PCSK9 antibody and use thereof

Abstract

Provided are a PCSK9 antibody and the use thereof. The PCSK9 antibody can be used in the preparation of drugs for reducing lipoprotein levels in blood and for preventing or treating cardiovascular diseases or disorders and thromboembolic diseases or disorders.

Description

A PCSK9 antibody and its application Technical Field

[0001] This invention relates to the field of therapeutic antibody formulations, specifically to a PCSK9 antibody and its applications. Background Technology

[0002] Serum low-density lipoprotein (LDL) levels are a major indicator of blood lipid levels. Elevated LDL is a major risk factor for atherosclerotic heart disease, inducing and promoting the development of atherosclerosis. Numerous studies have shown that PCSK9 (human proprotein convertase subtilisin 9) can bind to the low-density lipoprotein cholesterol receptor (LDL-R) on the cell surface and internalize it, guiding it to lysosomes for degradation and inhibiting its recirculation to the hepatocyte surface, thereby weakening the liver's ability to metabolize low-density lipoprotein cholesterol (LDL-C) in plasma. Among the many PCSK9 inhibitors under development, anti-PCSK9 monoclonal antibodies have attracted much attention. Currently, several large international pharmaceutical companies are actively developing monoclonal antibody drugs targeting huPCSK9.

[0003] Therapeutic macromolecules, such as antibodies, are prone to degradation or aggregation during formulation. Although PCSK9 antibody formulations are already on the market, there remains a need for formulations that are sufficiently stable and suitable for different types of PCSK9 antibodies. Summary of the Invention

[0004] To address the technical problem of the lack of a sufficiently stable formulation suitable for PCSK9 antibodies in the prior art, this invention provides a PCSK9 antibody and its application.

[0005] To solve the above-mentioned technical problems, one of the technical solutions provided by the present invention is: a PCSK9 antibody formulation, the formulation comprising a PCSK9 antibody, a buffer, a surfactant, and a stabilizer; the PCSK9 antibody includes a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising amino acid sequences such as VH-CDR1 as shown in SEQ ID NO:1, VH-CDR2 as shown in SEQ ID NO:2, and VH-CDR3 as shown in SEQ ID NO:3, or amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the above sequences; and the light chain variable region comprising amino acid sequences such as VL-CDR1 as shown in SEQ ID NO:4, VL-CDR2 as shown in SEQ ID NO:5, and VH-CDR3 as shown in SEQ ID NO:3. The VL-CDR3 shown in NO:6, or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the above sequence.

[0006] In a specific embodiment of the present invention, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:7, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it; and / or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:8, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. In the present invention, the variable region sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity does not involve changes to the CDR region sequence.

[0007] In a specific embodiment of the present invention, the PCSK9 antibody is a full-length antibody, Fab, Fab', F(ab')2 or Fv; the Fv is preferably scFv.

[0008] In a specific embodiment of the present invention, the antibody is a full-length antibody, whose heavy chain constant region and / or light chain constant region are derived from human antibodies. Preferably, the N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region, and the N-terminus of the light chain constant region is connected to the C-terminus of the heavy chain variable region.

[0009] In a specific embodiment of the present invention, the heavy chain constant region is derived from the human heavy chain IgG1 constant region; and / or, the light chain constant region is derived from the human light chain κ chain constant region.

[0010] In a specific embodiment of the present invention, the amino acid sequence of the heavy chain constant region of the antibody is as shown in SEQ ID NO:9, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it; and / or, the amino acid sequence of the light chain constant region of the antibody is as shown in SEQ ID NO:10, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. In a preferred embodiment, the N-terminus of the amino acid sequence of SEQ ID NO:9 in the heavy chain constant region of the antibody is connected to the C-terminus of the amino acid sequence of SEQ ID NO:7 in the heavy chain variable region of the antibody. In yet another preferred embodiment, the N-terminus of the amino acid sequence of SEQ ID NO:10 in the light chain constant region of the antibody is connected to the C-terminus of the amino acid sequence of SEQ ID NO:8 in the light chain variable region of the antibody.

[0011] In a specific embodiment of the present invention, the concentration of the PCSK9 antibody is 120-160 mg / mL, preferably 140-160 mg / mL.

[0012] In a specific embodiment of the present invention, the buffer is selected from one or more of histidine buffers, acetate buffers, citrate buffers, and phosphate buffers.

[0013] In a specific embodiment of the present invention, the buffer is a histidine salt buffer. In the present invention, a "histidine salt buffer" is an ion buffer system containing histidine, typically composed of histidine and its salts.

[0014] In a specific embodiment of the present invention, the histidine buffer is an L-histidine buffer, preferably an L-histidine / L-hydrochloric acid histidine, such as L-histidine monohydrate / L-hydrochloric acid histidine.

[0015] In a specific embodiment of the present invention, the concentration of the buffer is 15mM to 25mM, preferably 20mM to 25mM.

[0016] In a specific embodiment of the present invention, the surfactant is selected from one or more of polysorbate 20, polysorbate 80, polyhydroxyene, Triton, sodium dodecyl sulfonate, sodium lauryl sulfonate, sodium octyl glycoside, lauryl-, myristyl-, linoleyl-, stearyl-sulfobetaine, lauryl-, myristyl-, linoleyl-, stearyl-sarcosine, linoleyl-, myristyl-, cetyl-betaine, lauramidopropyl-, cocaramide-propyl-, linoleamide-propyl-, myristamide-propyl-, palmitamide-propyl-, isostearamide-propyl-betaine, myristamide-propyl-, palmitamide-propyl-, isostearamide-propyl-dimethylamine, sodium methyl cocoyl, sodium methyl oleyl taurate, polyethylene glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol.

[0017] In a specific embodiment of the present invention, the surfactant is polysorbate 20, also known as Tween 20.

