / or treating a disease, wherein the disease includes (i) an inherited coagulation factor deficiency disease, and (ii) a hyperlipidemia-related disease; the method for preventing and / or treating the inherited coagulation factor deficiency disease comprises administering to a subject a therapeutically effective amount of the liquid preparation of the aforementioned aspects, wherein the viral particles in the liquid preparation carry a coagulation factor VIII gene and / or a coagulation factor IX gene;

[0285] In another alternative embodiment, the administration of the preventive and / or therapeutic method includes intravenous injection and / or intramuscular injection.

[0286] In another alternative embodiment, the hereditary coagulation factor deficiency disease is hemophilia A, B or C.

[0287] In another optional example, the hereditary coagulation factor deficiency disease is a mammalian hereditary coagulation factor deficiency disease; optionally, the hereditary coagulation factor deficiency disease is a human hereditary coagulation factor deficiency disease.

[0288] Another aspect of the present disclosure provides a medicine kit, which contains a container and the liquid preparation according to the above aspect of the present disclosure located in the container.

[0289] Another aspect of the present disclosure provides a method for preparing a stable liquid formulation for improving the stability and / or dispersibility of viral particles, comprising the steps of:

[0290] (a) mixing virus particles, soluble salt substances, polyols and pH buffer;

[0291] (b) adjusting the pH of the mixture to a pH of 7.0 to 8.0 (e.g., 7.0 to 7.6, or 7.0 to 7.4);

[0292] Thus, a stable liquid preparation with improved stability and / or dispersibility of virus particles is obtained.

[0293] In another optional example, the step comprises mixing the components of the liquid preparation described in the above aspects, and then adjusting the pH value of the mixed mixture to a pH of 7.0 to 8.0, such as 7.0 to 7.6, or 7.0 to 7.4.

[0294] In another alternative, the stable liquid formulation for improving the stability and / or dispersibility of viral particles has one or more characteristics selected from the following group:

[0295] (a) the aggregate content of the liquid preparation is less than 1.5%, such as less than 1.0%, or less than 0.8%;

[0296] (b) the free nucleic acid content of the liquid preparation is less than 15 ppm, for example, less than 10 ppm, or less than 8 ppm;

[0297] (c) After a 7-day accelerated destruction test at 37°C, the liquid preparation showed no significant decrease in the intensity of the viral particle signal response and no significant decrease in viral activity;

[0298] (d) The liquid preparation undergoes a 7-day accelerated destruction test at 37° C., and the aggregate content is less than 1.5%, such as less than 1.0%, or less than 0.8%.

[0299] In another optional example, the method further comprises the step of mixing the virus particles, soluble salt substances, polyols and pH buffer with a surfactant.

[0300] In another alternative embodiment, the viral particles are a therapeutically effective amount of viral particles.

[0301] Liquid preparations

[0302] The liquid preparation disclosed herein for improving the stability and dispersibility of viral particles mainly comprises:

[0303] (i) a therapeutically effective amount of viral particles;

[0304] (ii) soluble salts;

[0305] (iii) polyols;

[0306] (iv) pH buffer;

[0307] The pH of the liquid preparation is 7.0 to 8.0 (eg, 7.0 to 7.6, or 7.0 to 7.4).

[0308] The therapeutically effective amount of viral particles present in the liquid formulation of the present disclosure is determined by considering the desired dosage volume and mode of administration. In the present disclosure, the concentration of viral particles is 5×10 11 ~5×10 14 vg / mL, for example 2×1012 ~2×10 14 vg / mL, or 1×10 13 -1×10 14 vg / mL, measured by the physical titer of the preparation, the concentration includes but is not limited to 5×10 11 vg / mL, 6×10 11 vg / mL, 7×10 11 vg / mL、8×10 11 vg / mL, 9×10 11 vg / mL, 1×10 12 vg / mL, 2×10 12 vg / mL, 3×10 12 vg / mL, 4×10 12 vg / mL, 5×10 12 vg / mL, 6×10 12 vg / mL, 7×10 12 vg / mL、8×10 12 vg / mL, 9×10 12 vg / mL, 1×10 13 vg / mL, 2×10 13 vg / mL, 3×10 13 vg / mL, 4×10 13 vg / mL, 5×10 13 vg / mL, 6×10 13 vg / mL, 7×10 13 vg / mL、8×10 13 vg / mL, 9×10 13 vg / mL, 1×10 14 vg / mL, 2×10 14 vg / mL, 3×10 14 vg / mL, 4×10 14 vg / mL or 5×10 14 The present disclosure includes ranges of values ​​using a combination of any of the above values ​​as upper and / or lower limits.

[0309] The buffer system used in the formulation of the present invention is a buffer system comprising a soluble salt substance, a polyol, and a pH buffer. In an alternative embodiment, the soluble salt substance of the present invention comprises a soluble monovalent metal salt. In an alternative embodiment, the soluble salt substance of the present invention comprises a soluble divalent metal salt.

[0310] In an alternative embodiment, the soluble monovalent metal salt includes a soluble sodium salt. In an alternative embodiment, the soluble divalent metal salt includes one or more of a soluble calcium salt, a soluble magnesium salt, a soluble zinc salt, a soluble copper salt, a soluble manganese salt, and a soluble chromium salt. In the present disclosure, the divalent metal ion in the soluble divalent metal salt can be selected from Ca 2+ Mg 2+ , and its concentration is 0.2-8mM, such as 0.5-5mM, or 0.8-2mM, including but not limited to 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM, 0.8mM, 0.9mM, 1.0mM, 1.5mM, 2.0mM, 2.5mM, 3.0mM, 3.5mM, 4.0mM, 4.5mM, 5.0mM, 5.5mM, 6.0mM, 6.5mM, 7.0mM, 7.5mM or 8.0mM.

[0311] In an optional embodiment, the concentration of the soluble monovalent metal salt in the soluble salt substance in the buffer system is 50-250mM, for example 80-200mM, or 100-190mM, or 100-160mM, including but not limited to 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 120mM, 140mM, 160mM, 180mM, 200mM, 220mM, 240mM or 250mM.

[0312] In an optional embodiment, the concentration (w / v) of the polyol in the buffer system is 0.5% to 20%, for example 0.8% to 10%, or 0.9% to 5%, based on the total weight volume percentage of the buffer system, including but not limited to 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5% or 10.0%.

