Anti-SOST antibody pharmaceutical composition and use thereof

Abstract

The present invention relates to an anti-SOST antibody pharmaceutical composition and uses thereof, particularly to a pharmaceutical composition comprising an anti-SOST antibody and an antioxidant, and uses thereof for treating a disease or condition associated with SOST.

Description

[Technical field]

[0001] The present disclosure is in the field of pharmaceutical formulations, and specifically relates to pharmaceutical compositions comprising anti-SOST antibodies and their use as medicines. [Background technology]

[0002] The statements herein are not necessarily intended to constitute prior art, but merely to provide background information relevant to the present disclosure.

[0003] Osteoporosis (OP), which includes postmenopausal osteoporosis (PMO) and senile osteoporosis, is a systemic bone metabolic disorder characterized by low bone mass and deterioration of bone microarchitecture, leading to decreased bone strength and increased bone fragility, and making bones susceptible to fractures.

[0004] Sclerostin (SOST) is a new biological target that has been used in drug development to treat osteoporosis by regulating the synthesis metabolism of osteoblasts. Sclerostin is a glycoprotein expressed and secreted by the SOST gene, and its amino acid sequence structure is characterized by a cyclic domain with 190 residues and cysteine. SOST is mainly expressed in bone cells, but its expression level in osteoblasts, cartilage, liver, kidney, bone marrow, heart, pancreas, etc. is very low. Research has shown that sclerostin inhibits Wnt signaling by binding to the low-density lipoprotein receptor LRP5 / 6, thereby regulating bone formation. Currently, monoclonal antibody drugs developed against this target have already entered clinical research, and all clinical data have demonstrated the safety and efficacy of this target in treating osteoporosis by regulating bone metabolism.

[0005] WO2016145961A1 and other publications disclose that anti-SOST antibodies are used to treat osteoporosis. However, because antibody drugs have large molecular weights and complex structures, research into stable formulations of antibody drugs is particularly important in order to achieve the therapeutic effects of antibodies. Summary of the Invention

[0006] The researchers of the present disclosure, through a large amount of research, have found that under the conditions of the anti-SOST antibody formulation disclosed in WO2019020069A1 (formulation method of the formulation: 10 mM acetic acid-sodium acetate buffer at pH 5.0, 100 mg / mL Ab-5 antibody, 80 mg / mL α,α-trehalose dihydrate, 0.4 mg / mL polysorbate 80, 4.5 mM calcium chloride) being left for a long period of time, a part of the Ab-5 antibody is cleaved at the fourth amino acid residue of the heavy chain HCDR3 (shown in SEQ ID NO: 5) to produce a fragment, which further affects the binding of the Ab-5 antibody to the antigen. The researchers of the present disclosure, through a large amount of research, have surprisingly found that the cleavage at the HCDR3 region of the Ab-5 antibody can be effectively reduced by adding an antioxidant to the above formulation. Therefore, the present disclosure provides an anti-SOST antibody pharmaceutical composition having the property of better preventing the cleavage at the HCDR3 region of the Ab-5 antibody and better stability. Specifically, The present disclosure provides a pharmaceutical composition comprising an anti-SOST antibody comprising an antibody heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an HCDR1 set forth in SEQ ID NO:3, an HCDR2 set forth in SEQ ID NO:9 or 4, and an HCDR3 set forth in SEQ ID NO:5, and the light chain variable region comprises an LCDR1 set forth in SEQ ID NO:6, an LCDR2 set forth in SEQ ID NO:7, and an LCDR3 set forth in SEQ ID NO:8, and an antioxidant.

[0007] In some embodiments of the pharmaceutical composition, the heavy chain variable region of the anti-SOST antibody comprises an HCDR1 set forth in SEQ ID NO:3, an HCDR2 set forth in SEQ ID NO:9, and an HCDR3 set forth in SEQ ID NO:5, and the light chain variable region comprises an LCDR1 set forth in SEQ ID NO:6, an LCDR2 set forth in SEQ ID NO:7, and an LCDR3 set forth in SEQ ID NO:8.

[0008] In some embodiments of the pharmaceutical composition, the anti-SOST antibody may be selected from a murine antibody, a chimeric antibody, and a humanized antibody, and in some embodiments, the anti-SOST antibody is a humanized antibody.

[0009] In some embodiments of the pharmaceutical composition, the heavy chain variable region of the anti-SOST antibody comprises the amino acid sequence of SEQ ID NO: 10, 11, or 12, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13, 14, or 15. In some embodiments, the heavy chain variable region of the anti-SOST antibody comprises the amino acid sequence of SEQ ID NO: 10, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13.

[0010] In some embodiments of the pharmaceutical composition, the light chain amino acid sequence of the anti-SOST antibody has at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the light chain of the Ab-5 antibody (SEQ ID NO:25), and the heavy chain amino acid sequence of the anti-SOST antibody has at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the Ab-5 antibody heavy chain (SEQ ID NO:22), in some embodiments, the heavy chain of the anti-SOST antibody comprises the amino acid sequence of SEQ ID NO:22, and the light chain comprises the amino acid sequence of SEQ ID NO:25.

[0011] In some embodiments of the pharmaceutical composition, the antioxidant is a free radical scavenger (e.g., ascorbic acid, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), sodium sulfite, para-aminobenzoic acid, glutathione, propyl gallate), a chelating agent (e.g., ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), nitrilotriacetic acid (NTA), N-2-acetamido-2-iminodiacetic acid (ADA), ethylene glycol bis(aminoethyl)ether, N,N,N',N'-tetraacetic acid (EGTA), transdiaminocyclohexanetetraacetic acid (DCTA), glutathione, propyl gallate), The glyceryl ester may be selected from the group consisting of cis- and aspartic acid, N-hydroxyethyliminodiacetic acid (HIMDA), N,N-bis-hydroxyethylglycine (bicine) and N-tris(hydroxymethyl)methylglycine (tricine), glycylglycine, sodium deoxycholate, ethylenediamine, propylenediamine, diethylenetriamine, triethylenetetramine (trien), EDTA disodium, EDTA calcium oxalate, malate, citric acid, citric acid monohydrate, trisodium citrate dihydrate, 8-hydroxyquinoline salts, amino acids, chain terminators (e.g., methionine, cysteine), and the like. In some embodiments, the antioxidant is selected from methionine, histidine, arginine, EDTA, citric acid, ascorbic acid, butylated hydroxytoluene, butylated hydroxyanisole, sodium sulfite, para-aminobenzoic acid, glutathione, propyl gallate, ethanol, and benzyl alcohol; in some embodiments, the antioxidant is selected from methionine, histidine, and arginine; in some embodiments, the antioxidant is methionine.

[0012] In some embodiments of the pharmaceutical composition, the concentration of the antioxidant is 1 to 100 mM. In some embodiments, the concentration of the antioxidant is 5 to 50 mM. In some embodiments, the concentration of the antioxidant is 5 to 25 mM. In some embodiments, the concentration of the antioxidant is 10 to 25 mM. In some embodiments, the concentration of the antioxidant is 5 to 15 mM. In some embodiments, the concentration of the antioxidant is about 10 mM. In some embodiments, the concentration of the antioxidant is about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 18 mM, about 20 mM, about 22 mM, about 24 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, or about 100 mM, or any range between these point values. The above "about" most preferably means a range of ±10%, and for example, when the concentration of the antioxidant is about 10 mM, the concentration of the antioxidant is 10 mM ±1 mM.

[0013] In some embodiments of the pharmaceutical composition, the concentration of the anti-SOST antibody is 1 to 300 mg / mL, in some embodiments, the concentration of the anti-SOST antibody is 60 to 250 mg / mL, in some embodiments, the concentration of the anti-SOST antibody is 80 to 200 mg / mL, in some embodiments, the concentration of the anti-SOST antibody is 80 to 180 mg / mL, in some embodiments, the concentration of the anti-SOST antibody is 80 to 150 mg / mL, in some embodiments, the concentration of the anti-SOST antibody is 50 to 200 mg / mL, and in some embodiments, the concentration of the anti-SOST antibody is 50 to 150 mg / mL. L, and in some embodiments, the concentration of the anti-SOST antibody is 100-200 mg / mL, and in some embodiments, the concentration of the anti-SOST antibody is 100-150 mg / mL, and in some embodiments, the concentration of the anti-SOST antibody is 60-120 mg / mL, and in some embodiments, the concentration of the anti-SOST antibody is 80-120 mg / mL, and in some embodiments, the concentration of the anti-SOST antibody is 90-110 mg / mL, and in some embodiments, the concentration of the anti-SOST antibody is about 100 mg / mL, and in some embodiments, the concentration of the anti-SOST antibody is about 150 mg / mL.In some embodiments, the concentration of the anti-SOST antibody is about 1 mg / mL, about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 85 mg / mL, about 90 mg / mL, about 91 mg / mL, about 92 mg / mL, about 93 mg / mL, or about 100 mg / mL. L, approx. 94 mg / mL, approx. 95 mg / mL, approx. 96 mg / mL, approx. 97 mg / mL, approx. 98 mg / mL, approx. 99 mg / mL, approx. 100 mg / mL, approx. 101 mg / mL, approx. 102 mg / m L, about 103 mg / mL, about 104 mg / mL, about 105 mg / mL, about 106 mg / mL, about 107 mg / mL, about 108 mg / mL, about 109 mg / mL, about 110 mg / mL, about 1 15mg / mL, approx. 120mg / mL, approx. 125mg / mL, approx. 130mg / mL, approx. 135mg / mL, approx. 140mg / mL, approx. 145mg / mL, approx. 150mg / mL, approx. 155mg / mL, approximately 160mg / mL, approximately 165mg / mL, approximately 170mg / mL, approximately 175mg / mL, approximately 180mg / mL, approximately 185mg / mL, approximately 190mg / mL, approximately 195mg / mL, The concentration of the protein is about 200 mg / mL, about 205 mg / mL, about 210 mg / mL, about 215 mg / mL, about 220 mg / mL, about 225 mg / mL, about 230 mg / mL, about 240 mg / mL, about 250 mg / mL, about 260 mg / mL, about 270 mg / mL, about 280 mg / mL, about 290 mg / mL, or about 300 mg / mL, or any range between these point values. The above about is most preferably within a range of ±10%, for example, if the protein concentration is about 100 mg / mL, it will be 100 mg / mL ±10 mg / mL.

[0014] In some embodiments of the pharmaceutical composition, the pH of the pharmaceutical composition is 4.8 to 5.5, in some embodiments, the pH of the pharmaceutical composition is 5.0 to 5.5, in some embodiments, the pH of the pharmaceutical composition is 4.8 to 5.2, in some embodiments, the pH of the pharmaceutical composition is 5.0 to 5.2, in some embodiments, the pH of the pharmaceutical composition is about 5.0. In some embodiments, the pH of the pharmaceutical composition is about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, or about 5.5, or any range between these point values. The "about" in this pH value is 0.05, for example, about 5.0 is 5.0±0.05.

