Stable formulation containing anti-PCSK9 antibody and preparation method and use thereof

Abstract

The present invention relates to a stable formulation containing a high concentration of an anti-PCSK9 antibody. The formulation comprises a therapeutically effective dose of an anti-PCSK9 antibody or an antigen-binding fragment thereof, a pharmaceutically acceptable buffer, an osmotic pressure regulator, and / or a surfactant, and exhibits long-term stability. When the formulation is a liquid formulation or a reconstituted liquid formulation of a lyophilized formulation, it exhibits low viscosity and long-term stability. The present invention also provides a method for preparing the formulation and its use in preparing a medicament for treating, preventing, and / or ameliorating any disease or symptom associated with PCSK9.

Description

[0001] This application claims priority to Chinese application number CN202011092207.8 filed on October 13, 2020, with the invention name being "Stable formulation containing anti-PCSK9 antibodies, preparation method and use thereof." Technical Field

[0002] The present invention relates to the field of medicine, and more specifically, to a stable formulation containing an anti-PCSK9 antibody and a preparation method thereof, as well as use of the stable formulation in a drug for treating, preventing or ameliorating diseases associated with PCSK9 activity. Background Art

[0003] Proprotein convertase subtilisin / kexin type 9 (PCSK9), also known as neural apoptosis-regulating convertase 1 (NARC-1), is a prohormone-to-proprotein convertase in the subtilisin (S8) family of serine proteases. It is expressed in cells capable of proliferation and differentiation, including hepatocytes, renal interstitial cells, ileal and colonic epithelial cells, and embryonic telencephalic neurons. Studies have shown that PCSK9 plays a role in the differentiation of hepatocytes and neurons. Not only does it specifically act on cholesterol biosynthesis or uptake, but circulating PCSK9 can directly bind to the low-density lipoprotein receptor (LDLR) on the surface of hepatocytes and, along with the LDLR, is phagocytosed by hepatocytes, promoting LDLR degradation in hepatocytes and hindering LDLR recycling, thereby increasing plasma LDL cholesterol (LDL-C). Elevated LDL-C expression is closely associated with human dyslipidemia and cardiovascular diseases. Thus, PCSK9 is a key regulator of cholesterol homeostasis and circulating low-density lipoprotein (LDL) levels. In addition, PCSK9 has multiple mutants, including S127R, N157K, F216L, R218S, and D374Y. Among them, S127R, F216L, and D374Y are associated with autosomal dominant hypercholesterolemia (ADH), an inherited metabolic disorder characterized by significantly increased LDL concentrations and cholesterol levels in plasma, leading to premature cardiovascular disease. When PCSK9 function is inhibited, the number of LDLRs on the surface of hepatocytes increases to clear more circulating LDL, and plasma cholesterol levels also decrease accordingly. Therefore, inhibiting PCSK9 can effectively reduce plasma LDL levels. As a result, PCSK9 has become a potential target for the treatment of diseases such as atherosclerosis, coronary heart disease, hypercholesterolemia, and dyslipidemia.

[0004] Anti-PCSK9 monoclonal antibodies specifically target PCSK9 and, by inhibiting its interaction with LDLR on the surface of hepatocytes, reduce LDLR endocytosis and degradation, significantly lowering circulating LDL-C concentrations. Anti-PCSK9 monoclonal antibodies offer advantages such as strong specificity, good safety, and convenient administration, and hold significant clinical promise in the treatment of hypercholesterolemia, hyperlipidemia, and related cardiovascular diseases.

[0005] CN201710816808.0 discloses a novel monoclonal antibody against proprotein convertase subtilisin-kexin type 9 (PCSK9).

[0006] Like other macromolecular therapeutics, therapeutic anti-PCSK9 antibodies not only need to be formulated into drug solutions suitable for administration, but also need to maintain their stability during manufacturing, storage, and subsequent use. However, therapeutic antibody solutions are subject to physical and chemical instability factors (such as aggregation, denaturation, cross-linking, deamidation, isomerization, oxidation, and shearing) during manufacturing or storage. Therefore, the stability of the prepared antibody liquid formulation depends not only on the type of excipients in the formulation, but also on the relative amounts and ratios of the active therapeutic ingredient and the excipients, as well as between the excipients.

[0007] Cardiovascular-related diseases are chronic diseases. If patients are able to take their own medication at home, subcutaneous injection of antibody drugs becomes the preferred method of administration.

[0008] Due to the presence of extracellular matrix, drug mobility is limited. Under normal circumstances, the subcutaneous injection volume is generally limited to 1-2mL. The concentration of antibody preparations needs to reach 100mg / mL or more to meet the requirements of clinical dosage. However, high-concentration antibody proteins will increase soluble and insoluble protein-protein aggregates and granular precipitates on the one hand, resulting in poor formulation stability. At the same time, the instability of protein conformation will also cause chemical properties, such as significant changes in charge heterogeneity, which will have an adverse effect on downstream processes (e.g., ultrafiltration and sterile filtration) and quality stability during storage. On the other hand, high-concentration protein will cause protein viscosity to increase due to protein as a macromolecular substance and intermolecular interactions, which limits the choice of protein drug delivery device and the many problems such as difficult push injection, injection pain and burning sensation caused by subcutaneous administration of drugs through injection devices, thereby affecting the patient's medication compliance. Thirdly, for liquid preparations of high-concentration proteins desired for subcutaneous administration, it is necessary to add high concentrations of stabilizers (e.g., sugars, polyols, amino acids, chelating agents, surfactants, etc.) to achieve long-term protein stability. However, the resulting solution preparations usually cause injection pain due to tissue damage.

[0009] CN201380035382.5 discloses a stable formulation comprising: at least one monoclonal antibody that specifically binds to PCSK9, wherein PCSK9 comprises the amino acid of SEQ ID NO.1 of the invention patent, and the amount of the monoclonal antibody is about 40 mg / ml to about 300 mg / ml; and an amount of a pharmaceutically acceptable buffering agent is about 0.05 mM to about 40 mM; and an amount of a pharmaceutically acceptable surfactant is about 0.01% w / v to about 20% w / v; and at least one pharmaceutically acceptable stabilizer is about 0.5% w / v to about 10% w / v, wherein the pH of the stable formulation is between about 4.0 and about 6.0.

[0010] CN201280037861.6 discloses a pharmaceutical formulation containing: (i) an antibody that specifically binds to human proprotein convertase subtilisin / kexin type 9 (human PCSK9); (ii) a buffer with a pH of 6.0±0.3; (iii) a non-ionic detergent; and (iv) a stabilizer.

[0011] Since the antibody described in CN201710816808.0 has significant differences in material properties from the antibodies in the prior art CN201380035382.5 and CN201280037861.6, it is difficult to solve the problems existing in the aforementioned subcutaneous injection liquid preparations by adopting the corresponding formulation scheme.

[0012] Therefore, there is an urgent need to develop an anti-PCSK9 antibody preparation that is high in concentration, low in viscosity, isotonic, has long-term stability, has a simple preparation process, is easy to control quality, and is convenient for subcutaneous administration to patients. Summary of the Invention

[0013] The present invention aims to address the deficiencies of the prior art and provides a stable formulation containing an anti-PCSK9 antibody, which has the characteristics of low viscosity, high antibody concentration suitable for subcutaneous injection, long-term stability and high patient compliance.

[0014] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0015] In one aspect, the present invention provides a stable formulation containing an anti-PCSK9 antibody, comprising:

[0016] (1) an anti-PCSK9 antibody or antigen-binding fragment thereof at a concentration of 50-200 mg / mL; and

[0017] (2) a pharmaceutically acceptable buffer at a concentration of 1-50 mmol / L (mM); and

[0018] (3) a pharmaceutically acceptable surfactant at a concentration of 0-0.1% w / w; and

[0019] (4) One or more other pharmaceutically acceptable excipients at a concentration of 100-400 mmol / L (mM) or 1-10% w / w.

[0020] The preparation may be a liquid preparation or a freeze-dried preparation that can be reconstituted into a liquid; preferably, it is a liquid preparation.

[0021] The anti-PCSK9 antibody or antigen-binding fragment thereof can specifically bind to the PCSK9 protein and can be either a polyclonal antibody or a monoclonal antibody. Preferably, the monoclonal antibody can be produced on a large scale and has homogeneity. The monoclonal antibody is not limited to monoclonal antibodies of human, mouse, rabbit, sheep, camel, monkey, etc., and can also be a recombinant antibody such as a chimeric antibody, a humanized antibody, or a fully human monoclonal antibody. Preferably, the fully human monoclonal antibody is produced by host cells or transgenic animals.

[0022] Specifically, the anti-PCSK9 antibodies or antigen-binding fragments thereof described herein comprise heavy chain complementary determining regions (HCDRs) and light chain complementary determining regions (LCDRs), wherein the HCDR1, HCDR2 and HCDR3 amino acid sequences comprise at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequences set forth in SEQ ID NOs: 1, 2 and 3; and the LCDR1, LCDR2 and LCDR3 comprise at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequences set forth in SEQ ID NOs: 4, 5 and 6.

[0023] The anti-PCSK9 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO:7; and the light chain variable region comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO:8.

[0024] The anti-PCSK9 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain connected by a disulfide bond, wherein the heavy chain comprises a heavy chain variable region and a heavy chain constant region, and the light chain comprises a light chain variable region and a light chain constant region, wherein the C-terminus of the heavy chain variable region is linked to the N-terminus of the heavy chain constant region, and the C-terminus of the light chain variable region is linked to the N-terminus of the light chain constant region, and the heavy chain variable region and light chain variable region comprise the amino acid sequences described above. The heavy chain constant region is selected from a human IgG1, IgG2, IgG3, or IgG4 constant region, preferably an IgG1 or IgG4 constant region, more preferably an IgG4 constant region, the amino acid sequence of which is set forth in SEQ ID NO:9; the light chain constant region is selected from a human κ light chain constant region or a λ light chain constant region, preferably a κ light chain constant region, the amino acid sequence of which is set forth in SEQ ID NO:10.

[0025] The anti-PCSK9 antibody or antigen-binding fragment thereof comprises or is composed of two heavy chains and two light chains, wherein each heavy chain comprises the aforementioned heavy chain constant region, heavy chain variable region, or CDR sequence, and each light chain comprises the aforementioned light chain constant region, light chain variable region, or CDR sequence. The antibody may also be a single-chain variable fragment (scFv) antibody or an antibody fragment (e.g., a Fab or F(ab')2 fragment). Preferably, the heavy chain of the anti-PCSK9 antibody or antigen-binding fragment thereof has the amino acid sequence set forth in SEQ ID NO:11, and the light chain has the amino acid sequence set forth in SEQ ID NO:12.

[0026] In one embodiment, the concentration of the anti-PCSK9 antibody or antigen-binding fragment thereof is 50-200 mg / mL. In another embodiment, the concentration of the anti-PCSK9 antibody or antigen-binding fragment thereof is 70-200 mg / mL. In another embodiment, the concentration of the anti-PCSK9 antibody or antigen-binding fragment thereof is 70 mg / mL. In a specific embodiment, the concentration of the anti-PCSK9 antibody or antigen-binding fragment thereof is 70 mg / mL. In a specific embodiment, the concentration of the anti-PCSK9 antibody or antigen-binding fragment thereof is 140 mg / mL. In a specific embodiment, the concentration of the anti-PCSK9 antibody or antigen-binding fragment thereof is 200 mg / mL.

[0027] The pharmaceutically acceptable buffer is selected from: histidine, acetate, succinate, or glutamate.

[0028] As a preferred technical solution of the present invention, the buffer is a histidine buffer. In a specific embodiment, the buffer is a histidine buffer composed of L-histidine and L-histidine hydrochloride, and the concentration (molar) ratio of L-histidine and L-histidine hydrochloride is 0.2:1-3:1. In one embodiment, the buffer is an acetate buffer. In a specific embodiment, the buffer is an acetate buffer composed of acetic acid and sodium acetate. When phosphate and citrate are selected as buffers for the preparation of the present invention, they are likely to cause pain reactions during subcutaneous injection, and the patient's medication compliance is therefore significantly affected.

[0029] The pharmaceutically acceptable buffer concentration is 1-50 mmol / L. In one embodiment, the buffer concentration is 5-50 mmol / L. In one embodiment, the buffer concentration is 10-30 mmol / L. In a specific embodiment, the buffer concentration is 20 mmol / L.

[0030] The pH value of the stable liquid formulation is 4.5-6.5. In one embodiment, the pH value of the stable liquid formulation is 4.5-5.5. In a specific embodiment, the pH value of the stable liquid formulation is 4.5, 5.0 or 6.0. In one embodiment, the pH value of the stable liquid formulation is 5.0-6.0. In a specific embodiment, the pH value of the stable liquid formulation is 5.0, 5.5 or 6.0. In one embodiment, the pH value of the stable liquid formulation is 5.5-6.5. In a specific embodiment, the pH value of the stable liquid formulation is 5.5, 6.0, 6.2, 6.3 or 6.5.

[0031] Histidine contains an imidazole group in its molecular structure, and its pKa is around 6.0, which is pH-sensitive. When the environmental pH is less than 6.0, protonation occurs, showing hydrophilicity; when the environmental pH is greater than 6.0, deprotonation occurs, showing hydrophobicity.