[0313] In an optional embodiment, the concentration (w / v) of the surfactant in the buffer system is 0.0005% to 0.002%, for example 0.0008% to 0.002%, including but not limited to 0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.001%, 0.0011%, 0.0012%, 0.0013%, 0.0014%, 0.0015%, 0.0016%, 0.0017%, 0.0018%, 0.0019% or 0.002%, based on the total weight volume percentage of the buffer system.

[0314] In an optional embodiment, the concentration of the pH buffer in the buffer system is 2 to 50 mM, for example 5 to 30 mM, or 8 to 20 mM, including but not limited to 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM or 50 mM.

[0315] The formulations of the present disclosure are optionally isotonic.The present disclosure includes ranges of values ​​using a combination of any of the above values ​​as upper and / or lower limits.

[0316] The amount of surfactant added in the present disclosure improves virus dispersion while also improving virus stability. An optional surfactant in the present disclosure is Poloxamer 188 (F68).

[0317] The present disclosure adjusts the pH value of the formulation by a buffer system to control the pH within the range of 7.0 to 8.0 (e.g., 7.0 to 7.6, or 7.0 to 7.4). In certain embodiments, the pH of the formulation is between 7.2 and 7.4. The present disclosure includes a range of values ​​using a combination of any of the above values ​​as the upper and / or lower limits.

[0318] In addition, after repeated experiments, the inventors conducted a large number of screenings on the components and contents of various buffer systems, and finally obtained the buffer system disclosed in the present invention from a large number of buffer systems. It was found that in this system, the stability and dispersibility of virus particles were significantly improved, and it can also prevent the virus from being damaged by capsid protein fragmentation, aggregation and packaging integrity caused by low temperature, multiple freeze-thaw cycles, and refrigerated and room temperature storage; disperse a larger number of virus particles; ensure isotonic pressure, realize more application scenarios, and avoid adverse reactions caused by high osmotic pressure. The osmotic pressure of the buffer system is 280-340mosm / kg, for example, 280-330mosm / kg, including but not limited to 280mosm / kg, 290mosm / kg, 300mosm / kg, 310mosm / kg, 320mosm / kg, 330mosm / kg or 340mosm / kg.

[0319] The liquid formulations of the present disclosure may include one or more other pharmaceutically acceptable carriers, excipients, or stabilizers, such as those described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980), as long as they do not adversely affect the desired characteristics of the formulation. Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosages and concentrations employed, and include other cosolvents; antioxidants including ascorbic acid and methionine; biodegradable polymers such as polyesters; and / or salt-forming counterions.

[0320] The preparation disclosed herein can be prepared by combining various components at certain concentrations using methods known in the art.

[0321] One optional method mainly includes the following steps:

[0322] Viral particles were concentrated by centrifugation (4500 rpm, 4-10°C) using Ultracel-30K ultrafiltration centrifuge tubes and exchanged into different formulation buffers. The viral particle concentration was adjusted to the desired concentration using pH buffer. The formulations were sterilized by filtration using a 0.22 μm Millex syringe filter. The prepared formulations were packaged in vials for ease of use.

[0323] In one aspect, the present disclosure provides a liquid preparation, wherein the liquid preparation comprises:

[0324] (i) a therapeutically effective amount of viral particles;

[0325] (ii) soluble salts;

[0326] (iii) polyols;

[0327] (iv) pH buffer.

[0328] In an optional embodiment, the pH value of the liquid preparation is 7.0-8.0, such as 7.0-7.6, or 7.0-7.4.

[0329] In an optional embodiment, the osmotic pressure of the liquid preparation is 280 to 330 mosm / kg, including but not limited to 280 mosm / kg, 290 mosm / kg, 300 mosm / kg, 310 mosm / kg, 320 mosm / kg or 330 mosm / kg.

[0330] In an optional embodiment, the soluble salt substance includes one or more soluble monovalent metal salts and / or one or more soluble divalent metal salts; the polyol includes one or more of fructose, glucose, maltose, sucrose, glycerol, trehalose, and mannitol; and the pH buffer is selected from PB buffer and / or HEPES buffer.

[0331] In an optional embodiment, the soluble salt substance includes one or more soluble monovalent metal salts and / or one or more soluble divalent metal salts; the polyol includes one or more of sucrose, glycerol, and mannitol; and the pH buffer is selected from PB buffer and / or HEPES buffer.

[0332] In an alternative embodiment, the soluble monovalent metal salt comprises a sodium salt, such as sodium chloride.

[0333] In an optional embodiment, the concentration of the soluble monovalent metal salt is 50-250 mM, such as 80-200 mM, or 100-190 mM, or 100-160 mM.

[0334] In an optional embodiment, the soluble monovalent metal salt is sodium chloride, and the concentration of the sodium chloride is 50-250 mM, such as 80-200 mM, or 100-190 mM, or 100-160 mM.

[0335] In an alternative embodiment, the soluble divalent metal salt comprises Ca 2+ Mg 2+ 、Zn 2+ 、Cu 2+ 、Mn 2+ and Cr 2+ One or more of 2+ and Mg 2+ .

[0336] In an optional embodiment, the concentration of the soluble divalent metal salt is 0.2-8 mM, such as 0.5-5 mM, or 0.8-2 mM.

[0337] In an optional embodiment, the soluble monovalent metal salt is sodium chloride, and the concentration of the sodium chloride is 50-250 mM, such as 80-200 mM, or 100-190 mM, or 100-160 mM. The soluble divalent metal salt is Ca 2+ and Mg 2+ , the Ca 2+ and Mg 2+ The concentration of is 0.2-8 mM, such as 0.5-5 mM, or 0.8-2 mM.

[0338] In an optional embodiment, the concentration of the polyol is 0.5% to 20%, such as 0.8% to 10%, or 0.9% to 5%, based on the total weight volume percentage of the buffer system; the concentration of the pH buffer is 2 to 50 mM, such as 5 to 30 mM, or 8 to 20 mM.

[0339] In an optional embodiment, the soluble monovalent metal salt is sodium chloride, and the concentration of the sodium chloride is 50-250 mM, such as 80-200 mM, or 100-190 mM, or 100-160 mM. The soluble divalent metal salt is Ca 2+ and Mg 2+ , the Ca 2+ and Mg 2+ The concentration of the polyol is sucrose, and the concentration of the sucrose is 0.5% to 20%, for example, 0.8% to 10%, or 0.9% to 5%. The pH buffer is PB buffer, and the concentration of the PB buffer is 2 to 50 mM, for example, 5 to 30 mM, or 8 to 20 mM.