[0015] In some embodiments of the pharmaceutical composition, the pharmaceutical composition further comprises a buffer, and in some embodiments, the buffer is selected from an acetate buffer, a histidine buffer, a phosphate buffer, and a succinate buffer, and in some embodiments, the buffer is selected from an acetate buffer or a histidine buffer, and in some embodiments, the buffer is selected from an acetate buffer, and in some embodiments, the buffer is an acetic acid-sodium acetate buffer. In some embodiments, the buffer concentration is 1-30 mM, in some embodiments, the buffer concentration is 5-20 mM, in some embodiments, the buffer concentration is 10-20 mM, in some embodiments, the buffer concentration is about 10 mM, in some embodiments, the buffer concentration is about 1 mM, about 5 mM, about 10 mM, about 12 mM, about 14 mM, about 15 mM, about 16 mM, about 18 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM, or any range between these point values. The above about is most preferably within a range of ±10%, for example, when the acetic acid-sodium acetate buffer concentration is about 10 mM, it is 10 mM ± 1 mM.

[0016] In some embodiments of the pharmaceutical composition, the pharmaceutical composition further comprises a sugar, in some embodiments the sugar is selected from a monosaccharide, a disaccharide, a trisaccharide, a polysaccharide, a sugar alcohol, in some embodiments the sugar is selected from a reducing sugar and a non-reducing sugar, in some embodiments the sugar is selected from glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol, mannitol, melibiose. , melezitose, raffinose, manninotriose, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol, iso-maltulose; in some embodiments, the sugar is selected from non-reducing disaccharides; in some embodiments, the sugar is selected from trehalose or sucrose; in some embodiments, the sugar is selected from trehalose (e.g., α,α-trehalose dihydrate); in some embodiments, the sugar is selected from sucrose. In some embodiments, the sugar concentration is 40-95 mg / mL, in some embodiments, the sugar concentration is 60-90 mg / mL, in some embodiments, the sugar concentration is about 75 mg / mL, in some embodiments, the sugar concentration is about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 55 mg / mL, about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 85 mg / mL, about 90 mg / mL or about 95 mg / mL, or any range between these point values. The above about is most preferably within a range of ±10%, for example, when the trehalose or sucrose concentration is about 75 mg / mL, it is 75 mg / mL ±7.5 mg / mL.

[0017] In some embodiments, the sugar is an osmolality adjusting agent, in some embodiments, the osmolality of the pharmaceutical composition is 270-330 mOsm, in some embodiments, the osmolality of the pharmaceutical composition is 280-320 mOsm, in some embodiments, the osmolality of the pharmaceutical composition is controlled to 290-310 mOsm, in some embodiments, the osmolality of the pharmaceutical composition is about 270 mOsm, about 280 mOsm, about 290 mOsm, about 295 mOsm, about 300 mOsm, about 305 mOsm, about 310 mOsm, about 320 mOsm or about 330 mOsm, and any range between these point values. In some embodiments, the osmolality of the pharmaceutical composition is about 300 mOsm.

[0018] In some embodiments of the pharmaceutical composition, the pharmaceutical composition further comprises a viscosity modifier, in some embodiments, the viscosity modifier is selected from calcium salts, sodium chloride, magnesium chloride, and arginine hydrochloride, in some embodiments, the viscosity modifier is selected from calcium chloride or calcium acetate, in some embodiments, the viscosity modifier is selected from calcium chloride. In some embodiments, the viscosity modifier has a concentration of 1 to 150 mM, in some embodiments, the viscosity modifier has a concentration of 1 to 20 mM, in some embodiments, the viscosity modifier has a concentration of 1 to 10 mM, in some embodiments, the viscosity modifier has a concentration of 2 to 10 mM, in some embodiments, the viscosity modifier has a concentration of 3 to 6 mM, in some embodiments, the viscosity modifier has a concentration of 4.5 to 20 mM, in some embodiments, the viscosity modifier has a concentration of 4.5 to 10 mM, and in some embodiments, the viscosity modifier has a concentration of about 4.5 mM. In some embodiments, the viscosity modifier concentration is about 1 mM, about 2 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about 30 mM, about 60 mM, about 90 mM, or about 150 mM, or any range between these points. Most preferably, the above "about" refers to a range of ±10%, e.g., when the calcium chloride or calcium acetate concentration is about 4.5 mM, it is 4.5 mM ±0.45 mM.

[0019] In some embodiments of the pharmaceutical composition, the pharmaceutical composition further comprises a surfactant. In some embodiments, the surfactant is a non-ionic surfactant, and in some embodiments, the surfactant is a poloxamer (e.g., poloxamer 188), polysorbate (e.g., polysorbate 20, polysorbate 80), poloxamer, Triton, sodium dodecyl sulfonate, sodium lauryl sulfonate, sodium octyl glucoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleic-sulfobetaine, stearic-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleic-sarcosine, stearic-sarcosine, linoleic-betaine, myristyl-betaine, cetyl-betaine, lauramidoprotease ... In some embodiments, the surfactant is selected from the group consisting of propyl-betaine, cocamidopropyl-betaine, linoleamidopropyl-betaine, myristamidopropyl-betaine, palmitamidopropyl-betaine, isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmitamidopropyl-dimethylamine, isostearamidopropyl-dimethylamine, sodium methyl cocoyl, sodium methyl oleyl taurate, polyethylene glycol, polypropylene glycol, copolymers of ethylene and propylene glycol, and the like; in some embodiments, the surfactant is a polysorbate or poloxamer; in some embodiments, the surfactant is polysorbate 80. In some embodiments, the concentration of the surfactant is 0.02-0.8 mg / mL, in some embodiments, the concentration of the surfactant is 0.2-0.6 mg / mL, in some embodiments, the concentration of the surfactant is 0.3-0.6 mg / mL, in some embodiments, the concentration of the surfactant is about 0.4 mg / mL, in some embodiments, the concentration of the surfactant is about 0.02 mg / mL, about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, or about 0.8 mg / mL, or any range between these point values.The above "about" most preferably means a range of ±10%. For example, when the polysorbate 80 concentration is about 0.4 mg / mL, the range is 0.4 mg / mL ±0.04 mg / mL.

[0020] In some embodiments of the pharmaceutical composition, the pharmaceutical composition comprises (a) 1 to 300 mg / mL of an anti-SOST antibody, preferably 50 to 200 mg / mL of an anti-SOST antibody, (b) 1 to 100 mM of methionine, histidine or arginine, (c) 40 to 95 mg / mL of trehalose or sucrose, (d) 1 to 30 mM, preferably 5 to 20 mM of an acetate buffer or histidine buffer, (e) 1 to 20 mM, preferably 4.5 to 20 mM, more preferably 2 to 10 mM, and most preferably 3 to 6 mM of a calcium salt, and (f) 0.02 to 0.8 mg / mL of a polysorbate, and the pH of the pharmaceutical composition is 4.8 to 5.5.

[0021] In some embodiments, the pharmaceutical composition comprises: (a) 1 to 300 mg / mL of the anti-SOST antibody; (b) 1 to 100 mM of methionine, histidine, or arginine; (c) 40 to 95 mg / mL of trehalose or sucrose; (d) 1 to 30 mM of acetate buffer or histidine buffer; (e) 4.5 to 20 mM of a calcium salt; and (f) 0.02 to 0.8 mg / mL of polysorbate, and the pH of the pharmaceutical composition is 4.8 to 5.5.

[0022] In some embodiments of the pharmaceutical composition, the pharmaceutical composition comprises (a) 1 to 150 mg / mL of an anti-SOST antibody, (b) 1 to 100 mM of methionine, histidine, or arginine, (c) 40 to 95 mg / mL of trehalose or sucrose, (d) 1 to 30 mM of an acetate buffer or a histidine buffer, (e) 4.5 to 20 mM of a calcium salt, and (f) 0.02 to 0.8 mg / mL of a polysorbate, and the pH of the pharmaceutical composition is 4.8 to 5.5.

[0023] In some embodiments of the pharmaceutical composition, the pharmaceutical composition comprises: (a) 50 to 200 mg / mL of an anti-SOST antibody comprising a heavy chain of SEQ ID NO: 22 and a light chain of SEQ ID NO: 25; (b) 5 to 25 mM methionine; (c) 60 to 90 mg / mL of trehalose or sucrose; (d) 10 to 20 mM acetate buffer; (e) 4.5 to 10 mM calcium chloride; and (f) 0.3 to 0.6 mg / mL of polysorbate 80; and the pH of the pharmaceutical composition is 5.0 to 5.2.

[0024] In some embodiments of the pharmaceutical composition, the pharmaceutical composition contains (a) 50-150 mg / mL of an anti-SOST antibody comprising a heavy chain of SEQ ID NO: 22 and a light chain of SEQ ID NO: 25, (b) 5-25 mM methionine, (c) 60-90 mg / mL of trehalose or sucrose, (d) 5-20 mM acetate buffer, (e) 1-10 mM calcium chloride, and (f) 0.3-0.6 mg / mL of polysorbate 80, and has a pH of 4.8-5.2. The trehalose is preferably α,α-trehalose dihydrate.

[0025] In some embodiments of the pharmaceutical composition, the pharmaceutical composition comprises (a) 80 to 120 mg / mL of an anti-SOST antibody comprising a heavy chain of SEQ ID NO: 22 and a light chain of SEQ ID NO: 25, (b) 5 to 25 mM methionine, (c) 60 to 90 mg / mL of trehalose or sucrose, (d) 10 to 20 mM acetate buffer, (e) 4.5 to 10 mM calcium chloride, and (f) 0.3 to 0.6 mg / mL of polysorbate 80, and the pH of the pharmaceutical composition is 5.0 to 5.2.

[0026] In some embodiments of the pharmaceutical composition, the pharmaceutical composition comprises (a) 80 to 120 mg / mL of an anti-SOST antibody comprising a heavy chain of SEQ ID NO: 22 and a light chain of SEQ ID NO: 25, (b) 5 to 15 mM methionine, (c) 60 to 90 mg / mL of trehalose or sucrose, (d) 5 to 20 mM acetate buffer, (e) 1 to 10 mM calcium chloride, and (f) 0.3 to 0.6 mg / mL of polysorbate 80, and the pH of the pharmaceutical composition is 4.8 to 5.2.

[0027] In some embodiments of the pharmaceutical composition, the pharmaceutical composition comprises: (a) 80-100 mg / mL of an anti-SOST antibody comprising a heavy chain of SEQ ID NO:22 and a light chain of SEQ ID NO:25; (b) about 10 mM methionine; (c) 70-80 mg / mL of trehalose (e.g., α,α-trehalose dihydrate) or sucrose; (d) about 10 mM acetate buffer; (e) about 4.5 mM calcium chloride; and (f) 0.3-0.6 mg / mL of polysorbate 80; and the pH of the pharmaceutical composition is 4.8-5.2.

[0028] In some embodiments of the pharmaceutical composition, the pharmaceutical composition comprises: (a) 80-100 mg / mL of an anti-SOST antibody comprising a heavy chain of SEQ ID NO: 22 and a light chain of SEQ ID NO: 25; (b) about 10 mM methionine; (c) 70-80 mg / mL of trehalose (e.g., α,α-trehalose dihydrate) or sucrose; (d) about 10 mM acetate buffer; (e) about 4.5 mM calcium chloride; and (f) 0.3-0.6 mg / mL of polysorbate 80; and the pH of the pharmaceutical composition is 5.0-5.2.

