Anti-PCSK9 Antibody Formulation and Its Applications

Abstract

The present invention relates to an anti-PCSK9 antibody preparation containing an anti-PCSK9 antibody and a buffer, and its pharmaceutical use. The anti-PCSK9 antibody preparation of the present invention can effectively suppress the aggregation of the anti-PCSK9 antibody, thereby preventing the degradation of the antibody product and obtaining a stable pharmaceutical preparation for injection.

Description

Technical Field

[0001] The present invention relates to the field of biological pharmaceuticals, and relates to anti-PCSK9 antibody preparations and their applications. Specifically, the present invention relates to anti-PCSK9 monoclonal antibody preparations.

Background Art

[0002] PCSK9 (proprotein Convertase Subtilisin Kexin Type 9) is a serine protease encoded by the PCSK9 gene and is mainly produced in the liver. PCSK9 binds to the LDL receptor (LDL-R) on the surface of hepatocytes, mediates the degradation of intracellular LDL-R, and increases plasma LDL-C levels. PCSK9 inhibitors (anti-PCSK9 antibodies) can interfere with the binding of PCSK9 and LDL-R, recycle LDL-R to the liver surface, promote more LDL-R expression in the liver, and reduce plasma LDL-C levels.

[0003] Anti-PCSK9 antibodies are used for the treatment of heterozygous familial hypercholesterolemia or clinical atherosclerotic cardiovascular disease, and for the treatment of patients who require further reduction of low-density lipoprotein cholesterol (LDL-C), and can also be used as adjuvants in the treatment of statin intolerance. Anti-PCSK9 antibodies may also improve cardiovascular diseases through other mechanisms such as reducing inflammation and oxidative stress in atherosclerotic plaques and inhibiting the thrombosis-promoting pathway, which is particularly important for patients with acute coronary syndrome.

[0004] The clinical significance of inhibiting PCSK9 lies in the fact that loss-of-function mutations in PCSK9 lead to a decrease in LDL-C levels and a significant reduction in the risk of cardiovascular events. Conversely, gain-of-function mutations in PCSK9 result in increased LDL-C levels and the risk of cardiovascular events. HMG-CoA reductase mutations and PCSK9 mutations reduce the LDL-C effect and have a cumulative effect on reducing the incidence of cardiovascular events, that is, the efficacy of anti-PCSK9 antibodies and statins may be additive.

[0005] Twenty-four randomized trials (n = 10,159) including various clinical cases such as familial hypercholesterolemia, other hypercholesterolemia, hypercholesterolemia with statin intolerance, intensive and non-intensive statin therapy, and no statin therapy were incorporated into the meta-analysis. The analysis found that anti-PCSK9 antibodies reduced all-cause mortality (OR 0.45, 95% CI 0.23 - 0.86), cardiovascular mortality (OR 0.50, CI 0.23 - 1.10), and myocardial infarction (OR 0.49, CI 0.26 - 0.93). The heterogeneity of the results incorporated in the trial was not statistically significant, indicating that anti-PCSK9 antibodies have a similar therapeutic effect on various clinical cases and risks based on cardiovascular diseases, similar to statins.

[0006] There are only two PCSK9 inhibitors approved for sale in the world, namely Evolocumab (trade name: Repatha) of Amgen and Alirocumab (trade name: Praluent) jointly developed by Sanofi and Regeneron. Both products are anti-PCSK9 monoclonal antibodies, and PCSK9 inhibitors as low-molecular chemical drugs have not been commercially available to date. The prevalence of dyslipidemia in Chinese adults has increased significantly. According to the "Report on Cardiovascular Diseases in China 2018 Edition", the prevalence of dyslipidemia in Chinese adults reached 40.4% in 2012. The awareness rate, treatment rate and control rate of dyslipidemia in Chinese adults still have much room for improvement. Anti-PCSK9 antibodies can meet the clinical needs of patients with dyslipidemia and inadequately controlled dyslipidemia. Therefore, in order to obtain a more stable, more effective and clinically convenient preparation, it is necessary to conduct in-depth research on this target antibody.

Summary of the Invention

[0007] An object of the present invention is to provide a stable anti-PCSK9 antibody preparation.

[0008] Specifically, the present invention relates to the following aspects:

[0009] 1. An anti-PCSK9 antibody preparation comprising an anti-PCSK9 antibody or an antigen-binding fragment thereof, at least one buffer, at least one stabilizer and at least one surfactant, wherein the anti-PCSK9 antibody comprises HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 8, and LCDR1, LCDR2 and LCDR3 contained in the light chain variable region shown in SEQ ID NO: 10. Preferably, according to the IMGT numbering system, the anti-PCSK9 antibody comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 3, LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5 and LCDR3 shown in SEQ ID NO: 6, and the sequence shown in SEQ ID NO: 5 is GVI. Preferably, the antigen-binding fragment is an anti-PCSK9 antibody preparation selected from a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fv fragment, an isolated CDR region, a single-chain Fv molecule (scFv), an F(ab')2, an Fd, a dAb, a Fab / c, a bivalent antibody, and a domain antibody.

[0010] 2. The buffer solution is selected from one or more of a histidine buffer solution, a citrate buffer solution, a succinate buffer solution, an acetate buffer solution, an arginine buffer solution, and a phosphate buffer solution. Preferably, the buffer solution is selected from 1-20 mM sodium citrate, 1-20 mM histidine, and / or histidine hydrochloride. Preferably, the content of histidine is 1-7.4 mM, preferably 3-5.4 mM, more preferably 4-4.4 mM, and most preferably 4.2 mM. Preferably, the content of histidine hydrochloride is 5-16.6 mM, preferably 8-13.6 mM, more preferably 10-11.6 mM, and most preferably 10.8 mM. Preferably, the pH value of the anti-PCSK9 antibody preparation is 5.0-6.0, preferably 5.2-5.8, and most preferably 5.5. The anti-PCSK9 antibody preparation according to item 1.

