Antibody drug against PCSK9 and its use

Abstract

FIELD: biotechnology.SUBSTANCE: stable formulation for storing anti-PCSK9 antibodies has been proposed. The composition contains an antibody against PCSK9 or its antigen-binding fragment, a buffer, a stabilizer and a surfactant and has a pH of 5.0–6.0. A method for obtaining the said composition is also proposed.EFFECT: proposed antibody composition can effectively inhibit the aggregation of anti-PCSK9 antibody, thereby preventing the degradation of the antibody and providing a stable injectable pharmaceutical preparation.10 cl, 23 tbl, 4 ex

Description

[0001] FIELD OF TECHNOLOGY

[0002] The present invention relates to the field of biological pharmaceuticals and, in particular, relates to the composition of an anti-PCSK9 antibody and its use. In particular, the present invention relates to the composition of a monoclonal antibody against PCSK9.

[0003] STATE OF THE ART

[0004] PCSK9 (proprotein convertase subtilisin-kexin type 9) is a serine protease encoded by the PCSK9 gene, which is produced primarily by the liver. PCSK9 binds to LDL receptors (LDL-Rs) on the surface of hepatocytes and mediates the intracellular degradation of LDL-Rs, leading to increased plasma LDL-C levels. PCSK9 inhibitors (anti-PCSK9 antibodies) can prevent PCSK9 binding to LDL-Rs, restore LDL-Rs on the liver surface, and promote the expression of more LDL-Rs in the liver, thereby reducing plasma LDL-C levels.

[0005] Anti-PCSK9 antibodies are used to treat heterozygous familial hypercholesterolemia or clinical atherosclerotic cardiovascular disease and in patients requiring additional low-density lipoprotein cholesterol (LDL-C) reduction. They can also be used as adjuvant therapy in cases of statin intolerance. Anti-PCSK9 antibodies may also improve cardiovascular health through other mechanisms, including reducing inflammation and oxidative stress in atherosclerotic plaques and inhibiting prothrombotic pathways, which is particularly important for patients with acute coronary syndrome.

[0006] The clinical significance of PCSK9 inhibition is that loss-of-function mutations in PCSK9 reduce LDL-C levels and significantly reduce the risk of cardiovascular events. Conversely, gain-of-function mutations in PCSK9 increase LDL-C levels and the risk of cardiovascular events. The reduction in LDL-C effects by HMG-CoA reductase mutations and PCSK9 mutations has a cumulative effect on reducing the incidence of cardiovascular events, suggesting that the efficacy of anti-PCSK9 antibodies and statins may be additive.

[0007] The meta-analysis included 24 randomized trials (n=10159) covering various clinical cases, including: cases with familial hypercholesterolemia; cases with other types of hypercholesterolemia; cases with statin intolerance and hypercholesterolemia; and cases with intensive and non-intensive statin therapy and without statin therapy. The analysis showed 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 of the included trials was not statistically significant, suggesting that anti-PCSK9 antibodies, like statins, have similar therapeutic effects across different clinical conditions and cardiovascular risk factors.

[0008] Only two PCSK9 inhibitors have been approved for sale globally: Amgen's evolocumab (trade name: Repatha) and alirocumab (trade name: Praluent), jointly developed by Sanofi and Regeneron. Both products are monoclonal antibodies against PCSK9. Currently, there is no PCSK9 inhibitor in the form of a small molecule chemical drug on the market. The prevalence of dyslipidemia among Chinese adults has increased significantly, and according to the "2018 China Cardiovascular Disease Report," the prevalence of dyslipidemia among Chinese adults reached 40.4% in 2012. There is still much room for improvement in the awareness level, treatment level, and control level of dyslipidemia among Chinese adults. Anti-PCSK9 antibodies can meet the clinical needs of people with dyslipidemia and poor control.Therefore, it is necessary to conduct in-depth research on this targeted antibody to obtain a more stable, more effective and clinically convenient formulation.

[0009] ESSENCE

[0010] The object of the present invention is to provide a stable formulation of an anti-PCSK9 antibody.

[0011] In particular, the present invention relates to the following aspects:

[0012] 1. A PCSK9 antibody composition comprising a PCSK9 antibody or an antigen-binding fragment thereof, at least one buffer, at least one stabilizer, and at least one surfactant, wherein the PCSK9 antibody comprises HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region indicated by SEQ ID NO: 8, and the antibody comprises LCDR1, LCDR2, and LCDR3 contained in the light chain variable region indicated by SEQ ID NO: 10, preferably the PCSK9 antibody comprises HCDR1 indicated by SEQ ID NO: 1, HCDR2 indicated by SEQ ID NO: 2, and HCDR3 indicated by SEQ ID NO: 3, and LCDR1 indicated by SEQ ID NO: 4, LCDR2 indicated by SEQ ID NO: 5, and LCDR3 indicated by SEQ ID NO: 6, according to the numbering system IMGT, wherein the sequence indicated under SEQ ID NO: 5 is GVI, and 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,single-chain Fv molecule (scFv), F(ab')2, Fd, dAb, Fab / c, divalent antibody and domain antibody.,

[0013] 2. The PCSK9 antibody formulation according to position 1, wherein the buffer is selected from one or more of a histidine buffer, a citrate buffer, a succinate buffer, an acetate buffer, an arginine buffer and a phosphate buffer, preferably the buffer is selected from 1-20 mM sodium citrate and 1-20 mM histidine and / or histidine hydrochloride, preferably the histidine content is 1-7.4 mM, preferably 3-5.4 mM, more preferably 4-4.4 mM and most preferably 4.2 mM, preferably the histidine hydrochloride content is 5-16.6 mM, preferably 8-13.6 mM, more preferably 10-11.6 mM and most preferably 10.8 mM, and preferably the PCSK9 antibody formulation has a pH of 5.0-6.0, preferably 5.2-5.8 and most preferably pH 5.5.