[0340] In an optional embodiment, the liquid preparation further includes a surfactant, which includes one or more of polyoxyethylene polyoxypropylene ether block copolymer (Poloxamer 188, F68), polysorbate (Tween), polyethylene glycol (PEG), polyethylene glycol p-isooctylphenyl ether (Triton), fatty acid sorbitan (Span), sucrose fatty acid ester (SE), polyoxyethylene fatty alcohol ether (Brij) and polyoxyethylene fatty acid ester (Myeij), such as F68.

[0341] In an optional embodiment, the concentration of the surfactant is 0.0005% to 0.002% (w / v).

[0342] In an optional embodiment, the soluble monovalent metal salt is sodium chloride, and the concentration of the sodium chloride is 50-250 mM, such as 80-200 mM, or 100-190 mM, or 100-160 mM. The soluble divalent metal salt is Ca 2+ and Mg 2+ , the Ca 2+ and Mg 2+The concentration of the polyol is sucrose, and the concentration of the sucrose is 0.5% to 20%, for example, 0.8% to 10%, or 0.9% to 5%. The pH buffer is PB buffer, and the concentration of the PB buffer is 2 to 50 mM, for example, 5 to 30 mM, or 8 to 20 mM. The surfactant is F68, and the concentration of F68 is 0.0005% to 0.002% (w / v).

[0343] In an optional embodiment, the viral particles include AAV virus, lentivirus or recombinant AAV virus, and the serotype of the AAV virus or recombinant AAV virus includes AAV2, AAV4, AAV5, AAV6, AAV6.2FF, AAV8, AAV9, AAVPHP.eB, AAVPHP.S or AAVPHP.B; optionally, the target gene carried by the viral particles includes a coagulation factor gene; optionally, the coagulation factor gene is selected from the FIX factor gene or the coagulation factor VIII gene; optionally, the nucleotide sequence of the FIX factor gene is as shown in SEQ ID No: 3.

[0344] In an alternative embodiment, the concentration of the virus particles is 5×10 11 ~5×10 14 vg / mL, for example 2×10 12 ~2×10 14 vg / mL, or 1×10 13 ~1×10 14 vg / mL.

[0345] In an alternative embodiment, the liquid formulation comprises (i) a therapeutically effective amount of viral particles; (ii) a soluble salt; (iii) a polyol; (iv) a pH buffer; and (V) a surfactant. The therapeutically effective amount of viral particles carries a FIX gene (as shown in SEQ ID No: 3), and the concentration of the viral particles is 5×10 11 ~5×10 14 vg / mL, for example 2×10 12 ~2×10 14 vg / mL, or 1×10 13 ~1×10 14 vg / mL, the soluble salt substances include soluble monovalent metal salts and soluble divalent metal salts, the soluble monovalent metal salt is sodium chloride, and the concentration of sodium chloride is 50-250mM, such as 80-200mM, or 100-190mM, or 100-160mM. The soluble divalent metal salt is Ca 2+ and Mg 2+ , the Ca2+ and Mg 2+ The concentration of the polyol is sucrose, and the concentration of the sucrose is 0.5% to 20%, for example, 0.8% to 10%, or 0.9% to 5%. The pH buffer is PB buffer, and the concentration of the PB buffer is 2 to 50 mM, for example, 5 to 30 mM, or 8 to 20 mM. The surfactant is F68, and the concentration of F68 is 0.0005% to 0.002% (w / v).

[0346] In an alternative embodiment, the liquid formulation has one or more characteristics selected from the following group:

[0347] (a) the aggregate content of the liquid preparation is less than 1.5%, such as less than 1.0%, or less than 0.8%, or less than 0.7%;

[0348] (b) the free nucleic acid content of the liquid preparation is less than 15 ppm, for example, less than 10 ppm, or less than 8 ppm;

[0349] (c) After a 7-day accelerated destruction test at 37°C, the liquid preparation showed no significant decrease in the intensity of the viral particle signal response and no significant decrease in viral activity;

[0350] (d) The liquid preparation undergoes a 7-day accelerated test, and the aggregate content is less than 1.5%, such as less than 1.0%, or less than 0.8%.

[0351] Another aspect of the present disclosure provides use of the aforementioned liquid preparation for preparing a medicament for preventing and / or treating hereditary coagulation factor deficiency.

[0352] In an alternative embodiment, the method of prevention and / or treatment comprises administering to a subject a therapeutically effective amount of the liquid preparation described in the aforementioned embodiment.

[0353] In an optional embodiment, the administration of the liquid preparation comprises intramuscular injection; optionally, the site of intramuscular injection is selected from the deltoid muscle, quadriceps femoris, biceps brachii, gastrocnemius muscle or soleus muscle.

[0354] In an optional embodiment, at least one injection point is selected for the intramuscular injection; optionally, more than two injection points are selected for the intramuscular injection, and the intervals between the injection points are at least 3 cm.

[0355] In an optional embodiment, the intramuscular injection dose is a single dose of 1 to 3×10 12 vg / kg, optional 2.5×10 12 vg / kg.

[0356] Another aspect of the present disclosure provides a liquid preparation for use as a medicament in preventing and / or treating hereditary coagulation factor deficiency. The liquid preparation has the same composition as the liquid preparation described in any of the aforementioned embodiments.

[0357] In an alternative embodiment, the method of prevention and / or treatment comprises administering to a subject a therapeutically effective amount of the liquid preparation described in the aforementioned embodiment.

[0358] In an optional embodiment, the administration of the liquid preparation comprises intramuscular injection; optionally, the site of intramuscular injection is selected from the deltoid muscle, quadriceps femoris, biceps brachii, gastrocnemius muscle or soleus muscle.

[0359] In an optional embodiment, at least one injection point is selected for the intramuscular injection; optionally, more than two injection points are selected for the intramuscular injection, and the intervals between the injection points are at least 3 cm.

[0360] In an optional embodiment, the intramuscular injection dose is a single dose of 1 to 3×10 12 vg / kg, optional 2.5×10 12 vg / kg.

[0361] Another aspect of the present disclosure provides a method for preventing and / or treating hereditary coagulation factor deficiency, comprising administering a therapeutically effective amount of the liquid preparation according to the aforementioned embodiment to a subject.