[0011] 3. The stabilizer is selected from one or more of sugars, amino acids, polyols, antioxidants, preservatives, cyclodextrins, polyethylene glycols (such as PEG3000, PEG3350, PEG4000, PEG6000), albumins (such as human serum albumin (HSA), bovine serum albumin (BSA)), salts (such as sodium chloride, calcium chloride), and chelating agents (such as EDTA). Preferably, the stabilizer is selected from one or more of saccharides. The saccharides may be selected from sucrose and / or trehalose. Preferably, the content of sucrose is 5 - 100 mg / mL, preferably 20 - 100 mg / mL, 40 - 80 mg / mL, more preferably 54 - 66 mg / mL, 55 - 65 mg / mL or 57 - 63 mg / mL, most preferably 60 mg / mL. The content of trehalose is 5 - 100 mg / mL, preferably 5 - 55 mg / mL, 15 - 45 mg / mL, more preferably 25 - 35 mg / mL, 27 - 33 mg / mL or 28.5 - 31.5 mg / mL, most preferably 30 mg / mL. Preferably, when sucrose and trehalose are used simultaneously, the total concentration of sucrose and trehalose is 85 - 95 mg / mL, preferably 90 mg / mL. The anti-PCSK9 antibody preparation according to any one of items 1 - 2.

[0012] 4. The surfactant is selected from polyoxyethylene sorbitan fatty acid esters (Tween) such as polysorbate 20 and polysorbate 80. Preferably, the content of polysorbate 80 is 0.02% - 0.08% (w / w), more preferably 0.025% - 0.075% or 0.04% - 0.06% (w / w), most preferably 0.05% (w / w). The anti-PCSK9 antibody preparation according to any one of items 1 - 3.

[0013] 5. The anti-PCSK9 antibody preparation according to any one of items 1 - 4, further comprising water for injection.

[0014] 6. The concentration of the anti-PCSK9 antibody or its antigen-binding fragment is 10 to 150 mg / mL, preferably 50 to 150 mg / mL or 80 to 120 mg / mL, more preferably 90 to 110 mg / mL, and most preferably 100 mg / mL. The anti-PCSK9 antibody preparation according to any one of items 1 to 5.

[0015] 7. The anti-PCSK9 antibody preparation according to any one of items 1 to 5, which contains 100 mg / mL of anti-PCSK9 antibody, 4.2 mM of histidine, 10.8 mM of histidine hydrochloride, 60 mg / mL of sucrose, 30 mg / mL of trehalose, and 0.05% (w / w) of polysorbate 80, and has a pH of 5.5.

[0016] 8. The anti-PCSK9 antibody preparation according to any one of items 1 to 6, which further contains a preservative, and the preservative is preferably used in an amount of about 0.001% to about 2% (w / v), and is preferably selected from ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl paraben, propyl paraben, or benzalkonium chloride.

[0017] 9. The anti-PCSK9 antibody preparation according to any one of items 1 to 6, which further contains an antibacterial agent and an antifungal agent, such as chlorobutanol and sorbic acid.

[0018] 10. The anti-PCSK9 antibody preparation according to any one of items 1 to 8, which is in a form suitable for injection (preferably subcutaneous injection).

[0019] 11. A method for preparing an anti-PCSK9 antibody preparation, which includes the step of mixing an anti-PCSK9 antibody or its antigen-binding fragment with at least one buffer, at least one stabilizer, and at least one surfactant, and adjusting the pH of the preparation to 5.0 to 6.0, preferably 5.2 to 5.8, and most preferably 5.5 with an acid or a base.

[0020] 12. The anti-PCSK9 antibody or antigen-binding fragment thereof is as described in item 1 or 6, the buffer solution is as described in item 2, the stabilizer is as described in item 3, and the surfactant is as described in item 4. The method according to item 11.

[0021] 13. The surfactant is a non-ionic surfactant (such as polysorbate 80), and the non-ionic surfactant is added last and the volume is made constant. The method according to item 11 or 12.

[0022] In the present invention, the expression "pharmaceutical preparation" also refers to an anti-PCSK9 antibody preparation.

[0023] In the pharmaceutical preparation described in the present invention, the anti-PCSK9 antibody or antigen-binding fragment thereof comprises any one or more CDR region sequences selected from the following, or an amino acid sequence having at least 85% (such as at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence homology with them and having the same activity:

[0024] In a specific embodiment, the anti-PCSK9 antibody comprises a heavy chain variable region and a light chain variable region, and the antibody comprises HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 8, and LCDR1, LCDR2 and LCDR3 contained in the light chain variable region shown in SEQ ID NO: 10. Preferably, the anti-PCSK9 antibody comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 3, LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5 and LCDR3 shown in SEQ ID NO: 6.

[0025] The amino acid sequence is shown in Table 1 below:

[0026]

Table 1

[0027] The DNA sequence of the heavy chain variable region of the anti-PCSK9 antibody is as follows (369 bp):

[0028] GAGGTGCAGCTGGTGGAGTCTGGAGGAGGCCTGGTGCAGCCCGGAAGATCTCTGAGACTGAGTTGCGCCGCTTCAGGATTCACCTTTAGCTCCTACAGCATGAACTGGGTGCGGCAGGCTCCTGGCAAGGGGCTGGAGTGGGTCTCCGGAATCTCTAGTTCAAGCTCCTACATTAGCTATGCAGACTCCGTCCAGGGAAGGTTCACCATCTCTCGCGATAACGGCAAGAACAGCCTGTATCTGCAGATGAACAGCCTGCGAGCAGAGGACACAGCCCTGTACTTCTGTGCCAGAGAATATGACTTCTGGTCCGCCTATTACGACGCCTTCGATGTCTGGGGACAGGGGACTATGGTCACTGTCTCAAGC (SEQ ID NO: 7)

[0029] The sequence of the heavy chain variable region encoded thereby is as follows (123 aa):

[0030] EVQLVESGGGLVQPGRSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSGISSSSSYISYADSVQGRFTISRDNGKNSLYLQMNSLRAEDTALYFCAREYDFWSAYYDAFDVWGQGTMVTVSS (SEQ ID NO: 8)

[0031] The DNA sequence of the light chain variable region of the anti-PCSK9 antibody is as follows (333 bp):

[0032] CAGAGCGAACTGACTCAGCCAAGAAGCGTCAGTGGATCACCTGGCCAGAGCGTGACAATCTCCTGCACCGGCACAAGCAGGAACATTGGCGGGGGAAATGACGTCCACTGGTACCAGCAGCATCCAGGGAAGGCCCCCAAACTGCTGATCTCCGGAGTGATTGAGCGGAGCTCCGGCGTCCCCGATAGATTCAGCGGGTCCAAGTCTGGAAACACAGCTTCTCTGACTATCAGTGGCCTGCAGGCAGAGGACGAAGCCGATTACTATTGCCAGTCTTTCGACGGCAGTCTGTCAGGGAGCGTGTTTGGCACTGGGACCGATGTGACCGTCCTG (SEQ ID NO: 9)

[0033] The sequence of the light chain variable region encoded thereby is as follows (111 aa):

[0034] QSELTQPRSVSGSPGQSVTISCTGTSRNIGGGNDVHWYQQHPGKAPKLLISGVIERSSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCQSFDGSLSGSVFGTGTDVTVL (SEQ ID NO: 10)

[0035] The anti-PCSK9 antibody preparation described in the present invention is a pharmaceutical preparation suitable for subcutaneous injection.

[0036] In one embodiment of the present invention, the anti-PCSK9 antibody preparation contains, or is composed of, an anti-PCSK9 antibody, a buffer, a saccharide, a surfactant, and optionally further contains water for injection.

[0037] In one embodiment of the present invention, the components of the anti-PCSK9 antibody preparation are preferably those shown in Table 2 below:

[0038]

Table 2

[0039] As used herein, the term "antibody" refers to an immunoglobulin, which is a tetrapeptide chain structure in which two identical heavy chains and two light chains are linked by interchain disulfide bonds.

[0040] The antibodies of the present invention include mouse-derived antibodies, chimeric antibodies, and humanized antibodies, preferably humanized antibodies.

[0041] The term "antigen-binding fragment" (or abbreviated as "antibody fragment") of an antibody refers to one or more fragments that maintain the ability to specifically bind to an antigen of the antibody (e.g., PCSK9). It has been shown that the antigen-binding function of an antibody can be exerted by using fragments of a full-length antibody. The antigen-binding portion can be generated by DNA recombination techniques or enzymatic or chemical cleavage of a complete immunoglobulin. In the present invention, examples of the antigen-binding fragment include Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, isolated CDR region, single-chain Fv molecule (scFv), F(ab')2, Fd, dAb, Fab / c, bispecific antibody, and domain antibody.

[0042] The term "CDR" refers to one of six hypervariable regions within the variable domain of an antibody that mainly facilitates antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat E.A. et al., (1991) Sequences of immunological Interests NIH Publication 91-3242. As used herein, the Kabat definition of CDR applies only to LCDR1, LCDR2, and LCDR3 of the light chain variable domain, and HCDR1, HCDR2, and HCDR3 of the heavy chain variable domain.

[0043] In the present invention, the antibodies described in the present invention may further include a light chain constant region containing a K chain, a λ chain, or a variant thereof derived from human or mouse.

[0044] In the present invention, the antibodies described in the present invention may further include a heavy chain constant region containing IgG1, IgG2, IgG3, IgG4, or a variant thereof derived from human or mouse.

[0045] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a mouse-derived antibody with the constant region of a human antibody, and can reduce the immune response induced by the mouse-derived antibody. A chimeric antibody is first established by establishing a hybridoma that secretes a specific monoclonal antibody derived from a mouse, then cloning the variable region gene from the mouse hybridoma cells, and further cloning the constant region gene of the human antibody as needed. The variable region gene of the mouse and the constant region gene of the human are combined into a chimeric gene and inserted into an expression vector, and finally the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic system to be established.

[0046] The term "humanized antibody", also called a CDR-grafted antibody, refers to an antibody generated by transplanting the CDR sequence of a mouse into the framework of the human antibody variable region, that is, an antibody generated from different types of human germline antibody framework sequences. It can overcome the heterologous reactions induced by chimeric antibodies containing a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases containing germline antibody gene sequences or published references.

[0047] The term "pharmaceutical formulation" or "formulation" refers to a product that is in a form that enables the biological activity of the active ingredient to be clearly effective and does not contain additional ingredients that are toxic to the subject to which the formulation is administered.

[0048] The term "liquid" as used herein in connection with the formulations of the present invention refers to a formulation that is liquid at a temperature of at least about 20°C to about 80°C at atmospheric pressure.

[0049] The term "stabilizer" refers to a pharmaceutically acceptable excipient that protects the active pharmaceutical ingredient and / or formulation from chemical and / or physical degradation during manufacturing, storage, and application. The chemical and physical degradation pathways of protein drugs are described by Cleland, J.L., M.F. Powell, et al. (1993). "The development of stable protein fomulations: a c10se look at protein aggregation, deamidation, and oxidation." Crit Rev Ther Drug Carrier Syst 10(4):307-77, Wang, W. (1999). "Instability, stabilization, and formulation of liquid protein pharmaceuticals." Int J Pharm 185(2):129-88., Wang, W. (2000). "Lyophilization and development of solid protein pharmaceuticals.Int J Pharm203(1-2):1-60" and Chi, E.Y., S.Kri shnan, et al. (2003). "Physical stability of proteins in aqueous solution: mechanism and driving forces in nonnative protein aggregation." Pharm Res 20(9):1325-36.

[0050] Preferably, the formulations of the present invention are "stable" formulations, which represent formulations in which the included protein (e.g., antibody) maintains its physical and chemical stability and its biological activity after storage.