[0032] Although histidine is pH-sensitive and easily oxidized, the inventors surprisingly discovered that when the formulation of the present invention uses L-histidine / L-histidine hydrochloride as a buffer system and maintains a pH value between 5.5 and 6.5, it has excellent compliance and quality stability. For example, compared with using an acetate buffer system with a lower pH value (pKa of 4.76), the patient's pain sensation can be significantly reduced during subcutaneous injection. For patients with chronic diseases who require long-term medication, the use of L-histidine / L-histidine hydrochloride liquid formulations can improve patient compliance. In addition, when the L-histidine / L-histidine hydrochloride buffer system is selected and the pH value is 5.5-6.5, the viscosity of the antibody in the liquid formulation of the present invention can be reduced to below 15 cp, or even below 10 cp, and the degradation rate of the antibody during storage can be reduced, resulting in better stability. However, when the pH is selected below 5.5, acid-catalyzed hydrolysis is accelerated, resulting in a faster rate of hydrolyzate formation.

[0033] The other excipients of the stable liquid preparation are selected from any one or a combination of proline, sucrose, trehalose, sodium chloride, sorbitol, mannitol, or arginine hydrochloride. In a specific embodiment, the other excipient is sucrose, and its concentration is 1%-10% (w / w), preferably 5%-10% (w / w), and more preferably 6% (w / w). As a preferred technical solution of the present invention, the other excipient is proline. In a specific embodiment, the other excipient is proline, and its concentration is 100-400mmol / L, preferably 200-300mmol / L, and more preferably 250mmol / L.

[0034] The stable liquid formulation has an osmotic pressure of 220-370 mOsmol / kg. In one embodiment, the stable liquid formulation has an osmotic pressure of 250-350 mOsm / kg. In one embodiment, the stable liquid formulation has an osmotic pressure of 300-330 mOsm / kg at 25°C.

[0035] Through extensive experiments, the present invention has discovered that when proline is selected as one of the excipients of the present invention, not only can the osmotic pressure of the liquid formulation of the present invention be maintained at 250-350 mOsm / kg due to its biocompatibility, but it can also form a hydration layer on the surface of the anti-PCSK9 antibody or antigen-binding fragment thereof, stabilizing the antibody protein without disrupting its structure. Furthermore, it can prevent the antibody protein from interacting with itself or acting on it with other substances, thereby reducing the viscosity and denaturation of the high-concentration antibody in the liquid formulation. This improves the stability and flowability of the liquid formulation, as well as patient compliance during subcutaneous administration. As one of the most suitable excipients for the formulation of the present invention, proline can avoid hemolysis or irritation during administration, effectively ensuring the safety and stability of the injectable formulation.

[0036] The surfactant of the stable liquid formulation is a nonionic surfactant selected from polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 80, polysorbate 60, polysorbate 40, or polysorbate 20), polyethylene-polypropylene copolymers, polyethylene-polypropylene glycol, polyoxyethylene stearate, polyoxyethylene alkyl ethers, alkylphenyl polyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymers (e.g., poloxamer, Pluronic), and sodium lauryl sulfate (SDS). As a preferred embodiment of the present invention, polysorbate 80 or polysorbate 20 is preferred, with polysorbate 80 being more preferred. The surfactant concentration is 0-1% (w / w), preferably 0.01-0.05% (w / w), and more preferably 0.01%, 0.02%, and 0.04% (w / w). In the present invention, the use of the nonionic surfactant in the aforementioned amount can prevent protein adsorption and interaction between proteins by reducing interfacial interactions, as well as prevent protein interaction with the inner surface of the packaging material, thereby achieving the purpose of stabilizing antibodies during preparation and storage.

[0037] Through extensive experiments, the present invention has discovered that the use of polysorbate 80 or polysorbate 20 as a surfactant in the liquid formulation of the present invention can reduce aggregation of the anti-PCSK9 antibody or antigen-binding fragment thereof during agitation and shaking, preventing adsorption of the antibody to the container surface, and thus meeting the requirements for subcutaneous administration of the liquid formulation of the present invention. Furthermore, polysorbate 80, due to its double bonds, has a low freezing point (it remains solid at room temperature), making it convenient to use. Furthermore, the ability of polysorbate 80 or polysorbate 20 to stabilize antibodies is also related to its concentration. In the liquid formulation of the present invention, polysorbate 80 or polysorbate 20 concentrations above 0.01% (w / w) can inhibit antibody aggregation caused by shaking. Concentrations below this concentration do not reduce protein instability caused by shaking, while concentrations above 0.05% (w / w) do not significantly improve antibody stability.

[0038] When the present invention selects the above-mentioned excipient types and concentrations, the viscosity of the stable liquid preparation at 25°C is about 1 to 20 centipoise (cp). As a preferred technical solution of the present invention, the viscosity of the stable liquid preparation at 25°C is less than 15cp. In one embodiment, the viscosity of the stable liquid preparation at 25°C is about 5 to 15cp. In one embodiment, the viscosity of the stable liquid preparation at 25°C is about 6 to 14cp. In a specific embodiment, the viscosity of the stable liquid preparation at 25°C is about 6.0cp, 8.0cp, 9.0cp, 10.0cp, 11.0cp, or 12.0cp. In a specific embodiment, the viscosity of the stable liquid preparation at 25°C is about 9.0cp.

[0039] The stable liquid formulation can be stored at about 5°C to about 40°C for at least 2 weeks to 36 months, for example, at about 5°C, about 25°C, or about 40°C for at least 2 weeks, at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 6 months, at least 9 months, at least 12 months, at least 18 months, at least 24 months, or 36 months. In one embodiment, the stable liquid formulation has an increase in aggregates of no more than 10%, for example, no more than 5%, 4%, 3%, 2%, 1.3%, 1.2%, 1.1%, or 2.5%, of the anti-PCSK9 antibody or antigen-binding fragment thereof, as detected by SEC-HPLC, after storage at 2-8°C for at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or longer, or at about 25°C for at least 2 weeks, at least 4 weeks, at least 12 weeks, or longer, or at 40°C for at least 2 weeks, at least 4 weeks, or longer. 1.0%, 0.8%, 0.7%, 0.6%, 0.5% or 0.1%, and the degradation products (e.g., fragments) of the anti-PCSK9 antibody or antigen-binding fragment thereof are increased by no more than 10%, such as no more than 5%, 4%, 3%, 2%, 1.3%, 1.2%, 1.1%, 1.0%, 0.8%, 0.7%, 0.6%, 0.5% or 0.1%. In one embodiment, the purity of the anti-PCSK9 antibody or antigen-binding fragment thereof is reduced by no more than 10%, e.g., no more than 5%, 4%, 3%, 2%, 1.0%, 0.5%, 0.2% or 0.1%, after storage at 2-8°C for at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 6 months, at least 9 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or longer, or at about 25°C for at least 2 weeks, at least 4 weeks, at least 12 weeks, or longer, or at 40°C for at least 2 weeks, at least 4 weeks, or longer, as detected by reduced CE-SDS (rCE-SDS) and non-reduced CE-SDS (nrCE-SDS). In one embodiment, the stable liquid formulation has no more than 50% acid-base charge variants of the anti-PCSK9 antibody or antigen-binding fragment thereof, as detected by CEX-HPLC, after storage at 2-8°C for at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 6 months, at least 9 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or longer, or at about 25°C for at least 2 weeks, at least 4 weeks, at least 12 weeks, or longer, or at 40°C for at least 2 weeks or longer. The content of basic charge variants is no more than about 30%, for example, no more than about 29%, 25%, or 22%, and the content of acidic charge variants is no more than 20%, for example, no more than 19%, 17%, 15%, or 10%.In one embodiment, the purity of the anti-PCSK9 antibody or antigen-binding fragment thereof decreases by no more than 5%, for example, no more than 5%, 4%, 3%, 2%, 1% or 0.5%, as detected by a non-reducing Caliper assay after storage of the stable liquid formulation at about 25° C. or about 40° C. for at least 2 weeks or 4 weeks.

[0040] The stable preparation is a liquid pharmaceutical preparation, preferably an injection, more preferably a subcutaneous injection or an intramuscular injection, and most preferably a subcutaneous injection.

[0041] In a preferred embodiment, the stable liquid formulation of the present invention comprises:

[0042] (1) an anti-PCSK9 antibody or antigen-binding fragment thereof at a concentration of 70-200 mg / mL; and

[0043] (2) a histidine buffer consisting of L-histidine and L-histidine hydrochloride in a concentration (molar) ratio of 0.2:1 to 3:1; and

[0044] (3) concentration of 0-0.05% w / w polysorbate 80 or polysorbate 20;

[0045] (4) The concentration is 200-300 mmol / L proline.

[0046] The concentration of the buffer is 1-50 mmol / L (mM), and the pH value of the liquid preparation is 5.5-6.5.

[0047] The anti-PCSK9 antibody or antigen-binding fragment thereof comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 of the amino acid sequence shown in SEQ ID NO: 2, HCDR3 of the amino acid sequence shown in SEQ ID NO: 3, LCDR1 of the amino acid sequence shown in SEQ ID NO: 4, LCDR2 of the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 6.

[0048] More specifically, the anti-PCSK9 antibody or antigen-binding fragment thereof comprises a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 7 and a light chain variable region with the amino acid sequence shown in SEQ ID NO: 8.

[0049] In a more preferred embodiment, the stable liquid formulation of the present invention comprises:

[0050] (1) an anti-PCSK9 antibody or antigen-binding fragment thereof at a concentration of 70 mg / mL or 140 mg / mL; and

[0051] (2) a histidine buffer consisting of L-histidine and L-histidine hydrochloride at a concentration of 20 mmol / L; and

[0052] (3) a concentration of 0.01-0.04% w / w polysorbate 80 or polysorbate 20; and

[0053] (4) The concentration is 250mmol / L proline.

[0054] The pH value of the liquid preparation is 5.5-6.5. In another aspect, the present invention provides a method for preparing a stable liquid preparation containing an anti-PCSK9 antibody, comprising the following steps:

[0055] a) providing an anti-PCSK9 antibody or an antigen-binding fragment thereof;

[0056] b) adding a buffer and an osmotic pressure regulator;

[0057] c) adding a surfactant;

[0058] d) sterile filtration;

[0059] e) Repackaging.

[0060] Wherein, the anti-PCSK9 antibody or antigen-binding fragment thereof in step a) is an antibody protein stock solution prepared by cell culture and obtained by separation and purification.

[0061] The buffer and osmotic pressure regulator in step b) are added to the anti-PCSK9 antibody or antigen-binding fragment thereof in step a) in the form of an ultrafiltrate, and the antibody or antigen-binding fragment thereof is ultrafiltrated and concentrated until the antibody or antigen-binding fragment thereof is completely replaced. Prior to step c), the concentration of the replaced anti-PCSK9 antibody or antigen-binding fragment thereof is adjusted.

[0062] The surfactant in step c) is provided in the form of an aqueous solution.

[0063] The storage device for the subpackaging in step e) is a pre-filled syringe. The anti-PCSK9 antibody or antigen-binding fragment thereof, buffer, osmotic pressure regulator and surfactant are as described above, and their dosage and / or concentration are as described above or can be appropriately determined.

[0064] In one embodiment, the method for preparing the stable liquid formulation of the present invention comprises the following steps:

[0065] a) separating and purifying the harvested anti-PCSK9 antibody or antigen-binding fragment thereof prepared by culture to obtain an antibody protein stock solution, wherein the anti-PCSK9 antibody or antigen-binding fragment thereof is as described above;

[0066] b) adding an ultrafiltrate composed of a buffer and an osmotic pressure regulator to perform ultrafiltration and concentration until the antibody protein is completely replaced, wherein the types, concentrations and pH values ​​of the buffer and the osmotic pressure regulator are as described above, preferably the buffer is a histidine buffer composed of L-histidine and L-histidine hydrochloride, and preferably the osmotic pressure regulator is proline;

[0067] c) adjusting the concentration of the replaced antibody protein to the concentration range defined by the stable liquid formulation of the present invention;

[0068] d) adding an aqueous surfactant solution, wherein the surfactant and its concentration are as described above, and preferably the surfactant is polysorbate 80 or polysorbate 20.

[0069] e) sterile filtering the solution of step d);

[0070] f) dispensing into prefilled syringes to obtain the stable liquid preparation of the present invention.

[0071] In a third aspect, the present invention provides a method for treating a stable liquid formulation containing an anti-PCSK9 antibody in the preparation of a drug for treating, preventing or ameliorating any disease or symptom associated with PCSK9.

[0072] The present invention further provides a prefilled syringe, wherein the prefilled syringe is loaded with the aforementioned stable preparation, which is a liquid preparation.

[0073] In one aspect, the present invention provides a method for treating a disease or condition associated with PCSK9 in a subject, the method comprising administering to the subject a therapeutically effective amount of a liquid formulation comprising an anti-PCSK9 antibody, or an antibody or antigen-binding fragment thereof, as described herein. In some embodiments, the activity of PCSK9 binding to LDLR is modulated by administering a liquid formulation comprising an anti-PCSK9 antibody, or an antibody or antigen-binding fragment thereof, as described herein. In some embodiments, a disease or condition treated or prevented in a subject using statins can also be treated or prevented by administering a liquid formulation comprising an anti-PCSK9 antibody, or an antibody or antigen-binding fragment thereof, as described herein. In some embodiments, a disease or condition treated by preventing cholesterol synthesis or increasing LDLR expression can also be treated by administering a liquid formulation comprising an anti-PCSK9 antibody, or an antibody or antigen-binding fragment thereof, as described herein. The present invention also provides use of a liquid formulation comprising an anti-PCSK9 antibody, or an anti-PCSK9 antibody or antigen-binding fragment thereof, as described herein, in the preparation of a medicament for treating a disease or condition associated with PCSK9.