[0018] In a specific embodiment of the present invention, the concentration of the surfactant is 0.05-0.15 mg / mL, preferably 0.1-0.15 mg / mL.

[0019] In a specific embodiment of the present invention, the stabilizer is a sugar.

[0020] In a specific embodiment of the present invention, the sugar is a conventional composition (CH2O). n Or its derivatives, such as one or more selected from monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars and non-reducing sugars.

[0021] In a specific embodiment of the present invention, the sugar is selected from one or more of glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, dextran, erythritol, glycerol, arabinitol, xylitol, sorbitol, mannitol, melibiose, pinotriose, melitriose, mannitol, stachyose, maltose, lactulose, sorbitol, maltitol, lactitol, and isomaltulose.

[0022] In a specific embodiment of the present invention, the stabilizer is sucrose.

[0023] In a specific embodiment of the present invention, the concentration of the stabilizer is 60-80 mg / mL, preferably 70-80 mg / mL.

[0024] In a specific embodiment of the present invention, the pH value of the formulation is 5.7-6.3; preferably 6.0-6.3. In the present invention, the pH value refers to the pH value of the actual formulation.

[0025] In a specific embodiment of the present invention, the formulation comprises 120-160 mg / mL of the PCSK9 antibody, 15-25 mM of L-histidine monohydrate / L-histidine hydrochloride, 0.05-0.15 mg / mL of polysorbate 20 and 60-80 mg / mL of sucrose, and the pH value of the formulation is 5.7-6.3.

[0026] In a specific embodiment of the present invention, the formulation comprises 140-160 mg / mL of the PCSK9 antibody, 20-25 mM of L-histidine monohydrate / L-histidine hydrochloride, 0.1-0.15 mg / mL of polysorbate 20 and 70-80 mg / mL of sucrose, and the pH value of the formulation is 5.7-6.3.

[0027] In a specific embodiment of the present invention, the formulation comprises 120 mg / mL of the PCSK9 antibody, 15 mM of L-histidine monohydrate / L-histidine hydrochloride, 0.05 mg / mL of polysorbate 20 and 80 mg / mL of sucrose, and the pH of the formulation is 5.7.

[0028] In a specific embodiment of the present invention, the formulation comprises 120 mg / mL of the PCSK9 antibody, 15 mM of L-histidine monohydrate / L-histidine hydrochloride, 0.15 mg / mL of polysorbate 20 and 60 mg / mL of sucrose, and the pH of the formulation is 6.3.

[0029] In a specific embodiment of the present invention, the formulation comprises 120 mg / mL of the PCSK9 antibody, 25 mM of L-histidine monohydrate / L-histidine hydrochloride, 0.05 mg / mL of polysorbate 20 and 60 mg / mL of sucrose, and the pH of the formulation is 6.3.

[0030] In a specific embodiment of the present invention, the formulation comprises 120 mg / mL of the PCSK9 antibody, 25 mM of L-histidine monohydrate / L-histidine hydrochloride, 0.15 mg / mL of polysorbate 20 and 80 mg / mL of sucrose, and the pH of the formulation is 5.7.

[0031] In a specific embodiment of the present invention, the formulation comprises 160 mg / mL of the PCSK9 antibody, 15 mM of L-histidine monohydrate / L-histidine hydrochloride, 0.05 mg / mL of polysorbate 20 and 80 mg / mL of sucrose, and the pH of the formulation is 6.3.

[0032] In a specific embodiment of the present invention, the formulation comprises 160 mg / mL of the PCSK9 antibody, 15 mM of L-histidine monohydrate / L-histidine hydrochloride, 0.15 mg / mL of polysorbate 20 and 60 mg / mL of sucrose, and the pH of the formulation is 5.7.

[0033] In a specific embodiment of the present invention, the formulation comprises 160 mg / mL of the PCSK9 antibody, 25 mM of L-histidine monohydrate / L-histidine hydrochloride, 0.05 mg / mL of polysorbate 20 and 60 mg / mL of sucrose, and the pH of the formulation is 5.7.

[0034] In a specific embodiment of the present invention, the formulation comprises 160 mg / mL of the PCSK9 antibody, 25 mM of L-histidine monohydrate / L-histidine hydrochloride, 0.15 mg / mL of polysorbate 20 and 80 mg / mL of sucrose, and the pH of the formulation is 6.3.

[0035] In a specific embodiment of the present invention, the formulation comprises 140 mg / mL of the PCSK9 antibody, 20 mM of L-histidine monohydrate / L-histidine hydrochloride, 0.1 mg / mL of polysorbate 20 and 70 mg / mL of sucrose, and the pH of the formulation is 6.0.

[0036] In a specific embodiment of the present invention, the formulation is 1 mL in size.

[0037] In a specific embodiment of the present invention, the preparation is a preparation administered via subcutaneous injection.

[0038] In a specific embodiment of the present invention, the viscosity of the preparation is less than 20 cp, preferably less than 10 cp.

[0039] To solve the above-mentioned technical problems, the second technical solution provided by the present invention is: a combination formulation, wherein the combination formulation comprises the formulation and the second therapeutic agent as described in the first technical solution of the present invention.

[0040] In a specific embodiment of the present invention, the second therapeutic agent comprises one or more selected from other antibodies, peptides, and small molecule drugs.

[0041] In this invention, the "other antibodies" are PCSK9 antibodies as defined in the formulations described in one of the technical solutions of this invention.

[0042] In a specific embodiment of the present invention, the other antibodies are anti-VEGF (human vascular endothelial growth factor) antibody and / or anti-CD20 antibody.