[0014] 3. The PCSK9 antibody formulation according to any one of provisions 1-2, wherein the stabilizer is selected from one or more of a saccharide, an amino acid, a polyhydric alcohol, an antioxidant, a preservative, a cyclodextrin, a polyethylene glycol (e.g., PEG3000, PEG3350, PEG4000, PEG6000), an albumin (e.g., human serum albumin (HSA), bovine serum albumin (BSA)), a salt (e.g., sodium chloride, calcium chloride) and an integrating agent (e.g., EDTA), preferably the stabilizer is selected from one or more saccharides, and the saccharide may be selected from sucrose and / or trehalose: preferably the sucrose content is 5-100 mg / ml, preferably 20-100 mg / ml or 40-80 mg / ml, more preferably 54-66 mg / ml, 55-65 mg / ml or 57-63 mg / ml, and most preferably 60 mg / ml, the trehalose content is 5-100 mg / ml, preferably 5-55 mg / ml or 15-45 mg / ml, more preferably 25-35 mg / ml, 27-33 mg / ml or 28.5-31.5 mg / ml, and most preferably 30 mg / ml,and preferably, when sucrose and trehalose are used simultaneously, sucrose and trehalose have a total concentration of 85-95 mg / ml, preferably 90 mg / ml.,

[0015] 4. The anti-PCSK9 antibody composition according to any one of 1 to 3, wherein the surfactant is selected from polyoxyethylene sorbitan fatty acid ester (Tween), such as polysorbate 20 and polysorbate 80, and 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), and most preferably 0.05% (w / w).

[0016] 5. The anti-PCSK9 antibody composition according to any one of provisions 1 to 4, wherein the anti-PCSK9 antibody composition further comprises water for injection.

[0017] 6. The composition of the anti-PCSK9 antibody according to any one of 1 to 5, wherein the anti-PCSK9 antibody or antigen-binding fragment thereof has a concentration of 10-150 mg / ml, preferably 50-150 mg / ml or 80-120 mg / ml, more preferably 90-110 mg / ml, and most preferably 100 mg / ml.

[0018] 7. The anti-PCSK9 antibody composition according to any one of provisions 1 to 5, wherein the anti-PCSK9 antibody composition comprises 100 mg / ml of anti-PCSK9 antibody, 4.2 mM histidine, 10.8 mM histidine hydrochloride, 60 mg / ml sucrose, 30 mg / ml trehalose, and 0.05% (w / w) polysorbate 80 with pH 5.5.

[0019] 8. The anti-PCSK9 antibody composition according to any one of provisions 1-6, wherein the anti-PCSK9 antibody composition further comprises a preservative, preferably the preservative is used in an amount of from about 0.001% to about 2% (w / v), preferably the preservative is selected from ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methylparaben, propylparaben or benzalkonium chloride.

[0020] 9. The anti-PCSK9 antibody composition according to any one of provisions 1 to 6, wherein the anti-PCSK9 antibody composition further comprises an antibacterial agent and an antifungal agent, for example, chlorobutanol and sorbic acid.

[0021] 10. The anti-PCSK9 antibody composition according to any one of provisions 1 to 8, wherein the anti-PCSK9 antibody composition is in a form suitable for injection (preferably subcutaneous injection).

[0022] 11. A method for producing an anti-PCSK9 antibody formulation comprising 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 formulation with an acid or base to 5.0-6.0, preferably 5.2-5.8, and most preferably pH 5.5.

[0023] 12. The method according to position 11, wherein the anti-PCSK9 antibody or antigen-binding fragment thereof is as described in position 1 or 6, the buffer is as described in position 2, the stabilizer is as described in position 3, and the surfactant is as described in position 4.

[0024] 13. The method according to position 11 or 12, wherein the surfactant is a non-ionic surfactant (e.g. polysorbate 80), and the non-ionic surfactant is added last, and then the volume is determined.

[0025] In the present description, the expression “drug composition” also means a composition of an anti-PCSK9 antibody.

[0026] In the medicinal composition described herein, the anti-PCSK9 antibody or antigen-binding fragment thereof comprises any one or more CDR region sequences selected from the following, or amino acid sequences having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence homology thereto and having the same activity:

[0027] in a specific embodiment, the anti-PCSK9 antibody comprises a heavy chain variable region and a light chain variable region, the antibody comprises HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region indicated by SEQ ID NO: 8, and the anti-PCSK9 antibody comprises LCDR1, LCDR2 and LCDR3 contained in the light chain variable region indicated by SEQ ID NO: 10, and preferably, the anti-PCSK9 antibody comprises HCDR1 indicated by SEQ ID NO: 1, HCDR2 indicated by SEQ ID NO: 2, and HCDR3 indicated by SEQ ID NO: 3, and LCDR1 indicated by SEQ ID NO: 4, LCDR2 indicated by SEQ ID NO: 5, and LCDR3 indicated by SEQ ID NO: 6.

[0028] The amino acid sequences are shown in Table 1-1 below:

[0029]

[0030] The DNA sequence of the heavy chain variable region of the anti-PCSK9 antibody is as follows (369 bp):

[0031] GAGGTGCAGCTGGTGGAGTCTGGAGGAGGCCTGGTGCAGCCCGGAAGATCTCTGAGACTGAGTTGCGCCGCTTCAGGATTCACCTTTAGCTCCTACAGCATGAACTGGGTGCGGCAGGCTCCTGGCAAGGGGCTGGAGTGGGTCTCCGGAATCTCTAGTTCAAGCTCCTACATTAGCTATG CAGACTCCGTCCAGGGAAGGTTCACCATCTCTCGCGATAACGGCAAGAACAGCCTGTATCTGCAGATGAACAGCCTGCGAGCAGAGGACACAGCCCTGTACTTCTGTGCCAGAGAATATGACTTCTGGTCCGCCTATTACGACGCCTTCGATGTCTGGGGACAGGGGACTATGGTCACTGTCTCAAGC (SEQ ID NO: 7)