[0074] In one aspect, the present invention provides a method for lowering cholesterol levels in a subject, comprising administering to the subject a therapeutically effective amount of a liquid formulation containing an anti-PCSK9 antibody, or an antibody or antigen-binding fragment thereof, as described herein. Cholesterol includes total cholesterol, LDL-C, and non-high-density lipoprotein (HDL) cholesterol. In one embodiment, the present invention provides a method for lowering LDL-C levels in a subject, comprising administering to the subject a therapeutically effective amount of a liquid formulation containing an anti-PCSK9 antibody, or an antibody or antigen-binding fragment thereof, as described herein. Following administration of the liquid formulation, antibody or antigen-binding fragment thereof, the LDL-C level decreases. In a specific embodiment, the present invention provides a method for lowering serum LDL-C levels in a subject, comprising administering to the subject a therapeutically effective amount of a liquid formulation containing an anti-PCSK9 antibody, or an antibody or antigen-binding fragment thereof, as described herein. Following administration of the liquid formulation, antibody or antigen-binding fragment thereof, the serum LDL-C level decreases. In one embodiment, the present invention provides a method for lowering total cholesterol levels in a subject, comprising administering to the subject a therapeutically effective amount of a liquid formulation containing an anti-PCSK9 antibody, or an antibody or antigen-binding fragment thereof, as described herein. After administration of the liquid formulation or the antibody or antigen-binding fragment thereof, the total cholesterol level is reduced. The present invention also provides a use of a liquid formulation containing an anti-PCSK9 antibody or the anti-PCSK9 antibody or antigen-binding fragment thereof in the preparation of a medicament for lowering cholesterol levels in a subject.

[0075] In one aspect, the present invention provides a method for treating and / or preventing cholesterol-related diseases in a subject, comprising administering to the subject a therapeutically effective amount of a liquid formulation containing an anti-PCSK9 antibody according to the present invention, or an anti-PCSK9 antibody or antigen-binding fragment thereof according to the present invention. The cholesterol-related diseases include any one or more of the following: hypercholesterolemia, heart disease, metabolic syndrome, diabetes, coronary heart disease, stroke, cardiovascular disease, Alzheimer's disease, and dyslipidemia (e.g., high levels of total serum cholesterol, high levels of LDL, high levels of triglycerides, high levels of very low-density lipoprotein (VLDL), and / or low levels of high-density lipoprotein (HDL)). In some embodiments, the present invention provides a method for treating and / or preventing hypercholesterolemia and / or dyslipidemia in a subject, comprising administering to the subject a therapeutically effective amount of a liquid formulation containing an anti-PCSK9 antibody according to the present invention, or an anti-PCSK9 antibody or antigen-binding fragment thereof according to the present invention. The present invention also provides a method for preparing a liquid formulation containing an anti-PCSK9 antibody, or an anti-PCSK9 antibody or antigen-binding fragment thereof according to the present invention for treating and / or preventing cholesterol-related diseases.

[0076] In one aspect, the present invention provides a method for administering a liquid formulation comprising an anti-PCSK9 antibody according to the present invention, or an anti-PCSK9 antibody or antigen-binding fragment thereof according to the present invention, in combination with another therapeutic agent. In some embodiments, an effective dose of the liquid formulation comprising an anti-PCSK9 antibody according to the present invention, or an anti-PCSK9 antibody or antigen-binding fragment thereof according to the present invention, is administered prior to administering at least one other therapeutic agent. In some embodiments, an effective dose of the liquid formulation comprising an anti-PCSK9 antibody according to the present invention, or an anti-PCSK9 antibody or antigen-binding fragment thereof according to the present invention, is administered simultaneously with administering at least one other therapeutic agent. In some embodiments, an effective dose of the liquid formulation comprising an anti-PCSK9 antibody according to the present invention, or an anti-PCSK9 antibody or antigen-binding fragment thereof according to the present invention, is administered after administering at least one other therapeutic agent. The other therapeutic agents include, but are not limited to, at least one drug that lowers cholesterol levels (including serum LDL-C and total cholesterol) or increases LDLR expression levels, such as statins (selected from: atorvastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, any one or any combination thereof). In some embodiments, the other therapeutic agents can be used to treat and / or treat hypercholesterolemia and / or dyslipidemia. In some embodiments, the other therapeutic agents can be used to treat and / or prevent atherosclerosis and / or cardiovascular disease and / or reduce recurrent cardiovascular events. In some embodiments, the other therapeutic agents can be used to increase HDL cholesterol levels.

[0077] The effective dosage range of the liquid preparation containing the anti-PCSK9 antibody or the anti-PCSK9 antibody or antigen-binding fragment thereof described above includes about 0.5-50 mg / kg.

[0078] In a fourth aspect, the present invention provides nucleotides encoding anti-PCSK9 antibodies or antigen-binding fragments thereof of the present invention. In some embodiments, the present invention provides vectors comprising the nucleotides, wherein the vectors include expression vectors. In some embodiments, the present invention also provides host cells for preparing the antibodies or antigen-binding fragments thereof, wherein the host cells are eukaryotic cells selected from mammalian cells, yeast cells, or other cells suitable for preparing antibodies or antigen-binding fragments thereof, preferably mammalian cells, including Chinese hamster ovary (CHO) cells, mouse NSO myeloma cells, COS cells, mouse myeloma SP2 / 0 cells, baby hamster kidney (BHK) cells, and 293 cells.

[0079] In another aspect, the present invention also provides a method for preparing the anti-PCSK9 antibody or antigen-binding fragment thereof described in the present invention, which comprises inserting a nucleotide encoding the antibody or antigen-binding fragment thereof described in the present invention into an expression vector so that the transcription and translation control elements in the expression vector can regulate the transcription and translation functions of the antibody or antigen-binding fragment thereof nucleotide, then transfecting the expression vector encoding the antibody or antigen-binding fragment thereof into a host cell for culturing, and isolating and purifying the antibody or antigen-binding fragment thereof prepared by the host cell.

[0080] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0081] (1) Provided is a liquid formulation containing a high concentration of anti-PCSK9 antibody, which has the characteristics of low viscosity, not only meeting the requirements of clinical dosage, but also meeting the antibody viscosity limit required for subcutaneous delivery using a syringe at ambient temperature, and is particularly suitable for subcutaneous injection or intramuscular injection;

[0082] (2) Provided is a liquid preparation containing a high concentration of anti-PCSK9 antibody, which has high physical and chemical stability and is not prone to the formation of aggregates, particle precipitates, and charged heterogeneities during long-term storage (e.g., more than 1 year, especially 2 or 3 years). It can be seen that the liquid preparation provided by the present invention is not only suitable for long-term storage, but also can ensure the biological activity of the antibody, and the quality safety, efficacy, and high consistency of the liquid preparation prepared as a clinical drug;

[0083] (3) Provided is a liquid preparation containing a high concentration of anti-PCSK9 antibody, which has simple ingredients, is safe and non-toxic, produces few aggregates and particulate precipitates, has high biological activity, and is easy to produce and easy to control quality;

[0084] (4) Provided is a liquid preparation containing a high concentration of anti-PCSK9 antibodies, which not only has the characteristics of high antibody concentration and low viscosity, but also maintains a pH value between 5.5-6.5 (especially 6.0-6.5), suitable for subcutaneous injection. It not only meets the needs of patients with chronic diseases to take medicine at home, but also can reduce the pain of administration to a lower value during the administration process, thereby improving patients' medication compliance. Detailed Description of the Invention

[0086] The following description of the present application is intended only to illustrate various embodiments of the present application. Therefore, the specific modifications discussed herein should not be construed as limiting the scope of the application. A person skilled in the art can easily derive multiple equivalents, variations, and modifications without departing from the scope of the present application, and it should be understood that such equivalent embodiments are included within the scope of the present invention. All documents cited in this application, including publications, patents, and patent applications, are incorporated by reference in their entirety.

[0087] definition

[0088] The terms used in the present invention have the following definitions. If no definition is given herein, the terms used in the present invention have the meanings commonly understood by those of ordinary skill in the art.

[0089] As used herein, the term "about," when applied to a specific value or range of values ​​listed, means that the value may vary from the specific value listed by ±20% or ±10%, including ±5%, ±1% and ±0.1%, and these variations are suitable for performing the disclosed methods.

[0090] To interpret this specification, the following definitions will apply, and wherever appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0091] The term "Proprotein convertase subtilisin / kexin type 9 (PCSK9)", "PCSK9" or "NARC-1" refers to a naturally occurring human proprotein convertase that belongs to the proteinase K subfamily of the secretory subtilisin family. PCSK9 is synthesized as a lysozyme, undergoes autocatalytic intramolecular processing in the endoplasmic reticulum, and is believed to function as a proprotein convertase. When used in this application, the term refers to any naturally occurring PCSK9, preferably any naturally occurring PCSK9 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). Representative amino acid sequences of human PCSK9 and representative nucleic acid sequences encoding them are disclosed by GenBank Accession Nos. NP_777596.2 and FJ525880.1, respectively, or the amino acid sequence of human PCSK9 is set forth in SEQ ID NO:21 herein. The term "PCSK9" encompasses "full-length" unprocessed PCSK9 as well as any form of PCSK9 produced by intracellular processing or any fragment thereof. The term also includes naturally occurring variants of PCSK9, e.g., splice variants, derivative variants, substitution variants, deletion variants and / or insertion variants or allelic variants, such as mutants D374Y, S127R, and F216L.

[0092] The term "antibody" includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody, or bispecific (bivalent) antibody that binds to a specific antigen. A naturally occurring intact antibody contains two heavy chains and two light chains. Each heavy chain consists of a variable region (VH) and three constant regions (CH1, CH2, and CH3), with a hinge region between CH1 and CH2. Each light chain consists of a variable region (VL) and a constant region (CL). Mammalian heavy chains are classified as α, δ, ε, γ, and μ, while mammalian light chains are classified as λ or κ. Antibodies are Y-shaped, with the neck of the Y consisting of the CH2 and CH3 of two heavy chains, joined by disulfide bonds at the hinge region. Each arm of the Y consists of the variable region and CH1 of one heavy chain, which are connected to the variable region and CL of one light chain by a disulfide bond. The variable regions of the light and heavy chains determine how the antibody binds to the antigen. The variable region of each chain contains three hypervariable regions (HVRs), called complementarity-determining regions (CDRs). The light chain (L) CDR region comprises LCDR1, LCDR2, and LCDR3, and the heavy chain (H) CDR region comprises HCDR1, HCDR2, and HCDR3. The CDR boundaries of the antibodies or antigen-binding fragments thereof disclosed in the present invention can be defined or identified using the Kabat, Chothia, IMGT, AbM, or Contact numbering systems / methods. (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927(1997); Chothia, C. et al., J Mol Biol. Dec 5; 186(3):651-63(1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196, 901 (1987); Chothia, C. et al., Nature. Dec 21-28; 342(6252):877-83 (1989); Kabat EA et al., National Institutes of Health, Bethesda, Md. (1991); Marie-Paule Lefranc. et al., Nucleic Acids Research, 1998, Vol. 26, No. 1 297–303). The three CDRs are separated by flanking contiguous portions called framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold supporting the hypervariable loops.Antibodies can be divided into several classes based on the amino acid sequence of their heavy chain constant regions. For example, depending on whether they contain α, δ, ε, γ, and μ heavy chains, antibodies can be divided into five major classes or isoforms: IgA, IgD, IgE, IgG, and IgM. Several major antibody classes are further divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), or IgG4 (γ4 heavy chain).

[0093] wherein each heavy chain complementarity determining region and / or each light chain complementarity determining region is defined according to the Kabat definition, the Chothia definition, a combination of the Kabat definition and the Chothia definition, the IMGT definition, the AbM definition, or the CDR contact definition; preferably, each CDR is defined according to the Kabat CDR definition or the Chothia CDR definition or a combination of the two.