[0362] In an optional embodiment, the administration of the liquid preparation comprises intramuscular injection; optionally, the site of intramuscular injection is selected from the deltoid muscle, quadriceps femoris, biceps brachii, gastrocnemius muscle or soleus muscle.

[0363] In an optional embodiment, at least one injection point is selected for the intramuscular injection; optionally, more than two injection points are selected for the intramuscular injection, and the intervals between the injection points are at least 3 cm.

[0364] In an optional embodiment, the intramuscular injection dose is a single dose of 1 to 3×10 12 vg / kg, optional 2.5×10 12 vg / kg.

[0365] Another aspect of the present disclosure provides a medicine kit comprising a container and the liquid preparation according to any one of the aforementioned embodiments located in the container.

[0366] The features described above, or the features described in the examples, may be combined in any combination. All features disclosed in this specification may be used in any combination, and each feature disclosed in this specification may be replaced by any alternative feature that serves the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the features disclosed are merely general examples of equivalent or similar features.

[0367] The main advantages of the present disclosure include:

[0368] 1. The present disclosure discovers for the first time that a buffer system containing soluble salts, polyols, and pH buffer can maintain the pH value of the solution at 7.0-8.0 (e.g., 7.0-7.6, or 7.0-7.4), and can significantly improve the stability and dispersibility of virus particles. Moreover, a liquid preparation containing soluble salts, polyols, and pH buffer can also significantly improve the stability and dispersibility of virus particles, reduce the aggregate content and the content of free nucleic acid, without a significant decrease in the signal response intensity of virus particles and a significant decrease in virus activity, and can significantly improve (e.g., enhance) the dispersed number and stability of virus particles.

[0369] 2. The buffer system or preparation disclosed herein can prevent damage to the virus capsid protein, such as fragmentation, aggregation, and packaging integrity, caused by low temperatures, repeated freeze-thaw cycles, and refrigerated and room temperature storage; disperse a larger number of virus particles; and ensure isotonic pressure, enabling more application scenarios and avoiding adverse reactions caused by high osmotic pressure.

[0370] 3. The buffer system or formulation disclosed herein can significantly reduce the aggregate content, such as an aggregate content of <1.5%, for example <1.0%, or <0.8%, or <0.7%.

[0371] 4. The buffer system or formulation disclosed herein can significantly reduce the content of free nucleic acids, such as a free nucleic acid content of <15 ppm, eg, <10 ppm, or <8 ppm.

[0372] 5. The AAV formulation disclosed herein can effectively prevent the capsid protein from breaking and the damage to the packaging integrity caused by the virus at low temperatures and repeated freezing and thawing. The integrity of this formulation did not show a significant decrease after 10 freeze-thaw cycles. Compared with the traditional formulation, it has better thermal stability (see Figures 1 and 8). It does not show obvious genome release for at least 24 hours at 37°C (see Figures 3 and 7). The number of viral particles is between 7 and 8 × 10 13vg / mL, and after continuous 168 hours at 37°C, no obvious damage to the virus's rigid structure was observed (data from SEC analysis, see Figure 13). Moreover, after 10 consecutive freeze-thaw cycles and 37°C destruction, no obvious decrease in virus activity was observed (data from activity analysis, see Table 3).

[0373] 6. The disclosed formula can disperse a larger number of virus particles, currently 7 to 8 × 10 13 No obvious turbidity or precipitation was found at a loading capacity of vg / mL (see Figure 9), enabling high-concentration injection.

[0374] 7. Compared with existing formulas, under the premise of ensuring stability and carrying capacity (physical titer), the formula disclosed in the present invention has a lower osmotic pressure, ensuring isotonic pressure (see Table 2), which can realize more application scenarios and avoid adverse reactions caused by high osmotic pressure (for example, the high osmotic pressure preparation caused by Tris buffer can cause pain to patients during injection).

[0375] The present disclosure will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present disclosure only and are not intended to limit the scope of the present disclosure. The test methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0376] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the disclosed methods. The preferred embodiments and materials described herein are for illustrative purposes only.

[0377] Unless otherwise specified, the materials and reagents used in the examples of this disclosure are all commercially available products.

[0378] General Methods

[0379] (1) Preparation of vector

[0380] The AAV that can be used in the present disclosure is not limited by its use and the target gene it carries. For example, the AAV can be used to treat, prevent and / or cure hereditary coagulation factor deficiency diseases; and / or (ii) drugs for preventing and / or treating hyperlipidemia-related diseases; the hereditary coagulation factor deficiency disease is hemophilia A, B or C; the hereditary coagulation factor deficiency disease is a mammalian hereditary coagulation factor deficiency disease; optionally, the hereditary coagulation factor deficiency disease is a human hereditary coagulation factor deficiency disease.

[0381] The AAV vector preparation production method is as follows: the purified AAV vector is dispersed into the buffer of Table 1 or Table 2, and a physical titer test (PCR method) is performed. The stock solution is diluted to 8×10 13 vg / mL (virus concentration), and the obtained AAV vector preparation was subjected to melting curve analysis (differential scanning calorimetry). The results are shown in Figures 1 and 8.

[0382] Table 1

[0383] Table 2

[0384] (2) Freeze-thaw test analysis method

[0385] Each group of formulations was subjected to 10 freeze-thaw cycles (freezing operation was carried out at -80°C, freezing time was not less than 6 hours, thawing operation was carried out at 23°C, thawing time was not less than 30 minutes). After each freeze-thaw cycle, a certain amount of sample was taken out for free nucleic acid detection (spectrophotometry, results see Figures 3 and 11), monomer and aggregate detection (size exclusion method, results see Figures 2, 9, and 10, among which Figure 9 shows that the degree of AAV solubility in different formulations is different, and the higher the signal intensity, the more virus particles in the solution), and AAV integrity detection (ion exchange method, results see Figures 4 and 12).

[0386] (3) Thermal stability test analysis method

[0387] Each formulation was placed at 37°C for 168 hours. A certain amount of samples were taken at 8, 24, 48, 72, 96 and 168 hours for free nucleic acid detection (spectrophotometry, see Figures 6 and 15 for results) and monomer and aggregate detection (size exclusion method, see Figures 5, 13 and 14 for results, Figure 13 shows that the higher the signal response direction and the more stable the change over time, the better the dispersion and stabilization effect on AAV.

[0388] The specific test procedures (SOP) for thermal stability testing, freeze-thaw testing, and 37°C accelerated testing are all routine operations.