[0032] The sequence of the heavy chain variable region encoded by the above DNA sequence is as follows (123 aa):

[0033] EVQLVESGGGLVQPGRSLRLSCAASGFFSSYSMNWVRQAPGKGLEWVSGISSSSSYISYADSVQGRFTISRDNGKNSLYLQMNSLRAEDTALYFCAREYDFWSAYYDAFDVWGQGTMVTVSS (SEQ ID NO: 8)

[0034] The DNA sequence of the light chain variable region of the anti-PCSK9 antibody is as follows (333 bp):

[0035] CAGAGCGAACTGACTCAGCCAAGAGCGTCAGTGGATCACCTGGCCAGAGCGTGACAATCTCCTGCACCGGCACAAGCAGGAACATTGGCGGGGGAAAATGACGTCCACTGGTACCAGCAGCATCCAGGGAAGGCCCCCAAACTGCTGATCTCCGG AGTGATTGAGCGGAGCTCCGGCGTCCCCGATAGATTCAGCGGGTCCAAGTCTGGAAACACAGCTTCTCTGACTATCAGTGGCCTGCAGGCAGAGGACGAAGCCGATTACTATTGCCAGTCTTTCGACGGCAGTCTGTCAGGGAGCGTGTTTGGCACTGGGAC CGATGTGACCGTCCTG (SEQ ID NO: 9)

[0036] The sequence of the light chain variable region encoded by the above DNA sequence is as follows (111 aa):

[0037] QSELTQPRSVSGSPGQSVTISCTGTSRNIGGGNDVHWYQQHPGKAPKLLISGVIERSSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCQSFDGSLSGSVFGTGTDVTVL (SEQ ID NO: 10).

[0038] The anti-PCSK9 antibody composition described in the present specification is a medicinal composition suitable for subcutaneous injection.

[0039] In one embodiment of the present invention, the anti-PCSK9 antibody formulation comprises or consists of an anti-PCSK9 antibody, a buffer, a saccharide, and a surfactant, and optionally further comprises water for injection.

[0040] In one embodiment of the present invention, the components of the anti-PCSK9 antibody composition are preferably as shown in Table 1-2 below:

[0041]

[0042] The "antibody" described herein refers to an immunoglobulin, which is a four-peptide chain structure formed by two identical heavy chains and two identical light chains linked via interchain disulfide bonds.

[0043] The antibody of the present invention includes a mouse antibody, a chimeric antibody and a humanized antibody, preferably a humanized antibody.

[0044] The term "antigen-binding fragment" of an antibody (or simply "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., PCSK9). It has been shown that fragments of a full-length antibody can be used to perform the antigen-binding function of an antibody. The antigen-binding portion can be obtained by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulin. In the context of the present invention, examples of the antigen-binding fragment include 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.

[0045] The term "CDR" refers to one of the six hypervariable regions in the variable domain of an antibody that contributes primarily to antigen binding. One of the most common definitions of the six CDRs is provided by Rabat E.A. et al., (1991) Sequences of proteins of immunity. NIH Publication 91-3242. In the present description, Rabat's 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.

[0046] Within the scope of the present invention, the antibody described herein may further comprise a light chain constant region, and the light chain constant region comprises a human or mouse k, A chain or a variant thereof.

[0047] Within the scope of the present invention, the antibody described herein may further comprise a heavy chain constant region, and the heavy chain constant region comprises human or mouse IgG1, IgG2, IgG3, IgG4 or a variant thereof.

[0048] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can reduce the immune response induced by the mouse antibody. A chimeric antibody is produced by first preparing a hybridoma cell secreting a specific mouse monoclonal antibody, then cloning the variable region gene from the mouse hybridoma cell, then cloning the constant region gene of the human antibody if necessary, ligating the mouse variable region gene and the human constant region gene into the chimeric gene, then inserting them into an expression vector, and finally expressing the chimeric antibody molecule in a eukaryotic system or a prokaryotic system.

[0049] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody produced by grafting a mouse CDR sequence onto the variable region framework of a human antibody, i.e., an antibody derived from framework sequences of various human germline antibody types. It can overcome the heterologous reaction caused by chimeric antibodies due to the transfer of a large number of mouse protein components. Such framework sequences can be obtained from publicly available DNA databases or published references containing germline antibody gene sequences.

[0050] The term "medicinal formulation" or "formulation" refers to such a product: it is in a form that allows the biological activity of the active ingredient to be demonstrably effective and does not contain additional components toxic to the individual to whom the formulation is to be administered.

[0051] The term "liquid" as used herein in relation to the composition of the present invention means a composition that is liquid at atmospheric pressure at a temperature of at least about 20°C to about 80°C.

[0052] The term "stabilizer" means a pharmaceutically acceptable excipient that protects the active pharmaceutical ingredient and / or formulation from chemical and / or physical degradation during manufacture, storage, and use. Pathways for chemical and physical degradation of protein drugs are reviewed by Cleland, J. L., M. F. Powell et al. (1993). "The development of stable protein formulations: a close 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" IJ Pharm 203(1-2): 1-60, and Chi, E.Y., S. Krishnan et al. (2003). “Physical stability of proteins in aqueous solution: mechanism and driving forces in nonnative protein aggregation.” Pharm Res 20(9): 1325-36.

[0053] Preferably, the composition of the present invention is a "stable" composition, which is a composition such that the protein (e.g., antibody) contained therein substantially retains its physical and chemical stability, and thus its biological activity, after storage.

[0054] For example, no significant change in the composition of the antibody of the present invention is observed under a refrigeration temperature of 2-8°C for at least 12 months, particularly for 2 years, and more particularly for 3 years. For example, the stability criteria are as follows: colorless to light yellow transparent liquid according to the visual method; not less than 95% of the main peak as a result of detection by size exclusion chromatography (SE-HPLC); not less than 95% of the sum of heavy chains and light chains as a result of detection by reduction capillary electrophoresis (rCE-SDS); not less than 85% of the main peak as a result of detection by non-reducible capillary electrophoresis (nrCE-SDS); and 50%-150% of the reference substance as a result of biological activity assay (ELISA).