[0051] For example, the antibody preparation of the present invention shows no significant changes at a refrigeration temperature of 2 to 8°C for at least 12 months, particularly for 2 years, and more particularly for 3 years. For example, the stability criteria are as follows: the detection result by visual inspection is a transparent liquid ranging from colorless to light yellow, the main peak of the size exclusion chromatography (SE-HPLC) detection result must be 95% or more, the sum of the heavy chain and the light chain of the reduced capillary electrophoresis (rCE-SDS) detection result must be 95% or more, the main peak of the non-reduced capillary electrophoresis (nrCE-SDS) detection result must be 85% or more, and the biological activity detection (ELISA) result must be 50% to 150% of the reference product.

[0052] The terms "protein PCSK9" and "PCSK9" are used interchangeably and include variants, isoforms, species homologs of human PCSK9, and analogs having at least one common epitope with PCSK9. The complete PCSK9 sequence can be retrieved according to NCBI Reference Sequence: Genbank ID: NP_000567.1.

[0053] The terms "anti-PCSK9 antibody", "antibody against PCSK9", and "antibody against PCSK9 protein" refer to antibodies that can bind to the PCSK9 protein with sufficient affinity and thus can be used as diagnostic and / or therapeutic agents targeting the PCSK9 protein. The term "binding to the PCSK9 protein" as used herein refers to the binding of an antibody to the PCSK9 protein in BIAcore measurement (Pharmacia Biosensor AB, Uppsala, Sweden) or ELISA.

[0054] The term "surfactant" as used herein refers to a pharmaceutically acceptable excipient for protecting protein preparations from physical stress (e.g., agitation and shear). Examples of pharmaceutically acceptable surfactants include polyoxyethylene sorbitan fatty acid esters (Tween) such as polysorbate 20 and polysorbate 80.

[0055] As used herein, the term "buffer" refers to a pharmaceutically acceptable excipient for stabilizing the pH of a pharmaceutical formulation. Suitable buffers are well known in the art and can be found in the literature. Preferred pharmaceutically acceptable buffers include, but are not limited to, histidine buffer, citrate buffer, succinate buffer, acetate buffer, arginine buffer, phosphate buffer, or mixtures thereof. A preferred buffer includes a histidine / hydrochloride histidine buffer that adjusts the pH with an acid or base known in the art. The above buffers are typically used in an amount of 1 to 100 mM, preferably 10 to 30 mM, and most preferably 20 mM. The pH of the pharmaceutical formulation can be adjusted to pH 5.0 to 6.0, particularly pH 5.2 to 5.8, and most particularly pH 5.5, with an acid or base known in the art (such as hydrochloric acid, acetic acid, phosphoric acid, sulfuric acid, citric acid, sodium hydroxide, and potassium hydroxide), independent of the buffer used.

[0056] In some embodiments, the pharmaceutical formulation of the present invention may further comprise an antioxidant as a second stabilizer. An "antioxidant" is a pharmaceutically acceptable excipient that inhibits the oxidation of the active pharmaceutical ingredient. Antioxidants include, but are not limited to, chelating agents such as EDTA, citric acid, ascorbic acid, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), sodium sulfite, para-aminobenzoic acid, glutathione, propyl gallate, cysteine, methionine, ethanol, benzyl alcohol, and n-acetylcysteine.

[0057] As used herein, the term "sugar" refers to a monosaccharide or an oligosaccharide. A monosaccharide is a monomeric carbohydrate that cannot be hydrolyzed by an acid and includes monosaccharides and their derivatives, such as amino sugars. Examples of monosaccharides include glucose, fructose, galactose, mannose, sorbose, ribose, deoxyribose, and neuraminic acid. An oligosaccharide is a branched or linear carbohydrate composed of multiple monomeric sugar units linked through glycosidic bonds. The monomeric sugar units within an oligosaccharide may be the same or different. Oligosaccharides are disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, etc., depending on the number of monomeric sugar units. Monosaccharides and oligosaccharides are water-soluble, unlike polysaccharides. Examples of oligosaccharides include sucrose, trehalose, lactose, maltose, and raffinose. Specifically, the sugar is selected from sucrose and trehalose.

[0058] As used herein, the term "amino acid" refers to a pharmaceutically acceptable organic molecule having an amino moiety located at the α-position of a carboxyl group. Examples of amino acids include arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, and proline. Amino acids are typically used in an amount of 5 to 200 mM, particularly 5 to 100 mM, and more particularly 5 to 70 mM.

[0059] The term "stabilizer" includes cryoprotectants. The term "cryoprotectant" refers to a pharmaceutically acceptable excipient that protects an active ingredient (e.g., a protein) that is unstable during the lyophilization process, subsequent storage, and reconstitution processes from destabilizing conditions. Cryoprotectants include, but are not limited to, sugars, polyols (e.g., sugar alcohols), and amino acids. Specifically, cryoprotectants can be selected from saccharides such as sucrose, trehalose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, neuraminic acid, amino sugars such as glucosamine, galactosamine, N-methylglucosamine ("meglumine"), polyols such as mannitol and sorbitol, and amino acids such as arginine and glycine or mixtures thereof. The cryoprotectant is preferably a disaccharide, specifically preferably sucrose and trehalose. The present invention has surprisingly discovered that disaccharides are superior to monosaccharides, polyols, and amino acids in terms of the stability of the formulation.

[0060] The pharmaceutical formulation may further contain an isotonic agent. As used herein, the term "isotonic agent" refers to a pharmaceutically acceptable isotonic agent for adjusting the tonicity of the formulation. The formulation may be hypotonic, isotonic, or hypertonic, preferably isotonic. Isotonicity generally relates to the relative osmotic pressure of a solution, often with respect to the osmotic pressure of human serum. An isotonic formulation is a liquid reconstituted from a liquid or solid form (e.g., a low-pressure lyophilized form) and has the same tonicity as other solutions (such as physiological saline solutions and sera) for comparison. Suitable isotonic agents include, but are not limited to, sodium chloride, potassium chloride, glycerin, and any component selected from amino acids, sugars, especially glucose. Isotonic agents are typically used in an amount of about 5 mM to about 500 mM. There is a group of compounds that can act in two ways on stabilizers and isotonic agents, i.e., they may be both stabilizers and isotonic agents at the same time. Examples of these can be found in the collection such as sugars, amino acids, polyols, cyclodextrins, polyglycols, and salts. An example of a sugar that can be both a stabilizer and an isotonic agent at the same time is trehalose.