[0029] In some embodiments of the pharmaceutical composition, the pharmaceutical composition comprises: (a) about 100 mg / mL of an anti-SOST antibody comprising a heavy chain of SEQ ID NO: 22 and a light chain of SEQ ID NO: 25; (b) about 10 mM methionine; (c) about 75 mg / mL of trehalose (e.g., α,α-trehalose dihydrate) or sucrose; (d) 10-20 mM acetic acid-sodium acetate buffer; (e) about 4.5 mM calcium chloride; and (f) about 0.4 mg / mL polysorbate 80, and the pH of the pharmaceutical composition is about 5.0.

[0030] In some embodiments of the pharmaceutical composition, the pharmaceutical composition comprises: (a) about 100 mg / mL of an anti-SOST antibody comprising a heavy chain of SEQ ID NO: 22 and a light chain of SEQ ID NO: 25; (b) about 10 mM methionine; (c) about 75 mg / mL of sucrose; (d) about 10 mM acetic acid-sodium acetate buffer; (e) about 4.5 mM calcium chloride; and (f) about 0.4 mg / mL of polysorbate 80, wherein the pH of the pharmaceutical composition is about 5.0.

[0031] In some embodiments of the pharmaceutical composition, the pharmaceutical composition comprises (a) 100 mg / mL ± 10 mg / mL of an anti-SOST antibody comprising a heavy chain of SEQ ID NO: 22 and a light chain of SEQ ID NO: 25, (b) 10 mM ± 1 mM methionine, (c) 75 mg / mL ± 7.5 mg / mL of sucrose, (d) 10 mM ± 1 mM acetic acid-sodium acetate buffer, (e) 4.5 mM ± 0.45 mM calcium chloride, and (f) 0.4 mg / mL ± 0.04 mg / mL of polysorbate 80, and the pH of the pharmaceutical composition is 5.0 ± 0.05.

[0032] In some embodiments of the pharmaceutical composition, the pharmaceutical composition comprises: (a) 150 mg / mL of an anti-SOST antibody comprising a heavy chain of SEQ ID NO: 22 and a light chain of SEQ ID NO: 25; (b) about 10 mM methionine; (c) about 75 mg / mL of sucrose; (d) about 10 mM acetic acid-sodium acetate buffer; (e) about 4.5 mM calcium chloride; and (f) about 0.4 mg / mL of polysorbate 80, wherein the pH of the pharmaceutical composition is about 5.0.

[0033] In some embodiments of the pharmaceutical composition, the pharmaceutical composition comprises: (a) about 100 mg / mL of an anti-SOST antibody comprising a heavy chain of SEQ ID NO: 22 and a light chain of SEQ ID NO: 25; (b) about 50 mM methionine; (c) about 70 mg / mL of sucrose or trehalose (e.g., α,α-trehalose dihydrate); (d) about 10 mM acetic acid-sodium acetate buffer; (e) about 4.5 mM calcium chloride; and (f) about 0.4 mg / mL polysorbate 80, wherein the pH of the pharmaceutical composition is about 5.0.

[0034] In some embodiments of the pharmaceutical composition, the pharmaceutical composition comprises: (a) about 100 mg / mL of an anti-SOST antibody comprising a heavy chain of SEQ ID NO: 22 and a light chain of SEQ ID NO: 25; (b) about 5 mM, about 10 mM, or about 25 mM methionine; (c) about 80 mg / mL of trehalose (e.g., α,α-trehalose dihydrate); (d) about 10 mM acetic acid-sodium acetate buffer; (e) about 4.5 mM calcium chloride; and (f) about 0.4 mg / mL of polysorbate 80, wherein the pH of the pharmaceutical composition is about 5.0.

[0035] In some embodiments of the pharmaceutical composition, the pharmaceutical composition comprises: (a) about 50 mg / mL of an anti-SOST antibody comprising a heavy chain of SEQ ID NO: 22 and a light chain of SEQ ID NO: 25; (b) about 10 mM methionine; (c) about 80 mg / mL trehalose (e.g., α,α-trehalose dihydrate); (d) about 10 mM acetic acid-sodium acetate buffer; (e) about 4.5 mM calcium chloride; and (f) about 0.4 mg / mL polysorbate 80, wherein the pH of the pharmaceutical composition is about 5.0.

[0036] In some embodiments of the pharmaceutical composition, the pharmaceutical composition comprises an intact anti-SOST antibody but does not comprise a truncated anti-SOST antibody, or the pharmaceutical composition comprises an intact anti-SOST antibody and a truncated anti-SOST antibody, the truncated anti-SOST antibody being produced by cleavage of HCDR3. In some embodiments, the truncated anti-SOST antibody is produced by cleavage at the fourth amino acid residue of HCDR3 of the anti-SOST antibody.In some embodiments, the pharmaceutical composition comprises an intact anti-SOST antibody but not a truncated anti-SOST antibody, the intact anti-SOST antibody having a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:22 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:25, the truncated anti-SOST antibody having a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:27, or the pharmaceutical composition comprises an intact anti-SOST antibody and a truncated anti-SOST antibody, the intact anti-SOST antibody being a truncated anti-SOST antibody. The ST antibody has a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:22 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:25, and the cleaved anti-SOST antibody has a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:27, and the amount of the cleaved anti-SOST antibody relative to the total antibody does not exceed 25% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1.0%, 1.5%, 2.0%, 3.0%, 4.0%, 4.5%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 11.0%, 12.0%, 13.0%, 14.0%, 15.0%, 16.0%, 17.0%, 18.0%, 19.0%, 20.0%, 21.0%, 22.0%, 23.0%, 24.0%, 25.0%, 26.0%, 27.0%, 28.0%, 29.0%, 30.0%, 31.0%, 32.0%, 33.0%, 34.0%, 35.0%, 36.0%, 37.0%, 38.0%, 39.0%, 40.0%, 41.0%, 42.0%, 43.0%, 44.0%, 45.0%, 46.0%, 47.0%, 48.0%, 49.0%, 50.0%, 51.0%, 52.0%, 53.0%, 54.0%, 55.0%, 56.0%, 57.0%, 58.0%, 59.0%, 60.0%, 61.0%, 62.0%, 63.0%, 6 or the amount of cleaved anti-SOST antibody relative to total antibody is in any range between these point values, e.g., the amount of cleaved anti-SOST antibody relative to total antibody is 0.1%-25%, 0.1%-20%, 0.3% to 16%, 0.3% to 10%, 0.3% to 5%, 0.3% to 2%, or 0.3% to 0.5%, etc.), in some embodiments, in the pharmaceutical composition, the amount of cleaved anti-SOST antibody whose heavy chain amino acid sequence is shown in SEQ ID NO: 27 relative to the whole antibody does not exceed 16%, and in some embodiments, in the pharmaceutical composition, the amount of cleaved anti-SOST antibody whose heavy chain amino acid sequence is shown in SEQ ID NO: 27 relative to the whole antibody does not exceed 10%. In some embodiments, in the pharmaceutical composition, the amount of cleaved anti-SOST antibody whose heavy chain amino acid sequence is shown in SEQ ID NO: 27 relative to the whole antibody does not exceed 2.0%. In some embodiments, in the pharmaceutical composition, the amount of cleaved anti-SOST antibody whose heavy chain amino acid sequence is shown in SEQ ID NO: 27 relative to the whole antibody does not exceed 0.3%.

[0037] The disclosure further provides pharmaceutical compositions comprising an intact anti-SOST antibody and a truncated anti-SOST antibody, wherein the intact anti-SOST antibody has a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 22 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 25. In some embodiments, the truncated anti-SOST antibody is produced by truncation of HCDR3. In some embodiments, the truncated anti-SOST antibody is produced by truncation at the fourth amino acid residue of HCDR3 of the anti-SOST antibody.

[0038] In some embodiments of the pharmaceutical composition, the pharmaceutical composition comprises an intact anti-SOST antibody and a truncated anti-SOST antibody, wherein the intact anti-SOST antibody has a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:22 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:25, and the truncated anti-SOST antibody has a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:27, and the amount of the truncated anti-SOST antibody relative to the total antibody does not exceed 0.1% to 25% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1.0%, 1.5%, 2.0%, 3.0%, 4.0%, 4.5%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 9.5%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 8 %, 23%, 24% or 25%, or any value between these point values, or the amount of the cleaved anti-SOST antibody relative to the whole antibody is in any range between these point values, e.g., the amount of the cleaved anti-SOST antibody relative to the whole antibody is 0.1% to 25%, 0.1% to 20%, 0.3% to 16%, 0.3% to 10%, 0.3% to 5%, 0.3% to 2%, 0.3% to 0.5%, etc.), in some embodiments, in the pharmaceutical composition, the amount of the cleaved anti-SOST antibody whose heavy chain amino acid sequence is set forth in SEQ ID NO:27 relative to the whole antibody does not exceed 16%, and in some embodiments, in the pharmaceutical composition, the amount of the cleaved anti-SOST antibody whose heavy chain amino acid sequence is set forth in SEQ ID NO:27 relative to the whole antibody does not exceed 10%. In some embodiments, in the pharmaceutical composition, the amount of the cleaved anti-SOST antibody whose heavy chain amino acid sequence is set forth in SEQ ID NO:27 relative to the whole antibody does not exceed 2.0%. In some embodiments, in the pharmaceutical composition, the amount of cleaved anti-SOST antibody having a heavy chain amino acid sequence set forth in SEQ ID NO:27 relative to the whole antibody does not exceed 0.3%.

[0039] In some embodiments of the pharmaceutical composition, the pharmaceutical composition comprises an intact anti-SOST antibody and a truncated anti-SOST antibody, wherein the intact anti-SOST antibody has a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:22 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:25, and the truncated anti-SOST antibody has a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:26, and the amount of the truncated anti-SOST antibody relative to the total antibody does not exceed 4% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9 ... or the amount of cleaved anti-SOST antibody relative to the total antibody is in any range between these point values, e.g., the amount of cleaved anti-SOST antibody relative to the total antibody is 0.1%-4%, 0.1%-2.8%, 0.1%-2.5%, 0.1%-1.5%, or 0.4%-1.4%, etc.); in some embodiments, the amount of cleaved anti-SOST antibody whose heavy chain amino acid sequence is set forth in SEQ ID NO:26 relative to the total antibody does not exceed 2.3%, and in some embodiments, the amount of cleaved anti-SOST antibody whose heavy chain amino acid sequence is set forth in SEQ ID NO:26 relative to the total antibody does not exceed 1.4%. In some embodiments, the amount of cleaved anti-SOST antibody whose heavy chain amino acid sequence is set forth in SEQ ID NO:26 relative to the total antibody does not exceed 0.7%. In some embodiments, in the pharmaceutical composition, the amount of cleaved anti-SOST antibody having a heavy chain amino acid sequence set forth in SEQ ID NO:26 relative to the whole antibody does not exceed 0.4%.