[0055] The terms "PCSK9 protein" and "PCSK9" are used interchangeably and include variants, isoforms, species homologs, and analogs that share at least one epitope with human PCSK9. The complete PCSK9 sequence can be found according to the NCBI reference sequence: Genbank ID: NP 000567.1.

[0056] The terms “anti-PCSK9 antibody,” “anti-PCSK9 antibody,” and “anti-PCSK9 protein antibody” refer to an antibody that is capable of binding to the PCSK9 protein with sufficient affinity so that the antibody can be used as a diagnostic agent and / or therapeutic agent for targeting the PCSK9 protein. The term “binding to the PCSK9 protein,” as used herein, refers to binding of the antibody to the PCSK9 protein in the BIAcore assay (Pharmacia Biosensor AB, Uppsala, Sweden) or in ELISA.

[0057] The term "surfactant," as used herein, refers to a pharmaceutically acceptable excipient used to protect a protein formulation from physical stress (e.g., agitation and shear). Examples of pharmaceutically acceptable surfactants include polyoxyethylene sorbitan fatty acid ester (Tween). Examples of polyoxyethylene sorbitan fatty acid esters include polysorbate 20 and polysorbate 80.

[0058] The term "buffer" as used herein refers to a pharmaceutically acceptable excipient that stabilizes the pH of a drug 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, a histidine buffer, a citrate buffer, a succinate buffer, an acetate buffer, an arginine buffer, a phosphate buffer, or a mixture thereof. A preferred buffer includes a histidine / histidine hydrochloride buffer whose pH is adjusted using an acid or base known in the art. The above buffer is typically used in an amount of 1-100 mM, preferably 10-30 mM, and most preferably 20 mM.The pH of the medicinal composition can be adjusted to pH 5.0-6.0, in particular to pH 5.2-5.8 and most preferably to pH 5.5, using an acid or base known in the art (e.g. hydrochloric acid, acetic acid, phosphoric acid, sulfuric acid and citric acid, sodium hydroxide and potassium hydroxide), regardless of the buffer used.

[0059] 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 prevents oxidation of the active pharmaceutical ingredient. The antioxidant includes, but is not limited to: integrating agents such as EDTA, citric acid, ascorbic acid, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), sodium sulfite, p-aminobenzoic acid, glutathione, propyl gallate, cysteine, methionine, ethanol, benzyl alcohol, and n-acetylcysteine.

[0060] The term "saccharide" as used herein refers to a monosaccharide or oligosaccharide. A monosaccharide is a monomeric carbohydrate that cannot be hydrolyzed by acid, including a monosaccharide and its derivative, such as an amino sugar. Examples of monosaccharides include glucose, fructose, galactose, mannose, sorbose, ribose, deoxyribose, and neuraminic acid. An oligosaccharide is a branched or linear carbohydrate consisting of more than one monomeric sugar unit attached via a glycosidic bond. The monomeric sugar units in an oligosaccharide may be the same or different. Depending on the number of monomeric sugar units, an oligosaccharide is a disaccharide, trisaccharide, tetrasaccharide, pentasaccharide, etc. Unlike polysaccharides, monosaccharides and oligosaccharides are water-soluble. Examples of oligosaccharides include sucrose, trehalose, lactose, maltose, and raffinose. Specifically, a monosaccharide is selected from sucrose and trehalose.

[0061] The term "amino acid" as used herein refers to a pharmaceutically acceptable organic molecule having an amino moiety located at the α-site of the 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. The amino acid is typically used in the following amount: an amount of 5-200 mM, in particular an amount of 5-100 mM, and more specifically an amount of 5-70 mM.

[0062] The term "stabilizer" also includes a cryoprotectant. The term "cryoprotectant" means a pharmaceutically acceptable excipient that protects an unstable active ingredient (e.g., a protein) from destabilization during freeze-drying, subsequent storage, and reconstitution. The cryoprotectant includes, but is not limited to, a saccharide, a polyhydric alcohol (e.g., a sugar alcohol), and an amino acid. In particular, the cryoprotectant can be selected from: a saccharide such as sucrose, trehalose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, neuraminic acid, an amino sugar such as glucosamine, galactosamine, N-methylglucosamine ("meglumine"), a polyhydric alcohol such as mannitol and sorbitol, and an amino acid such as arginine and glycine, or mixtures thereof. The cryoprotectant is preferably a disaccharide, and is particularly preferably sucrose and trehalose.The inventors unexpectedly discovered that the disaccharide is superior to the monosaccharide, polyhydric alcohol and amino acid in terms of compositional stability.

[0063] The medicinal formulation may also contain a tonicity regulator. The term "tonicity regulator," as used herein, refers to a pharmaceutically acceptable tonicity regulator for adjusting the tonicity of the formulation. The formulation may be hypotonic, isotonic, or hypertonic, preferably isotonic. Isotonicity typically refers to the relative osmotic pressure of a solution, often relative to the osmotic pressure of human serum. An isotonic formulation is a liquid or a liquid reconstituted from a solid form (e.g., from a lyophilized form) and refers to a solution having the same tonicity as any other solution to which it is compared (e.g., saline and serum). Suitable tonicity regulators include, but are not limited to, sodium chloride, potassium chloride, glycerol, and any component selected from the following: an amino acid, a saccharide, especially glucose.Tonicity regulators are typically used in amounts ranging from approximately 5 mM to approximately 500 mM. Among stabilizers and tonicity regulators, there is a group of compounds that can act in two ways, that is, they can act as both a stabilizer and a tonicity regulator. Examples of these include: saccharide, amino acid, polyhydric alcohol, cyclodextrin, polyglycol, and salt. An example of a saccharide that can act as both a stabilizer and a tonicity regulator is trehalose.