[0061] The pharmaceutical preparation may contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. By means of sterilization operation and the inclusion of different antibacterial and antifungal agents (such as methylparaben, trichlorobutanol, phenol, sorbic acid, etc.), it is possible to ensure the prevention of the presence of microorganisms. Preservatives are usually used in an amount of about 0.001% to about 2% (w / v). Preservatives include, but are not limited to, ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methylparaben or propylparaben, benzalkonium chloride.

[0062] The pharmaceutical preparation of the anti-PCSK9 antibody according to the present invention is used for the treatment of heterozygous familial hypercholesterolemia or clinical atherosclerotic cardiovascular disease, and for the treatment of adults who require further reduction of low-density lipoprotein cholesterol (LDL-C), and can also be used as an adjuvant in the treatment of statin intolerance. The anti-PCSK9 antibody may improve cardiovascular diseases through other mechanisms such as reducing inflammation and oxidative stress in atherosclerotic plaques and inhibiting the thrombus formation promoting pathway.

[0063] The pharmaceutical preparation of the anti-PCSK9 antibody according to the present invention can be administered by intravenous (i.v.), subcutaneous (s.c.) or any other parenteral administration means (such as methods known in the pharmaceutical field).

[0064] Considering their high stability, the pharmaceutical preparation according to the present invention can be administered subcutaneously without the need for an in-line filter.

[0065] The preparation for in vivo administration must be sterile. This can be effectively achieved by an aseptic manufacturing process that is not terminally sterilized.

[0066] The pharmaceutical preparation of the anti-PCSK9 antibody according to the present invention can be prepared by known methods in the art such as ultrafiltration-diafiltration, dialysis, addition mixing, reconstitution and combinations thereof. Preparation examples of the preparation according to the present invention can be found below.

Best Mode for Carrying Out the Invention

[0067] The embodiments of the present invention will be described in detail below by combining examples. Those skilled in the art will understand that the following examples are only for explaining the present invention and should not be regarded as limiting the scope of the present invention. For examples where specific techniques or conditions are not specified, the techniques or conditions described in the literature of the technical field (for example, refer to the third edition of "Molecular Cloning: A Laboratory Manual" written by J. Sambrook et al., translated by Huang Peitang et al., and published by Science Press) or the product specifications are followed. The reagents or instruments used are commercially available conventional products when the manufacturer is not specified.

[0068] Preparation Example 1. Preparation of Humanized Anti-PCSK9 Antibody 1. Antibody Design To prepare monoclonal antibody MAB1, the inventor artificially designed a series of antibody sequences based on the existing PCSK9 protein sequence (NP_777596.2) and the three-dimensional crystal structure of the protein. Through extensive screening and detection, antibody MAB1 that specifically binds to PCSK9 was finally obtained. The amino acid sequences of the heavy chain variable region and the light chain variable region of this monoclonal antibody and their coding sequences are shown in SEQ ID NOs: 1 to 4 below.

[0069] The DNA sequence of the designed MAB1 heavy chain VH is as follows (369 bp):

[0070] GAGGTGCAGCTGGTGGAGTCTGGAGGAGGCCTGGTGCAGCCCGGAAGATCTCTGAGACTGAGTTGCGCCGCTTCAGGATTCACCTTTAGCTCCTACAGCATGAACTGGGTGCGGCAGGCTCCTGGCAAGGGGCTGGAGTGGGTCTCCGGAATCTCTAGTTCAAGCTCCTACATTAGCTATGCAGACTCCGTCCAGGGAAGGTTCACCATCTCTCGCGATAACGGCAAGAACAGCCTGTATCTGCAGATGAACAGCCTGCGAGCAGAGGACACAGCCCTGTACTTCTGTGCCAGAGAATATGACTTCTGGTCCGCCTATTACGACGCCTTCGATGTCTGGGGACAGGGGACTATGGTCACTGTCTCAAGC (SEQ ID NO: 7)

[0071] The VH protein sequence encoded thereby is as follows (123 aa):

[0072] EVQLVESGGGLVQPGRSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSGISSSSSYISYADSVQGRFTISRDNGKNSLYLQMNSLRAEDTALYFCAREYDFWSAYYDAFDVWGQGTMVTVSS (SEQ ID NO: 8)

[0073] The DNA sequence of the VL of MAB1 light chain is as follows (333 bp):

[0074] CAGAGCGAACTGACTCAGCCAAGAAGCGTCAGTGGATCACCTGGCCAGAGCGTGACAATCTCCTGCACCGGCACAAGCAGGAACATTGGCGGGGGAAATGACGTCCACTGGTACCAGCAGCATCCAGGGAAGGCCCCCAAACTGCTGATCTCCGGAGTGATTGAGCGGAGCTCCGGCGTCCCCGATAGATTCAGCGGGTCCAAGTCTGGAAACACAGCTTCTCTGACTATCAGTGGCCTGCAGGCAGAGGACGAAGCCGATTACTATTGCCAGTCTTTCGACGGCAGTCTGTCAGGGAGCGTGTTTGGCACTGGGACCGATGTGACCGTCCTG (SEQ ID NO: 9)

[0075] The VL protein sequence encoded thereby is as follows (111 aa):

[0076] QSELTQPRSVSGSPGQSVTISCTGTSRNIGGGNDVHWYQQHPGKAPKLLISGVIERSSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCQSFDGSLSGSVFGTGTDVTVL (SEQ ID NO: 10)

[0077] The CDR sequences of said antibody are as follows:

[0078] HCDR1: GFTFSSYS (SEQ ID NO: 1), HCDR2: ISSSSSYI (SEQ ID NO: 2), HCDR3: AREYDFWSAYYDAFDV (SEQ ID NO: 3), LCDR1: SRNIGGGND (SEQ ID NO: 4), LCDR2: GVI (SEQ ID NO: 5), and LCDR3: QSFDGSLSGSV (SEQ ID NO: 6).