[0043] In a specific embodiment of the present invention, the anti-VEGF antibody is MIL60 (bevacizumab, source: Beijing Tianguangshi Biotechnology Co., Ltd.), preferably with the heavy chain sequence shown in SEQ ID NO:11 and the light chain sequence shown in SEQ ID NO:12.

[0044] In a specific embodiment of the present invention, the anti-CD20 antibody is MIL62 (source: Beijing Tianguangshi Biotechnology Co., Ltd.), preferably with the heavy chain sequence as shown in SEQ ID NO:13 and the light chain sequence as shown in SEQ ID NO:14.

[0045] In a specific embodiment of the present invention, the amino acid sequence of the polypeptide is shown in SEQ ID NO:15 or SEQ ID NO:16.

[0046] In a specific embodiment of the present invention, the small molecule drug is a G protein-coupled receptor kinase activator.

[0047] In a specific embodiment of the present invention, the G protein-coupled receptor kinase activator is deoxycholic acid.

[0048] In a specific embodiment of the present invention, the structural formula of the deoxycholic acid is:

[0049] To solve the above-mentioned technical problems, the third technical solution provided by the present invention is: the use of the formulation as described in the first technical solution of the present invention and / or the combination formulation as described in the second technical solution of the present invention in the preparation of the following drugs:

[0050] Medications for the prevention or treatment of cardiovascular diseases or disorders and / or thromboembolic diseases or disorders;

[0051] Medications that lower blood lipoprotein levels;

[0052] Drugs that specifically bind to PCSK9;

[0053] Drugs that block the binding of PCSK9 to LDL-R;

[0054] Drugs that increase the number of LDL-R on the cell surface or the level of LDL-R in plasma;

[0055] Drugs that lower plasma LDL or LDL-c levels;

[0056] Drugs that inhibit the accumulation of LDL in plasma;

[0057] Drugs that inhibit PCSK9-mediated LDL-R degradation; or,

[0058] Drugs that increase the metabolic levels of cholesterol and / or triglycerides carried by LDL.

[0059] In a specific embodiment of the present invention, the cardiovascular disease or disorder is selected from dyslipidemia, coronary atherosclerotic heart disease, acute myocardial infarction, asymptomatic carotid atherosclerosis, stroke, and peripheral artery occlusive disease.

[0060] In a specific embodiment of the present invention, the dyslipidemia is selected from elevated cholesterol, elevated triglycerides, elevated low-density lipoprotein (LDL), and decreased high-density lipoprotein (HDL) in the blood, such as hyperlipidemia.

[0061] In a specific embodiment of the present invention, the thromboembolic disease or disorder is selected from pulmonary embolism and central retinal vein embolism.

[0062] To solve the above-mentioned technical problems, the fourth technical solution provided by the present invention is: a method for preventing or treating cardiovascular diseases or disorders and / or thromboembolic diseases or disorders, or for reducing the level of lipoproteins in the blood, the method comprising administering to a subject in need an effective amount of the preparation as described in the first technical solution of the present invention and / or the combination preparation as described in the second technical solution of the present invention.

[0063] In a specific embodiment of the present invention, the cardiovascular disease or disorder is selected from dyslipidemia, coronary atherosclerotic heart disease, acute myocardial infarction, asymptomatic carotid atherosclerosis, stroke, and peripheral artery occlusive disease.

[0064] In a specific embodiment of the present invention, the dyslipidemia is selected from elevated cholesterol, elevated triglycerides, elevated low-density lipoprotein (LDL), and decreased high-density lipoprotein (HDL) in the blood, such as hyperlipidemia.

[0065] In a specific embodiment of the present invention, the thromboembolic disease or disorder is selected from pulmonary embolism and central retinal vein embolism.

[0066] In a specific embodiment of the present invention, the formulation or the combination formulation is administered via subcutaneous injection.

[0067] To solve the above-mentioned technical problems, the fifth technical solution provided by the present invention is: a method in vivo or in vitro, the method comprising using an effective amount of the formulation as described in the first technical solution of the present invention and / or the combination formulation as described in the second technical solution of the present invention, wherein the method is selected from the following:

[0068] Methods for specific binding to PCSK9;

[0069] Methods to block PCSK9 from binding to LDL-R;

[0070] Methods to increase the number of LDL-R on the cell surface or the level of LDL-R in plasma;

[0071] Methods to lower LDL or LDL-c levels in plasma;

[0072] Methods to inhibit LDL accumulation in plasma;

[0073] Methods to inhibit PCSK9-mediated LDL-R degradation; or,

[0074] Methods to improve the metabolic levels of cholesterol and / or triglycerides carried by LDL.

[0075] In a specific embodiment of the present invention, the method is not for diagnostic, preventive and / or therapeutic purposes.

[0076] To solve the above-mentioned technical problems, the sixth technical solution provided by the present invention is: the formulation as described in the first technical solution of the present invention and / or the combination formulation as described in the second technical solution of the present invention, which is used for:

[0077] Prevention or treatment of cardiovascular diseases or disorders and / or thromboembolic diseases or disorders;

[0078] Lowering blood lipoprotein levels;

[0079] Specifically binds to PCSK9;

[0080] Blocking the binding of PCSK9 to LDL-R;

[0081] Increase the number of LDL-R on the cell surface or the level of LDL-R in plasma;

[0082] Lowering plasma LDL or LDL-c levels;

[0083] Inhibit the accumulation of LDL in plasma;

[0084] Inhibit PCSK9-mediated LDL-R degradation; or,

[0085] Increase the metabolic levels of cholesterol and / or triglycerides carried by LDL.

[0086] In a specific embodiment of the present invention, the cardiovascular disease or disorder is selected from dyslipidemia, coronary atherosclerotic heart disease, acute myocardial infarction, asymptomatic carotid atherosclerosis, stroke, and peripheral artery occlusive disease.