[0094] The term "antigen-binding fragment" refers to an antibody fragment formed by the portion of an antibody containing one or more CDRs, or any other antibody fragment that binds to an antigen but lacks the structure of a complete antibody. Antigen-binding fragments can bind to the same antigen as intact antibodies. In certain embodiments, an antigen-binding fragment may contain one or more CDRs from a specific human antibody grafted onto framework regions from one or more different human antibodies. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (dsdiabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), bivalent single-chain antibodies (bsFv), camelized single-domain antibodies, nanobodies, domain antibodies, and diabodies. For example, an antibody "Fab" fragment is composed of a light chain (including the light chain variable region and light chain constant region) linked to the variable region and CH1 of a heavy chain via a disulfide bond. A "Fab'" fragment is a Fab fragment that includes part of the hinge region. "F(ab')2" refers to a Fab dimer. An antibody "Fc" fragment is composed of the CH2 and CH3 of the heavy chain linked by a disulfide bond. The Fc fragment of an antibody is responsible for various effector functions, such as determining the serum half-life of the antibody in vivo and mediating immune responses, such as antibody-dependent cell-mediated cytotoxicity (ADCC), activation of complement-dependent cytotoxicity (CDC), or antibody-dependent cell-mediated phagocytosis (ADCP), but does not participate in antigen binding. An antibody "Fv" fragment is the smallest antibody fragment that contains a complete antigen-binding site. An Fv fragment consists of the variable region of one light chain and one heavy chain. A "single-chain Fv antibody (scFv)" refers to an engineered antibody in which the light and heavy chain variable regions are linked directly or via a peptide chain (Huston JS et al., Proc Natl Acad Sci USA, 85:5879 (1988)). A "(dsFv)2" contains three peptide chains: two VH groups are linked by a polypeptide linker and bound to two VL groups via disulfide bonds.A "bispecific ds-type bifunctional antibody" contains VL1-VH2 (connected by a polypeptide linker) and VH1-VL2 (also connected by a polypeptide linker), which are bound by a disulfide bond between VH1 and VL1. A "bispecific dsFv" or "dsFv-dsFv" contains three polypeptide chains: VH1-VH2 fragments, the heavy chains of which are connected by a polypeptide linker (e.g., a long flexible linker) and bound to the VL1 and VL2 fragments, respectively, by disulfide bonds. Each pair of disulfide-bonded heavy and light chains has different antigenic specificities. A "scFv dimer" is a bivalent bifunctional antibody or bivalent single-chain antibody (BsFv) containing two dimerized VH-VL fragments (connected by a polypeptide linker). The VH of one fragment cooperates with the VL of the other fragment to form two binding sites. These two binding sites can target the same antigen (or antigen-binding epitope) or different antigens (or antigen-binding epitopes). In other embodiments, a "scFv dimer" is a bispecific, bifunctional antibody comprising interconnected VL1-VH2 (connected by a polypeptide linker) and VH1-VL2 (connected by a polypeptide linker), wherein VH1 and VL1 collaborate, and VH2 and VL2 collaborate, and each collaborative pairing has a different antigenic specificity. A "single-chain antibody Fv-Fc (scFv-Fc)" refers to an engineered antibody composed of an scFv and an antibody Fc fragment. "Camelized single-domain antibody", "heavy-chain antibody" or "HCAb (Heavy-chain-only antibodies, HCAb)" refers to an antibody containing two VH domains but no light chain (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10; 231(1-2): 25-38 (1999); Muyldermans S., J Biotechnol. Jun; 74(4): 277-302 (2001); WO94 / 04678; WO94 / 25591; US ​​Patent No. 6,005,079). Heavy-chain antibodies were originally discovered in Camelidae (including camels, dromedaries and llamas).Although camelized antibodies lack light chains, they possess all the functions of antigen binding (Hamers-Casterman C. et al., Nature. Jun 3; 363(6428): 446-8 (1993); Nguyen VK. et al., "Heavy-chain antibodies in Camelidae: a case of evolutionary innovation," Immunogenetics. Apr; 54(1): 39-47 (2002); Nguyen VK. et al., Immunology. May; 109(1): 93-101 (2003)). The variable region (VHH domain) of a heavy chain antibody is the smallest known antigen-binding unit produced by adaptive immunity (Koch-Nolte F. et al., FASEB J. Nov; 21(13): 3490-8. Epub 2007 Jun 15 (2007)). "Nanobodies" are antibody fragments consisting of a VHH domain from a heavy chain antibody and two constant regions, CH2 and CH3. "Domain antibodies" are antibody fragments that contain only one heavy chain variable region or one light chain variable region. In some cases, two or more VH domains are covalently linked by a polypeptide linker to form a bivalent domain antibody. The two VH domains of a bivalent domain antibody can target the same or different antigens. "Diabodies" include small antibody fragments with two antigen-binding sites, which contain a VH domain and a VL domain connected on the same polypeptide chain (such as VH-VL or VL-VH) (Holliger P. et al., Proc Natl Acad Sci USA. Jul 15; 90(14): 6444-8 (1993); EP404097; WO93 / 11161). The linker between the two domains is very short, so that the two domains on the same chain cannot pair with each other, thereby forcing the two domains to pair with the complementary domains of the other chain to form two antibody binding sites. The two antibody binding sites can target the same or different antigens (or antigen binding epitopes).

[0095] The term "fully human antibody or antigen-binding fragment" means that the amino acid sequence of the antibody or antigen-binding fragment corresponds to the amino acid sequence of an antibody produced by a human or human immune cell, or derived from a non-human source such as a transgenic non-human animal using a human antibody library, or other sequences encoding human antibodies. In some embodiments, the fully human antibody does not contain amino acid residues (particularly antigen-binding residues) derived from non-human antibodies. In some embodiments, the fully human antibody is prepared using recombinant methods. For example, a transgenic animal such as a mouse can be prepared to carry a transgene or transchromosome of a human immunoglobulin gene and thus be able to produce fully human antibodies after immunization with a suitable antigen such as human PCSK9. Fully human antibodies can be isolated from the transgenic animal, or alternatively, can be prepared by hybridoma technology, where the spleen cells of the transgenic animal are fused with an immortal cell line to generate hybridoma cells that secrete the fully human antibodies. Exemplary transgenic animals include, but are not limited to, Omni rats, whose endogenous rat immunoglobulin genes are knocked out and inactivated, and are genetically engineered to contain functional recombinant human immunoglobulin loci; Omni mice, whose endogenous mouse immunoglobulin genes are knocked out and inactivated, and are genetically engineered to contain recombinant human immunoglobulin loci with J-locus deletion and C-kappa mutation; OmniFilc, which are transgenic rats, whose endogenous rat immunoglobulin genes are knocked out and inactivated, and are genetically engineered to contain recombinant human immunoglobulin loci with a single common, recombinant VkJk light chain and a functional heavy chain (Osborn M. et al, Journal of Immunology, 2013, 190: 1481-90; Ma B. et al, Journal of Immunological Methods 400-401 (2013) 78-86; Geurts A. et al, Journal of Immunological Methods 400-401 (2013) 78-86; Geurts A. et al, Journal of Immunological Methods 400-401 (2013) 78-86). al, Science, 2009, 325: 433; U.S. Patent US8,907,157; ​​European Patent EP2152880B1; European Patent EP2336329B1).Other suitable transgenic animals can also be used, for example, HuMab mice (Lonberg, N. et al. Nature 368(6474):856-859 (1994)), Xeno mice (Mendez et al. Nat Genet., 1997, 15:146-156), TransChromo mice (Ishida et al. Cloning Stem Cells, 2002, 4:91-102), VelocImmune mice (Murphy et al. Proc Natl Acad Sci USA, 2014, 111:5153-5158), Kymouse transgenic mice (Lee et al. Nat Biotechnol, 2014, 32:356-363), or transgenic rabbits (Flisikowska et al. PLoS One, 2011, 6:e21045).

[0096] The term "humanized antibody or antigen-binding fragment" refers to an antibody or antigen-binding fragment comprising a CDR derived from a non-human animal, a FR region derived from a human, and a constant region derived from a human. Because humanized antibodies or antigen-binding fragments have reduced immunogenicity, they can be used as therapeutic agents for humans. In some embodiments, the non-human animal is a mammal such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster. In some embodiments, the humanized antibody or antigen-binding fragment, except that the CDR sequence is non-human, the rest of the antibody is substantially entirely composed of human sequences. In some embodiments, the FR region derived from a human may include the same amino acid sequence as the human antibody from which it is derived, or it may include some amino acid changes, for example, no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid changes. In some embodiments, the amino acid changes may be present only in the heavy chain FR region, only in the light chain FR region, or in both chains simultaneously.

[0097] The term "chimeric" refers to an antibody or antigen-binding fragment having a portion of a heavy chain and / or light chain derived from one species, and the remainder of the heavy chain and / or light chain derived from a different species. In an illustrative example, a chimeric antibody can include a constant region derived from a human and a variable region derived from a non-human animal, such as a mouse.

[0098] The term "specific binding" refers to the reaction between an antibody and an antigen. In some embodiments, the antibodies or antigen-binding fragments of the present invention specifically bind to rat, human, and / or monkey PCSK9, and their binding affinity (K D )≤10 -6 M(eg:≤5×10 -7M, preferably ≤ 1.0×10 -8 M, more preferably 1.0×10 -9 M). K in this application D It is the ratio of the dissociation rate to the association rate (k off / k on ), can be measured by surface plasmon resonance, for example, using a Biacore instrument.

[0099] The term "antibody isotype" refers to the class of antibody encoded by the heavy chain constant region gene. For example, an "IgG isotype antibody" refers to the IgG form to which the heavy chain constant region of an antibody belongs. The heavy chain constant region of all antibodies of the same isotype is the same, while the heavy chain constant region of antibodies of different isotypes may differ. For example, an antibody of the IgG1 isotype refers to an antibody whose heavy chain constant region Ig domain is an IgG1 Ig domain.

[0100] The term "percent sequence identity" refers to the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to those in a reference sequence, after aligning the amino acid sequences (or nucleic acid sequences) and introducing gaps, if necessary, to maximize the number of identical amino acids (or nucleic acids). Conservative substitutions of amino acid residues may or may not be considered identical residues. Sequences can be aligned to determine the percentage sequence identity of amino acid (or nucleic acid) sequences using tools disclosed in the art, such as BLASTN, BLASTp (National Center for Biotechnology Information (NCBI), see also Altschul SF et al., J. Mol. Biol., 215: 403-410 (1990); Stephen F et al., Nucleic Acids Res., 25: 3389-3402 (1997)), ClustalW2 (European Bioinformatics Institute, see also Higgins DG et al., Methods in Enzymology, 266: 383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, England), 23(21): 2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. Those skilled in the art can use the default parameters of the tool or appropriately adjust the parameters according to the needs of the comparison, for example, by selecting a suitable algorithm.

[0101] The term "LDL receptor (LDLR)" is a cell surface chimeric protein with 839 amino acids (after removing the 21 amino acid signal peptide) that mediates the endocytosis of LDL-C and removes LDL-C from the blood. The representative amino acid sequence of human LDL-R and the mRNA nucleic acid sequence encoding it are disclosed by GenBank accession numbers P01130.1 and NM_000527.4, respectively. When PCSK9 binds to the LDL receptor, the antibody is destroyed and cannot remove LDL-C from the blood. In contrast, when PCSK9 is blocked, there will be more LDL receptors on the surface of the liver and more LDL cholesterol will be removed from the blood. "Anti-PCSK9 antibody" as used in this application refers to an antibody that can specifically bind to PCSK9 (e.g., human or monkey PCSK9) with an affinity sufficient to provide diagnostic and / or therapeutic uses.

[0102] The term "vector" refers to a vehicle into which a polynucleotide encoding a protein can be operatively inserted and the protein can be expressed. A vector can be used to transform, transduce, or transfect a host cell so that the genetic material elements it carries are expressed in the host cell. Such vectors are also referred to as "expression vectors" in this application. Vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. Animal virus species used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector can also contain a replication initiation site. A vector can also include components that assist its entry into cells, including but not limited to viral particles, liposomes, or protein coats.

[0103] The term "host cell" refers to a cell into which an exogenous polynucleotide and / or vector has been introduced. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. This application includes mutant progeny screened or selected for the same function or biological activity as the initially transformed cell. Preferably, mammalian host cells, for example, SV40-transformed monkey kidney cell CV1 line (COS-7, ATCC CRL1651), human embryonic kidney cell line (293 or suspension cultured 293 cell subclone, Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical cancer cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), Buffalo rat liver cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human hepatocytes (HepG2, HB 8065), mouse mammary tumor (MMT060562, ATCC CCL51), TRI cells (Mathe r et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)), MRC5 cells, FS4 cells, or human hepatoma cell line (Hep G2).

[0104] The term "LDL-C" refers to low-density lipoprotein cholesterol. "HDL-C" refers to high-density lipoprotein cholesterol. LDL and HDL belong to five major lipoprotein groups: chylomicrons, very low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) (in order from largest particles to densest / smallest particles). LDL (also known as the "bad" cholesterol containing particles) can transport lipid / sterol molecules such as cholesterol (i.e., LDL-C) to the artery wall, attracting macrophages and thus inducing atherosclerosis. In contrast, HDL (also known as the "good" cholesterol containing particles) can remove lipid molecules such as cholesterol (i.e., HDL-C) from macrophages on the artery wall. Therefore, high levels of LDL-C are a major risk factor for cardiovascular diseases (CVD), such as peripheral artery disease, coronary artery disease (CAD, such as angina pectoris, myocardial infarction (commonly known as heart disease), hyperlipidemia, hypercholesterolemia, hypertriglyceridemia), atherosclerosis, stroke, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, arrhythmias, congenital heart disease, valvular heart disease, myocarditis, aortic aneurysm, peripheral artery disease, obesity, hepatobiliary disease, nephrotic syndrome, hypothyroidism, and venous thrombosis.

[0105] "Treatment" of a disease or symptom means alleviating the disease or symptom, reducing the rate of onset or development of a disease or symptom, reducing the risk of developing a disease or symptom, or delaying the development of symptoms associated with a disease or symptom, reducing or stopping symptoms associated with a disease or symptom, producing complete or partial reversal of a disease or symptom, curing a disease or symptom, or a combination of the above.

[0106] "Prevention" includes the inhibition of the development or progression of a disease or condition or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of the disease are candidates for a preventative regimen. Generally, the term "prevention" refers to the administration of a drug before signs or symptoms of a disease occur, particularly in a subject at risk.

[0107] "PCSK9-associated disease or symptom" refers to a disease or symptom caused or characterized by changes in PCSK9, such as changes in expression levels, activity, and / or the presence of variants or mutations of PCSK9. Examples of PCSK9-associated diseases or symptoms include, but are not limited to, hyperlipoproteinemia, hyperlipidemia; dyslipidemia (e.g., elevated total cholesterol, elevated LDL, elevated triglycerides, elevated VLDL, and / or decreased HDL); hypercholesterolemia, heart disease, stroke, coronary heart disease, atherosclerosis, peripheral vascular disease, claudication, type II diabetes, hypertension, cardiovascular disease or symptoms, inflammation, or autoimmune disease. Methods for identifying / diagnosing the above-mentioned diseases or symptoms are known in the art.

[0108] The term "subject" includes any human or non-human animal. "Non-human animals" include all vertebrates, such as mammals (including but not limited to domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., monkeys), rabbits, and rodents (e.g., mice and rats)) and non-mammals (e.g., poultry, amphibians, and reptiles). In some embodiments, the subject is a human.

