Formulations containing ActRIIA protein variants

Abstract

In certain aspects, the present disclosure provides a stable liquid pharmaceutical formulation comprising a recombinant fusion protein comprising the extracellular domain (ECD) of human activin receptor type IIA (ActRIIA) protein or a variant thereof linked to a constant domain of an immunoglobulin, such as a human IgG1 Fc domain, and one or more pharmaceutical additives and / or excipients.

Description

[Technical Field]

[0001] Cross-reference to related applications This application claims priority from U.S. Provisional Application No. 63 / 451,195, filed on 9 March 2023, and U.S. Provisional Application No. 63 / 626,394, filed on 29 January 2024. The aforementioned applications are incorporated herein by reference in their entirety.

[0002] References to electronically submitted sequence listings This application includes a sequence listing submitted electronically in XML format, which is incorporated herein by reference in its entirety. The XML file was created on March 6, 2024, and its filename is 1848179-0002-170-WO1_Sequence_Listing.xml, with a size of 51.2 KB.

[0003] This specification describes a stable liquid pharmaceutical formulation comprising a recombinant fusion protein containing the extracellular domain (ECD) or a variant thereof of a human activin receptor type IIA (ActRIIA) protein linked to a constant domain of an immunoglobulin such as the human IgG1 Fc domain, and one or more pharmaceutical excipients and / or additives. [Background technology]

[0004] Pulmonary hypertension (PH) is a disease characterized by high blood pressure in the pulmonary vascular system, including the pulmonary arteries, pulmonary veins, and pulmonary capillaries. Generally, PH is defined as a mean pulmonary artery (PA) pressure at rest ≥ 25 mmHg or a mean pulmonary artery (PA) pressure during exercise ≥ 30 mmHg (Hill et al., Respiratory Care 54(7):958-68 (2009)). The main symptom of PH is dyspnea or shortness of breath, while other symptoms include fatigue, dizziness, syncope, peripheral edema (swelling of the feet, legs, or ankles), bluish lips and skin, chest pain, angina, unsteadiness during exercise, unproductive cough, rapid heartbeat, and palpitations. Pulmonary hypertension is a serious disease that can lead to heart failure, which is one of the most common causes of death in people with pulmonary hypertension. Postoperative pulmonary hypertension can occur as a complication of many types of surgery or procedures and presents a challenge with a high mortality rate.

[0005] PH can be grouped based on various signs of diseases that share similarities in pathophysiological mechanisms, clinical symptoms, and therapeutic approaches ((Simonneau et al., JACC 54(1):S44-54 (2009)). The clinical classification of PH was first proposed in 1973, and the latest clinical classification was approved by the World Health Organization (WHO) in 2008. According to the latest clinical classification of PH, there are five main groups of PH: (1) pulmonary arterial hypertension (PAH) characterized by PA wedge pressure ≤ 15 mmHg; (2) PH resulting from left heart disease (also known as pulmonary venous hypertension or congestive heart failure); (3) PH characterized by PA wedge pressure > 15 mmHg; (4) PH resulting from lung disease and / or hypoxia; chronic thromboembolic PH; and (5) PH of unknown or multifactorial etiology ((Simonneau et al., JACC 54(1):S44-54 (2009); Hill et al., Respiratory Care 54(7):958-68 (2009)). PAH is further classified into: idiopathic PAH (IPAH) [which is a sporadic disorder with no family history of PAH and no identified risk factors]; hereditary PAH; drug or toxin-induced PAH; PAH associated with connective tissue disease, HIV infection, portal hypertension, congenital heart disease, schistosomiasis and chronic hemolytic anemia; and persistent PH in newborns (Simonneau et al., JACC 54(1):S44-54 (2009)). A series of tests are required to diagnose the various types of PH.