[0087] In a specific embodiment of the present invention, the dyslipidemia is selected from elevated cholesterol, elevated triglycerides, elevated low-density lipoprotein (LDL), and decreased high-density lipoprotein (HDL) in the blood, such as hyperlipidemia.

[0088] In a specific embodiment of the present invention, the thromboembolic disease or disorder is selected from pulmonary embolism and central retinal vein embolism.

[0089] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0090] The reagents and raw materials used in this invention are all commercially available.

[0091] The positive and progressive effects of this invention are reflected in the following:

[0092] The formulations and combinations thereof of the present invention exhibit good stability under shaking and freeze-thaw conditions; and also have good high-temperature physical stability and photophysical stability.

[0093] The formulation of the present invention has a solution viscosity of less than 10 cp at room temperature, which meets the requirement of being a low viscosity solution (<20 cp) and can be used as a subcutaneous injection solution. The combination formulation of the present invention has no interaction between the components, maintaining the stability of the combined drug composition, and can be used to establish robustness and reliability for potential therapeutic applications. Attached Figure Description

[0094] Figure 1 shows the correlation curves between temperature and viscosity of the MIL86 antibody preparation. Detailed Implementation

[0095] In this application, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the operational procedures used herein, such as molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA, are all routine procedures widely used in their respective fields. To better understand this application, definitions and explanations of relevant terms are provided below:

[0096] The term "antibody" in this article is intended to include full-length antibodies of IgG, IgA, IgD, IgE, and IgM, as well as any antigen-binding fragments (i.e., antigen-binding portions). A full-length antibody is a glycoprotein containing at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can also be divided into hypervariable regions called complementarity-determining regions (CDRs), separated by more conserved backbone regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies can mediate the binding of immunoglobulins to host tissues or factors, including binding to various immune system cells (e.g., effector cells) and the first component (C1q) of the conventional complement system.

[0097] The heavy chain variable region (CDR) and light chain variable region (CDR) of the antibody or its antigen-binding moiety in this application were determined using the Kabat numbering / definition method, and the SEQ ID NO of the amino acid sequence of the CDR region, together with the SEQ ID NO of the heavy / light chain amino acid sequence, are listed in Table 11. As is well known in the art, the heavy chain variable region and light chain variable region (CDR) can also be determined using numbering / definition methods such as Chothia, IMGT, AbM, or Contact.

[0098] The active ingredient of the formulation of this invention is the recombinant fully human monoclonal antibody MIL86, and its active target is the proprotein convertase subtilisin 9 (PCSK9). Numerous studies have demonstrated that PCSK9 can mediate the degradation of low-density lipoprotein receptor (LDLR). MIL86 reduces and regulates plasma low-density lipoprotein cholesterol (LDL-C) levels by inhibiting the interaction between PCSK9 and LDLR.

[0099] The formulation of this invention can be produced using suspension cell culture technology, with antibody expression performed using Chinese hamster ovary cells (CHOK1). After steps including chromatographic purification, virus inactivation / removal, and ultrafiltration / concentration, a stock solution is obtained, which is then sterilely filtered and dispensed into the formulation.

[0100] The formulation of this invention uses recombinant fully human monoclonal antibody MIL86 as the main drug; polysorbate 20 as the surfactant; L-histidine / L-histidine hydrochloride (monohydrate) as the buffer component; and sucrose as the stabilizer. Specifically, sucrose can competitively bind with water, preventing the formation of water of crystallization and acting as a cryoprotectant to protect the protein; at the same time, a certain concentration of sucrose can also supplement the osmotic pressure of the injection solution, playing an isotonic adjustment role.

[0101] Based on the results of previous formulation research experiments, the inventors investigated the effects of protein concentration, sucrose concentration, polysorbate 20 concentration, L-histidine / L-histidine hydrochloride (monohydrate) buffer concentration, and solution pH on the stability of the formulation through "formulation screening test" and "formulation confirmation test", thereby obtaining a formulation suitable for this antibody product and with robustness.

[0102] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0103] Example 1: Screening Test for MIL86 Antibody Formulation

[0104] To understand the relationship between the parameters of each formulation component and the key stability parameters of MIL86, a Design of Experiment (DoE) experiment was conducted using JMP10 software based on the principles of Partial Factorial Design (FFD). Five main effects were investigated, using protein concentration, histidine concentration, polysorbate 20 concentration, sucrose concentration, and pH as the investigation factors. The pH was chosen with pH 6.0 as the midpoint, with the investigation range within ±0.3 units (5.7–6.3). The antibody concentration was 120–160 mg / ml, the L-histidine / L-histidine hydrochloride concentration was 4–8 mM, the sucrose concentration was 80–120 mg / ml, and the polysorbate 20 concentration was 0.05–0.15 mg / ml. A total of 8 formulations (DOE-1 to DOE-8), plus the preliminary design (DOE-9), were designed, resulting in 9 experimental formulations, as shown in Table 1 below.

[0105] Table 1 Preliminary formulation of MIL86 antibody injection Note: "0" represents the designed concentration of the formulation component; "+" and "-" indicate that the concentration of the protein component, L-histidine / L-histidine hydrochloride, polysorbate 20, pH value, and sucrose concentration are higher or lower than the corresponding designed formulation, respectively.

[0106] Test Example 1: High Temperature Experiment

[0107] The above formulations were placed in a constant temperature incubator at 40℃±2℃ and 75%±5%RH for 4 weeks, and samples were taken for testing afterward. First, an appearance inspection was performed, and a small amount of visible particles were found in all nine formulations. From the size exclusion chromatography (SEC-HPLC) results, after 4 weeks of high-temperature degradation, the SEC purity of each formulation decreased by 3.8% to 8.5%. Regarding the change in non-reduced CE-SDS purity, the changes varied significantly among the formulations, with DOE-7 showing the lowest (1.6%) and DOE-1 the highest (21.1%). The charge variable peak content decreased by 20.0% to 33.9% across all formulations. This result indicates that 4 weeks of high-temperature degradation significantly affects the purity of the MIL86 antibody peak, indicating a clear chemical degradation reaction. The reductions in SEC purity, non-reduced CE-SDS purity, and charge variable peak content of DOE-4 and DOE-7 were all lower than those of other formulations, as shown in Table 2 below.