[0109] The term "therapeutically effective amount" or "effective dose" refers to a dose or concentration that is effective to prevent or ameliorate symptoms associated with a disease or condition and / or reduce the severity of the disease or condition, at a desired dosage and for a desired period of time. The therapeutically effective amount of an agent, antibody, or antigen-binding fragment thereof, or composition of the invention can vary depending on a variety of factors, such as the disease state, the age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also considered to be such that any toxic or deleterious effects of the agent, antibody, or antigen-binding fragment thereof, or composition are outweighed by the therapeutically beneficial effects.

[0110] The term "preparation" refers to a composition comprising at least one active ingredient and at least one inactive ingredient suitable for administration to animals, preferably mammals (including humans). "Liquid preparation" refers to a preparation in liquid form. The liquid preparation of the present invention comprises (1) an anti-PCSK9 antibody or an antigen-binding fragment thereof; (2) a buffer; (3) an osmotic pressure regulator; and / or (4) a surfactant. The composition of the preparation of the present invention may be as shown in the liquid preparation embodiment described above. The liquid preparation of the present invention is preferably an injection, more preferably a subcutaneous injection or an intramuscular injection, and most preferably a subcutaneous injection.

[0111] The term "buffer" refers to a pH buffer. Preferably, the buffer is capable of maintaining the pH of the liquid formulation of the present invention at about 4.5-6.5, preferably about 5.5-6.5. The concentration of the buffer in the liquid formulation is about 1-50 mmol / L, preferably 5-50 mmol / L, and more preferably 10-30 mmol / L. The buffer is selected from histidine, acetate, succinate, or glutamate, with histidine being the preferred buffer.

[0112] "Histidine buffer" refers to a buffer containing histidine. Histidine buffers include histidine hydrochloride, histidine acetate, histidine phosphate, and histidine sulfate. In one embodiment, the histidine buffer is composed of L-histidine and L-histidine hydrochloride, with the concentration ratio of L-histidine to L-histidine hydrochloride being 0.2:1-3:1.

[0113] The term "excipient" refers to a non-therapeutic agent that can be added to a formulation to provide desired properties (e.g., consistency, improved stability) and / or to adjust osmotic pressure. Such non-therapeutic agents are added to the formulation to stabilize the physicochemical and biological properties of the active ingredient. Examples of commonly used excipients include, but are not limited to, carbohydrates, polyols, amino acids, surfactants, and polymers.

[0114] The term "osmotic pressure regulator" refers to a molecule that increases the osmotic pressure of a solution. The term "osmotic pressure regulator" herein refers to a molecule capable of adjusting, altering, or optimizing the osmotic pressure of the anti-PCSK9 antibody liquid formulation of the present invention. Preferably, the osmotic pressure of the liquid formulation of the present invention is adjusted to not only maintain the isotonicity of the liquid formulation but also maximize the stability of the anti-PCSK9 antibody or antigen-binding fragment thereof, while also minimizing patient discomfort during administration. Examples of osmotic pressure regulators suitable for altering osmotic pressure include, but are not limited to, amino acids (proline, arginine, cysteine, histidine, etc.), salts (sodium chloride, potassium chloride, calcium chloride, etc.), and / or sugars (sucrose, trehalose, glucose, mannitol, sorbitol, and their analogs). Preferred osmotic pressure regulators are proline or sucrose, with proline being more preferred.

[0115] The term "surfactant" refers to a substance that can significantly change the interfacial state of its solution system after adding a small amount. For example, it can protect proteins (such as antibodies) from the stress induced by the air / solution interface, the stress induced by the solution / surface, thereby reducing the aggregation of proteins or the formation of particulate matter in the preparation. Exemplary surfactants include but are not limited to nonionic surfactants, such as polyoxyethylene sorbitan fatty acid esters (such as polysorbate 80, polysorbate 60, polysorbate 40 or polysorbate 20), polyethylene-polypropylene copolymers, polyethylene-polypropylene glycol, polyoxyethylene-stearate, polyoxyethylene alkyl ethers, such as polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ether (Triton-X), polyoxyethylene-polyoxypropylene copolymers (poloxamers, Pluronic), sodium lauryl sulfate (SDS). The preferred surfactant of the present invention is a nonionic surfactant, such as polysorbate 80 or polysorbate 20. The concentration of the surfactant in the liquid preparation is 0.001-1% (w / w), preferably 0.01-0.05% (w / w), more preferably 0.01% (w / w), 0.02% (w / w) and 0.04% (w / w).

[0116] The term "viscosity" may be "kinematic viscosity" or "absolute viscosity". "Kinematic viscosity" is a measure of a fluid's resistance to flow under the influence of gravity. When two equal volumes of fluid are placed in two identical capillary viscometers and allowed to flow by gravity, the viscous fluid takes longer to flow through the capillary than the less viscous fluid. "Absolute viscosity", sometimes called dynamic viscosity or simple viscosity, is the product of kinematic viscosity and the density of the fluid (absolute viscosity = kinematic viscosity x density). The dimension of kinematic viscosity is L 2 / T (where L is length and T is time). Usually, kinematic viscosity is expressed in centistokes (cSt). The SI unit of kinematic viscosity is mm 2 Absolute viscosity is expressed in centipoise (cP). The SI unit of absolute viscosity is milliPascal-second (mPa-s), where 1 cP = 1 mPa-s.

[0117] The term "low-level viscosity" refers to an absolute viscosity of less than about 15 cp. "Medium-level viscosity" refers to an absolute viscosity between about 15 cp and about 35 cp. When measured using standard viscosity measurement techniques, the absolute viscosity of the antibody liquid formulation of the present invention is about 6.0 cp, about 8.0 cp, about 9.0 cp, about 10.0 cp, about 11.0 cp, or about 12.0 cp, indicating that the liquid formulation of the present invention has a "low-level viscosity". In some embodiments, the inventors of the present application have surprisingly found that proline can not only serve as an osmotic pressure regulator, but also can reduce the viscosity of the antibody liquid formulation of the present invention when formulated with an anti-PCSK9 antibody and a buffer.

[0118] The term "isotonic" refers to a liquid formulation having an osmotic pressure substantially the same as that of human blood. Isotonic formulations typically have an osmotic pressure of approximately 250 to 350 mOsm. Isotonicity can be measured using a vapor pressure or freezing point depression osmometer.

[0119] The term "solvent" refers to a substance used to mix, disperse, or dissolve a test substance or a control substance without affecting the test results. Solvents that can be used in the present invention include, but are not limited to, water for injection, salts (e.g., normal saline), sugars (e.g., glucose injection), organic solvents for injection (e.g., including but not limited to oil for injection, ethanol, propylene glycol, etc.), or combinations thereof.

[0120] A "stable" antibody preparation refers to an antibody preparation that substantially maintains its physicochemical stability and / or biological activity during the manufacturing process and / or storage. Even if the antibody contained therein fails to maintain 100% of its physicochemical properties or biological function after a certain period of storage, the antibody preparation can be considered stable. For example, after a certain period of storage, if the preparation can maintain more than 90% of the antibody structure or function, it can also be considered a "stable" preparation. Stability standards include, for example, liquid preparations that are colorless or clear to slightly milky white when observed with the naked eye; changes in the antibody concentration, pH value and osmotic pressure of the preparation do not exceed ±10%; the biological activity of the antibody is 60-140%, preferably 80-120%, of the reference antibody; the degradation of the antibody monomer in the preparation does not exceed 10%, preferably 5%; the aggregates formed in the preparation do not exceed 10%, preferably 5%; or the hydrolyzates formed in the preparation do not exceed 10%, preferably 5%.

[0121] If the color and / or clarity of the preparation is observed by naked eye, or there is no significant increase in the aggregation, precipitation and / or denaturation of the antibody in the preparation as detected by differential scanning calorimetry (DSC), size exclusion chromatography (SEC-HPLC) and dynamic light scattering (DLS), it is indicated that the antibody in the preparation "maintains its physical stability". If the antibody in the preparation does not undergo significant chemical changes and its chemical structure remains intact, it is indicated that the antibody "maintains its chemical stability". Most of the destruction of chemical stability can be attributed to the formation of covalent modifications (e.g., covalent aggregates, degradation products or charge isomers) of the protein and non-covalent modifications (e.g., non-covalent aggregates) of the protein. Changes in the chemical structure of the antibody protein (e.g., variants of different molecular weight or charge) can be determined using methods known to those skilled in the art. The methods include, but are not limited to, using SEC-HPLC and sodium dodecyl sulfate capillary gel electrophoresis (CE-SDS) to detect antibody hydrolysates, using non-reducing Caliper to detect degradation fragments of the antibody and other protein molecules with a molecular weight smaller than the antibody, or using cation exchange chromatography (CE-HPLC) to detect antibody charge isomers. If, during the storage period, the biological activity of the antibody in the formulation remains within the range of biological activity exhibited at the time of preparation, the antibody "retains its biological activity." Methods for detecting the biological activity of the antibody include, but are not limited to, antigen binding ELISA assays or antibody cell activity assays.

[0122] "Pharmaceutically acceptable" means that the referenced carriers, vehicles, diluents, excipients and / or salts are generally chemically and / or physically compatible with the other ingredients of the formulation and physiologically compatible with the subject.

[0123] Anti-PCSK9 antibodies

[0124] Exemplary anti-PCSK9 antibodies contained in the liquid formulation of the present invention are described in detail in the published patent application CN201710816808.0, the disclosure of which is incorporated herein by reference in its entirety. The anti-PCSK9 antibodies of the present invention are preferably fully human antibodies, and preferably, the antibodies are prepared by recombinant methods. In one embodiment, human PCSK9 protein is used to immunize transgenic rats containing human immunoglobulin variable region genes. (OMT) to obtain a fully human antibody. The non-limiting, exemplary antibody used in the Examples of the present invention is referred to as "Antibody A," which is a fully human antibody that specifically binds to human PCSK9. The amino acid and nucleotide sequences of its CDR regions, heavy chain variable regions, and light chain variable regions are shown in Table 1. The CDR regions in Table 1-1 are defined using the Kabat method, while Table 1-2 shows the CDR region amino acid sequences defined by different definition methods:

[0125] Table 1-1 Amino acid sequences and sequence numbers of the CDR regions, heavy chain variable regions, and light chain variable regions of anti-PCSK9 antibody (Antibody A)

[0126]

[0127]

[0128] Table 1-2 CDR region amino acid sequences defined by different definition methods

[0129]

[0130]

[0131] In one embodiment, the anti-PCSK9 antibody or antigen-binding fragment thereof of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 2, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 3, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 6. In one embodiment, the anti-PCSK9 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the amino acid sequence of VH is shown in SEQ ID NO: 7, and the amino acid sequence of VL is shown in SEQ ID NO: 8. In some embodiments, the anti-PCSK9 antibody or antigen-binding fragment thereof comprises one or more CDR sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the CDR sequences listed in Table 1, while retaining a binding affinity to human PCSK9 that is similar to or even greater than that of the parent antibody (e.g., Antibody A), wherein the parent antibody has substantially the same sequence, but its corresponding CDR sequence has 100% sequence identity with the sequences listed in Table 1. In some embodiments, the anti-PCSK9 antibody or antigen-binding fragment thereof has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the VH or VL sequence listed in Table 1, while retaining a binding affinity to human PCSK9 that is similar to or even greater than that of the parent antibody.

[0132] Preparation and purification of anti-PCSK9 antibodies

[0133] The present invention also provides polynucleotides encoding anti-PCSK9 antibodies or antigen-binding fragments thereof. In some embodiments, the polynucleotides comprise one or more nucleotide sequences as shown in Table 1, encoding CDR region sequences, heavy chain variable region sequences, and / or light chain variable region sequences as shown in Table 1.

[0134] In some embodiments, the polynucleotide sequence encoding the heavy chain variable region is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 19 shown in Table 1. In some embodiments, the polynucleotide sequence encoding the light chain variable region is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 19 shown in Table 1. In some embodiments, the percentage of identity is due to the degeneracy of the genetic code, while the encoded protein sequence remains unchanged.

[0135] In some embodiments, a vector comprising a polynucleotide encoding the anti-PCSK9 antibody or antigen-binding fragment thereof (e.g., the sequence shown in Table 1) can be introduced into a host cell for cloning (amplification of DNA) or gene expression using recombinant techniques known in the art. In some embodiments, the antibody or antigen-binding fragment thereof can be produced by homologous recombination methods known in the art. The DNA encoding the antibody or antigen-binding fragment thereof can be isolated and sequenced by conventional methods, for example, using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody or antigen-binding fragment thereof. As described above, a variety of vectors are available. Vector elements generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer sequence, a promoter (e.g., SV40, CMV, or EF-1α), a transcription sequence, and a termination sequence. In some embodiments, the vector system includes mammalian, bacterial, yeast systems, etc., and suitable vectors may include plasmids or viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses). The plasmids include, but are not limited to, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2, etc., which can be obtained from the laboratory or commercially available.

[0136] A vector comprising a polynucleotide encoding the anti-PCSK9 antibody or antigen-binding fragment thereof can be introduced into a host cell for cloning or gene expression. In the present invention, the host cell suitable for cloning or expressing the DNA in the vector can be a prokaryotic cell, yeast, or the aforementioned eukaryotic cells, preferably a eukaryotic cell, and more preferably a mammalian host cell. In some preferred embodiments, the host cell is a CHO cell or a 293 cell.

[0137] Host cells are transformed with the above-mentioned vectors for expression or cloning of anti-PCSK9 antibodies or antigen-binding fragments thereof and cultured in a conventional nutrient medium that has been modified to be suitable for inducing promoters, selecting transformed cells, or amplifying genes encoding target sequences.