[0079] 2. Expression and purification of the antibody The heavy-chain cDNA sequence of MAB1 (the coding sequence of VH is shown in SEQ ID NO: 7, the constant region is Ig gamma-1 chain C region, ACCESSION: P01857) and the cDNA sequence of the light chain (the coding sequence of VL is shown in SEQ ID NO: 9, the constant region is Ig lambda-2 chain C regions, ACCESSION: P0CG05.1) were respectively cloned into the pUC57simple vector (provided by GenScript), and the pUC57simple-MAB1H and pUC57simple-MAB1L plasmids were obtained respectively. The plasmids pUC57simple-MAB1H and pUC57simple-MAB1L were respectively cleaved with enzymes (HindIII&EcoRI), and the recycled heavy chain and light chain by electrophoresis were respectively subcloned into the pcDNA3.1 vector, the recombinant plasmid was extracted and co-transfected into 293F cells. After culturing the cells for 7 days, the culture medium was centrifuged at high speed, the supernatant was concentrated and loaded onto a HiTrap MabSelect SuRe column, the protein was eluted in one step using an Elution Buffer, the target sample was recycled, and the culture medium was exchanged with PBS to obtain an anti-PCSK9 antibody. In the examples, the expression "anti-PCSK9 antibody" refers to the MAB1 antibody.

[0080] Example 1: Preparation process of the formulation The composition of the anti-PCSK9 antibody formulation is as follows: This formulation consists of a solute and a solvent. The solute is an anti-PCSK9 antibody, histidine, histidine hydrochloride, sucrose, trehalose and polysorbate 80, and the solvent is water for injection. The concentration of the anti-PCSK9 antibody in the formulation is 100 mg / mL, the concentration of histidine in the formulation is 4.2 mM, the concentration of histidine hydrochloride in the formulation is 10.8 mM, the concentration of sucrose in the formulation is 60 mg / mL, the concentration of trehalose in the formulation is 30 mg / mL, the concentration of polysorbate 80 in the formulation is 0.05% (w / v), and the pH is 5.5.

[0081] When preparing an anti-PCSK9 antibody formulation, the nonionic surfactant polysorbate 80 in the solute is added last and the volume is made up, and the addition of other solutes is not carried out in sequence. The anti-PCSK9 antibody formulation is aseptically dispensed into a penicillin bottle and capped with a rubber stopper and an aluminum plastic cover, or the anti-PCSK9 antibody formulation is aseptically dispensed into a liquid storage bag (Sartorius Flexboy) to obtain the finished product of this formulation.

[0082] Example 2: Stability experiment of anti-PCSK9 antibody formulation A stability experiment was conducted on the anti-PCSK9 antibody formulation prepared in Example 1. Using the changes in the content of the main component and the content of the main peak of protein purity as the determination means, through size exclusion chromatography (SEC-HPLC), capillary isoelectric focusing electrophoresis (icIEF), reduced CE-SDS, non-reduced CE-SDS, activity and observation of the appearance of the formulation, the chemical stability of the anti-PCSK9 antibody in the formulation under various environmental conditions was characterized respectively, and the stability of the formulation under conditions such as high temperature, light, freezing and thawing, and vibration was investigated.

[0083]

Table 3

[0084]

Table 4

[0085]

Table 5

[0086]

Table 6

[0087] The anti-PCSK9 antibody preparation prepared in Preparation Example 1 was analyzed by size exclusion chromatography SEC-HPLC, capillary isoelectric focusing icIEF, reduced CE-SDS, non-reduced CE-SDS and activity (ELISA). Freezing and thawing were performed 3 cycles under the experimental conditions of -20°C / 25°C, shaken at 300 rpm for 3 days, stored at a high temperature of 40°C for 30 days, and stored under light of 4500±500 Lux for 15 days. The changes in protein content, protein purity and purity of charge inhibitor are shown in Tables 1-4.

[0088] According to the results in Table 1, the change in protein content and protein purity of the anti-PCSK9 antibody preparation decreased with the increase in storage time under the high temperature condition of 40°C. In the SE-HPLC measurement on the 30th day, the main peak was 92.3%, and the antibody monomer was decomposed by 10% or less. Also, by visual analysis, it is a pale yellow transparent liquid consistent with the normal condition. The protein concentration of the preparation is 98.2 mg / mL, which is within + / -10% of the protein concentration under normal conditions. This indicates that this preparation is still a stable liquid antibody preparation after being stored at a high temperature of 40°C for 30 days.

[0089] According to the results in Table 2, the protein content and protein purity of the anti-PCSK9 antibody preparation did not change significantly after freezing and thawing. This indicates that this preparation is still a stable liquid antibody preparation after 3 cycles of freezing and thawing.

[0090] According to the results in Table 3, the change in protein content and protein purity of the anti-PCSK9 antibody preparation decreased with the increase in storage time under light conditions. In the SE-HPLC measurement on the 15th day, the main peak was 94.4%, and the antibody monomer was decomposed by 10% or less. Also, by visual analysis, it is a pale yellow transparent liquid consistent with the normal condition. The protein concentration of the preparation is 97.3 mg / mL, which is within + / -10% of the protein concentration under normal conditions. This indicates that this preparation is still a stable liquid antibody preparation after being stored under light conditions for 15 days.

[0091] According to the results in Table 4, the protein content and protein purity of the anti-PCSK9 antibody formulation did not change significantly after the shaking treatment. This indicates that this formulation is still a stable liquid antibody formulation even after shaking at 300 rpm for 3 days.