[0108] Table 2 Thermal stability of MIL86 injection formulations

[0109] Test Example 2 Oscillation Experiment

[0110] After shaking and disruption, an appearance inspection was performed first, and a small amount of visible particles were found in all nine formulations. Size exclusion chromatography (SEC-HPLC) results showed no significant changes in any formulation. Non-reducing CE-SDS analysis showed that only DOE-6 exhibited a purity reduction >1.0%, while charge variable analysis showed minimal changes in all formulations (maximum 2.7%). Overall, all results indicate that each formulation, under shaking conditions, protects the physical and chemical stability of the MIL86 antibody, as shown in Table 3 below.

[0111] Table 3. Results of oscillation stress test for MIL86 injectable formulation

[0112] Test Example 3: Illumination Experiment

[0113] Light exposure test: Samples were taken after being placed at 5℃±3℃ and 4500Lx±500Lx for 10 days (240 hours). After light exposure, an appearance inspection was performed first, and a small amount of visible particles were found in all nine formulations. Regarding the reduction in purity according to size exclusion chromatography, the changes were significant across the formulations. Only the DOE-4 group showed a reduction of less than 10.0%, while DOE-2 and DOE-5 both showed a reduction greater than 20.0%. In terms of the reduction in non-reduced CE-SDS purity, DOE-4 also showed the smallest change (5.5%). The results of both tests indicate that light exposure caused significant higher-order structural changes in the MIL86 protein in all formulations.

[0114] Regarding the reduction in the content of the main peak of charge variable, the changes in each formulation ranged from 9.8% to 15.1%, indicating that light damage can cause significant changes in the charge variable of this product, as shown in Table 4 below.

[0115] Table 4. Photostability Formulation of MIL86 Antibody Preparation

[0116] Test Example 4: Repeated Freeze-Thaw Experiment

[0117] Samples were taken after being frozen at -20℃ for 2 days and placed at 25℃±2℃ for 2 days, and this freeze-thaw cycle was repeated 3 times. After freeze-thaw destruction, only the DOE-6 group was clear and transparent, while the other formulations showed a small amount of visible particles. From the size exclusion chromatography (SEC-HPLC) results, no significant changes were detected in any formulation. In the non-reducing CE-SDS, only the purity of DOE-2 decreased by more than 4.5%, while no significant changes were observed in the other formulations. Regarding charge variables, the DOE-2 group also showed the largest change in the main peak content, which decreased by 6.5% compared to the control, indicating that the DOE-2 group formulation was more prone to physicochemical changes under freeze-thaw conditions than the other formulations, as shown in Table 5 below.

[0118] Table 5 Freeze-thaw stability test of MIL86 antibody preparation

[0119] Summarize:

[0120] The above experimental results show that after oscillation destruction (72h) and repeated freeze-thaw cycles (3 cycles), the designed formulation DOE-9 and the other 8 formulations DOE-1 to DOE-8 showed little change in SEC-HPLC monomer purity, charge variable main peak content, and non-reduced CE-SDS monomer purity, and no key formulation factors were investigated; however, the stability of this product was significantly affected after high temperature destruction and light destruction.

[0121] Statistical analysis of the reduction in monomer purity, charge variable peak content, and non-reducing CE-SDS monomer purity by SEC-HPLC was performed using JMP10 software. The results showed that histidine concentration was a significant factor affecting the stability under high temperature and light conditions, while pH was a significant factor affecting stability under high temperature conditions. After treatment under high temperature and light conditions, changes in histidine concentration within the investigated range (4 mM–8 mM) were positively correlated with changes in SEC-HPLC monomer purity and charge variable peak content of MIL86 protein; pH was negatively correlated with changes in SEC-HPLC monomer purity of MIL86 protein under high temperature conditions. Therefore, it is recommended to further investigate lower pH ranges and higher histidine concentrations to improve the physicochemical stability of the formulation.

[0122] Example 2: MIL86 Antibody Formulation Validation Test

[0123] To confirm the relationship between the pH and histidine content of the formulation solution and key stability parameters of MIL86, and to examine whether the formulation can maintain its robustness within a range of variation, a formulation validation study of MIL86 was conducted. The experiment was designed using JMP10 software based on the FFD principle, with protein concentration, histidine concentration, polysorbate concentration, sucrose concentration, and pH as the main effects factors, conducting a five-factor main effect study. In the previous "Formulation Screening Study," pH was identified as a key factor for high-temperature stability; therefore, this experiment used the actual formulation pH of 6.0 as the midpoint, with the range of pH within ±0.3 units (5.7–6.3).

[0124] Through "formulation screening studies," histidine concentration was confirmed to be a key factor positively correlated with light and high-temperature stability. Therefore, the concentration of L-histidine / L-histidine hydrochloride was increased to 15mM–25mM in this experiment. Sucrose concentration was not a key factor, and increasing sucrose concentration would increase the viscosity of the product; therefore, the sucrose concentration was reduced to 60–80 mg / ml. Regarding other factors, the antibody concentration was 120–160 mg / ml, and the polysorbate 20 concentration was 0.05–0.15 mg / ml. A total of 8 formulations (DOE-1 to DOE-8) were designed, plus the preliminary design formulation DOE-9, for a total of 9 experimental formulations, as shown in Table 6 below.