[0389] (IV) Freeze-thaw test operation method

[0390] For each group of formulations, 10 encapsulated preparations (1 mL / vial) were taken and frozen at -80°C. The freezing time was not less than 8 hours. 9 preparations were taken out and placed at room temperature until they were completely thawed (the thawing time was not less than 40 minutes). After they were completely thawed, they were frozen again at -80°C. The above cycle was followed by a total of 9 freeze-thaw operations until sampling and testing were performed. All the preparations were taken out and placed at room temperature for complete thaw, completing 10 freeze-thaw tests.

[0391] (V) 37℃ accelerated destruction test operation method:

[0392] For each group of formulations, 4 to 6 encapsulated preparations (1 mL / vial) were taken and placed in a 37°C environment. The timer was started, and one corresponding formulation preparation was taken out at the corresponding time point. After taking out, the preparations were frozen in a -80°C environment until sampling and testing.

[0393] (VI) SEC analysis (aggregate detection) operation method:

[0394] Sample processing: No processing is required, sampling can be directly analyzed on the machine

[0395] Chromatographic column: Saifen, STR-500, 4.6×300mm, 5μm,

[0396] Elution solution: PBS, ethanol;

[0397] Detection wavelength: 260nm, 280nm;

[0398] Flow rate: 0.3 mL / min.

[0399] (VII) AEX analysis (full load factor detection) method:

[0400] Sample processing: No processing is required, samples can be directly analyzed on the machine;

[0401] Chromatographic column: Nanospectroscopy, 4.6×100 mm, 5 μm, non-porous;

[0402] Detection wavelength: excitation wavelength 280nm, emission wavelength 350nm;

[0403] Flow rate: 0.8 mL / min;

[0404] Elution solution: Bis-Tris propane, pH = 9.0, tetramethylammonium chloride.

[0405] (8) Free nucleic acid detection method:

[0406] Sample preparation: Take 100 μL of sample / standard control, add 100 μL of 1× ssDNA buffer, mix well, and shake in the dark at room temperature for several minutes before detection.

[0407] Detection conditions: excitation wavelength 480 nm, emission wavelength 520 nm.

[0408] (IX) Thermal stability test method:

[0409] Sample processing: Take the titer as 1×10 11 vg / mL test sample 15 μL, add 5 μL 1% SYPRO Orange dye, mix well, and add 25 μL D-PBS.

[0410] Q-PCR parameter setting: temperature increased from 35°C to 95°C, with a heating rate of 0.05°C / s.

[0411] Detection wavelength: excitation wavelength 485nm, emission wavelength 625nm.

[0412] (10) Activity detection method:

[0413] Sample processing: Take 1×10 10 vg / well virus was inoculated into the cells. After 24 hours of inoculation, the cell culture supernatant was taken and added into the well plate containing the enrichment antibody, and incubated with the labeled detection antibody. After that, all the liquid was discarded and the cells were washed repeatedly, and the color developer was added.

[0414] Example

[0415] The instruments, equipment, reagents and consumables used in the experiment are described as follows:

[0416] Instruments and Equipment

[0417] Reagents and consumables

[0418] Sample Information

[0419] Among them, carrier 1 is the product numbered KL001 by the applicant, and its structure is shown in the HA-04 structure diagram in Figure 1 of the patent (application number: CN202310394646.1; application date: 2023-04-13).

[0420] Example 1: Preliminary screening of formulations

[0421] The vector 1 product was replaced with buffer solutions of different formulations. After multiple freeze-thaw tests or high-temperature accelerated destruction tests, aggregation, integrity and other indicators were monitored to screen and optimize the optimal formulation conditions.

[0422] Screen the main types and proportions of additives in the formula, and preliminarily confirm the formulation by testing aggregation and integrity.

[0423] 1.1 Experimental plan

[0424] 1.2 Test results

[0425] 1.2.1 Thermal stability results

[0426] As shown in Figure 1, the thermal stability of all ten formulations was around 79.5°C. Formulations 6, 7, 8, and 9 performed relatively well, all above the average. This is likely due to the addition of sugar. Furthermore, as the amount of F68 in formulations 1, 2, 3, and 4 decreased, the thermal stability of the virus increased. This suggests that excessive amounts of F68, as a surfactant, should be avoided, as this would accelerate viral destruction.

[0427] 1.2.2 Freeze-thaw test results

[0428] Ten formulations were subjected to 10 freeze-thaw cycles. Figure 2 shows that formulations 6 through 9 exhibited significantly better aggregation than the remaining formulations, indicating that the addition of sugars or polyols to the formulation significantly mitigates aggregation caused by freeze-thaw cycles. Formulations 6, 7, and 9 exhibited the best aggregation, while formulation 8 performed poorly. This may be due to the fact that mannitol has some protective effect against viruses during freeze-thaw cycles, but the effect is less pronounced than that of sucrose and glycerol. Comparing formulations 4 and 5, aggregation was significantly improved with increasing sodium chloride concentrations, suggesting that increasing sodium chloride concentrations can help mitigate viral aggregation. This was evident in formulations 1 through 4 (where sodium chloride concentrations were gradually increased). Comparing formulations 2 and 5, aggregation was essentially the same throughout the entire process. While formulation 2 contained less sodium chloride than formulation 5, the results did not show that formulation 5 exhibited better aggregation than formulation 2. This may be due to the presence of F68 in formulation 2, which contained a higher concentration than formulation 5. This may be due to the higher concentration of F68 in formulation 2, which, at a relatively low sodium chloride concentration, can better correct viral aggregation.

[0429] Figure 3 shows the results of free nucleic acid after 10 freeze-thaw cycles for different formulations. Formulations 6, 7, and 9 performed best. Protected by sucrose or glycerol, the virus remained relatively intact after multiple freeze-thaw cycles, with no excess nucleic acid released. Increasing the sodium chloride concentration in formulations 0 through 5 helped stabilize viral integrity, while excessive F68 could cause the virus to become unstable.

[0430] The percentage of full shells after 10 freeze-thaw cycles was essentially consistent with the aforementioned parameters, as shown in Figure 4. Formulations 6 to 9 all performed best. Comparing Formulations 1 to 5, the results were consistent with those described above, indicating that a relatively high concentration of sodium chloride helps stabilize viral integrity.

[0431] 1.2.3 Accelerated test results

[0432] In the accelerated test, there was no significant difference in aggregation between the different formulations, and the overall performance was relatively consistent. The results are shown in Figure 5.