[0138] The host cells used to produce the antibodies or antigen-binding fragments thereof of the present invention can be cultured in a variety of culture media. Commercially available culture media such as Ham's F10 (Sigma), minimum essential medium such as MEM (Sigma), RPMI-1640 (Sigma), or Dulbecco's Modified Eagle's Medium (DMEM, Sigma) can be used to culture the host cells. In addition, any culture medium described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469, WO 90 / 03430, WO 87 / 00195, or U.S. Patent Application Re. 30,985 can be used as culture media for the host cells. These media may be supplemented with necessary hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium chloride, magnesium chloride, and phosphate), buffers (such as HEPES), nucleotides (such as adenylate and thymidine), antibiotics (such as gentamicin), trace elements (defined as inorganic compounds whose final concentrations are generally in the micromolar range), and glucose or an equivalent energy source. The media may also contain any other necessary additives at appropriate concentrations known in the art. The conditions of the media, such as temperature, pH, and the like, are those previously used for the host cells selected for expression and are well known to those of ordinary skill in the art.

[0139] When using recombinant techniques, the antibody or its antigen-binding fragment can be produced intracellularly, in the periplasmic space, or directly secreted into the culture medium. If the antibody is produced intracellularly, the particulate debris of the host cells or lysed fragments is first removed, for example, by centrifugation or ultrasound. Carter et al., Bio / Technology 10:163-167 (1992) describes a method for isolating antibodies secreted into the periplasmic space of Escherichia coli. Briefly, a cell paste is dissolved in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for approximately 30 minutes or more. Cell debris is removed by centrifugation. If the antibody or its antigen-binding fragment is secreted into the cell culture medium, the supernatant of the expression system is typically first concentrated using a commercially available protein filter. Protease inhibitors such as PMSF may be added in any of the aforementioned steps to inhibit protein degradation, as well as antibiotics to prevent the growth of accidental contaminants.

[0140] The antibodies or antigen-binding fragments thereof produced from the cells can be purified using purification methods such as hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography columns, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique. The type of antibody and the presence of any immunoglobulin Fc domain in the antibody determine whether Protein A is suitable as its affinity ligand. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is suitable for all murine isoforms and human γ3 (Guss et al., EMBO J. 5:1567-1575 (1986)). Agarose is the most commonly used matrix for attachment of affinity ligands, but other matrices can also be used. Mechanically stable matrices such as controlled pore glass or poly(styrene)benzene can achieve faster flow rates and shorter processing times than using agarose. If the antibody contains a CH3 domain, it can be purified using Bakerbond ABX.™ resin (JT Baker, Phillipsburg, NJ). Other protein purification techniques can also be determined based on the desired antibody, such as fractionation on an ion exchange column, ethanol precipitation, reversed-phase HPLC, silica gel chromatography, heparin-sepharose chromatography based on anion or cation exchange resins (e.g., polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

[0141] After any preliminary purification steps, the mixture containing the antibody of interest and impurities can be treated by low pH hydrophobic interaction chromatography using a wash buffer having a pH of about 2.5-4.5, preferably at a low salt concentration (e.g., from about 0 to 0.25 M salt concentration). Analytical methods for evaluating the stability of liquid formulations containing anti-PCSK9 antibodies.

[0142] The antibody liquid formulation of the present invention contains a high concentration of anti-PCSK9 antibody, a pharmaceutically acceptable buffer, and an excipient. The antibody formulation should be formulated to maintain the physicochemical properties and biological activity of the antibody. Methods for evaluating the physicochemical stability of the antibody formulation include, but are not limited to, DSC, DLS, UV spectrophotometry, SEC-HPLC, CE-SDS, CEX-HPLC, and / or non-reducing Caliper. Among them, DSC (Pharm. Res., 15:200, 1998; Pharm. Res., 9:109, 1982) detects the denaturation temperature and glass transition temperature of the protein to analyze the stability of the protein in the natural (folded) state; DLS (American Lab., Nov. 1991) detects the average diffusion coefficient of the solution and analyzes the content of soluble and insoluble aggregates; UV spectrophotometry detects the absorbance of the preparation at 278 nm to analyze the concentration of the antibody protein; SEC-HPLC can separate higher molecular weight aggregates or lower molecular weight degradation products (such as fragments) from natural antibodies and analyze the percentage of natural antibodies (or antibody monomers), soluble hydrolysates (or degradation products with low molecular weight, such as fragments ) and the percentage of aggregates (i.e., determined based on the percentage of the peak area of ​​the native antibody, hydrolysate or aggregate compared to the total peak area of ​​all antibodies); CE-SDS is used to detect hydrolysates in the preparation, wherein reducing CE-SDS (rCE-SDS) can detect antibody heavy chains, non-glycosylated heavy chains, light chains and degradation fragments; non-reducing CE-SDS (nrCE-SDS) can detect antibody monomers and degradation fragments; CEX-HPLC can be used to separate the charge isomers of native antibodies in the preparation from native antibodies, and the peak eluted from the CEX-HPLC column earlier than the main peak (i.e., the main charged form) is an acidic peak, and the peak eluted from the CEX-HPLC column later than the main peak is a basic peak. The charge isomers of natural antibodies are determined based on the changes in the relative percentages of the ratios of the areas of the main peak, acidic peak, and basic peak to the total area of ​​all antibody peaks. The stability of the antibody is inversely proportional to the percentage of the acidic form of the antibody. The non-reducing Caliper assay detects degradation fragments of the antibody in the preparation and other protein molecules with a molecular weight smaller than the antibody. The potency or biological activity of the anti-PCSK9 antibody can be evaluated by its ability to bind to the antigen PCSK9. The specific binding ability of the antibody to the antigen can be quantitatively detected using methods known to those skilled in the art, such as immunoassays such as ELISA (enzyme-linked immunosorbent assay). DETAILED DESCRIPTION

[0143] The present invention will be further described in detail below with reference to examples, but the embodiments of the present invention are not limited thereto.

[0144] The meaning of the Chinese and English abbreviations:

[0145] DSC (Differential Scanning Calorimetry): Differential Scanning Calorimetry

[0146] DLS (Dynamic light scattering): Dynamic light scattering

[0147] HPLC (High Performance Liquid Chromatography)

[0148] CEX (Cation-exchange): cation exchange

[0149] SEC (Size-Exclusion Chromatography): Size Exclusion Chromatography

[0150] CEX-HPLC: Cation Exchange Chromatography

[0151] SEC-HPLC: Size Exclusion Chromatography

[0152] CE (Capillary Electrophoresis): Capillary Electrophoresis

[0153] SDS (Sodium Dodecyl Sulfate): Sodium Dodecyl Sulfate

[0154] CE-SDS: sodium dodecyl sulfate capillary gel electrophoresis

[0155] The anti-PCSK9 antibody used in the examples of the present invention was screened using the method for antibody 18.156.8 in Examples 1-3 of the patent publication CN201710816808.0, and a CHO cell line capable of stably expressing the anti-PCSK9 antibody of the present invention was constructed. After cell culture, the supernatant was obtained and purified by affinity chromatography, ion exchange, and ultrafiltration to obtain a stock solution of the anti-PCSK9 antibody of the present invention (also referred to as Antibody A in the present invention).

[0156] Example 1 Buffer system and pH value screening experiment

[0157] The present invention prepares anti-PCSK9 antibody preparations in different buffer systems according to the formulation in Table 2. By conducting a high-temperature accelerated stability test at 40±2°C, the stability of a 70 mg / mL anti-PCSK9 antibody (e.g., Antibody A) in three buffer systems with different ratios of acetic acid / sodium acetate, histidine / histidine hydrochloride, and succinic acid / sodium succinate and at different pH values ​​was investigated. The anti-PCSK9 antibody samples in different buffer systems were tested for appearance, pH, DSC, protein content, SEC-HPLC, CEX-HPLC, non-reducing Caliper, etc. to screen for the optimal buffer system suitable for the anti-PCSK9 antibody of the present invention.

[0158] The method for preparing anti-PCSK9 antibody preparations under different buffer systems is as follows: add the anti-PCSK9 antibody (antibody A) stock solution into an ultrafiltration centrifuge tube (molecular weight cut-off 30K D ), centrifuged and concentrated, then added with buffer solutions of varying pH values ​​as shown in Table 2, diluted, and then concentrated again three or more times until the anti-PCSK9 antibody was completely replaced. The replaced antibody concentration was adjusted to 70 mg / ml, sterilized and filtered, and the vials were individually filled and capped to create samples F1-F7.

[0159] Table 2. Anti-PCSK9 antibody formulations in different buffer systems

[0160]

[0161] Each sample was placed at 40±2°C and taken out for analysis and testing at week 0, week 2, and week 4, respectively. The specific test items are shown in Table 3. The protein charge properties were examined to see whether they were altered due to aggregates, degradation, and modification. SEC-HPLC was used to detect the percentages of native (protein monomers) and aggregated anti-PCSK9 antibodies; CEX-HPLC was used to detect the percentages of acidic and basic antibodies (main protein peaks); and non-reducing Caliper was used to detect the percentages of anti-PCSK9 antibodies, their degradation fragments, and other protein molecules with a molecular weight smaller than the antibodies.

[0162] SEC-HPLC method: A gel chromatography column (5 μm, 7.8×300 mm) was used, 20 μl of the diluted sample was injected, and isocratic elution was performed.

[0163] CEX-HPLC method: A weak cation chromatographic column (10 μm, 4×250 mm) was used. 100 μl of the diluted sample was injected, and 4 mM 2-Methylpiperazine, 4 mM Imidazole, and 4 mM Tris were used as the mobile phase for pH gradient elution.

[0164] Non-reducing Caliper: Samples were analyzed using a PerkinElmer LabChip GXII Touch HT instrument and HTProtein Express LabChip. Samples were diluted and mixed with denaturing buffer (containing 100 mM NEM and 10% SDS) and incubated for 10 minutes. The samples were then transferred to a 96-well plate and analyzed.

[0165] Table 3 Anti-PCSK9 antibody sample detection items under different buffer systems

[0166]

[0167] (X indicates that the test item is appearance, pH value, protein content, SEC-HPLC, CEX-HPLC, non-reducing Caliper; Y indicates that the test item is DSC).

[0168] Table 4 Stability test results of anti-PCSK9 antibody samples in different buffer systems at 40±2°C

[0169]

[0170]

[0171] The test results are shown in Table 4. When the buffer system consisted of histidine / histidine hydrochloride at pH 5.5-6.5 (samples F4-F6), after 4 weeks at 40±2°C / 75%±5% RH, SEC-HPLC analysis revealed that the main peak content of all samples was above 94%, with a decrease of approximately 1.5% in the main peak content. Non-reducing Caliper analysis revealed that the anti-PCSK9 antibody purity decreased within 1.5%. Compared to other formulations, when the buffer system consisted of histidine / histidine hydrochloride buffer at pH 6.0 (sample F5), after 4 weeks at 40±2°C / 75%±5% RH, the SEC-HPLC main peak (or protein monomer) content was the highest (approximately 94.8%), with the lowest decrease in main peak content (approximately 1.5%). Non-reducing Caliper analysis also revealed the lowest decrease in purity (approximately 0.9%). Furthermore, DSC and CEX-HPLC analysis revealed that F5 also exhibited excellent stability. When succinic acid / sodium succinate was used as the buffer system, the sample had slight flocculent precipitation, and when stored at 40±2°C, the SEC-HPLC main peak content and CEX-HPLC main peak content decreased the most (approximately 4.5% and 5.0%, respectively). When acetic acid / sodium acetate was used as the buffer system, the sample was inferior to the buffer system of histidine / histidine hydrochloride in terms of non-reducing caliper purity, DSC stability, and SEC-HPLC protein monomer content. Based on these results, a histidine / histidine hydrochloride buffer with a pH of 5.5-6.5 was selected as the buffer system for the anti-PCSK9 antibody liquid formulation of the present invention.

[0172] Example 2 Excipient Screening Experiment

[0173] Based on the experimental results of buffer system screening in Example 1, the present invention screened nine different excipient formulations and compared their effects on the stability, viscosity, and osmotic pressure of high-concentration anti-PCSK9 antibodies through accelerated tests at room temperature (25±2°C) and high temperature (40±2°C). The specific excipient formulations and information items for their corresponding test items are shown in Table 5. The different excipient formulations were prepared by adding the different excipients or combinations thereof shown in Table 5 to a solution containing 140 mg / mL anti-PCSK9 antibody (Antibody A) and 20 mM histidine / histidine hydrochloride buffer (pH 6.0) or acetic acid / sodium acetate buffer (pH 5.0). The surfactants described in Table 5 were then added to the solution. The prepared formulations were dispensed into vials, stoppered, and capped. The vials were then stored at 25°C and 40°C, respectively. The vials were removed and analyzed for antibody stability, formulation viscosity, osmotic pressure, etc., after two and four weeks.

[0174] ELISA was used to determine biological activity: anti-PCSK9 antibody was diluted to 1.5 μg / ml with coating solution, and 100 μl / well was added to a 96-well ELISA plate, and coated overnight at 2-8°C. After washing and blocking, the standard and test samples were diluted into the same series of concentrations, added to the ELISA plate at 100 μl / well, sealed with a sealing membrane, and incubated at 25°C, 220 rpm for 1 hour. After washing, horseradish peroxidase-labeled anti-human immunoglobulin was added to each reaction well of the ELISA plate at 100 μl / well, sealed with a sealing membrane, and incubated at 25°C, 220 rpm for 1 hour. After washing, TMB substrate solution was added for color development, and after color development, 2M sulfuric acid was used to terminate the reaction and measure A. 450 . Perform four-parameter curve fitting based on the EC values ​​of the standard and test samples. 50 The biological activity was calculated and the test results are shown in Table 5.