[0125] Table 6 MIL86 antibody formulation Note: "0" represents the designed concentration of the formulation component; "+" and "-" indicate that the concentration of the protein component, L-histidine / L-histidine hydrochloride, polysorbate 20, pH value, and sucrose concentration are higher or lower than the corresponding designed formulation, respectively.

[0126] Test Example 1: High Temperature Experiment

[0127] Samples were placed in a constant temperature incubator at 40℃±2℃ and 75%±5%RH, and samples were taken after 14 and 28 days. All formulations were then subjected to high-temperature conditions, and samples were taken after 14 and 28 days for analysis. First, an appearance inspection was performed; all nine formulations were slightly light brown in color and contained no visible particles. Referring to Table 7-2, the size exclusion chromatography (SEC-HPLC) results showed slight changes in SEC purity for each formulation after 28 days at high temperature, but the reduction was less than 4.0%, with the central design formulation DOE-9 showing the lowest reduction (2.9%). Regarding the change in non-reducing CE-SDS purity, the purity reduction for each formulation ranged from 1.5% to 2.5% after 14 days of high-temperature degradation, and from 2.8% to 4.2% after 28 days, with minimal differences between formulations. These results indicate that the designed formulations possess good high-temperature physical stability and maintain robustness even under changes in formulation factors (DOE-1 to DOE-8).

[0128] Regarding the changes in the charge variable peak, after 14 days of high-temperature degradation (see Table 7-1), the reduction in the peak value of each formulation ranged from 8.3% to 11.2%, and after 28 days, the reduction in purity ranged from 17.6% to 21.0%, showing significant variations. This result indicates that high-temperature degradation has a significant impact on the purity of the MIL86 antibody peak in each formulation, indicating a clear chemical degradation reaction. Among them, the change in the charge variable peak value of DOE-9 was relatively lower than that of other formulations, at 8.3% after 14 days and 18.0% after 28 days. Compared with the performance of the formulations examined in the "Formulation Screening Study" (23.2%, see Table 2), its chemical stability was significantly improved.

[0129] Table 7-1 Results of Thermal Stress Test on MIL86 Antibody Formulation

[0130] Table 7-2 Results of Thermal Stress Test on MIL86 Antibody Formulation

[0131] Test Example 2 Oscillation Experiment

[0132] Samples were taken after shaking at 25℃±2℃ and 180rpm for 72 hours. The results after shaking were shown in Table 8. All nine formulations showed a slightly light brown tint and no visible particles. Size exclusion chromatography (SEC-HPLC) and non-reducing CE-SDS analysis showed no significant changes in any formulation. In charge variable analysis, all formulations showed changes of less than 2.0%, indicating very small variations. Overall, the results suggest that each formulation, under shaking conditions, protects the physical and chemical stability of the MIL86 antibody.

[0133] Table 8 Results of vibration stress test on MIL86 formulation

[0134] Test Example 3: Illumination Experiment

[0135] Samples were taken after being placed at 5℃±3℃ and 4500Lx±500Lx for 10 days (240 hours). After light exposure, all nine formulations were slightly light brown in color and contained no visible particles. Regarding the reduction in purity under size exclusion chromatography, there were significant differences among the formulations, with the lowest being 2.5% for DOE-4 and the highest being 7.3% for DOE-5. Similarly, regarding the reduction in purity of non-reducing CE-SDS, DOE-4 showed the smallest change (2.0%), while DOE-5 showed the largest change (4.5%). Compared to the light exposure results of Test Example 3 in the "Formulation Screening Study" of Example 1, the physical stability of each formulation under light conditions in this experiment was significantly improved.

[0136] Regarding charge variables, there were significant variations in each group of formulations, with the content of the main charge variable peak decreasing by 10.7% to 19.7%, indicating that light damage can cause a relatively obvious chemical degradation reaction in this product, as shown in Table 9 below.

[0137] Table 9. Results of photostability tests on MIL86 formulations

[0138] Test Example 4: Repeated Freeze-Thaw Study

[0139] Samples were taken after being frozen at -20℃ for 2 days and then placed at 25℃±2℃ for 2 days, repeating this freeze-thaw cycle three times. The test results after repeated freeze-thaw cycles are shown in Table 10. All nine formulations showed a slightly light brown tint and no visible particles. Size exclusion chromatography (SEC-HPLC) and non-reducing CE-SDS results showed no significant changes in any formulation. In the charge variable analysis, all formulations showed changes of less than 3.0%, indicating very small variations. Overall, all test results suggest that each formulation, under shaking conditions, protects the physical and chemical stability of the MIL86 antibody.

[0140] Table 10 Results of freeze-thaw stability tests for MIL86 formulations

[0141] Summary of Prescription Validation Experimental Study:

[0142] The above experimental results show that after shaking and repeated freeze-thaw cycles, the designed formulation DOE-9 and the other eight formulations DOE-1 to DOE-8 showed very little change in terms of monomer purity, charge variable main peak content, and non-reduced CE-SDS monomer purity under SEC-HPLC conditions. Each formulation exhibited good stability under shaking and freeze-thaw conditions.

[0143] In the high-temperature test, the purity of monomers in SEC-HPLC and the purity of non-reduced CE-SDS monomers of each formulation showed little change, indicating good high-temperature physical stability. From the results of charge variable, the chemical stability of the formulations designed in this experiment was significantly improved compared with the formulations in the "Formulation Screening Study".

[0144] In the light irradiation experiments, the results of SEC-HPLC monomer purity and non-reduced CE-SDS monomer purity showed that, compared with the "formulation screening study," the physical stability of each formulation under light irradiation was significantly improved in this experiment. The results of charge variable analysis indicated that light irradiation caused a relatively significant chemical degradation reaction in the product. Key factor analysis revealed that histidine concentration, protein concentration, and sucrose concentration were the key factors affecting the stability of the MIL86 formulation.