[0433] However, the free nucleic acid performance of different formulations in the accelerated destruction test varied significantly. As shown in Figure 6, Formulation 6 performed best, with significantly lower free nucleic acid levels than the other formulations in the 24-hour accelerated test, and the change in free nucleic acid increment was minimal. Comparing Formulation 6 with Formulation 7, the addition of calcium ions significantly improved the stability of viral particles.

[0434] In the results of fully loaded viruses of different recipes, it was found that recipe 6 in Figure 7 performed the best, and the full load did not decrease significantly over time. The remaining recipes showed a significant decrease in both full load rate and full load rate over time.

[0435] 1.2.4 Test conclusion

[0436] After 10 freeze-thaw cycles and 24-hour accelerated testing of 10 formulations, formulation 6 performed best, outperforming the other formulations in both freeze-thaw stability and 24-hour accelerated stability. Therefore, formulation 6 was selected for further optimization and adjustment. Furthermore, through multiple formulation comparisons, it was found that the concentration of F68 should not be too high, as excessive F68 content would accelerate the destabilization of viral particles. While high sodium chloride also contributes to viral particle stability, excessive sodium chloride concentrations can result in high osmotic pressure and require sucrose addition, further increasing the formulation's osmotic pressure, making it unsuitable for large-scale intramuscular injection. At the same time, the addition of calcium ions can further enhance viral particle stability, thanks to the complexation of divalent calcium ions with glutamate on the outside of viral particles, improving viral particle stability. Magnesium ions were also studied in subsequent experiments. At the same time, it is necessary to comprehensively consider osmotic pressure requirements and adjust the amounts of sodium chloride, polyols, and divalent ions.

[0437] Example 2 formula collocation again screening

[0438] Screen and confirm the addition ratio of key additives in the formula.

[0439] 2.1 Experimental plan

[0440] 2.2 Calculation of magnesium ion precipitation

[0441] Magnesium ions are added to this formula. Considering that magnesium phosphate is insoluble in water, the amount of magnesium ions added needs to be calculated:

[0442] The pH of the formula is controlled at 7.2-7.4, calculated based on pH=7.3

[0443] The secondary dissociation constant of phosphoric acid, pKa, is 7.2. According to formula (1), under this environment, c About 1×10 -7 mol / L( The concentration is calculated with the upper limit of 10mM), and according to formula (2), when Mg 2+ When the addition amount is 1mM, the amount of magnesium phosphate precipitation required is The minimum concentration is 1×10 -6 mol / L, the concentration is less than the Therefore, when Mg 2+ When the addition amount is only 1 mM, no precipitation will occur.

[0444] 2.3 Test results

[0445] Table 3

[0446] Note: Formulations 1 to 7 in Table 3 correspond to formulations 1 to 7 in Table 1 and Table 2, respectively.

[0447] Table 3 shows that the freeze-thaw results from the second screening test indicate that sodium chloride concentration is a key indicator affecting biological activity. Formulations 1 and 2, both with sodium chloride concentrations of 110 mM, exhibited discrepancies in viral activity, suggesting that appropriately increasing the sodium chloride concentration can improve viral activity. Formulations 1 and 2 exhibited relatively low activity in the 72-hour 37°C accelerated test, suggesting that appropriately increasing the sodium chloride concentration can help improve viral activity. Activity testing alone does not reveal much information, so further characterization data will be needed to determine the optimal formulation.

[0448] 2.3.1 Thermal stability results

[0449] The thermal stability performance of different formulations is basically the same, all at 79.8±0.5℃, and there is no obvious difference between different formulations. The results are shown in Figure 8.

[0450] 2.3.2 Freeze-thaw results

[0451] After 10 freeze-thaw cycles, the monomer signal intensity of each formulation did not show a significant variation trend, but the overall signal intensity of the different formulations was significantly different. The signal intensity of formulations 1 and 2 was significantly lower than that of the other formulations, indicating that the number of virus particles in formulations 1 and 2 was significantly less than that in the other formulations. This is most likely because 110 mM sodium chloride is not enough to completely disperse 8 × 10 13 vg / mL of virus, and compared with Formula 1 and Formula 2, appropriately increasing the sucrose content helps to better disperse the virus particles, as shown in Figure 9.

[0452] After repeated freeze-thaw cycles, different formulations all showed good anti-aggregation effects, with no large-scale aggregation and only a small amount of aggregation, and the differences between the formulations were not obvious, as shown in Figure 10.

[0453] In the free nucleic acid test, no serious virus particle destruction results were found, but there were obvious differences between different formulas. Formula 4 and Formula 5 performed poorly, with relatively more free nucleic acid. This is most likely because the osmotic pressure of Formula 4 and Formula 5 is higher than that of other formulas, which affects the stability of virus particles, causing virus particles to break or nucleic acid to overflow. Therefore, it can be seen that relatively low osmotic pressure can better maintain the integrity of virus particles, as shown in Figure 11.

[0454] After 10 freeze-thaw cycles, the virus particles showed no obvious differences among different formulations, and the percentage of full shells was basically the same, which was relatively consistent with the performance of free nucleic acids, as shown in Figure 12.

[0455] 2.3.3 Accelerated test results

[0456] After 7 days of accelerated destruction test at 37°C, the virus particle response intensity of Formulas 1 to 4 showed a significant downward trend as the acceleration time prolonged, while Formulas 5 to 7 performed well and did not suffer excessive losses as the acceleration time prolonged. Among them, Formulas 6 and 7 performed best, and Formula 7 was slightly better than Formula 6. This indicates that the addition of magnesium ions can better stabilize the virus particles from being destroyed. The results are shown in Figure 13.

[0457] After 7 days of accelerated testing, the aggregation of each formulation was good, with the aggregation amount exceeding 1%. Among them, formulations 1 and 2 performed best, both maintaining below 0.5%, as shown in Figure 14.

[0458] Different formulations exhibited different rupture patterns during the 7-day accelerated destruction test. The free nucleic acid concentration of all formulations increased with the extension of the acceleration time. Among them, formulations 6 and 7 performed best in terms of overall free nucleic acid concentration and amplification rate, as shown in Figure 15.