[0175] Table 5 Screening formulas for different excipients and their test items (X indicates that the test item is appearance, pH value, protein content, SEC-HPLC, CEX-HPLC, non-reducing Caliper, DLS; Y indicates that the test item is DSC, osmotic pressure, viscosity; Z indicates that the test item is activity; "[]" indicates optional test items)

[0176]

[0177] Table 6 Stability test results of anti-PCSK9 antibody samples with different excipients at 25±2℃ and 40±2℃

[0178]

[0179]

[0180]

[0181]

[0182] As shown in Table 6, when the excipient is proline, not only can the osmotic pressure of the antibody liquid preparation be maintained in the range of 300-340 mOsm / Kg, but the viscosity of the preparation can be significantly reduced (<15 cp), the average particle size of the anti-PCSK9 antibody is <15 d.nm, and the antibody has strong thermal stability. That is, after the liquid preparation is placed at a high temperature of 40°C for 4 weeks, the antibody monomer (or main peak) content detected by SEC-HPLC is above 94%, and the monomer content decreases by about 1.6-3.2%; the antibody charge main peak content detected by CEX-HPLC is above 47%, and the charge main peak content decreases by about 0.2-0.5%; the antibody purity detected by non-reducing Caliper is above 97%, and the purity decreases by about 1.3-1.7%. When sucrose was used as the excipient, the viscosity, osmotic pressure, stopper sliding performance, average particle size of the antibody, CEX-HPLC charge main peak content decrease rate after 4 weeks at 40°C, and non-reducing Caliper antibody purity decrease rate of the antibody liquid preparation were all higher than those of the preparation using proline as the excipient.

[0183] Further, comprehensive analysis of various data showed that when the excipient of the antibody liquid formulation was proline, the buffer system was histidine / histidine hydrochloride buffer, and the pH was 6.0 (i.e., the prescription of samples F13 and F16), the fluidity of the formulation and the patient's compliance with the medication were most suitable and the stability of the antibody was the strongest. Specifically, the osmotic pressure of the liquid formulation was approximately 330mOsm / Kg, the antibody viscosity was <10cp, and after the antibody liquid formulation was placed at 40°C for 4 weeks, the SEC-HPLC antibody monomer content decreased by approximately 1.6% and 2.2%, respectively, the CEX-HPLC main peak content decreased by approximately 0.3% and 0.5%, respectively, and the non-reducing Caliper purity decreased by approximately 1.3% and 1.5%, respectively.

[0184] Again, when the excipient of the antibody liquid formulation was proline and the buffer system was acetic acid / sodium acetate buffer at pH 5.0 (i.e., the formulation of sample F18), the viscosity of the formulation (approximately 11.63 cp) was slightly higher than that of F13 and F16, and the rubber stopper sliding performance was inferior to that of F13 and F16. After being placed at 40°C for 4 weeks, the SEC-HPLC antibody monomer content decreased by approximately 3.2%, which was higher than the antibody monomer content decrease rate of F13 and F16. The non-reducing Caliper purity decrease rate was also slightly higher than that of F13 and F16, indicating that acid catalysis may be the main reason for the formation of hydrolyzates. In addition, the liquid formulation with acetic acid / sodium acetate buffer at pH 5.0 can cause subcutaneous injection pain, reducing patient compliance during medication.

[0185] In addition, when the excipient of the antibody liquid preparation contains only proline at a concentration of 200 to 300 mM, and the buffer system is a histidine / histidine hydrochloride buffer with a concentration ratio of L-histidine to L-histidine hydrochloride of 0.2:1-3:1, not only can the liquid preparation of the present invention be ensured to be an isotonic solution, but the pH value of the liquid preparation is also maintained within the range of 5.5-6.5, and the liquid preparation no longer contains other stabilizer components, so that the auxiliary material components of the liquid preparation of the present invention are simple and easy to control quality.

[0186] Thus, when proline is used as an excipient in the antibody liquid formulation of the present invention, it can serve not only as a stabilizer but also as a viscosity reducer and osmotic pressure regulator. In particular, when the buffer system is a histidine / histidine hydrochloride buffer and proline is used as an excipient, it can significantly reduce the degradation and aggregation of the high-concentration anti-PCSK9 antibody of the present invention, ensuring antibody stability, while also significantly reducing the viscosity of the high-concentration antibody. Furthermore, it can ensure that the liquid formulation of the present invention is an isotonic solution and that the antibody protein is uniformly distributed, thereby significantly improving patient compliance during medication.

[0187] Example 3 Surfactant screening experiment

[0188] Based on the aforementioned buffer system and excipient screening, the present invention further conducted surfactant screening research. According to the formulation formula shown in Table 7, 0.01% (w / w) polysorbate 80 was added to the formulation containing 20mM histidine / histidine hydrochloride (pH 6.0), excipients (250mM proline or 6% (w / w) sucrose), and 140mg / mL of anti-PCSK9 antibody (Antibody A), or no polysorbate 80 was added. The stability of the antibody was analyzed by freeze-thaw cycle experiments at low temperature of -70±10°C and room temperature to investigate whether the antibody liquid formulation of the present invention requires the addition of a surfactant.

[0189] Table 7 Antibody liquid formulation formula for surfactant screening experiment

[0190]

[0191] Table 8 Results of freeze-thaw cycle experiments investigating the effects of surfactants on antibody stability

[0192]

[0193]

[0194] As shown in Table 8, samples F19-F21 showed no significant changes in appearance, protein content, pH, SEC-HPLC main peak purity, non-reducing Caliper purity, and CEX-HPLC charge main peak purity after three and five freeze-thaw cycles, respectively, compared to pre-freeze-thaw cycles, and there were no significant differences between the samples. After five freeze-thaw cycles, the antibody activities of F19, F20, and F21 remained between 80% and 120%, with no significant differences between the samples. After five freeze-thaw cycles, the amount of insoluble particulate matter in sample F20 was slightly less than that in formulation F21. This indicates that the antibody liquid formulation of the present invention can ensure antibody stability without the presence of a surfactant. Since the liquid formulation of the present invention contains a high concentration of antibody, the addition of a surfactant to the liquid formulation can protect the antibody from air / solution interface-induced stress and solution / surface-induced stress during storage and transportation, thereby reducing antibody aggregation or the formation of particulate matter in the formulation, further improving antibody stability.

[0195] In addition, the present invention also screened and analyzed the concentration of surfactants (such as polysorbate 80) through a room temperature shaking stability test. That is, at room temperature and a rotation speed of 300 rpm, the sample solution was prepared according to the prescription shown in Table 9, and the sample solution was filled with a prefilled syringe and shaken for 1 day and 3 days to simulate the storage and transportation process. The samples were taken out and tested and analyzed. The test results are shown in Table 10.

[0196] Table 9 Prescriptions of anti-PCSK9 antibody liquid preparations containing different concentrations of surfactants

[0197]

[0198]

[0199] Table 10 Stability test results of anti-PCSK9 antibody liquid formulations containing different concentrations of surfactants

[0200]

[0201] As shown in Table 10, samples F22, F23, and F24 maintained good antibody stability after shaking at room temperature and 300 rpm for 1 and 3 days, and there were no significant differences among the samples in appearance, protein content, pH, SEC-HPLC main peak purity, non-reducing Caliper purity, and CEX-HPLC charge main peak purity. This indicates that the inclusion of 0.01%-0.04% (w / w) polysorbate 80 in the antibody liquid formulation of the present invention can effectively maintain antibody stability.

[0202] In addition, polysorbate 20 can also reduce the aggregation of the antibody of the present invention during the shaking process, prevent the antibody from being adsorbed to the surface of the container, and meet the requirements of the subcutaneous injection of the liquid preparation of the present invention.

[0203] Example 4 Stability Verification Study of Liquid Formulations Containing Anti-PCSK9 Antibodies

[0204] A semi-finished product was prepared according to the formulation of Sample F13 in Example 2 (i.e., 140 mg / mL anti-PCSK9 antibody protein, 20 mM histidine / histidine hydrochloride buffer system, pH 6.0, 250 mM proline, and 0.01% polysorbate 80). The semi-finished product was then filled into a 1 mL slender prefilled syringe (with an injection needle) with a matching rubber stopper. Three-month storage stability, three-month accelerated stability, and one-month high-temperature stability studies were conducted at 5±3°C, 25±2°C, and 40±2°C, respectively. The specific study details are shown in Table 11. Reduced CE-SDS electrophoresis was used to determine the sum of the antibody heavy chain, non-glycosylated heavy chain, and light chain contents, while non-reduced CE-SDS electrophoresis was used to determine the main peak content. The stability test results are shown in Tables 12-14.

[0205] Table 11 Antibody Liquid Formulation Stability Validation Study Plan

[0206]

[0207] (X indicates that the test items are appearance, pH value, protein content, SEC-HPLC, CEX-HPLC, reducing CE-SDS, and non-reducing CE-SDS; Y indicates that the test items are osmotic pressure and viscosity; Z indicates that the test items are insoluble particles (MFI) and rubber stopper sliding performance test; W indicates that the test item is activity; "[]" indicates whether to decide whether to test based on the test results at the next time point).

[0208] Table 12 Results of 3-month storage stability test of liquid formulation of anti-PCSK9 antibody at 5±3°C

[0209]

[0210]

[0211] Table 13 Results of 3-month accelerated stability test of liquid formulations of anti-PCSK9 antibodies at 25±2°C

[0212]

[0213] Table 14 Results of a one-month high temperature stability test at 40±2°C for liquid formulations of anti-PCSK9 antibodies

[0214]

[0215]

[0216] As shown in Table 12, the antibody liquid preparation of the present invention was stored at 5±3°C and protected from light for 3 months. The stability of all test items was good, proving that the antibody liquid preparation of the present invention meets the requirements of drug quality stability. From the experimental results in Table 13, it can be seen that the antibody liquid preparation of the present invention was stored at room temperature of 25±2°C for 3 months. The sample protein content, appearance, pH value, insoluble particles, and biological activity test results all showed good stability. The antibody monomer content (SEC-HPLC), reduced CE-SDS purity, and non-reduced CE-SDS purity showed a slight decrease of 0.6%, 0.5%, and 1.2%, respectively. However, the degree of decrease was less than 5%, indicating that the liquid preparation of the present invention remained stable at room temperature for at least 3 months. As can be seen from the experimental results in Table 14, the antibody liquid preparation of the present invention was stored at a high temperature of 40±2°C for one month. The protein content, appearance, pH value, insoluble particles, and biological activity test results of the sample all showed good stability. The antibody monomer content (SEC-HPLC), reduced CE-SDS purity, and non-reduced CE-SDS purity all showed a downward trend, reaching 1.7%, 1%, and 1.4%, respectively. This indicates that the antibody liquid preparation of the present invention can be stably stored for a short period of time under high temperature conditions.

[0217] Example 5 Long-term stability study of liquid formulations containing anti-PCSK9 antibodies

[0218] Based on the stability verification experiment in Example 4, the present invention further studied the long-term stability of three batches of antibody liquid preparations at 5±3°C. The research results are shown in Tables 15-17.

[0219] Table 15 Results of long-term stability study of anti-PCSK9 antibody liquid formulation (Batch No.: Antibody A-001) at 5±3°C

[0220]

[0221]

[0222] Table 16 Results of long-term stability study of anti-PCSK9 antibody liquid formulation (Batch No.: Antibody A-002) at 5±3°C

[0223]

[0224]

[0225] Table 17 Results of long-term stability study of anti-PCSK9 antibody liquid formulation (Batch No.: Antibody A-003) at 5±3°C

[0226]

[0227] As can be seen from Tables 15-17, the three batches of antibody liquid preparations of the present invention were stored at 5±3°C for 3 months, 6 months, 12 months, 18 months, 24 months and 36 months. There was no significant change in their appearance, pH value, protein content, SEC-HPLC, non-reducing CE-SDS and biological activity; visible foreign matter, incompatible particles and sterility all met the requirements of the pharmacopoeia; CEX-HPLC detection (samples were treated with CPB enzymes) showed slight changes in antibody charge heterogeneity, which were mainly caused by deamidation and heavy chain N-terminal cyclization. These changes are general modifications and conformational changes of antibodies, and these changes have no effect on their biological activity. It can be seen that the antibody liquid preparation of the present invention can be stored at a low temperature of 5±3°C for up to 36 months, ensuring the long-term stability of the drug at low temperature storage, and the batch-to-batch differences of the preparations are small, the antibody biological activity is high, and it meets the quality and safety requirements for long-term medication for patients.