[0040] In some embodiments of the pharmaceutical composition, the antibody is Ab-5, and the pharmaceutical composition has a property of better preventing cleavage of HCDR3 of the heavy chain compared to a pharmaceutical composition free of an antioxidant (e.g., methionine). In some embodiments, the pharmaceutical composition has a purity percentage of complete antibody monomer of 70% or more (e.g., 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more) after being placed under high temperature conditions at 40° C. for 7 days or 30 days, or under conditions at 25° C. or 4° C. for 1, 3, 6, 12, 15, or 18 months. or 100%), and in some embodiments, the purity of the complete antibody monomer of the pharmaceutical composition after being placed under high temperature conditions at 40° C. for 30 days is 70% or more (e.g., 70% or more, e.g., 75% or more, 80% or more, 85% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%). In some embodiments, the purity of the complete antibody monomer is detected by a method known in the art, including, but not limited to, SEC size exclusion chromatography, NR-CE capillary gel electrophoresis, and the like.

[0041] In some embodiments, the present disclosure further provides a lyophilized formulation that can be reconstituted to form any one of the pharmaceutical compositions described above.

[0042] In some embodiments, the present disclosure further provides a lyophilized formulation, which is a lyophilized form of any one of the pharmaceutical compositions described above.

[0043] In some embodiments, the present disclosure further provides a lyophilized formulation obtained by lyophilizing any one of the pharmaceutical compositions described above.

[0044] In some embodiments, the present disclosure further provides a method for preparing a lyophilized formulation, the method comprising the step of lyophilizing any one of the pharmaceutical compositions described above. In some embodiments, the lyophilization step comprises the steps of pre-freezing, primary drying, and secondary drying, in that order. In some embodiments, the pre-freezing step is performed by freezing at 5°C to -40 to -50°C, most preferably -45°C, and the degree of vacuum is not limited, the primary drying temperature is -30 to 0°C, most preferably -27°C, the degree of vacuum is 0.05 to 0.2 mBar, most preferably 0.1 mBar, the secondary drying temperature is 20 to 30°C, most preferably 25°C, and the degree of vacuum is reduced from 0.05 to 0.2 mBar, most preferably 0.1 mBar, to a vacuum of 0.005 to 0.02 mBar, most preferably 0.01 mBar.

[0045] The present disclosure further provides a lyophilized formulation prepared by the above-described method for preparing a lyophilized formulation.

[0046] In some embodiments, the present disclosure further provides a reconstituted solution prepared by reconstituting any one of the lyophilized formulations described above.

[0047] In some embodiments, the present disclosure further provides a reconstituted solution, which is a reconstituted form of any one of the lyophilized formulations described above.

[0048] In some embodiments, the present disclosure further provides a method for preparing any one of the above reconstituted solutions, comprising the step of reconstituting the lyophilized formulation, the solution used for the reconstitution being selected from, but not limited to, water for injection, saline, or glucose solution. The reconstitution can be carried out by dissolving the lyophilized protein formulation under low pressure in a diluent (selected from, but not limited to, water for injection, saline, or glucose solution) and dispersing the protein in the reconstituted solution to obtain the corresponding reconstituted solution.

[0049] In some embodiments, the present disclosure further provides a reconstituted solution obtained by the above method for preparing a reconstituted solution.

[0050] In some embodiments, the pharmaceutical composition or reconstituted solution described in any one of the above is an intravenous injection preparation, a subcutaneous injection preparation, an intraperitoneal injection preparation, or an intramuscular injection preparation, and in some embodiments, the pharmaceutical composition or reconstituted solution described in any one of the above is an intravenous injection preparation. In some embodiments, the pharmaceutical composition or reconstituted solution described in any one of the above is applied to intravenous injection, subcutaneous injection, intraperitoneal injection, or intramuscular injection. In some embodiments, the pharmaceutical composition or reconstituted solution or lyophilized formulation described in any one of the above is used to prepare a drug for intravenous injection, subcutaneous injection, intraperitoneal injection, or intramuscular injection.

[0051] In some embodiments, the present disclosure further provides an article of manufacture comprising a container containing any one of the pharmaceutical compositions, lyophilized formulations, or reconstituted solutions described above. In some embodiments, the container is an injection vial made of neutral borosilicate glass tubing.

[0052] In some embodiments, the present disclosure further provides a method for treating a disease or condition associated with SOST, the method comprising administering to a subject in need thereof a therapeutically effective amount of the following substances: (A) a pharmaceutical composition described in any one of the above, (B) a lyophilized formulation described in any one of the above, (C) a reconstituted solution described in any one of the above, or (D) a product described in any one of the above.

[0053] In some embodiments, the present disclosure further provides a use of any one of the pharmaceutical compositions, lyophilized formulations, reconstituted solutions, or products described above in the preparation of a medicament for treating a disease or condition associated with SOST.

[0054] In some embodiments, the present disclosure further provides any one of the above pharmaceutical compositions, lyophilized formulations, reconstituted solutions, or products for treating a disease or condition associated with SOST.

[0055] In some embodiments, the SOST-related disease or condition described in any one of the above is selected from osteoporosis, osteopenia, osteoarthritis, rheumatoid arthritis, periodontal disease, and multiple myeloma, and in some embodiments, the SOST-related disease or condition is osteoporosis. [Brief description of the drawings]

[0056] [Figure 1] NR-CE spectrum of Ab-5 formulation 1. [Diagram 2] 1 is an R-CE spectrum of Ab-5 formulation 1. [Diagram 3] 13 is a molecular weight spectrum of fragment 1 of Ab-5 preparation 2. [Figure 4] 13 is a molecular weight spectrum of fragment 2 of Ab-5 preparation 2. [Diagram 5] Peptide map of Ab-5 formulation 2 after high temperature degradation (placed at 40° C. for 30 days). [Figure 6] 1 is a peptide map of Ab-5 formulation 2 before high-temperature degradation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] term In order that the present disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless expressly defined otherwise herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art.

[0058] An "antioxidant" is an agent that prevents or reduces the oxidation of an active drug ingredient. Antioxidants include free radical scavengers (e.g., ascorbic acid, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), sodium sulfite, para-aminobenzoic acid, glutathione, propyl gallate, etc.), chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), nitrilotriacetic acid (NTA), N-2-acetamido-2-iminodiacetic acid (ADA), ethylene glycol bis(aminoethyl) ether, N,N,N',N'-tetraacetic acid (EGTA), trans-diaminocyclohexanetetraacetic acid (DCTA), amino acids (glutamic acid and asparagine), and the like. The antioxidants include, but are not limited to, N-hydroxyethyliminodiacetic acid (HIMDA), N,N-bis-hydroxyethylglycine (bicine) and N-tris(hydroxymethyl)methylglycine (tricine), glycylglycine, sodium deoxycholate, ethylenediamine, propylenediamine, diethylenetriamine, triethylenetetramine (trien), EDTA disodium, EDTA calcium oxalate, malate, citric acid, citric acid monohydrate and trisodium citrate dihydrate, 8-hydroxyquinolinate, chain terminators (e.g., methionine, cysteine, etc.) and the like. In some embodiments, the antioxidant may be selected from methionine, histidine, arginine, EDTA, citric acid, ascorbic acid, butylated hydroxytoluene, butylated hydroxyanisole, sodium sulfite, para-aminobenzoic acid, glutathione, propyl gallate, ethanol, benzyl alcohol, etc. In some embodiments, the antioxidant is selected from methionine, histidine, and arginine. In some embodiments, the antioxidant is selected from methionine. In some embodiments, the antioxidant can effectively reduce cleavage of HCDR3 of Ab-5 antibody. In some embodiments, the antioxidant can effectively prevent cleavage at the fourth amino acid residue of HCDR3 of Ab-5 antibody heavy chain from occurring.

[0059] A "buffer" is a buffering agent that resists changes in pH by the action of its acid-base conjugate components. Examples of buffers that control pH within the appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate (also called phosphate), tartrate, fumarate, glycylglycine and other organic acid buffers.

[0060] An "acetate buffer" is a buffer that contains acetate ions. Examples of acetate buffers include acetic acid-sodium acetate, histidine-histidine acetate, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate, etc. A preferred acetate buffer is acetic acid-sodium acetate.

[0061] "Histidine buffer" refers to a buffer containing histidine. Examples of histidine buffer include histidine-histidine hydrochloride, histidine-histidine acetate, histidine-histidine phosphate, histidine-histidine sulfate, and the like, preferably histidine-histidine hydrochloride. Histidine-histidine hydrochloride buffer may be prepared from histidine and hydrochloric acid, or may be prepared from histidine and histidine hydrochloride.

[0062] A "citrate buffer" or "citrate buffer" is a buffer that contains citrate ions. Examples of citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, etc. A preferred citrate buffer is citric acid-sodium citrate.

[0063] A "succinate buffer" is a buffer containing succinate ions. Examples of succinate buffers include succinic acid-sodium salt, succinic acid-potassium salt, succinic acid-calcium salt, etc. A preferred succinate buffer is succinic acid-sodium salt. Exemplarily, the succinic acid-sodium salt may be prepared from succinic acid and sodium hydroxide, or from succinic acid and sodium salt.

[0064] A "phosphate buffer" is a buffer that contains phosphate ions. Examples of phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-citric acid, etc. A preferred phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.

[0065] "Viscosity modifier" is a common medicinal material added to adjust the viscosity of the formulation. The viscosity modifier can be an inorganic salt and an amino acid salt, preferably the inorganic salt is selected from sodium chloride, calcium chloride, magnesium chloride or calcium acetate, and preferably the amino acid salt is selected from arginine hydrochloride, histidine hydrochloride, glycine hydrochloride, histidine acetate, etc.

[0066] "Replacement" refers to the replacement of a solvent system that dissolves the antibody protein, for example, replacing a high salt or high osmolarity solvent system containing the antibody protein by a physical manipulation method, including, but not limited to, ultrafiltration, dialysis, or centrifugation followed by redissolution, with a buffer system that stabilizes the formulation so that the antibody protein is in a stable formulation.

[0067] A "pharmaceutical composition" refers to a mixture of one or more of the antibodies described herein (e.g., anti-SOST antibodies) or physiologically / pharmaceutical acceptable salts or prodrugs thereof with other chemical components, and other components such as physiologically / pharmaceutical acceptable vectors and excipients, to facilitate administration to a living body and contribute to the absorption of the active ingredients to further exert biological activity.

[0068] A "pharmaceutically acceptable vector" or "pharmaceutically acceptable excipient" includes any material that, when combined with an active ingredient, allows the ingredient to retain its biological activity and does not react with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical vector, such as phosphate buffered saline solution, water, emulsions such as oil / water emulsions, and various wetting agents. In some embodiments, the diluent used for aerosol or parenteral administration is phosphate buffered saline (PBS) or normal (0.9%) saline. Compositions containing such vectors are prepared by conventional methods well known in the art (see, for example, Remington's Pharmaceutical Sciences, 18th edition, edited by A. Gennaro, Mack Publishing Co., Easton, PA, 1990, and R Remington, The Science and Practice of Pharmacy, 20th edition, Mack Publishing, 2000).