[0459] 2.3.4 Test conclusion

[0460] After considering the different ratios of key additives, it can be seen that the amount of sodium chloride added depends on the concentration of virus particles. Currently, it is 8×10 13vg / mL of virus, 110mM sodium chloride is not enough to completely disperse the virus, but by increasing the sucrose concentration slightly, the situation can be appropriately improved. On the other hand, adjusting the content of sodium chloride and sucrose will have a significant impact on the osmotic pressure. From the test results, it is shown that higher osmotic pressure is not friendly to the integrity of the virus particles. The addition of calcium and magnesium ions can better stabilize the virus particles from being destroyed, and magnesium ions have a better effect. The study found that the simultaneous addition of calcium and magnesium ions (both 1mM) can further improve stability. At the same time, it is also necessary to consider whether increasing the magnesium ion content can also achieve the desired effect.

[0461] 2.3.5 Conclusion

[0462] A series of studies have shown that increasing the amount of sodium chloride and sugar can improve the stability and dispersibility of viral particles. Sodium chloride focuses more on improving the dispersion of viral particles, while sugar focuses more on protecting viral particles from damage. Sucrose has been found to be more protective of viral particles than mannitol and is more user-friendly for weighing and preparation than glycerol. The addition of both sucrose and sodium chloride significantly increases osmotic pressure, and high osmotic pressure is not conducive to the stability of viral particles or the use of formulations. Therefore, it is best to maintain isotonic osmotic pressure. The amount of surfactant bolusamer 188 (F68) added should be controlled and should not be excessive, as excessive F68 will accelerate the destruction of the virus. The study found that the addition of calcium and magnesium ions would have a positive effect on the stability of virus particles. In comparison, the improvement effect of magnesium ions was more obvious. However, whether to add magnesium ions and the amount to be added in the presence of phosphate buffer should be determined according to the specific situation. The 10mM PB buffer used in the current study had no effect when 1mM magnesium was added, and the performance was also good in the absence of magnesium ions, without large-scale aggregation and fragmentation.

[0463] Example 3 Calcium and magnesium formula optimization (the following is supplementary data)

[0464] According to Table 3.1, liquid preparations were prepared for different serotypes of AAV viruses carrying different target genes to improve their stability and dispersion ability.

[0465] Table 3.1

[0466] Among them, AAV6.2FF-EGFP represents a recombinant serotype AAV6.2FF virus carrying the target gene EGFP (Enhanced Green Fluorescent Protein), the nucleotide sequence of the target gene EGFP is shown in SEQ ID No: 1, and the amino acid sequence is shown in SEQ ID No: 2; the vector structure of AAV6.2FF-ScFv is shown in the patent (application number: CN202311296135.2; application date: 2023-09-28) as shown in No. 10 in Figure 5: "AAV6.2FF-CAG-UTR-tpa-scFv (EA) -igG4-WPRE"; the vector structure of KL001 is shown in the HA-04 structure diagram in Figure 1 of the patent (application number: CN202310394646.1; application date: 2023-04-13), and the nucleotide sequence of the FIX gene therein is shown in SEQ ID No: 3.

[0467] SEQ ID No: 1 (nucleotide sequence of EGFP)

[0468] SEQ ID No: 2 (amino acid sequence of EGFP)

[0469] SEQ ID No: 3 (nucleotide sequence of FIX gene)

[0470] The above formulations were subjected to multiple performance tests with reference to Examples 1 and 2, and the results are shown in Table 3.2 and Figures 16 to 21.

[0471] 3.1 Effects of calcium, magnesium, and F68 on the thermal stability of AAV virus

[0472] As can be seen from Figures 16, 17, and 20, the addition of calcium, magnesium, and F68 does not affect the stability of AAV virus particles during repeated freeze-thaw cycles. Across different formulations, neither the AAV virus particle concentration (Figure 16) nor the integrity of the AAV virus particles (Figures 17 and 20) showed significant changes, and no significant differences were observed in the consistency ratios between different formulations, indicating that calcium, magnesium, and F68 are key factors in stabilizing virus particles during the non-freeze-thaw process.

[0473] 3.2 Effects of calcium, magnesium, and F68 on AAV virus acceleration assay

[0474] As can be seen from Figures 18, 19 and 21, the importance of adding calcium and magnesium under accelerated conditions. Comparing Formula 2 and Formula 3 in Figure 19 and Figure 21, it can be seen that the addition of calcium and magnesium is beneficial to the stability of AAV particles under non-low temperature conditions. In Figure 19, in the formula without the addition of calcium and magnesium, the purity of the virus continued to decline significantly during the long-term continuous heat treatment process, while in the continuous process of Figure 21, a large amount of free nucleic acid continued to be produced due to the cracking of the virus or the squeezing of the environment, resulting in impaired integrity. This proves that the addition of calcium and magnesium can better stabilize and ensure the integrity of AAV particles. As can be seen from the comparison of Formula 6 and Formula 7 in Figure 18, the addition of F68 can improve the dispersion and adsorption of virus particles to a certain extent, but this is not significant.

[0475] The results of 10 freeze-thaw cycles showed that the consistency of different formulations was good and no significant differences were observed between different formulations, as shown in Table 3.2.

[0476] Table 3.2

[0477] It can be seen from the above test results that, regardless of the freeze-thaw process or the 37°C accelerated process, the formulation provided in this example can stabilize the integrity of AAV particles at above 98% (Formula 1 has an initial integrity of only about 90%, but a series of processes do not cause its integrity to decline, which can also be explained by the RSD data of the results. In good cases, the RSD is greater than 99%).

[0478] 3.3 Conclusion

[0479] Formulas 1-2, 4-7, and 5-6 of this example all exhibit broad applicability for different AAV6.2FF AAV viruses containing different target genes, stabilizing diverse serotypes and packaged gene vectors. None exhibited significant structural damage in continuous destructive studies. Furthermore, these formulas stabilized AAV particles for extended periods at 2-8°C and for at least several weeks to months at room temperature.

[0480] Example 4 Hemophilia B Clinical Trial

[0481] In this example, two hemophilia B patients (patient 1: LZPE and patient 2: MFTA) were treated with formula 3 in Table 3.1.

[0482] 4.1 Administration:

[0483] Before administration, give the patient an intravenous push or infusion of the FIX protein product (50-100 IU / kg) used for daily treatment to prevent bleeding events when KL001 is administered intramuscularly, and then inject the KL001 product. KL001 can be injected into muscles such as the deltoid, quadriceps, biceps, gastrocnemius, and soleus under ultrasound guidance. This embodiment uses a 1-inch or 1.5-inch 22-gauge needle and a syringe with an accuracy of 0.01 ml to draw 1 ml of KL001. Each injection point is at least 3 cm apart, and 1 ml of KL001 injection is injected at each point. The injection is slowly pushed to prevent drug overflow. If the last point is less than 1 ml, it is drawn according to the actual volume. After each bottle of drug is injected, a new syringe should be replaced and must not be reused.