[0145] In summary, all formulations exhibited good stability across varying concentrations of MIL86 protein, L-histidine / L-histidine hydrochloride (monohydrate), polysorbate 20, sucrose, and pH. Therefore, this formulation design is considered robust. However, experiments show that this product is sensitive to high temperatures and light; therefore, careful attention should be paid to storage conditions, using outer packaging to avoid light exposure, and storing and transporting it at low temperatures.

[0146] Based on the above prescription validation experiments, the preferred formulation was determined to be the designed formulation DOE-9, as follows:

[0147] MIL86 protein 140mg / ml, L-histidine / L-histidine hydrochloride (monohydrate) 20mM, sucrose 70mg / ml (w / v), polysorbate 20 0.1mg / ml (w / v), pH 6.0, specification 1ml: 140mg.

[0148] Test Example 5: Viscosity Experiment

[0149] To confirm whether the formulation was suitable for subcutaneous application, the viscosity of the preferred formulation was tested.

[0150] Formulation viscosity is an important indicator for subcutaneous formulations. Excessive viscosity can make injection difficult and cause pain to patients during infusion. Therefore, the viscosity of the injection solution should be avoided to exceed 20 cp (according to the Hagen-Poiseuille equation, 1 ml of injection solution should be injected in 10 seconds using a 13 mm needle syringe of the corresponding volume).

[0151] Figure 1 shows the temperature-viscosity correlation curve of the DOE-9 formulation. Viscosity testing confirmed that the viscosity of the solution in this embodiment at room temperature is below 10 cp, meeting the requirement of a low-viscosity solution (<20 cp), and can be used as a subcutaneous injection solution.

[0152] Example 3: Development of Combination Formulations of MIL86

[0153] In the context of combination drug formulations, a combination formulation comprising a PSCK9 antibody is developed, including the PSCK9 antibody and a second therapeutic agent, such as a second antibody, peptide, or small molecule. The second antibody is, for example, a recombinant humanized monoclonal antibody designated as MIL60 (bevacizumab) or MIL62. The peptide is, for example, a peptide compound specifically identified as peptide 1 (amino acid sequence as shown in SEQ ID NO:15) and peptide 2 (amino acid sequence as shown in SEQ ID NO:16). The small molecule is, for example, a G protein-coupled receptor kinase activator, specifically, for example, deoxycholic acid with the following chemical structure. The stability of these combination formulations is tested under the experimental conditions of Test Examples 2-4 in Examples 1 or 2, including high-temperature experiments, shaking experiments, and repeated freeze-thaw experiments, and long-term stability tests are conducted on each combination formulation (long-term stability test conditions are 2-8°C, protected from light, for 24 months). Following the rigorous evaluation described above, the appearance, size exclusion chromatography, non-reducing CE-SDS assay, and charge variable assay results all indicate that the physicochemical properties of these compositions remain unchanged, their bioefficacy is preserved, and there are no significant deviations. Notably, no interactions were observed between the formulation components, thus maintaining the stability profile of the combined pharmaceutical composition. These findings are crucial for establishing the robustness and reliability of this formulation for potential therapeutic applications.

[0154] The sequences used in this application are shown in Table 11 below:

[0155] Table 11 Sequence List

[0156] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A formulation of a PCSK9 antibody, characterized in that, The formulation comprises a PCSK9 antibody, a buffer, a surfactant, and a stabilizer; the PCSK9 antibody includes a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequences VH-CDR1 (as shown in SEQ ID NO:1), VH-CDR2 (as shown in SEQ ID NO:2), and VH-CDR3 (as shown in SEQ ID NO:3); and the light chain variable region comprising the amino acid sequences VL-CDR1 (as shown in SEQ ID NO:4), VL-CDR2 (as shown in SEQ ID NO:5), and VL-CDR3 (as shown in SEQ ID NO:6).

2. The formulation of claim 1, wherein, The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:7, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, and / or the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:8, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it; And / or, the concentration of the PCSK9 antibody is 120-160 mg / mL, preferably 140-160 mg / mL.

3. The preparation according to claim 1 or 2, characterized in that The PCSK9 antibody is a full-length antibody, Fab, Fab', F(ab')2, or Fv; the Fv is preferably scFv. Preferably, the antibody is a full-length antibody, the heavy chain constant region and / or light chain constant region of which are derived from human antibodies; More preferably, the heavy chain constant region is derived from the human heavy chain IgG1 constant region; and / or, the light chain constant region is derived from the human light chain κ chain constant region; More preferably, the amino acid sequence of the heavy chain constant region of the antibody is as shown in SEQ ID NO:9, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it; and / or, the amino acid sequence of the light chain constant region of the antibody is as shown in SEQ ID NO:10, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

4. Formulations according to any one of claims 1 to 3, characterized in that The buffer is a histidine buffer; and / or the concentration of the buffer is 15 mM to 25 mM, preferably 20 mM to 25 mM; Preferably, the histidine buffer is an L-histidine buffer, more preferably an L-histidine / L-hydrochloric acid histidine, such as L-histidine monohydrate / L-hydrochloric acid histidine.

5. The preparation according to any one of claims 1 to 4, characterized in that The surfactant is polysorbate 20; and / or the concentration of the surfactant is 0.05-0.15 mg / mL, preferably 0.1-0.15 mg / mL.

6. The preparation according to any one of claims 1 to 5, characterized in that The stabilizer is sucrose; and / or the concentration of the stabilizer is 60-80 mg / mL, preferably 70-80 mg / mL.

7. The preparation according to any one of claims 1 to 6, characterized in that The pH value of the preparation is 5.7-6.3; preferably 6.0-6.

3.