[0064] The dosage form may also contain an auxiliary substance such as a preservative, wetting agent, emulsifier, and dispersing agent. Prevention of the presence of microorganisms can be ensured through sterilization operations and by including various antibacterial and antifungal agents (e.g., methylparaben, trichlorobutanol, phenol, sorbic acid, etc.). The preservative is usually used in an amount of from about 0.001% to about 2% (w / v). The preservative includes, but is not limited to: ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methylparaben or propylparaben, and benzalkonium chloride.

[0065] The anti-PCSK9 antibody formulation of the present invention can be used for the treatment of heterozygous familial hypercholesterolemia or clinical atherosclerotic cardiovascular disease and for adults requiring additional reduction in low-density lipoprotein cholesterol (LDL-C), and can also be used as an adjuvant in cases of statin therapy intolerance. The anti-PCSK9 antibody can improve cardiovascular conditions through other mechanisms, including reducing inflammation and oxidative stress in atherosclerotic plaques and inhibiting prothrombotic pathways.

[0066] The anti-PCSK9 antibody drug formulation of the present invention can be administered intravenously (IV), subcutaneously (SC), or by any other parenteral administration route such as those known in the pharmaceutical field.

[0067] In view of its high stability, the medicinal composition according to the present invention can be administered subcutaneously without the need for using a filter (built-in filter).

[0068] The composition for in vivo administration must be sterile. This can be effectively achieved through an aseptic manufacturing process that does not constitute terminal sterilization.

[0069] The anti-PCSK9 antibody formulation of the present invention can be prepared by methods known in the art, such as ultrafiltration-diafiltration, dialysis, addition and mixing, reconstitution, and combinations thereof. Examples of the preparation of the formulation according to the present invention can be found below.

[0070] DETAILED DESCRIPTION

[0071] The embodiments of the present invention are described in detail below using examples. It will be understood by those skilled in the art that the following examples are intended only to illustrate the present invention and should not be construed as limiting the scope of the present invention. Examples in which specific technologies or conditions are not indicated are carried out in accordance with the technologies or conditions described in publications in the field (for example, see Molecular Cloning: A Laboratory Manual, authored by J. Sambrook et al., and translated by Huang Peitang et al., third edition, Science Press) or in accordance with the package insert. The reagents or devices used are commercially available conventional products unless their manufacturers are indicated.

[0072] Production Example 1. Production of humanized anti-PCSK9 antibody

[0073] 1. Construction of antibodies To produce the monoclonal antibody MAB1, the inventors constructed a series of antibody sequences based on the known PCSK9 protein sequence (NP 777596.2) and its three-dimensional crystal structure. Through extensive screening and analysis, the MAB1 antibody, which specifically binds to PCSK9, was ultimately obtained. The amino acid sequences of the variable regions of the heavy chain and light chain of the monoclonal antibody and their coding sequences are provided in SEQ ID NOs: 1-4 below.

[0075] The DNA sequence of the heavy chain VH of the constructed MAB1 is as follows (369 bp):

[0076] GAGGTGCAGCTGGTGGAGTCTGGAGGAGGCCTGGTGCAGCCCGGAAGATCTCTGAGACTGAGTTGCGCCGCTTCAGGATTCACCTTTAGCTCCTACAGCATGAACTGGGTGCGGCAGGCTCCTGGCAAGGGGCTGGAGTGGGTCTCCGGAATCTCTAGTTCAAGCTCCTACATTAGCTATG CAGACTCCGTCCAGGGAAGGTTCACCATCTCTCGCGATAACGGCAAGAACAGCCTGTATCTGCAGATGAACAGCCTGCGAGCAGAGGACACAGCCCTGTACTTCTGTGCCAGAGAATATGACTTCTGGTCCGCCTATTACGACGCCTTCGATGTCTGGGGACAGGGGACTATGGTCACTGTCTCAAGC (SEQ ID NO: 7)

[0077] The VH protein sequence encoded by the above DNA sequence is as follows (123 aa):

[0078] EVQLVESGGGLVQPGRSLRLSCAASGFFSSYSMNWVRQAPGKGLEWVSGISSSSSYISYADSVQGRFTISRDNGKNSLYLQMNSLRAEDTALYFCAREYDFWSAYYDAFDVWGQGTMVTVSS(SEQ ID NO: 8)

[0079] The DNA sequence of the VL light chain of MAB1 is as follows (333 bp):

[0080] CAGAGCGAACTGACTCAGCCAAGAAGCGTCAGTGGATCACCTGGCCAGAGCGTGACAATCTCCTGCACCGGCACAAGCAGGAACATTGGCGGGGGAAAATGACGTCCACTGGTACCAGCAGCATCCAGGGAAGGCCCCCAAACTGCTGATCTCCGGAGTGATTG AGCGGAGCTCCGGCGTCCCCGATAGATTCAGCGGGTCCAAGTCTGGAAACACAGCTTCTCTGACTATCAGTGGCCTGCAGGCAGAGGACGAAGCCGATTACTATTGCCAGTCTTTCGACGGCAGTCTGTCAGGGAGCGTGTTTGGCACTGGGACCGATGTGACCGTCCTG (SEQ ID NO: 9)

[0081] The VL protein sequence encoded by the above DNA sequence is as follows (111 aa):

[0082] QSELTQPRSVSGSPGQSVTISCTGTSRNIGGGNDVHWYQQHPGKAPKLLISGVIERSSGVP DRFSGSKSGNTASLTISGLQAEDEADYYCQSFDGSLSGSSVFGTGTDVTVL (SEQ ID NO: 10)

[0083] The CDR sequences of the antibodies are as follows:

[0084] HCDR1: GFTFSSYS (SEQ ID NO: 1);

[0085] HCDR2: ISSSSSYI (SEQ ID NO: 2);

[0086] HCDR3: AREYDFWSAYYDAFDV (SEQ ID NO: 3);

[0087] LCDR1: SRNIGGGND (SEQ ID NO: 4);

[0088] LCDR2: GVI (SEQ ID NO: 5); And

[0089] LCDR3: QSFDGSLSGSV (SEQ ID NO: 6).