[0069] "Lyophilized formulation" refers to a formulation or pharmaceutical composition obtained after performing a vacuum freeze-drying step on a pharmaceutical composition in liquid or solution form or a liquid or solution formulation. Generally, freeze-drying includes pre-freezing, primary drying, and secondary drying. Pre-freezing is to freeze the product to obtain a crystalline solid, and in some embodiments, the pre-freezing temperature is set to -45°C and the pre-freezing rate is set to 1°C / min. Primary drying, also called main drying, is the main step of sample freeze-drying, and is to remove ice in the product while maintaining the shape of the product and minimizing damage to the product. If the temperature and vacuum degree of primary drying are not properly selected, it will lead to the collapse of the product. At higher temperatures and vacuum degrees, the efficiency of freeze-drying will increase, but at the same time, the risk of product collapse will also increase. In some embodiments, the temperature of primary drying may be a normal temperature in this field, for example, -30 to 0°C. Secondary drying, also called decomposition drying, is the main step of removing bound water from the product by pulling the ultimate vacuum (0.01 mbar) and increasing the temperature (20 to 40°C). Since most biological products are temperature sensitive, the temperature of secondary drying is often selected at the lower end of the temperature range, e.g., 25° C. The time of lyophilization is related to the freezer, the volume of the lyophilized formulation, and the container of the lyophilized drug. Such time adjustments are well known to those skilled in the art.

[0070] In the solution forms of the pharmaceutical compositions described in the present disclosure, the solvent therein is water, unless otherwise specified.

[0071] In this disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0072] The pharmaceutical compositions described herein are capable of achieving a stable effect, i.e., an antibody therein (e.g., an anti-SOST antibody) essentially retains its physical and / or chemical stability and / or biological activity after storage, and preferably, the pharmaceutical composition essentially retains its physical and chemical stability and biological activity after storage. The storage period is generally selected based on the desired shelf life of the pharmaceutical composition. Currently, there are several analytical techniques for measuring protein stability, which can measure stability after storage at a given temperature for a given period of time.

[0073] In the present disclosure, a stable formulation includes a formulation that does not show significant changes when stored at refrigerated temperatures (2-8°C) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably 2 years. Stable liquid formulations also include liquid formulations that exhibit the desired characteristics after storage at temperatures including 25°C for periods including 1 month, 3 months, or 6 months. Typical examples of stability include anti-SOST antibodies that typically exhibit no more than about 10%, preferably no more than about 5%, aggregation or degradation as measured by SEC-HPLC. Upon visual analysis, the formulation is a pale yellow, almost colorless, or colorless, transparent liquid, or clear to slightly milky white. The concentration, pH, and weight molality of the formulation vary by no more than ±10%. Typically, no more than about 10% and preferably no more than about 5% decrease is observed. Typically, no more than about 10% and preferably no more than about 5% aggregation is formed. In some embodiments, an anti-SOST antibody formulation of the present disclosure has a SEC monomer % of 90% or more (e.g., 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, etc.) and / or a CE-SDS(NR) % of 70% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more, etc.) after being placed under high temperature conditions at 40°C for one month.

[0074] An anti-SOST antibody "retains its physical stability" in a pharmaceutical formulation if it does not exhibit significant increased aggregation, precipitation, and / or denaturation as determined by visual inspection for color and / or clarity or by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS). Changes in protein conformation can be assessed by fluorescence spectroscopy (which determines the tertiary structure of a protein) and by FTIR spectroscopy (which determines the secondary structure of a protein).

[0075] An anti-SOST antibody "retains its chemical stability" in a pharmaceutical formulation if the anti-SOST antibody does not undergo significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered proteins. Degradation processes that constantly change the chemical structure of proteins include hydrolysis or cleavage (e.g., assessed by methods such as size exclusion chromatography and CE-SDS), oxidation (e.g., assessed by methods such as peptide mapping coupled with mass spectrometry or MALDI / TOF / MS), deamidation (e.g., assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartic acid measurement), and isomerization (e.g., isoaspartic acid content measurement, peptide mapping, etc.).

[0076] An anti-SOST antibody "retains its biological activity" in a pharmaceutical formulation if the biological activity at a given time is within a given range of biological activity exhibited when the pharmaceutical formulation is prepared.

[0077] The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p3558 (1968).

[0078] As used in this disclosure, the singular forms "a," "an," and "the above" include plural referents unless the context clearly dictates otherwise.

[0079] Unless the context clearly indicates otherwise, in the patent specification and claims, the words "containing," "having," "including," and the like, are to be understood in the sense of "including, but not limited to," rather than in the exclusive or exhaustive sense.

[0080] The term "and / or" is intended to include the two meanings "and" and "or." For example, the phrase "A, B and / or C" is intended to cover each of A, B and C, A, B or C, A or C, A or B, B or C, A and C, A and B, B and C, A (alone), B (alone), and C (alone).

[0081] "Optionally" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes cases where the event or circumstance occurs and cases where it does not. For example, "optionally comprising a heavy chain variable region of SEQ ID NO: 11" means that an antibody heavy chain variable region of a particular sequence may be present, but is not necessarily present.

[0082] The terms "about" and "approximately" as used herein mean that a numerical value is within an acceptable range of error of a particular value as determined by one of ordinary skill in the art, as determined by how it is measured or determined (i.e., the limitations of the measurement system), and in this application, unless otherwise specified (e.g., pH ranges), the term "about" can refer to a range of ±10% of the particular value that follows, optionally, a range of ±10%, ±9%, ±8%, ±7%, ±6%, or ±5% of the particular value that follows.

[0083] Although the present disclosure provides content ranges or content values, it will be understood by those of skill in the art that the content ranges or content values ​​cover the acceptable error range of the specific values ​​measured.

[0084] The term "sclerostin" or "SOST" or "SOST protein" or "Sclerostin" refers to the sclerostin (or SOST) gene expression product (protein), and in this disclosure refers to human SOST (h-SOST), unless specifically referred to mouse SOST (m-SOST) or cynomolgus SOST (cyno-SOST). The human, mouse, and cynomolgus SOST used in this disclosure all obtain their nucleotide sequences from GenBank, e.g., NP_079513.1 provides the human SOST protein sequence.

[0085] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that act in the same manner as naturally occurring amino acids. Naturally occurring amino acids are those amino acids that are coded for by codons, and those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid (i.e., an α carbon attached to a hydrogen, a carboxy group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that act in the same manner as a naturally occurring amino acid.

[0086] The term "amino acid mutation" includes amino acid substitution (also called amino acid replacement), deletion, insertion and modification. Any combination of substitution, deletion, insertion and modification can be performed to achieve the final construct, provided that the final construct has the desired properties, such as reduced binding to Fc receptors. Deletion and insertion of amino acid sequences include deletion and insertion at the amino and / or carboxy termini of the polypeptide chain. A specific amino acid mutation may be an amino acid substitution. In one embodiment, the amino acid mutation is a non-conservative amino acid substitution, i.e., replacing one amino acid with another amino acid having different structure and / or chemical properties. Amino acid substitutions include replacement with non-naturally occurring amino acids or derivatives of the 20 natural amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated by genetic or chemical methods known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, etc. It is anticipated that methods of modifying amino acid side groups other than genetic engineering, such as chemical modification, may also be available. In the present application, various names may be used to refer to the same amino acid mutation. In the present application, the amino acid residue at a particular site may be designated in the form of position + amino acid residue, for example, 366W indicates that the amino acid residue at site 366 is W. T366W indicates that the amino acid residue at site 366 has been mutated from the original T to W.

[0087] The term "antibody" is used in the broadest sense and covers a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antigen-binding fragments (or antigen-binding portions) provided that they exhibit the desired antigen-binding activity. A "native antibody" is a naturally occurring immunoglobulin molecule. For example, a native IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From N-terminus to C-terminus, each heavy chain has one variable region (VH), also called variable heavy domain, heavy chain variable region, followed by a heavy chain constant region; an IgG heavy chain constant region (CH) generally contains three constant domains (CH1, CH2 and CH3); similarly, from N-terminus to C-terminus, each light chain has one variable region (VL), also called variable light domain or light chain variable domain, followed by a constant light chain domain (light chain constant region, CL). The terms "full length antibody", "complete antibody" and "whole antibody" may be used interchangeably herein and refer to an antibody having a structure substantially similar to that of a native antibody or having a heavy chain of the Fc region as defined herein. The light chain of a native whole antibody comprises a light chain variable domain, VL, and a constant domain, CL, where VL is at the amino terminus of the light chain, and the light chain constant domain comprises a kappa chain and a lambda chain; the heavy chain comprises a variable domain, VH, and constant domains (CH1, CH2, and CH3), where VH is at the amino terminus of the heavy chain and the constant domain is at the carboxy terminus, of which CH3 is closest to the carboxy terminus of the polypeptide; and the heavy chain may belong to any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0088] The term "variable region" or "variable domain" of an antibody refers to a domain in the heavy or light chain of the antibody that is involved in binding the antibody to an antigen. In the present application, the heavy chain variable region (VH) and the light chain variable region (VL) of an antibody each contain four conserved framework regions (FR) and three complementarity determining regions (CDR). Among them, the term "complementarity determining region" or "CDR" refers to the region in the variable domain that mainly promotes binding to an antigen, and "framework" or "FR" refers to the variable domain residues excluding the CDR residues. VH contains three CDR regions, HCDR1, HCDR2, and HCDR3, and VL contains three CDR regions, LCDR1, LCDR2, and LCDR3. Each of VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus (also called N-terminus) to the carboxy terminus (also called C-terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0089] The boundaries of the amino acid sequences of the CDRs can be determined by various known methods, such as the "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering convention, the "ABM" numbering convention, the "contact" numbering convention (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains[J]. 2001), and the ImMunoGenTics (IMGT) numbering convention (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77(2003); Front Immunol. 2018 Oct 16;9:2278), and the correspondence between various numbering systems is well known to those skilled in the art and is illustratively shown in Table 1 below.

[0090] [Table 1]

[0091] Unless otherwise stated, all variable region and CDR sequences in the examples of this disclosure follow the "Kabat" numbering convention.

[0092] The term "truncated antibody" refers to a molecule different from a complete antibody and is a part of a complete antibody. In the present disclosure, in some embodiments, the truncated anti-SOST antibody is an antibody produced by truncating the HCDR3 of an antibody, in some embodiments, the truncated anti-SOST antibody is an antibody produced by truncating the fourth amino acid residue of the HCDR3 of Ab-5, in some embodiments, the truncated antibody is a truncated anti-SOST antibody whose heavy chain amino acid sequence is shown in SEQ ID NO: 26, in some embodiments, the truncated antibody is a truncated anti-SOST antibody whose heavy chain amino acid sequence is shown in SEQ ID NO: 27, and the C-terminus of the heavy chain of the truncated anti-SOST antibody may or may not have a K because the K at the end of the heavy chain sequence of the Ab-5 humanized antibody may be removed during antibody expression.

[0093] In the present disclosure, unless otherwise specified, the amount of cleaved anti-SOST antibody relative to the total antibody is expressed by the non-reduced CE purity of the cleaved anti-SOST antibody, and the amount of cleaved anti-SOST antibody relative to the total antibody = cleaved anti-SOST antibody peak / total antibody peak x 100% (wherein the cleaved anti-SOST antibody peak is the peak area of ​​the cleaved anti-SOST antibody in the sample, and the total antibody peak is the sum of the peak areas of all antibodies in the sample). In some embodiments, in the above pharmaceutical composition, the amount of the cleaved anti-SOST antibody relative to the total antibody is the amount of the cleaved anti-SOST antibody relative to the total antibody after the pharmaceutical composition is placed under a high temperature condition of 40°C for 7 days or 30 days, or the amount of the anti-SOST antibody relative to the total antibody after the pharmaceutical composition is placed under a condition of 25°C or 4°C for 1, 3, 6, 12 or 18 months, or the amount of the cleaved anti-SOST antibody relative to the total antibody after being placed under other conditions.