8. The preparation according to any one of claims 1 to 7, characterized in that The formulation comprises 120-160 mg / mL of the PCSK9 antibody, 15-25 mM of L-histidine monohydrate / L-histidine hydrochloride, 0.05-0.15 mg / mL of polysorbate 20 and 60-80 mg / mL of sucrose, and the pH of the formulation is 5.7-6.

3. Preferably, the formulation comprises 140-160 mg / mL of the PCSK9 antibody, 20-25 mM of L-histidine monohydrate / L-histidine hydrochloride, 0.1-0.15 mg / mL of polysorbate 20 and 70-80 mg / mL of sucrose, and the pH of the formulation is 5.7-6.

3.

9. The formulation of claim 8, wherein, The formulation comprises 140 mg / mL of the PCSK9 antibody, 20 mM of L-histidine monohydrate / L-histidine hydrochloride, 0.1 mg / mL of polysorbate 20 and 70 mg / mL of sucrose, and the pH of the formulation is 6.

0. Alternatively, the formulation comprises 120 mg / mL of the PCSK9 antibody, 15 mM of L-histidine monohydrate / L-histidine hydrochloride, 0.05 mg / mL of polysorbate 20 and 80 mg / mL of sucrose, and the pH of the formulation is 5.

7. Alternatively, the formulation comprises 120 mg / mL of the PCSK9 antibody, 15 mM of L-histidine monohydrate / L-histidine hydrochloride, 0.15 mg / mL of polysorbate 20 and 60 mg / mL of sucrose, and the pH of the formulation is 6.

3. Alternatively, the formulation comprises 120 mg / mL of the PCSK9 antibody, 25 mM of L-histidine monohydrate / L-histidine hydrochloride, 0.05 mg / mL of polysorbate 20 and 60 mg / mL of sucrose, and the pH of the formulation is 6.

3. Alternatively, the formulation comprises 120 mg / mL of the PCSK9 antibody, 25 mM of L-histidine monohydrate / L-histidine hydrochloride, 0.15 mg / mL of polysorbate 20 and 80 mg / mL of sucrose, and the pH of the formulation is 5.

7. Alternatively, the formulation comprises 160 mg / mL of the PCSK9 antibody, 15 mM of L-histidine monohydrate / L-histidine hydrochloride, 0.05 mg / mL of polysorbate 20 and 80 mg / mL of sucrose, and the pH of the formulation is 6.

3. Alternatively, the formulation comprises 160 mg / mL of the PCSK9 antibody, 15 mM of L-histidine monohydrate / L-histidine hydrochloride, 0.15 mg / mL of polysorbate 20 and 60 mg / mL of sucrose, and the pH of the formulation is 5.

7. Alternatively, the formulation comprises 160 mg / mL of the PCSK9 antibody, 25 mM of L-histidine monohydrate / L-histidine hydrochloride, 0.05 mg / mL of polysorbate 20 and 60 mg / mL of sucrose, and the pH of the formulation is 5.

7. Alternatively, the formulation comprises 160 mg / mL of the PCSK9 antibody, 25 mM of L-histidine monohydrate / L-histidine hydrochloride, 0.15 mg / mL of polysorbate 20, and 80 mg / mL of sucrose, and the pH of the formulation is 6.

3.

10. The preparation according to any one of claims 1 to 9, characterized in that The formulation satisfies one or more of the following conditions: (1) The specification of the preparation is 1 mL; (2) The formulation is a formulation administered by subcutaneous injection; and, (3) The viscosity of the preparation is less than 20 cp, preferably less than 10 cp.

11. A combination formulation, characterized in that, The combination formulation comprises the formulation and the second therapeutic agent as described in any one of claims 1 to 10; Preferably, the second therapeutic agent comprises one or more selected from other antibodies, peptides, and small molecule drugs.

12. The combination preparation of claim 11, wherein The second therapeutic agent meets one or more of the following conditions: (a) The other antibody is an anti-VEGF antibody, such as MIL60, preferably with a heavy chain sequence as shown in SEQ ID NO:11 and a light chain sequence as shown in SEQ ID NO:12; and / or, the other antibody is an anti-CD20 antibody, such as MIL62, preferably with a heavy chain sequence as shown in SEQ ID NO:13 and a light chain sequence as shown in SEQ ID NO:14; (b) The amino acid sequence of the polypeptide is shown in SEQ ID NO:15 or SEQ ID NO:16; and, (c) the small molecule drug is a G protein-coupled receptor kinase activator, for example is deoxycholic acid, preferably the deoxycholic acid has the structural formula:

13. Use of the formulation according to any one of claims 1 to 10 and / or the combination formulation according to claim 11 or 12 in the preparation of the following pharmaceutical products: Medications for the prevention or treatment of cardiovascular diseases or disorders and / or thromboembolic diseases or disorders; Medications that lower blood lipoprotein levels; Drugs that specifically bind to PCSK9; Drugs that block the binding of PCSK9 to LDL-R; Drugs that increase the number of LDL-R on the cell surface or the level of LDL-R in plasma; Drugs that lower plasma LDL or LDL-c levels; Drugs that inhibit the accumulation of LDL in plasma; Drugs that inhibit PCSK9-mediated LDL-R degradation; or, Drugs that increase the metabolic levels of cholesterol and / or triglycerides carried by LDL. Preferably, the cardiovascular disease or disorder is selected from dyslipidemia, coronary atherosclerotic heart disease, acute myocardial infarction, asymptomatic carotid atherosclerosis, stroke, and peripheral artery occlusive disease; and / or, the thromboembolic disease or disorder is selected from pulmonary embolism and central retinal vein embolism. More preferably, the dyslipidemia is selected from elevated cholesterol, elevated triglycerides, elevated low-density lipoprotein and decreased high-density lipoprotein in the blood, such as hyperlipidemia.

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