[0092] Summary: The results in Tables 1 to 4 indicate that this formulation has sufficient stability under the above storage conditions. This is because the formulation of this anti-PCSK9 antibody can slow down the chemical degradation rate of the anti-PCSK9 antibody contained therein and enhance the physical and chemical stability of the anti-PCSK9 antibody under various conditions that accelerate the chemical decomposition and physical changes of the anti-PCSK9 antibody, indicating that this monoclonal antibody can stably exist in the formulation of the preparation.

[0093] Example 3. Comparison of the compositions and pH values of different formulations

[0094] (1) Compositions with different formulations shown in Table 5 were used respectively, and the purity of the formulation at different times was tested by SEC-HPLC, and the results are shown in Table 6. AK102 represents an anti-PCSK9 antibody-MAB1 antibody at a concentration of 100 mg / mL.

[0095] [Table 7]

[0096] As can be seen from Table 6, all formulations within the range of the component content of the present invention guaranteed good stability.

[0097] (2) The pH of the solution is one of the most important parameters that affect the physical and chemical stability of biopharmaceuticals. pH can adjust the charge distribution on the protein surface, thereby affecting the intramolecular and intermolecular forces of biopharmaceuticals, and at the same time, it can also affect the chemical modification rate of antibody drugs, which is one of the most important attributes of biopharmaceutical formulations.

[0098] According to the preparation formula in Table 7, perform the Tm test and high-temperature destruction test to compare the differences in stability of different formulas.

[0099]

Table 8

[0100] (2.1) Test of Tm value The melting temperature (Tm) of a protein reflects the thermal stability of a biopharmaceutical in an aqueous solution. The higher the Tm, the lower the possibility of structural changes under actual storage conditions. The Tm values of the anti-PCSK9 antibody in buffers with different pH values were investigated. The formulations in Table 8 were prepared by using 5 μL of the anti-PCSK9 antibody in the formulation of Table 7 respectively, and adding 5 μL of SYPRO Orange (20X) and 90 μL of buffer. The Tm values of the samples were measured by a method similar to differential scanning fluorescence technology (DSF). The results are shown in Table 8.

[0101]

Table 9

[0102] (2.2) High-temperature accelerated experiment of samples with different formulations The formulations of different prescriptions in Table 7 were placed in a sample test chamber at 40 ± 2 °C for 3 days of accelerated destruction, and the stability of different formulations was investigated by appearance and SEC-HPLC.

[0103] The detection results were summarized in Table 9 and Table 10.

[0104]

Table 10

[0105] According to the Tm measurement results and the high-temperature accelerated test results, only the appearance of formulation F8 remains clear even after acceleration, while the other formulations are all turbid, so it is not suitable for the storage of AK102. At the same time, the SEC-HPLC purity indicates that formulation F8 does not change significantly after being purified after 3 days of accelerated degradation, and the stability of AK102 under the conditions of a polyhydroxyl protecting agent and pH 5.5 is preliminarily evaluated. Based on this result, the polyhydroxyl structure protecting agent was screened and optimized.

[0106] Screening and Optimization of Protecting Agents According to the general protecting agents for antibody pharmaceuticals, three formulations in Table 11 were designed, and the samples of different formulations were placed under the condition of 40±2°C to investigate the stability, and the effects of different protecting agents on the stability of AK102 were compared to select the optimal formulation.

[0107]

Table 11

[0108] After the samples were destroyed under the condition of 40±2°C, sampling was carried out after 1 week, 2 weeks, 4 weeks and 8 weeks, and SEC-HPLC and CE-SDS analyses were performed to compare the change situations of the three formulations. The detection results are shown in Table 12 and Table 13.

[0109]

Table 12

[0110] Summary: When accelerating for 8 weeks, the detection analysis related to formulation F11 was not carried out. From the results of SEC-HPLC and CE-SDS SEC-HPLC, the stability of F12 (trehalose) is significantly better than that of formulation F13 (sorbitol), and the results indicate that AK102 has better stability in the trehalose formulation.

[0111] From the perspective of process cost, a mixture of sucrose and trehalose was selected as the protective agent for AK102, and the ratio of the mixture was studied. As shown in Table 14, formulations of different ratios of the mixed saccharide protective agent were designed, and the concentration of the AK102 sample in the formulation was 100 mg / ml in all cases, which is the same as the finally proposed packaging concentration.

[0112] [Table 13]

[0113] Example 4. Stability analysis of different formulation prescriptions at different time points Different formulations were prepared according to Table 16.

[0114] [Table 14]

[0115] 1 L of ultrafiltration buffer for sample F1-19 (containing no polysorbate 80 and antibody) was prepared according to the prescription in Table 16, 1.9 mL of 6 M hydrochloric acid was added, and the protein solution in Table 16 was prepared using an ultrafiltration concentration system. Then, 10% of the polysorbate 80 (II) solution in the corresponding sample in Table 16 was added to prepare approximately 90 ml of each formulation.

[0116] Filtration was performed using a 0.22 μm filter membrane. For each prescription, 30 tubes (1.5 ml / tube) of samples were prepared. For each prescription, 10 tubes of samples were taken for detection at the T0 time point (T0 indicates the direct detection time point after placement), and 20 tubes of samples were placed in a stability test chamber at 25 °C for an accelerated test (25 °C 3M indicates being left at 25 °C for 3 months). Refer to Table 17 for the detection items. The detection results are shown in Tables 18-1, 18-2 (detection results at T0) and Tables 18-3, 18-4 (detection results at 25 °C 3M).

[0117] [Table 15]

[0118]

Table 16

[0119]

Table 17

[0120]

Table 18

[0121]

Table 19

[0122] According to the results of Table 18, the protein purity of the anti-PCSK9 antibody formulation within the formulation prescription range of Table 16 decreased with the increase of storage time under the condition of 25°C. In the SE-HPLC detection at the third month, all the main peaks exceeded 96%, and the antibody monomer was decomposed by 10% or less. Also, through visual analysis, the appearance is a light yellow transparent liquid consistent with the normal conditions. This indicates that this formulation is a stable liquid antibody formulation even after being stored at 25°C for 30 days.