[0228] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention. Sequence Listing <110> Xinlitai (Chengdu) Biotechnology Co., Ltd. <120> Stable formulation containing anti-PCSK9 antibody and preparation method and use thereof <150> 2020110922078 <151> 2020-10-13 <160> twenty one <170> SIPOSequenceListing 1.0 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <400> 1 Ser Tyr Gly Met His 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial Sequence <400> 2 Val Ile Trp Tyr Asp Gly Thr Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 3 <211> 5 <212> PRT <213> Artificial Sequence <400> 3 Glu Lys Gly Leu Asp 1 5 <210> 4 <211> 17 <212> PRT <213> Artificial Sequence <400> 4 Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Thr Asn Lys Asn Tyr Leu 1 5 10 15 Val <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <400> 5 Trp Ala Ser Thr Arg Glu Ser 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <400> 6 Gln Gln Tyr Tyr Ser Thr Pro Trp Thr 1 5 <210> 7 <211> 114 <212> PRT <213> Artificial Sequence <400> 7 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Ala Val Ile Trp Tyr Asp Gly Thr Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Lys Gly Leu Asp Trp Gly Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser <210> 8 <211> 113 <212> PRT <213> Artificial Sequence <400> 8 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30 Ser Thr Asn Lys Asn Tyr Leu Val Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Thr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys <210> 9 <211> 327 <212> PRT <213> Homo sapiens (Artificial Sequence) <400> 9 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 10 <211> 107 <212> PRT <213> Homo sapiens (Artificial Sequence) <400> 10 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 11<00006六百六十一><211> 441 <212> PRT <213> Artificial Sequence <400> 11 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30<六百六十九>Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 It should be noted that there seems to be an incorrect tag value in the original text as "<00006六百六十一>" which is likely a typo. This might cause issues in a proper interpretation of the content. The above translation attempts to handle the text as accurately as possible while maintaining the integrity of the tags and sequences.Ala Val Ile Trp Tyr Asp Gly Thr Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Lys Gly Leu Asp Trp Gly Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys 115 120 125 Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys 130 135 140 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu 145 150 155 160 Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 165 170 175 Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 180 185 190 Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val 195 200 205 Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 210 215 220 Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 225 230 235 240 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 245 250 255 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 260 265 270 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 275 280 285 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 290 295 300 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 305 310 315 320 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 325 330 335 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 340 345 350 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 355 360 365 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 370 375 380 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 385 390 395 400 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 405 410 415 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 420 425 430 Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440<0000�21><210> 12 <211> 220 <212> PRT <213> Artificial Sequence <400> 12 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30 Ser Thr Asn Lys Asn Tyr Leu Val Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val<00007३३>50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Thr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 <210> 13 <211> 15 <212> DNA <213> Artificial Sequence <400> 13 agctatggca tgcac 15 <210> 14 <211> 51 <212> DNA <213> Artificial Sequence <400> 14 gttatatggt atgatggaac taataaatac tatgcagact ccgtgaaggg c 51 <210> 15 <211> 15 <212> DNA <213> Artificial Sequence <400> 15 gagaaggggc tggac 15 <210> 16 <211> 51 <212> DNA <213> Artificial Sequence <400> 16 aagtccagcc agagtgtttt atacagctcc accaataaga actacttagt t 51 <210> 17 <211> twenty one <212> DNA <213> Artificial Sequence <400> 17 tgggcatcta cccgggaatc c 21 <210> 18 <211> 27 <212> DNA <213> Artificial Sequence <400> 18 cagcaatatt atagtactcc gtggacg 27 <210> 19 <211> 342 <212> DNA <213> Artificial Sequence <400> 19 caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cgtctggatt caccttcagt agctatggca tgcactgggt ccgccaggct 120 ccaggcaagg ggctggagtg gatggcagtt atatggtatg atggaactaa taaatactat 180 gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacggtgtat 240 ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagagagaag 300 gggctggact ggggccaggg aaccctggtc accgtctcct ca 342 <210> 20 <211> 339 <212> DNA <213> Artificial Sequence <400> 20 gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60 atcaactgca agtccagcca gagtgtttta tacagctcca ccaataagaa ctacttagtt 120 tggtaccagc agaaaccagg acagcctcct aagctgctca tttactgggc atctacccgg 180 gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240 atcagcagcc tgcaggctga agatgtggca gtttattact gtcagcaata ttatagtact 300 ccgtggacgt tcggccaagg gaccaaggtg gaaatcaaa 339 <210> 21 <211> 692 <212> PRT <213> Homo sapiens (Artificial Sequence) <400> 21 Met Gly Thr Val Ser Ser Arg Arg Ser Trp Trp Pro Leu Pro Leu Leu 1 5 10 15 Leu Leu Leu Leu Leu Leu Leu Gly Pro Ala Gly Ala Arg Ala Gln Glu 20 25 30 Asp Glu Asp Gly Asp Tyr Glu Glu Leu Val Leu Ala Leu Arg Ser Glu 35 40 45 Glu Asp Gly Leu Ala Glu Ala Pro Glu His Gly Thr Thr Ala Thr Phe 50 55 60 His Arg Cys Ala Lys Asp Pro Trp Arg Leu Pro Gly Thr Tyr Val Val 65 70 75 80 Val Leu Lys Glu Glu Thr His Leu Ser Gln Ser Glu Arg Thr Ala Arg 85 90 95 Arg Leu Gln Ala Gln Ala Ala Arg Arg Gly Tyr Leu Thr Lys Ile Leu 100 105 110 His Val Phe His Gly Leu Leu Pro Gly Phe Leu Val Lys Met Ser Gly 115 120 125 Asp Leu Leu Glu Leu Ala Leu Lys Leu Pro His Val Asp Tyr Ile Glu 130 135 140 Glu Asp Ser Ser Val Phe Ala Gln Ser Ile Pro Trp Asn Leu Glu Arg 145 150 155 160 Ile Thr Pro Pro Arg Tyr Arg Ala Asp Glu Tyr Gln Pro Pro Asp Gly 165 170 175 Gly Ser Leu Val Glu Val Tyr Leu Leu Asp Thr Ser Ile Gln Ser Asp 180 185 190 His Arg Glu Ile Glu Gly Arg Val Met Val Thr Asp Phe Glu Asn Val 195 200 205 Pro Glu Glu Asp Gly Thr Arg Phe His Arg Gln Ala Ser Lys Cys Asp 210 215 220 Ser His Gly Thr His Leu Ala Gly Val Val Ser Gly Arg Asp Ala Gly 225 230 235 240 Val Ala Lys Gly Ala Ser Met Arg Ser Leu Arg Val Leu Asn Cys Gln 245 250 255 Gly Lys Gly Thr Val Ser Gly Thr Leu Ile Gly Leu Glu Phe Ile Arg 260 265 270 Lys Ser Gln Leu Val Gln Pro Val Gly Pro Leu Val Val Leu Leu Pro 275 280 285 Leu Ala Gly Gly Tyr Ser Arg Val Leu Asn Ala Ala Cys Gln Arg Leu 290 295 300 Ala Arg Ala Gly Val Val Leu Val Thr Ala Ala Gly Asn Phe Arg Asp 305 310 315 320 Asp Ala Cys Leu Tyr Ser Pro Ala Ser Ala Pro Glu Val Ile Thr Val 325 330 335 Gly Ala Thr Asn Ala Gln Asp Gln Pro Val Thr Leu Gly Thr Leu Gly 340 345 350 Thr Asn Phe Gly Arg Cys Val Asp Leu Phe Ala Pro Gly Glu Asp Ile 355 360 365 Ile Gly Ala Ser Ser Asp Cys Ser Thr Cys Phe Val Ser Gln Ser Gly 370 375 380 Thr Ser Gln Ala Ala Ala His Val Ala Gly Ile Ala Ala Met Met Leu 385 390 395 400 Ser Ala Glu Pro Glu Leu Thr Leu Ala Glu Leu Arg Gln Arg Leu Ile 405 410 415 His Phe Ser Ala Lys Asp Val Ile Asn Glu Ala Trp Phe Pro Glu Asp 420 425 430 Gln Arg Val Leu Thr Pro Asn Leu Val Ala Ala Leu Pro Pro Ser Thr 435 440 445 His Gly Ala Gly Trp Gln Leu Phe Cys Arg Thr Val Trp Ser Ala His 450 455 460 Ser Gly Pro Thr Arg Met Ala Thr Ala Val Ala Arg Cys Ala Pro Asp 465 470 475 480 Glu Glu Leu Leu Ser Cys Ser Ser Phe Ser Arg Ser Gly Lys Arg Arg 485 490 495 Gly Glu Arg Met Glu Ala Gln Gly Gly Lys Leu Val Cys Arg Ala His 500 505 510 Asn Ala Phe Gly Gly Glu Gly Val Tyr Ala Ile Ala Arg Cys Cys Leu 515 520 525 Leu Pro Gln Ala Asn Cys Ser Val His Thr Ala Pro Pro Ala Glu Ala 530 535 540 Ser Met Gly Thr Arg Val His Cys His Gln Gln Gly His Val Leu Thr 545 550 555 560 Gly Cys Ser Ser His Trp Glu Val Glu Asp Leu Gly Thr His Lys Pro 565 570 575 Pro Val Leu Arg Pro Arg Gly Gln Pro Asn Gln Cys Val Gly His Arg 580 585 590 Glu Ala Ser Ile His Ala Ser Cys Cys His Ala Pro Gly Leu Glu Cys 595 600 605 Lys Val Lys Glu His Gly Ile Pro Ala Pro Gln Glu Gln Val Thr Val 610 615 620 Ala Cys Glu Glu Gly Trp Thr Leu Thr Gly Cys Ser Ala Leu Pro Gly 625 630 635 640 Thr Ser His Val Leu Gly Ala Tyr Ala Val Asp Asn Thr Cys Val Val 645 650 655 Arg Ser Arg Asp Val Ser Thr Thr Gly Ser Thr Ser Glu Gly Ala Val 660 665 670 Thr Ala Val Ala Ile Cys Cys Arg Ser Arg His Leu Ala Gln Ala Ser 675 680 685 Gln Glu Leu Gln 690

Claims

1. A stable preparation comprising: (1) an anti-PCSK9 antibody or antigen-binding fragment thereof at a concentration of 70-140 mg / mL; and (2) a pharmaceutically acceptable buffer at a concentration of 20 mmol / L, wherein the buffer is a histidine buffer composed of L-histidine and L-histidine hydrochloride at a concentration ratio of 0.2:1-3:1, and a pH of 5.5-6.5; and (3) a pharmaceutically acceptable surfactant selected from polysorbate 80 at a concentration of 0.01-0.04% w / w; and (4) other pharmaceutically acceptable excipients at a concentration of 250 mmol / L, wherein the excipient is selected from proline; wherein the anti-PCSK9 antibody or antigen-binding fragment thereof comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 of the amino acid sequence shown in SEQ ID NO: 2, HCDR3 of the amino acid sequence shown in SEQ ID NO: 3, LCDR1 of the amino acid sequence shown in SEQ ID NO: 4, LCDR2 of the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 of the amino acid sequence shown in SEQ ID NO:

6.

2. The stable formulation according to claim 1, wherein The anti-PCSK9 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least 85% identical to the amino acid sequence shown in SEQ ID NO:7; and the light chain variable region comprises an amino acid sequence that is at least 85% identical to the amino acid sequence shown in SEQ ID NO:

8.

3. The stable formulation according to claim 2, wherein The heavy chain variable region comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:7; the light chain variable region comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:

8.

4. The stable formulation according to any one of claims 1 to 3, wherein The anti-PCSK9 antibody or antigen-binding fragment thereof comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is selected from human IgG1, IgG2, IgG3 or IgG4 constant region; and the light chain constant region is selected from human κ light chain constant region or λ light chain constant region.

5. The stable formulation according to claim 4, wherein The heavy chain constant region is selected from the IgG4 constant region, the amino acid sequence of the IgG4 constant region is shown in SEQ ID NO: 9, and the amino acid sequence of the kappa light chain constant region is shown in SEQ ID NO:

10.

6. The stable formulation according to any one of claims 1 to 3, wherein The anti-PCSK9 antibody or antigen-binding fragment thereof comprises two heavy chains and two light chains or consists of two heavy chains and two light chains, wherein the heavy chain has the amino acid sequence shown in SEQ ID NO: 11, and the light chain has the amino acid sequence shown in SEQ ID NO:

12.

7. The stable formulation according to claim 1, wherein The preparation is a liquid preparation, the viscosity of the liquid preparation at 25° C. is less than 15 cp, and the osmotic pressure of the liquid preparation at 25° C. is 220 to 370 mOsm / Kg.

8. The stable formulation according to claim 7, wherein The viscosity of the liquid preparation at 25° C. is less than 10 cp, and the osmotic pressure is 250-350 mOsm / Kg.

9. The stable formulation according to claim 1, wherein The liquid formulation can be stably stored at about 5° C. to about 40° C. for at least 2 weeks.

10. The stable formulation according to claim 9, wherein The liquid formulation can be stored stably at about 5° C. to about 40° C. for at least 6 months.

11. The stable formulation according to claim 1, wherein When the preparation is stored at 2-8°C for at least 3 months, the purity of the anti-PCSK9 antibody or its antigen-binding fragment decreases by no more than 10%, and / or the acid-base charge heterogeneity does not exceed 50%, and / or the aggregates increase by no more than 10%, and / or the degradation products increase by no more than 10%.

12. The stable formulation according to claim 11, wherein When the preparation is stored at 2-8°C for at least 12 months, the purity of the anti-PCSK9 antibody or its antigen-binding fragment decreases by no more than 5%, and / or the aggregates increase by no more than 5%, and / or the degradation products increase by no more than 5%.

13. The stable formulation according to claim 1, wherein The preparation is for parenteral administration, and the parenteral administration is selected from subcutaneous or intramuscular injection.

14. A method for preparing a stable formulation according to any one of claims 1 to 13, characterized in that: The preparation method comprises the following steps: a) providing an anti-PCSK9 antibody or an antigen-binding fragment thereof; b) adding a buffer and an osmotic pressure regulator; c) adding a surfactant; d) sterile filtration; e) Repackaging.

15. A prefilled syringe, characterized in that The pre-filled syringe is loaded with the stable preparation according to any one of claims 1 to 13, which is a liquid preparation.

16. Use of the stable formulation according to any one of claims 1 to 13 in the preparation of a medicament for treating, preventing or ameliorating any disease or symptom associated with PCSK9, wherein the disease or condition associated with PCSK9 is selected from hyperlipidemia, hypercholesterolemia, stroke, atherosclerosis, and heart disease.

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