[0090] 2. Expression and purification of antibodies

[0091] The cDNA sequence of the heavy chain (the coding sequence of VH is listed as SEQ ID NO: 7; the constant region was the C region of the Ig gamma-1 chain, accession number: P01857) and the cDNA sequence of the light chain (the coding sequence of VL is listed as SEQ ID NO: 9; the constant region was the C region of the Ig lambda-2 chain; accession number: P0CG05.1) of MAB1 were cloned separately into pUC57simple vectors (provided by GenScript) to obtain plasmids pUC57simple-MAB1H and pUC57simple-MAB1L, respectively. Plasmids pUC57simple-MAB1H and pUC57simple-MAB1L were digested with HindIII and EcoRI, and the heavy and light chains, isolated by electrophoresis, were separately subcloned into pcDNA3.1 vectors. The recombinant plasmids were extracted for co-transfection of 293F cells. After cell culturing for 7 days, the culture solution was centrifuged at high speed. The supernatant was concentrated and loaded onto a HiTrap MabSelect SuRe column.The protein was eluted in a single step using elution buffer. The target sample was isolated, and the buffer was exchanged with PBS to yield the anti-PCSK9 antibody. In this example, the MAB1 antibody is designated as the anti-PCSK9 antibody.

[0092] Example 1: The process of obtaining the composition

[0093] The composition of the anti-PCSK9 antibody formulation was as follows: the formulation consisted of a solute and a solvent, wherein the solute was an anti-PCSK9 antibody, histidine, histidine hydrochloride, sucrose, trehalose, and polysorbate 80, and the solvent was water for injection; and the concentration of the anti-PCSK9 antibody in the formulation was 100 mg / mL, the concentration of the histidine in the formulation was 4.2 mM, the concentration of the histidine hydrochloride in the formulation was 10.8 mM, the concentration of sucrose in the formulation was 60 mg / mL, the concentration of trehalose in the formulation was 30 mg / mL, the concentration of polysorbate 80 in the formulation was 0.05% (w / v), and the pH value was 5.5.

[0094] When formulating the anti-PCSK9 antibody, the non-ionic surfactant polysorbate 80 was added to the solution last, and then the volume was determined. The addition of other solutes was performed out of order. The anti-PCSK9 antibody formulation was aseptically repackaged into a penicillin vial and sealed with a rubber stopper and aluminum-plastic cap, or the anti-PCSK9 antibody formulation was aseptically repackaged into a liquid storage bag (Sartorius Flexboy) to obtain the finished formulation product.

[0095] Example 2: Stability Experiment of Anti-PCSK9 Antibody Formulation

[0096] The stability experiment was performed on the anti-PCSK9 antibody formulation obtained according to Example 1. The stability of the formulation under high temperature, light, freeze-thaw, stirring and other conditions was examined using SEC-HPLC size exclusion chromatography, icIEF capillary isoelectric focusing electrophoresis, CE-SDS with reduction, CE-SDS without reduction, observation of the activity and appearance of the formulation to characterize the chemical stability of the anti-PCSK9 antibody in the formulation under various environmental conditions separately, using the change in the percentage of the main component and the content of the main peak as methods for determining the purity of the protein.

[0097]

[0098] The anti-PCSK9 antibody obtained in Production Example 1 was analyzed by SEC-HPLC size exclusion chromatography, icIEF capillary isoelectric focusing electrophoresis, CE-SDS with reduction, CE-SDS without reduction, and activity assay (ELISA) under the following experimental conditions: repeated freezing and thawing at -20°C / 25°C for 3 cycles; stirring at 300 rpm for 3 days; and storing at 40°C for 30 days; and the results of change in protein content, change in protein purity, and change in charge inhibitor purity upon storage under illumination of 4500±500 lux for 15 days are shown in Tables 2-1-4.

[0099] According to the results in Table 2-1, the change in protein content and protein purity of the anti-PCSK9 antibody formulation decreased with increasing storage time at a high temperature of 40°C. When measured by SE-HPLC on day 30, the main peak was 92.3%, and the antibody monomer was degraded by no more than 10%. In addition, a light yellow transparent liquid was observed by visual analysis, which was consistent with the observation under normal conditions. The protein concentration of the formulation was 98.2 mg / mL, no more than ±10% of the protein concentration under normal conditions. It was shown that the formulation was still a stable liquid antibody formulation after storage for 30 days at a high temperature of 40°C.

[0100] According to the results in Table 2-2, the protein content and protein purity of the anti-PCSK9 antibody formulation did not change significantly after freeze-thaw treatment. It was shown that the formulation remained a stable liquid antibody formulation after three freeze-thaw cycles.

[0101] According to the results in Table 3, the change in protein content and protein purity of the anti-PCSK9 antibody formulation decreased with increasing storage time under light. In the SE-HPLC measurement on day 15, the main peak was 94.4%, and the antibody monomer was degraded by no more than 10%. In addition, a light yellow transparent liquid was detected by visual analysis, which was consistent with observations under normal conditions. The protein concentration in the formulation was 97.3 mg / mL, no more than ±10% of the protein concentration under normal conditions. It was indicated that the formulation was still a stable liquid antibody formulation after storage for 15 days under light.

[0102] According to the results in Table 4, the protein content and protein purity of the anti-PCSK9 antibody formulation did not change significantly after stirring. It was shown that the formulation remained a stable liquid antibody formulation after stirring at 300 rpm for 3 days.