[0484] 4.2 Preliminary Clinical Results

[0485] Two patients received a low dose (2.5 × 10 12 vg / kg) of KL001. Preliminary results showed that the activity of vector-derived FIX in plasma was significantly increased after treatment compared to baseline. At 2 weeks after administration, the activity of vector-derived FIX exceeded 5%, and at 5 and 8 weeks after administration, the FIX activity reached a peak value of greater than 20%, which improved the clinical symptoms to a mild degree. In patient 1, the vector-derived FIX activity was relatively stable within 10 weeks after administration, with no obvious downward trend. In patient 2, the FIX activity began to fluctuate after 5 weeks of treatment, and decreased from the peak value, but the overall activity remained at a high level.

[0486] All documents mentioned in this disclosure are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of this disclosure, those skilled in the art may make various changes or modifications to this disclosure, and that these equivalents also fall within the scope of the claims appended hereto.

Claims

1. A liquid preparation, wherein: The liquid preparation comprises: (i) a therapeutically effective amount of viral particles; (ii) soluble salts; (iii) polyols; (iv) pH buffer.

2. The liquid preparation according to claim 1, wherein The pH value of the liquid preparation is 7.0 to 8.0, such as 7.0 to 7.6, or 7.0 to 7.

4.

3. The liquid preparation according to claim 1 or 2, wherein The osmotic pressure of the liquid preparation is 280 to 330 mosm / kg.

4. The liquid preparation according to any one of claims 1 to 3, wherein The soluble salt substances include one or more soluble monovalent metal salts and / or one or more soluble divalent metal salts; The polyol includes one or more of fructose, glucose, maltose, sucrose, glycerol, trehalose and mannitol; The pH buffer is selected from PB buffer and / or HEPES buffer.

5. The liquid preparation according to claim 4, wherein The soluble monovalent metal salts include sodium salts, such as sodium chloride.

6. The liquid preparation according to claim 4 or 5, wherein The concentration of the soluble monovalent metal salt is 50-250 mM, such as 80-200 mM, or 100-190 mM, or 100-160 mM.

7. The liquid preparation according to any one of claims 4 to 6, wherein The soluble divalent metal salts include Ca 2+ Mg 2+ 、Zn 2+ , Cu 2+ , Mn 2+ and Cr 2+ One or more of 2+ and Mg 2+ .

8. The liquid preparation according to any one of claims 4 to 7, wherein The concentration of the soluble divalent metal salt is 0.2-8 mM, such as 0.5-5 mM, or 0.8-2 mM.

9. The liquid preparation according to any one of claims 1 to 8, wherein The concentration of the polyol is 0.5% to 20%, such as 0.8% to 10%, or 0.9% to 5%, based on the total weight volume percentage of the buffer system; The concentration of the pH buffer is 2-50 mM, such as 5-30 mM, or 8-20 mM.

10. The liquid preparation according to any one of claims 1 to 9, wherein The liquid preparation also includes a surfactant, which includes one or more of polyoxyethylene polyoxypropylene ether block copolymer (Poloxamer 188, F68), polysorbate (Tween, Tween), polyethylene glycol (PEG), polyethylene glycol p-isooctylphenyl ether (Triton), fatty acid sorbitan (Span), sucrose fatty acid ester (SE), polyoxyethylene fatty alcohol ether (Brij) and polyoxyethylene fatty acid ester (Myeij), such as F68.

11. The liquid preparation according to claim 10, wherein The concentration of the surfactant is 0.0005% to 0.002% (w / v).

12. The liquid preparation according to any one of claims 1 to 11, wherein The virus particle includes an AAV virus, a lentivirus or a recombinant AAV virus, and the serotype of the AAV virus or the recombinant AAV virus includes AAV2, AAV4, AAV5, AAV6, AAV6.2FF, AAV8, AAV9, AAVPHP.eB, AAVPHP.S or AAVPHP.B; Optionally, the target gene carried by the viral particle includes a coagulation factor gene; Optionally, the coagulation factor gene is selected from a FIX factor gene or a coagulation factor VIII gene; Optionally, the nucleotide sequence of the FIX factor gene is shown in SEQ ID No:

3.

13. The liquid preparation according to any one of claims 1 to 12, wherein The concentration of the virus particles was 5×10 11 ~5×10 14 vg / mL, for example 2×10 12 ~2×10 14 vg / mL, or 1×10 13 ~1×10 14 vg / mL.

14. The liquid preparation according to any one of claims 1 to 13, wherein The liquid formulation has one or more characteristics selected from the group consisting of: (a) the aggregate content of the liquid preparation is less than 1.5%, such as less than 1.0%, or less than 0.8%, or less than 0.7%; (b) the free nucleic acid content of the liquid preparation is less than 15 ppm, such as less than 10 ppm, or less than 8 ppm; (c) After a 7-day accelerated destruction test at 37°C, the liquid preparation showed no significant decrease in the intensity of the virus particle signal response and no significant decrease in the virus activity; (d) After a 7-day accelerated test, the liquid preparation has an aggregate content of <1.5%, such as <1.0%, or <0.8%.

15. Use of the liquid preparation according to any one of claims 1 to 14 for preparing a drug for preventing and / or treating hereditary coagulation factor deficiency.

16. The use according to claim 15, wherein The method of prevention and / or treatment comprises administering a therapeutically effective amount of the liquid preparation according to any one of claims 1 to 14 to a subject.

17. The use according to claim 16, wherein The administration method of the liquid preparation includes intramuscular injection; Optionally, the site of intramuscular injection is selected from the deltoid muscle, quadriceps femoris, biceps brachii, gastrocnemius muscle or soleus muscle.

18. The use according to claim 17, wherein The intramuscular injection selects at least one injection point; Optionally, the intramuscular injection is performed at two or more injection points, and the intervals between the injection points are at least 3 cm.

19. The use according to claim 17 or 18, wherein The intramuscular injection dose is 1 to 3 × 10 12 vg / kg, optional 2.5×10 12 vg / kg.

20. A medicine kit comprising a container and the liquid preparation according to any one of claims 1 to 14 in the container.

Images