[0094] The term "antigen-binding fragment" refers to a molecule distinct from a complete antibody and includes a portion of the complete antibody that binds to the antigen to which the complete antibody binds. Examples of antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, single domain antibodies, single chain Fab (scFab), diabody, linear antibody, single chain antibody molecule (e.g., scFv), and multispecific antibodies consisting of antigen-binding fragments. In some embodiments, the antigen-binding fragment of the antibody is a monovalent Fab (i.e., Fab), a bivalent Fab (F(ab)2), a trivalent Fab fragment (F(ab)3), a multivalent Fab (two or more Fabs), and may be a monospecific, bispecific, or multispecific antigen-binding fragment that includes at least one Fab fragment.

[0095] The term "monoclonal antibody" refers to a population of essentially homogeneous antibodies, i.e., the amino acid sequences of the antibody molecules in the population are the same, except for minor natural mutations that may exist. In contrast, a polyclonal antibody preparation usually contains a number of different antibodies having different amino acid sequences in their variable domains that are generally specific for different epitopes. "Monoclonal" characterizes the antibody as being obtained from an essentially homogeneous antibody population, and should not be construed as requiring the antibody to be produced by any particular method. In some embodiments, the antibodies provided by the present disclosure are monoclonal antibodies.

[0096] The antibodies of the present disclosure may be of animal origin (e.g., antibodies of mouse, bird, rabbit, camel, monkey, etc. origin), chimeric antibodies, or humanized antibodies.

[0097] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from another, different source or species.

[0098] The term "humanized" antibody is an antibody that retains the reactivity of a non-human antibody while at the same time having relatively low immunogenicity in humans, which may be achieved, for example, by retaining the non-human CDR regions and replacing the remainder of the antibody with its human counterparts (i.e., the constant and variable region framework region portions).

[0099] The term "affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise specified, as used herein, binding "affinity" refers to internal binding affinity and reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its ligand Y can generally be expressed as a dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein.

[0100] As used herein, the term "kassoc" or "ka" refers to the association rate of a particular antibody-antigen interaction, and the term "kdis" or "kd" refers to the dissociation rate of a particular antibody-antigen interaction. The term "KD" refers to the dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd / ka) and is expressed as a molar concentration (M). The KD value of an antibody can be measured by methods known in the art. For example, it can be measured by a biosensing system such as a surface plasmon resonance system, or by solution equilibrium titration (SET) to measure affinity in solution. In some embodiments, the KD value is detected by Biacore.

[0101] The term "antigen" refers to a molecule or portion of a molecule that can be bound by a selective binding agent, such as an antigen binding protein (e.g., an antibody), and can then be used in an animal to produce antibodies capable of binding to that antigen. An antigen may have one or more epitopes that are capable of interacting with different antigen binding proteins (e.g., antibodies).

[0102] The term "epitope" refers to an area or region on an antigen capable of specific binding to an antibody or antigen-binding fragment thereof. An epitope may be formed by a contiguous string of amino acids (linear epitope) or may comprise non-contiguous amino acids in spatial proximity (conformational epitope), for example due to antigen folding (i.e., tertiary folding of the antigen due to proteinaceous properties). Conformational and linear epitopes differ in that antibody binding to conformational epitopes is lost in the presence of denaturing solvents. An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a specific epitope (i.e., those that bind to the same epitope) can be performed by methods routine in the art, including, but not limited to, alanine scanning, peptide blots, peptide truncation analysis, epitope excision, epitope extraction, chemical modification of the antigen (see Prot. Sci. 9 (2000) 487-496), and cross-blocking.

[0103] The terms "capable of specifically binding," "specifically binds," or "binding" refer to the ability of an antibody (e.g., an anti-SOST antibody) to bind to an antigen or an epitope within that antigen with greater affinity than to other antigens or epitopes. Generally, antibodies have a binding affinity of about 1×10 -7The antibody binds to an antigen or an epitope within the antigen with an equilibrium dissociation constant (KD) of M or less (e.g., less than 10 nM, 3 nM, 1 nM, 0.3 nM, 0.1 nM or less). In some embodiments, the KD of the antibody binding to the antigen is 10% or less (e.g., 1%) of the KD of the antibody binding to a non-specific antigen (e.g., BSA, casein). The KD can be measured by known methods, for example, by BIACORE® surface plasmon resonance measurement. However, antibodies that specifically bind to an antigen or an epitope within an antigen may be cross-reactive to other related antigens, for example to the corresponding antigen from other species (homo) (e.g., humans or monkeys such as Macaca fascicularis (cynomolgus, cyno), Pan troglodytes (chimpanzee, chimp) or Callithrix jacchus (common marmoset, marmoset).

[0104] The term sequence "identity" refers to the degree (percentage) of amino acids / nucleic acids of two sequences that are the same at equivalent positions when optimally aligned to obtain the maximum percentage of sequence identity, with gaps introduced as necessary, and without any conservative substitutions being considered as part of the sequence identity. To measure the percentage of sequence identity, alignment can be achieved by techniques known in the art, such as publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters to be applied to measure alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences to be compared.

[0105] "Administration," "giving," and "treatment," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to the contact of an exogenous agent, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administration," "giving," and "treatment" may refer, for example, to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of cells includes contact of a reagent with a cell, and contact of a reagent with a fluid, where the fluid contacts the cell. "Administration," "giving," and "treatment" also refer to treating, for example, cells with a reagent, diagnostic, binding composition, or through another type of cell, ex vivo and in vitro. "Treatment," when applied to a human, veterinary, or research subject, refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic uses.

[0106] "Treatment" refers to providing an internal or external therapeutic agent, including, for example, any one of the pharmaceutical compositions of the present disclosure, to a patient, the patient having one or more disease symptoms, and the therapeutic agent is known to have a therapeutic effect on those symptoms. Typically, the patient or population being treated is provided with a therapeutic agent in an amount that effectively relieves one or more disease symptoms, thereby inducing the resolution of those symptoms, or inhibiting those symptoms from progressing to any clinically measurable extent. The amount of therapeutic agent that effectively relieves any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on a variety of factors, including the disease state, age and weight of the patient, and the ability of the drug to produce the required therapeutic effect in the patient. Whether the disease symptom has been reduced can be evaluated by any clinical detection method commonly used by a physician or other professional health care provider to evaluate the severity or progression of the condition. An embodiment of the present disclosure (e.g., a method of treatment or product) may be ineffective in alleviating each target disease symptom, but should reduce the target disease symptom in a statistically significant number of patients, as determined by any statistical testing method known in the art, such as Student's t-test, chi-square test, Mann and Whitney U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0107] An "effective amount" includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical disorder. Effective amount also refers to an amount sufficient to permit or facilitate diagnosis. The effective amount used in a subject can vary depending on factors such as, for example, the condition being treated, the overall health of the subject, the route and dose of administration, and the severity of side effects. An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects. The subject of the present disclosure may be an animal or human subject.

[0108] The pharmaceutical compositions of the present disclosure may be administered by any suitable means, including parenteral, pulmonary, and intranasal administration, and may be administered intralesionally if localized treatment is required. Parenteral injections include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration may be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection. Various dosing schedules are contemplated herein, including, but not limited to, a single dose or multiple doses at multiple time points, bolus administration, and pulse infusion. In some embodiments, the pharmaceutical compositions of the present disclosure are administered intravenously.

[0109] The pharmaceutical compositions of the present disclosure are prepared, dosed and administered in a manner consistent with good medical practice. Factors to consider in this context include the specific condition being treated, the specific mammal being treated, the clinical status of the individual patient, the cause of the condition, the site of delivery of the reagent, the method of administration, the administration schedule and other factors known to medical practitioners. Optionally, the pharmaceutical composition may be prepared in combination with one or more other reagents used in the prevention or treatment of the above-mentioned condition. The effective amount of such other reagents depends on the amount of antigen-binding molecule present in the pharmaceutical composition, the type of condition or treatment and other factors. They may be used in the same doses and routes of administration as described herein, or at about 1% to 99% of the doses described herein, or at any dose and by any route empirically / clinically confirmed to be appropriate.

[0110] The following will be further described in conjunction with Examples or Test Examples, but these Examples or Test Examples are not intended to limit the scope. For experimental methods where specific conditions are not specified in the Examples or Test Examples, generally follow normal conditions or the conditions recommended by the raw material or product manufacturer. Refer to Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, and Current Protocols in Molecular Biology, Ausubel et al., Greene Publishing Associates, Wiley Interscience, NY. Reagents for which specific sources are not specified are ordinary commercially available reagents.

[0111] The method for preparing the SOST antibody in the present application is described in the patent application WO2016145961A1, and this disclosure is incorporated herein by reference in its entirety. Example 1: Production of anti-human SOST monoclonal antibody

[0112] The anti-human SOST monoclonal antibody was produced by immunizing mice. The experimental SJL white mice were female and 6 weeks old (Beijing Vital River Laboratory Animal Technology Co., Ltd., animal production license number: SCXK(Jing)2012-0001). Breeding environment: SPF grade. After purchasing the mice, they were adjusted to a 12 / 12 hour light / dark cycle and bred in a laboratory environment with a temperature of 20-25°C and a humidity of 40%-60% for 1 week. The mice adapted to the environment were divided into 2 groups (A / B) of 10 mice per group.

[0113] The immunization antigen was SOST recombinant protein with a His tag (His-h-SOST). Group A was emulsified with Freund's adjuvant (sigma Lot Num: F5881 / F5506): Complete Freund's adjuvant (CFA) was used for the primary immunization, and Incomplete Freund's adjuvant (IFA) was used for the remaining booster immunizations. The antigen-to-adjuvant ratio was 1:1, 200 μL / 200 μg / mouse (primary immunization), 200 μL / 100 μg / mouse (booster immunization). Group B was cross-immunized with Titermax (sigma Lot Num: T2684) and Alum (Thremo Lot Num: 77161). The ratio of antigen to adjuvant (titermax) was 1:1, and the ratio of antigen to adjuvant (Alum) was 3:1, with 200μL / 200μg / mouse (primary immunization) and 200μL / 100μg / mouse (booster immunization). After antigen emulsification, the mice were vaccinated on days 0, 14, 28, 42, and 56.

[0114] On day 0, emulsified antigen was injected intraperitoneally (IP) to group A / B at 50 μg / mouse. On day 14, it was injected subcutaneously (sc) at multiple points (usually 6 to 8 points on the back) at 25 μg / mouse. On days 28 and 42, antigen was injected either at the back or intraperitoneally depending on the dorsal mass and abdominal swelling. Booster immunization was performed 3 days before splenocyte fusion, and antigen solution prepared with saline was injected intraperitoneally (IP) at 200 μg / mouse.