[0103] Summary: The results in Tables 2-1-4 show that the formulation has sufficient stability under the above storage conditions. It is shown that under various conditions that accelerate the chemical degradation and physical changes of the anti-PCSK9 antibody, the formulation can help reduce the chemical degradation rate of the contained anti-PCSK9 antibody and improve the physicochemical stability of the anti-PCSK9 antibody, so that the monoclonal antibody can stably exist in this formulation.

[0104] Example 3. Comparison of different formulations and pH

[0105] (1) The purity of the formulations at different times was tested by the SEC-HPLC method using different compositions of the formulations shown in Table 5, respectively, and the results are shown in Table 6. AK102 refers to the anti-PCSK9 antibody MAB1 antibody at a concentration of 100 mg / mL.

[0106]

[0107] As can be seen from Table 6, all the formulations in the range of component contents of the present invention provided good stability.

[0108] (2) The pH of a solution is one of the most important parameters affecting the physicochemical stability of biopharmaceuticals. pH can regulate the charge distribution on the protein surface, thereby influencing the intramolecular and intermolecular forces of biopharmaceuticals and simultaneously influencing the degree of chemical modification of antibodies. This is one of the most important properties of biopharmaceutical formulations.

[0109] According to the compositions of the formulations in Table 7, Tm and high temperature degradation tests were conducted to identify the differences in stability of different formulations.

[0110]

[0111] (2.1) Study of Tm value

[0112] The protein melting temperature (Tm) reflects the thermal stability of biopharmaceuticals in aqueous solutions. The higher the Tm, the lower the probability of conformational changes under actual storage conditions. The Tm values ​​of the anti-PCSK9 antibody in buffer at different pH values ​​were studied. The formulations in Table 8 were prepared using 5 μL of the anti-PCSK9 antibody in the formulations in Table 7 and with the addition of 5 μL of SYPRO Orange (20X) and 90 μL of buffer, respectively. The Tm value of the sample was determined using a method similar to differential scanning fluorimetry (DSF). The results are presented in Table 8.

[0113]

[0114] (2.2) High-temperature accelerated test study on specimens of different compositions

[0115] The formulations with different compositions shown in Table 7 were placed in a sample testing chamber at 40±2°C for accelerated damage for 3 days, and the stability of different formulations was examined by appearance and SEC-HPLC.

[0116] The detection results are summarized in Table 9 and Table 10.

[0117]

[0118] According to the Tm measurement results and the high-temperature accelerated test results, only the F8 compound remained transparent after exposure to accelerated conditions, while the other compounds were cloudy, which was unsuitable for preserving AK102. Meanwhile, the SEC-HPLC purity analysis showed that the purity of the F8 compound did not change significantly after 3 days of accelerated damage. It was preliminarily estimated that AK102 had good stability under the conditions of a polyhydroxyl protective agent and pH 5.5. Based on this result, the polyhydroxyl structural protective agent was screened and optimized.

[0119] Screening and optimization of protective equipment

[0120] According to the common protective agents for antibody drugs, three formulations shown in Table 11 were developed, samples of different formulations were placed under the condition of 40±2°C for stability study, and the effects of different protective agents on the stability of AK102 were compared to select the optimal formulation.

[0121]

[0122] After the samples were damaged at 40±2°C, they were collected at 1 week, 2 weeks, 4 weeks, and 8 weeks for SEC-HPLC and CE-SDS analysis, and the changes in the three compositions were compared. The detection results are presented in Tables 12 and 13.

[0123]

[0124] Summary: At week 8 of acceleration, no corresponding detection analysis was performed for formulation F11. According to the SEC-HPLC and CE-SDS results, the stability of F12 (trehalose) was significantly better than that of formulation F13 (sorbitol), and the results suggested that AK102 has better stability in the trehalose formulation.

[0125] Based on process cost, a mixture of sucrose and trehalose was selected as the preservative for AK102, and the mixing ratio was studied. Mixed saccharide preservative formulations with different ratios were developed, as shown in Table 14. The concentration of AK102 samples in all formulations was 100 mg / mL, which was the same as the final proposed concentration in the packaging.

[0126]

[0127] Example 4. Analysis of the stability of various formulations at different points in time

[0128] Different compositions were made according to Table 16

[0129]

[0130] 1 L of ultrafiltration buffer (without polysorbate 80 and antibody) for each of the F1-19 samples was prepared according to the compositions in Table 16, and 1.9 mL of 6 M hydrochloric acid was added. The protein solutions in Table 16 were prepared using an ultrafiltration system, and 10% polysorbate 80 (II) solution was added to the corresponding samples in Table 16. Approximately 90 mL of each composition was obtained.

[0131] Filtration was performed using a 0.22 μm membrane filter. A total of 30 samples (1.5 mL / sample) of each formulation were obtained. 10 samples of each formulation were sent for detection at time T0 (T0 refers to the time point for direct testing after formulation), and 20 samples were placed in a stability test chamber under accelerated test conditions at 25°C (25°C 3M refers to aging for 3 months at 25°C). See Table 17 for the parameters to be determined. The detection results are presented in Tables 18-1, 18-2 (detection results at T0) and Tables 18-3 and 18-4 (detection results at 25°C 3M).

[0132]

[0133]

[0134] According to the results in Table 18, the protein purity of the anti-PCSK9 antibody formulations within the formulation composition range shown in Table 16 decreased with increasing storage time at a high temperature of 25°C. When detected by SE-HPLC after 3 months, all major peaks were above 96%, and the antibody monomer was degraded by no more than 10%. In addition, a light yellow transparent liquid was observed upon visual analysis, which was consistent with that under normal conditions. It was shown that after 30 days of storage at 25°C, the formulation still represented a stable liquid antibody formulation.