Claims

**Claim 1** An anti-PCSK9 antibody preparation comprising an anti-PCSK9 antibody or an antigen-binding fragment thereof, at least one buffer, at least one stabilizer, and at least one surfactant, wherein the anti-PCSK9 antibody comprises HCDR1, HCDR2, and HCDR3 contained in the heavy-chain variable region shown in SEQ ID NO: 8, and LCDR1, LCDR2, and LCDR3 contained in the light-chain variable region shown in SEQ ID NO: 10, and preferably, according to the IMGT numbering system, the anti-PCSK9 antibody comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 3, LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5, and LCDR3 shown in SEQ ID NO: 6, and the sequence shown in SEQ ID NO: 5 is GVI, Preferably, the antigen-binding fragment is selected from a Fab fragment, a Fab' fragment, an F(ab') fragment, an Fv fragment, an isolated CDR region, a single-chain Fv molecule (scFv), F(ab') ２ , Fd, dAb, Fab / c, a bivalent antibody, and a domain antibody, an anti-PCSK9 antibody preparation. **Claim 2** The buffer is selected from one or more of histidine buffer, citrate buffer, succinate buffer, acetate buffer, arginine buffer, and phosphate buffer. Preferably, the buffer is selected from 1-20 mM sodium citrate, 1-20 mM histidine, and / or histidine hydrochloride. Preferably, the content of histidine is 1-7.4 mM, preferably 3-5.4 mM, more preferably 4-4.4 mM, and most preferably 4.2 mM. Preferably, the content of histidine hydrochloride is 5-16.6 mM, preferably 8-13.6 mM, more preferably 10-11.6 mM, and most preferably 10.8 mM. Preferably, the pH value of the anti-PCSK9 antibody preparation is 5.0-6.0, preferably 5.2-5.8, and most preferably 5.

5. The anti-PCSK9 antibody preparation according to claim 1. **Claim 3** The stabilizer is selected from one or more of sugars, amino acids, polyols, antioxidants, preservatives, cyclodextrins, polyethylene glycols (such as PEG3000, PEG3350, PEG4000, PEG6000), albumins (such as human serum albumin (HSA), bovine serum albumin (BSA)), salts (such as sodium chloride, calcium chloride) and chelating agents (such as EDTA). Preferably, the stabilizer is selected from one or more of saccharides. The saccharides may be selected from sucrose and / or trehalose. Preferably, the content of sucrose is 5 to 100 mg / mL, preferably 20 to 100 mg / mL, 40 to 80 mg / mL, more preferably 54 to 66 mg / mL, 55 to 65 mg / mL or 57 to 63 mg / mL, most preferably 60 mg / mL. The content of trehalose is 5 to 100 mg / mL, preferably 5 to 55 mg / mL, 15 to 45 mg / mL, more preferably 25 to 35 mg / mL, 27 to 33 mg / mL or 28.5 to 31.5 mg / mL, most preferably 30 mg / mL. Preferably, when sucrose and trehalose are used simultaneously, the total concentration of sucrose and trehalose is 85 to 95 mg / mL, preferably 90 mg / mL. The anti-PCSK9 antibody preparation according to any one of claims 1 to 2.

4. The surfactant is selected from polyoxyethylene sorbitan fatty acid esters (Tween) such as polysorbate 20 and polysorbate 80. Preferably, the content of polysorbate 80 is 0.02% to 0.08% (w / w), more preferably 0.025% to 0.075% or 0.04% to 0.06% (w / w), most preferably 0.05% (w / w). The anti-PCSK9 antibody preparation according to any one of claims 1 to 3.

5. The anti-PCSK9 antibody preparation according to any one of claims 1 to 4, further comprising water for injection.

6. The concentration of the anti-PCSK9 antibody or its antigen-binding fragment is 10 to 150 mg / mL, preferably 50 to 150 mg / mL or 80 to 120 mg / mL, more preferably 90 to 110 mg / mL, most preferably 100 mg / mL. The anti-PCSK9 antibody preparation according to any one of claims 1 to 5.

7. An anti-PCSK9 antibody preparation according to any one of claims 1 to 5, containing 100 mg / mL of anti-PCSK9 antibody, 4.2 mM of histidine, 10.8 mM of histidine hydrochloride, 60 mg / mL of sucrose, 30 mg / mL of trehalose, 0.05% (w / w) of polysorbate 80, and having a pH of 5.

5.

8. Further comprising a preservative, preferably, the preservative is used in an amount of about 0.001% to about 2% (w / v), preferably selected from ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl paraben, propyl paraben or benzalkonium chloride, an anti-PCSK9 antibody preparation according to any one of claims 1 to 6.

9. An anti-PCSK9 antibody preparation according to any one of claims 1 to 6, further comprising an antibacterial agent and an antifungal agent, such as chlorobutanol and sorbic acid.

10. An anti-PCSK9 antibody preparation according to any one of claims 1 to 8, which is in a form suitable for injection (preferably subcutaneous injection).

11. A method for preparing an anti-PCSK9 antibody preparation, comprising the step of mixing an anti-PCSK9 antibody or an antigen-binding fragment thereof with at least one buffer, at least one stabilizer and at least one surfactant, and adjusting the pH of the preparation to 5.0 to 6.0, preferably 5.2 to 5.8, most preferably 5.5 with an acid or a base.

12. The method according to claim 11, wherein the anti-PCSK9 antibody or an antigen-binding fragment thereof is as described in claim 1 or 6, the buffer is as described in claim 2, the stabilizer is as described in claim 3, and the surfactant is as described in claim 4.

13. The method according to claim 11 or 12, wherein the surfactant is a nonionic surfactant (such as polysorbate 80), and the nonionic surfactant is added last and made up to volume.

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