[0115] Blood tests were performed on days 22, 36, 50, and 64, and the binding activity of mouse serum to human sclerostin was detected by ELISA. After the fourth immunization, mice with a tendency for the antibody titer in the serum to be high and stable were selected for splenocyte fusion, and the spleen lymphocytes were fused with myeloma cells Sp2 / 0 cells (ATCC® CRL-8287®) using an optimized PEG-mediated fusion step to obtain hybridoma cells. In addition, the blocking activity of anti-SOST antibodies in mouse serum against the binding of human SOST and human LRP-6 was detected to select a monoclonal hybridoma cell line Ab-1 with good in vitro activity, and the activity detection results are shown in Table 2:

[0116] [Table 2] Example 2: Humanization of a mouse anti-human sclerostin antibody

[0117] A monoclonal hybridoma cell line Ab-1 with good in vitro activity was selected, and the sequence of the monoclonal antibody therein was cloned, and then humanized, recombinantly expressed and activity evaluated.

[0118] The process of cloning the sequence from the hybridoma is as follows: hybridoma cells in the logarithmic growth phase are collected, and RNA is extracted with Trizol (Invitrogen, 15596-018) (following the procedure in the instruction manual of the reagent kit), reverse transcribed (PrimeScript™ Reverse Transcriptase, Takara, cat#2680A), and the cDNA obtained by reverse transcription is PCR amplified with mouse Ig-Primer Set (Novagen, TB326 Rev.B 0503), and then sent to a sequencing company for sequencing, and the amino acid sequence of the variable region of the hybridoma cell line Ab-1 was obtained: [ka] [ka]

[0119] Methods for humanizing mouse anti-human sclerostin monoclonal antibodies are known in the art, i.e., by substituting human constant domains for the parent (mouse antibody) constant domains, and selecting highly homologous human germline templates according to the homology between mouse and human antibodies, for humanization, specifically as follows: 1. CDR region of mouse anti-sclerostin antibody The amino acid residues of the CDRs of the antibody VH / VL are determined and annotated by the Kabat numbering system, and the CDR sequences of the murine antibody Ab-1 are shown in Table 3:

[0120] [Table 3]

[0121] 2. Selection of human germline FR region sequences Based on the typical structures of CDRs of the obtained mouse antibody VH / VL, the sequences of the heavy and light chain variable regions were compared with the antibody GermLine database to obtain highly homologous human germline templates. Among them, the human germline light chain framework region is derived from a human kappa light chain gene, and in the present disclosure, the human germline light chain template IGKV1-39*01 or IGKV4-1*01 is preferred. The human germline heavy chain framework region is obtained from a human heavy chain, and in the antibody of the present disclosure, the human germline heavy chain template IGHV1-18*01 is preferred. The CDR regions of the mouse antibody Ab-1 were grafted into the selected humanized template, replacing the humanized variable region, and recombined with the human constant region. Furthermore, based on the three-dimensional structure of the mouse antibody, buried residues, residues that directly interact with the CDR region, and residues that have an important effect on the conformation of VL and VH were back-mutated, and in some embodiments, chemically unstable amino acid residues in the CDR region, including optimization of the heavy chain HCDR2, were optimized to obtain a new heavy chain HCDR2 sequence: DIDPNDGDILYNQKFRD (SEQ ID NO: 9). The obtained humanized antibody has a heavy chain variable region shown in SEQ ID NOs: 10 to 12, which is fused to any one of the heavy chain constant regions selected from SEQ ID NOs: 16 to 18 to form a humanized antibody heavy chain, and a light chain variable region shown in SEQ ID NOs: 13 to 15, which is fused to the light chain constant region shown in SEQ ID NO: 19 to form a humanized antibody light chain.

[0122] 1) Humanized antibody heavy chain variable region: [ka] [ka] [ka] 2) The heavy chain constant region of the humanized antibody is optionally chosen from the following sequences: [ka] [ka] [ka] 3) Light chain variable region: [ka] [ka] [ka] [ka] The above antibodies were cloned, expressed and purified by gene cloning and recombinant expression methods, and then subjected to binding ELISA, antigen receptor binding blocking experiments, Biacore, cell activity detection and other methods. As a result, the humanized antibodies Ab-5, Ab-9 and Ab-10 with the best activity were selected, and their sequences are as follows: [ka] [ka] [ka] [ka] [ka] [ka] The amino acid sequence of the antibody fragment produced by truncation of the fourth amino acid residue in the HCDR3 region of the heavy chain of Ab-5 is as follows: [ka] [ka] Example 3: Anti-SOST antibody formulations

[0123] The instruments, antibodies and methods used during preparation and detection of the formulations are as follows: SEC Size Exclusion Chromatography: This is an analytical method that separates solutes based on the correlation between the pore size of gel pores and the coil dimensions of polymer sample molecules.

[0124] SEC% (SEC monomer content percentage) = A monomer / A total × 100% (A monomer is the peak area of ​​the main peak monomer in the sample, and A total is the sum of all peak areas.) SEC measurement equipment: Agilent HPLC1260, column: water, XBridge BEH200Å SEC (7.8×300mM 3.5μm) NR-CE (also called CE-SDS(NR)) Capillary Gel Electrophoresis: This is an electrophoresis method in which a gel is moved into a capillary as a support medium, and samples are separated according to their molecular weight at a constant voltage.

[0125] Percentage purity of non-reduced CE = A main peak / A total x 100% (A main peak is the peak area of ​​the main peak in the sample, and A total is the sum of all peak areas.) CE measurement equipment: Beckman capillary electrophoresis apparatus model plus800. R-CE (also called Reduced-CE, CE-SDS(R), CE) Capillary Gel Electrophoresis:

[0126] This is an electrophoresis method in which a gel is moved into a capillary as a support medium, and samples are separated according to their molecular weight at a constant voltage.

[0127] Equipment for R-CE measurements: Beckman capillary electrophoresis apparatus, model no. plus800. iCIEF Imaging Capillary Isoelectric Focusing:

[0128] This is a technology that separates proteins based on their isoelectric points (pI).

[0129] iCIEF neutral peak content percentage=neutral peak area / total area×100% (total area is the sum of the acidic peak, neutral peak and basic peak areas).

[0130] Equipment for iCIEF measurement: simple protein, model number muarice. Osmolality measurement:

[0131] Osmotic pressure was measured by the freezing point method. Based on the fact that the freezing point depression value is directly proportional to the molar concentration of the solution, the freezing point of the solution was measured using a highly sensitive temperature sensor, and the result was converted into osmotic pressure using an electrical quantity.

[0132] Equipment for measuring osmolarity: Loser, model number OM815. Determination of protein concentration:

[0133] The anti-SOST antibody used in the following formulation examples was Ab-5. The antibody concentration was calculated using the protein concentration calculation method.

[0134] Equipment for measuring protein concentration: UV-visible spectrophotometer, model number: Nano Drop oneC, light path 1 mm. Example 4: Ab-5 antibody fragment identification experiment

[0135] An anti-SOST antibody (Ab-5) solution formulation 1 was prepared having a protein concentration of 100 mg / mL, a calcium chloride concentration of 4.5 mM, an α,α-trehalose dihydrate concentration of 80 mg / mL, and a polysorbate 80 concentration of 0.4 mg / mL in an acetic acid-sodium acetate buffer system of 10 mM at pH 5.0.

[0136] In addition, anti-SOST antibody (Ab-5) solution formulation 2 was prepared having a protein concentration of 50 mg / mL, an α,α-trehalose dihydrate concentration of 80 mg / mL, and a polysorbate 80 concentration of 0.4 mg / mL in a 10 mM acetic acid-sodium acetate buffer system at pH 5.0.

[0137] Formulation 1 was filled into 2 mL vials at 1.0 mL per vial, a laminated rubber stopper was inserted, and the vials were left at 40° C. for 30 days, and the antibody fragments in the formulation were preliminarily identified by NR-CE and R-CE. The experimental results are shown in FIG. 1 and FIG. 2.

[0138] The experimental results showed that, compared with formulation 1 on day 0, after formulation 1 was placed under high temperature conditions of 40°C for 30 days, antibody fragment 1 and fragment 2 were produced in the formulation. Here, in the NR-CE spectrum, the molecular weight of fragment 1 was slightly larger than 10 kDa, and in the R-CE spectrum, fragment 2 appeared between the light chain and the heavy chain, suggesting that the fragments were produced by cleavage of the antibody heavy chain.

[0139] In addition, Formulation 2 was filled into 2 mL vials at 1.0 mL per vial, a laminated rubber stopper was inserted, and the vials were left at 40°C for 30 days to further confirm whether the production of Fragment 1 and Fragment 2 was due to cleavage of the antibody heavy chain based on the deglycosylated reduced molecular weight and peptide map. The experimental results are shown in Figures 3, 4, 5, and 6.

[0140] The experimental results of deglycosylated reduced molecular weight showed that the molecular weight of fragment 1 was 11.39144 kDa and that of fragment 2 was 37.51025 kDa, which is consistent with the experimental results of formulation 1. The experimental results of peptide map showed that, compared with formulation 2 on day 0, formulation 2 decomposed at a high temperature of 40°C produced non-enzymatically cleaved peptide fragment 1 (polypeptide formed from amino acid residues 1 to 101 of the Ab-5 heavy chain (amino acid sequence shown in SEQ ID NO: 26), hereinafter referred to as antibody fragment H1) and peptide fragment 2 (polypeptide formed from amino acid residues 103 to 443 of the Ab-5 heavy chain (amino acid sequence shown in SEQ ID NO: 27), hereinafter referred to as antibody fragment H2), suggesting that the 4th amino acid residue of HCDR3 of the Ab-5 antibody heavy chain (amino acid residue site at 102 (natural order numbering) of the heavy chain) is a site that is easily cleaved. Calculations revealed that the theoretical molecular weight of antibody fragment H1 was 11.39356 kDa, and that of antibody fragment H2 was 37.50691 kDa, which is consistent with the molecular weights of fragment 1 and fragment 2 in Figures 3 and 4.

[0141] As described above, the Ab-5 antibody is easily cleaved before or after the fourth amino acid residue of HCDR3 of the heavy chain to produce antibody fragments. Example 5: Inhibition experiment of HCDR3 cleavage of Ab-5 antibody by antioxidants

[0142] An anti-SOST antibody (Ab-5) solution was prepared in a 10 mM acetic acid-sodium acetate buffer system at pH 5.0, containing 100 mg / mL protein, 4.5 mM calcium chloride, 80 mg / mL α,α-trehalose dihydrate (72 mg / mL anhydrous trehalose), 0.4 mg / mL polysorbate 80, and one of the following antioxidants: (1) N / A (no antioxidants added) (2) 50 mM histidine (3) 50 mM methionine (4) 50 mM arginine The formulation was filled into 2 mL vials at 1.0 mL / bottle and sealed with a laminated chlorinated butyl rubber stopper. The formulation was then placed at high temperature (40°C) and the stability of the formulation was monitored using appearance and NR-CE as evaluation indices.

[0143] The experimental results are shown in Table 4. As can be seen from the experimental results, after 30 days under high temperature conditions at 40°C, the arginine group had a small amount of opalescence, while the other groups were all transparent in appearance. The NR-CE experiment results showed that the antibody purity NR-CE percentage of the groups containing methionine, histidine or arginine was significantly better than that of the group without antioxidants, and the antibody fragments produced by cleavage of the fourth amino acid residue of HCDR3 of Ab-5 were significantly reduced. Antioxidants such as methionine can inhibit the cleavage of the fourth amino acid residue of HCDR3 of Ab-5 antibody.