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[0412] < / insdfeature>

[0413] < / INSDSeq_feature-table>

[0414] <INSDSeq_sequence>ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT

[0415] FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS

[0416] VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL

[0417] HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV

[0418] EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK< /

[0419] INSDSeq_sequence>

[0420] < / insdseq>

[0421] < / sequencedata>

[0422] <sequencedata sequenceidnumber="12">

[0423] <insdseq>

[0424] <INSDSeq_length>106< / INSDSeq_length>

[0425] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0426] <INSDSeq_division>PAT< / INSDSeq_division>

[0427] <INSDSeq_feature-table>

[0428] <insdfeature>

[0429] <INSDFeature_key>source< / INSDFeature_key>

[0430] <INSDFeature_location>1..106< / INSDFeature_location>

[0431] <INSDFeature_quals>

[0432] <insdqualifier>

[0433] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0434] <INSDQualifier_value>protein< / INSDQualifier_value>

[0435] < / insdqualifier>

[0436] <insdqualifier id="q14">

[0437] <INSDQualifier_name>organism< / INSDQualifier_name>

[0438] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0439] < / insdqualifier>

[0440] < / INSDFeature_quals>

[0441] < / insdfeature>

[0442] < / INSDSeq_feature-table>

[0443] <INSDSeq_sequence>GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAG

[0444] VETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS< / INSDSeq_sequen

[0445] ce>

[0446] < / insdseq>

[0447] < / sequencedata>

[0448]

[0449] <---

Claims

1. A stable formulation for storing an anti-PCSK9 antibody, comprising an anti-PCSK9 antibody or an antigen-binding fragment thereof, a buffer, a stabilizer, and a surfactant, wherein the anti-PCSK9 antibody comprises a heavy chain variable region as indicated by SEQ ID NO: 8, and the antibody comprises a light chain variable region as indicated by SEQ ID NO: 10, and the antigen-binding fragment is selected from a Fab fragment, a Fab' fragment, an F(ab)' fragment, a single-chain Fv molecule (scFv), F(ab')2, Fab / c, or a divalent antibody, a buffer consisting of 1-20 mM histidine and / or histidine hydrochloride, a stabilizer consisting of 90 mg / ml sucrose or trehalose, or sucrose and trehalose in a total concentration of 85-95 mg / ml, a surfactant which is polysorbate 80, and the content of polysorbate 80 is 0.02-0.08% (w / w), and wherein the anti-PCSK9 antibody composition has a pH of 5.0-6.

0.

2. The composition of the anti-PCSK9 antibody according to claim 1, wherein the anti-PCSK9 antibodies comprise the constant region of the Ig heavy chain gamma-1 chain C under the accession number: P01857, represented by SEQ ID NO: 11, and the constant region of the Ig light chain lambda-2 chain C under the accession number: P0CG05.1, represented by SEQ ID NO:

12.

3. The composition of the anti-PCSK9 antibody according to claim 1 or 2, wherein the anti-PCSK9 antibody composition has a pH of 5.3-6.0, or the buffer is 15-20 mM histidine and / or histidine hydrochloride; or when sucrose and trehalose are used simultaneously, their total concentration is 90 mg / ml, or the surfactant content is 0.04-0.06% (w / w), or the composition of the antibody against PCSK9 additionally contains a preservative, or the composition of the anti-PCSK9 antibody additionally contains antibacterial and antifungal agents, or the PCSK9 antibody composition is in a form suitable for injection, or the concentration of the antibody against PCSK9 or its antigen-binding fragment is 10-150 mg / ml, or additionally contains water for injection.

4. The composition of the anti-PCSK9 antibody according to claim 2, wherein the anti-PCSK9 antibody composition has a pH of 5.2-5.8, or the histidine content is 1-7.4 mM, or the histidine hydrochloride content is 5-16.6 mM, or the surfactant content is 0.05% (w / w), or the preservative is used in an amount of 0.001 to 2% (w / v), or the composition of the antibody against PCSK9 additionally contains chlorobutanol and sorbic acid, or the anti-PCSK9 antibody composition is in a form suitable for subcutaneous administration, or the concentration of antibodies against PCSK9 or their antigen-binding fragment is 50-150 mg / ml.

5. The composition of the anti-PCSK9 antibody according to claim 4, wherein the anti-PCSK9 antibody formulation has a pH of 5.5, or the histidine content is 3-5.4 mM, or the histidine hydrochloride content is 8-13.6 mM, or the preservative is selected from ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methylparaben, propylparaben or benzalkonium chloride, or the concentration of antibodies against PCSK9 or their antigen-binding fragment is 80-120 mg / ml.

6. The composition of the anti-PCSK9 antibody according to claim 2, wherein the histidine content is 4-4.4 mM, or the histidine hydrochloride content is 10-11.6 mM, or the concentration of antibodies against PCSK9 or their antigen-binding fragment is 90-110 mg / ml.

7. The composition of the anti-PCSK9 antibody according to any one of claims 1-5, wherein the histidine content is 4.2 mM, and the histidine hydrochloride content is 10.8 mM; or the PCSK9 antibody or its antigen-binding fragment has a concentration of 100 mg / ml.

8. The anti-PCSK9 antibody formulation of any one of claims 1-5, wherein the anti-PCSK9 antibody formulation comprises 100 mg / ml anti-PCSK9 antibody, 4.2 mM histidine, 10.8 mM histidine hydrochloride, 60 mg / ml sucrose, 30 mg / ml trehalose, and 0.05% (w / w) polysorbate 80, pH 5.

5.

9. A method for producing a composition according to any one of claims 1-8, comprising mixing an antibody against PCSK9 or an antigen-binding fragment thereof with a buffer comprising 1-20 mM histidine and / or histidine hydrochloride, a stabilizer comprising 90 mg / ml sucrose or trehalose, or sucrose and trehalose at a total concentration of 85-95 mg / ml, and a surfactant comprising polysorbate 80, wherein the content of polysorbate 80 is 0.02-0.08% (w / w), and adjusting the pH of the composition with an acid or base to 5.0-6.

0.

10. The method of claim 9, wherein the surfactant is polysorbate 80 and the surfactant is added last and then the volume is determined, or the pH of the composition is adjusted with an acid or alkali to a value of 5.5.

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