[0144] [Table 4]

[0145] Note: In the table, "antibody fragment H1+H2" is the NR-CE% of the peaks of antibody fragment H1 (amino acid sequence shown in SEQ ID NO: 26) and antibody fragment H2 (amino acid sequence shown in SEQ ID NO: 27) produced by cleavage of the fourth amino acid residue of HCDR3 of Ab-5, "other fragments" refers to antibody fragment H1 and other Ab-5 antibody fragments other than antibody fragment H1, and "NA" indicates that the content was too small to be measured. Example 6: Screening of antioxidant concentrations

[0146] Anti-SOST antibody (Ab-5) solutions were prepared containing 100 mg / mL protein, 4.5 mM calcium chloride, 80 mg / mL α,α-trehalose dihydrate, 0.4 mg / mL polysorbate 80, and 5 mM, 10 mM, and 25 mM methionine in a 10 mM acetic acid-sodium acetate buffer system at pH 5.0.

[0147] The formulation was filled into 2 mL vials at 1.0 mL / bottle and sealed with a laminated chlorinated butyl rubber stopper. The formulation was then placed at high temperature (40°C) and the stability of the formulation was monitored using appearance and NR-CE as evaluation indices.

[0148] The experimental results are shown in Table 5. It can be seen that the addition of methionine significantly improved the stability of the samples, and the stability of the preparations improved slightly with increasing amounts of methionine. The NR-CE percentage of Ab-5 purity reached 85% for the preparation containing 10 mM methionine after 30 days at a high temperature of 40°C.

[0149] [Table 5] Example 7: Screening of sugars in formulations

[0150] Anti-SOST antibody (Ab-5) solutions were prepared in a 10 mM acetic acid-sodium acetate buffer system at pH 5.0, with a protein concentration of 100 mg / mL, calcium chloride concentration of 4.5 mM, sugar concentration of 70 mg / mL, polysorbate 80 concentration of 0.4 mg / mL, and methionine concentration of 50 mM, each containing the following types of sugar: (1) Sucrose (2) α,α-Trehalose dihydrate The formulation was filled into 2 mL vials at 1.0 mL / bottle and sealed with a laminated chlorinated butyl rubber stopper. The vials were then placed at high temperature (40°C) and the stability of the formulation was monitored using appearance, NR-CE, SEC, and IEC as evaluation indicators.

[0151] The experimental results are shown in Table 6. As can be seen from the experimental results, after 7 days under high temperature conditions of 40℃, the appearance of each formulation is transparent, and there is no significant difference in the purity. In addition, when the concentration of sucrose is 75mg / mL, the preparation sample has an osmotic pressure of about 300mOsm and is close to isotonicity, so 75mg / mL sucrose was selected for further study.

[0152] [Table 6] Example 8: Formulation stability considerations

[0153] An anti-SOST antibody (Ab-5) solution was prepared in a 10 mM acetic acid-sodium acetate buffer system at pH 5.0, with a protein concentration of 100 mg / mL, calcium chloride concentration of 4.5 mM, sucrose concentration of 75 mg / mL, polysorbate 80 concentration of 0.4 mg / mL, and methionine concentration of 10 mM.

[0154] The formulation was filled into 1 mL prefilled syringes at 1.0 mL per syringe, a laminated rubber stopper was inserted, and the syringes were stored at 25°C or 4°C. The stability of the formulation was monitored using appearance and NR-CE as evaluation indices.

[0155] The experimental results are shown in Table 7. As can be seen from the experimental results, after being stored at 25°C for 3 months or at 4°C for 18 months, both the appearance and NR-CE were stable, and the stability of the formulation was good.

[0156] [Table 7]

[0157] Note: In the table, ΔNR-CE% is the difference between the NR-CE% of the antibody at the start of the experiment (0:00) and the NR-CE% of the antibody at the end of the experiment (25°C for 3 months, 4°C for 18 months). Example 9: Stability considerations of high concentration formulations

[0158] An anti-SOST antibody (Ab-5) solution was prepared in a 10 mM acetic acid-sodium acetate buffer system at pH 5.0, with a protein concentration of 150 mg / mL, calcium chloride concentration of 4.5 mM, sucrose concentration of 75 mg / mL, polysorbate 80 concentration of 0.4 mg / mL, and methionine concentration of 10 mM.

[0159] The formulation was filled into a 1 mL prefilled syringe at 1.0 mL per syringe, a laminated rubber stopper was inserted, and the formulation was stored at 25°C or 4°C (see Table 8 for storage time), and the stability of the formulation was monitored using appearance and NR-CE as evaluation indexes. The experimental results are shown in Table 8.

[0160] [Table 8]

[0161] Note: In the table, for example, "25°C 6 months" means that it is left for 6 months under the condition of 25°C, and "NA" means that the content is too small to measure.

[0162] As is evident from the experimental results, the formulation with a high protein concentration of 150 mg / mL containing methionine has good stability after being stored at 25° C. for 6 months or at 4° C. for 15 months.

[0163] Although specific embodiments of the present disclosure have been described above, those skilled in the art should understand that these are merely illustrative and that various changes and modifications can be made to these embodiments without departing from the principles and spirit of the present disclosure. Accordingly, the scope of the present disclosure is limited by the appended claims.

Claims

1. 1. A pharmaceutical composition comprising an anti-SOST antibody comprising an antibody heavy chain variable region and a light chain variable region, and an antioxidant, the heavy chain variable region comprises an HCDR1 represented by SEQ ID NO: 3, an HCDR2 represented by SEQ ID NO: 9 or 4, and an HCDR3 represented by SEQ ID NO: 5, and the light chain variable region comprises an LCDR1 represented by SEQ ID NO: 6, an LCDR2 represented by SEQ ID NO: 7, and an LCDR3 represented by SEQ ID NO: 8; Preferably, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13; More preferably, the anti-SOST antibody comprises a heavy chain set forth in SEQ ID NO: 22 and a light chain set forth in SEQ ID NO:

25. Pharmaceutical compositions.

2. the antioxidant is selected from methionine, histidine, and arginine; Preferably, the antioxidant is methionine. The pharmaceutical composition of claim 1.

3. The concentration of the antioxidant is 1 to 100 mM; Preferably, the concentration of the antioxidant is 5 to 50 mM; More preferably, the concentration of the antioxidant is 5 to 25 mM; Most preferably, the concentration of the antioxidant is about 10 mM. The pharmaceutical composition of claim 1.

4. the concentration of the anti-SOST antibody is 1 to 300 mg / mL; Preferably, the concentration of the anti-SOST antibody is 50-150 mg / mL; More preferably, the concentration of the anti-SOST antibody is 100-150 mg / mL. The pharmaceutical composition of claim 1.

5. the pH of the pharmaceutical composition is 4.8 to 5.5; Preferably, the pH of the pharmaceutical composition is 4.8 to 5.2; More preferably, the pH of the pharmaceutical composition is about 5.

0. The pharmaceutical composition of claim 1.

6. The pharmaceutical composition further comprises a buffer, and the buffer is preferably selected from acetate buffer, histidine buffer, phosphate buffer and succinate buffer, and more preferably acetic acid-sodium acetate buffer; Preferably, the concentration of the buffer is 1 to 30 mM; More preferably, the concentration of the buffer is 5 to 20 mM; Most preferably, the concentration of the buffer is about 10 mM. The pharmaceutical composition of claim 1.

7. the pharmaceutical composition further comprises a sugar, preferably trehalose or sucrose, more preferably α,α-trehalose dihydrate; Preferably, the sugar has a concentration of 40 to 95 mg / mL; More preferably, the sugar concentration is 60 to 90 mg / mL; Most preferably, the sugar concentration is about 75 mg / mL. The pharmaceutical composition of claim 1.

8. The pharmaceutical composition further comprises a viscosity modifier, and the viscosity modifier is preferably selected from calcium salts, sodium chloride, magnesium chloride, and arginine hydrochloride, and more preferably calcium chloride or calcium acetate; Preferably, the concentration of the viscosity modifier is 1 to 150 mM; More preferably, the concentration of the viscosity modifier is 1 to 10 mM; Most preferably, the concentration of the viscosity modifier is about 4.5 mM. The pharmaceutical composition of claim 1.

9. The pharmaceutical composition further comprises a surfactant, preferably a polysorbate or a poloxamer, more preferably a polysorbate 80; Preferably, the concentration of the surfactant is 0.02 to 0.8 mg / mL; More preferably, the concentration of the surfactant is 0.3 to 0.6 mg / mL; Most preferably, the concentration of the surfactant is about 0.4 mg / mL. The pharmaceutical composition of claim 1.

10. (a) 1 to 300 mg / mL of the anti-SOST antibody; (b) 1 to 100 mM of methionine, histidine, or arginine; (c) 40 to 95 mg / mL of trehalose or sucrose; (d) 1 to 30 mM of acetate or histidine buffer; (e) 1 to 20 mM of a calcium salt; and (f) 0.02 to 0.8 mg / mL of polysorbate, wherein the pH of the pharmaceutical composition is 4.8 to 5.5; Preferably, the pharmaceutical composition comprises: (a) 50-150 mg / mL of an anti-SOST antibody comprising a heavy chain of SEQ ID NO: 22 and a light chain of SEQ ID NO: 25; (b) 5-25 mM methionine; (c) 60-90 mg / mL of trehalose or sucrose; (d) 5-20 mM acetate buffer; (e) 1-10 mM calcium chloride; and (f) 0.3-0.6 mg / mL of polysorbate 80, wherein the pH of the pharmaceutical composition is 4.8-5.2; More preferably, the pharmaceutical composition comprises: (a) about 100 mg / mL of an anti-SOST antibody comprising a heavy chain of SEQ ID NO: 22 and a light chain of SEQ ID NO: 25; (b) about 10 mM methionine; (c) about 75 mg / mL of trehalose or sucrose; (d) 10-20 mM acetic acid-sodium acetate buffer; (e) about 4.5 mM calcium chloride; and (f) about 0.4 mg / mL of polysorbate 80, wherein the pH of the pharmaceutical composition is about 5.0; Most preferably, the pharmaceutical composition comprises: (a) about 100 mg / mL of an anti-SOST antibody comprising a heavy chain of SEQ ID NO: 22 and a light chain of SEQ ID NO: 25; (b) about 10 mM methionine; (c) about 75 mg / mL of sucrose; (d) about 10 mM acetic acid-sodium acetate buffer; (e) about 4.5 mM calcium chloride; and (f) about 0.4 mg / mL of polysorbate 80, wherein the pH of the pharmaceutical composition is about 5.

0. The pharmaceutical composition of claim 1.

11. A pharmaceutical composition according to any one of claims 1 to 10, obtained by freeze-drying the pharmaceutical composition. Lyophilized formulation.

12. 1. A method of treating a disease or condition associated with SOST, said method comprising administering to a subject in need thereof a therapeutically effective amount of one of the following: (A) a pharmaceutical composition according to any one of claims 1 to 10, or (B) The freeze-dried preparation according to claim 11. administering Preferably, the SOST-associated disease or condition is selected from the group consisting of osteoporosis, osteopenia or osteoarthritis, rheumatoid arthritis, periodontal disease, or multiple myeloma; More preferably, the SOST-associated disease or condition is osteoporosis. method.

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