[0006] Generally, the treatment of pulmonary hypertension (PH) depends on its cause or classification. When PH is caused by a known drug or medical condition, it is known as secondary PH, and its treatment is usually directed towards the underlying disease. Treatment for pulmonary venous hypertension generally involves optimizing left ventricular function with the administration of diuretics, beta-blockers, and ACE inhibitors, or repairing or replacing the mitral or aortic valve. PAH treatments include pulmonary vasodilators, digoxin, diuretics, anticoagulants, and oxygen therapy. Pulmonary vasodilators target various pathways, including the prostacyclin pathway (e.g., prostacyclins, e.g., intravenous epoprostenol, subcutaneous or intravenous treprostinil, and inhaled iloprost), the nitric oxide pathway (e.g., phosphodiesterase-5 inhibitors, e.g., sildenafil and tadalafil), and the endothelin-1 pathway (e.g., endothelin receptor antagonists, e.g., oral bosentan and oral ambrisentan) (Humbert, M. Am. J. Respir. Crit. Care Med. 179:650-6 (2009); Hill et al., Respiratory Care 54(7):958-68 (2009)). However, current treatments do not reverse PH, and they do not directly treat the underlying vascular remodeling and muscularization seen in many PH patients. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Hill et al., Respiratory Care 54(7):958-68 (2009) [Non-Patent Document 2] Simonneau et al., JACC 54(1):S44-54 (2009) [Non-Patent Document 3] Humbert, M. Am. J. Respir. Crit. Care Med. 179:650-6 (2009) [Overview of the project] [Problems that the invention aims to solve]

[0008] Accordingly, the object of this disclosure is to provide a stable liquid pharmaceutical formulation comprising an ActRIIa fusion protein, and corresponding methods for treating, preventing or mitigating the rate of progression and / or severity of PH, in particular, corresponding methods for treating, preventing or mitigating the rate of progression and / or severity of one or more PH-related complications. [Means for solving the problem]

[0009] summary Provided herein are stable liquid pharmaceutical formulations comprising a recombinant fusion protein containing the extracellular domain (ECD) or a variant thereof of human activin receptor type IIA (ActRIIa) linked to a constant domain of an immunoglobulin such as the human IgG1 Fc domain, and one or more pharmaceutical excipients and / or additives.

[0010] In some embodiments, the ActRIIa protein begins with any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of SEQ ID NO: 9 and is also one of amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 12 The ActRIIa protein includes an amino acid sequence that is at least 70% (e.g., at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence ending at any one of 8, 129, 130, 131, 132, 133, 134, or 135. In some embodiments, the ActRIIa protein includes an amino acid sequence that is at least 70% (e.g., at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the ActRIIa protein contains an amino acid sequence that is at least 70% (e.g., at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 11.

[0011] In some embodiments, the ActRIIa protein is an ActRIIa fusion protein comprising an ActRIIa extracellular domain and one or more heterologous protein domains relative to ActRIIa. In some embodiments, the ActRIIa protein is a fusion protein comprising an immunoglobulin Fc domain. In some embodiments, the immunoglobulin Fc domain is the Fc domain of IgG1 immunoglobulin. In some embodiments, the ActRIIa fusion protein further comprises a linker domain positioned between the ActRIIa protein domain and the one or more heterologous domains (e.g., the Fc immunoglobulin domain). In some embodiments, the linker domain is selected from the group consisting of: TGGG (SEQ ID NO: 23), TGGGG (SEQ ID NO: 21), SGGGG (SEQ ID NO: 22), GGGGS (SEQ ID NO: 25), GGG (SEQ ID NO: 19), GGGG (SEQ ID NO: 20), and SGGG (SEQ ID NO: 24). In some embodiments, the ActRIIa fusion protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 32. In some embodiments, the ActRIIa fusion protein comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the ActRIIa fusion protein comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the ActRIIa fusion protein comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, the ActRIIa fusion protein comprises the amino acid sequence of SEQ ID NO: 32 or 41. In some embodiments, the ActRIIa fusion protein comprises a variant of the amino acid sequence described in SEQ ID NO: 32, wherein the sequence lacks the C-terminal lysine residue of SEQ ID NO: 32. In some embodiments, the ActRIIa fusion protein variant lacking the C-terminal lysine residue contains or consists of the amino acid sequence of SEQ ID NO: 41. In some embodiments, the ActRIIa fusion protein is part of a homodimeric protein complex.In some embodiments, the ActRIIa fusion protein is glycosylated. In some embodiments, the ActRIIa fusion protein has a glycosylation pattern obtained by expression in Chinese hamster ovary cells.

[0012] In certain embodiments, the pharmaceutical formulations described herein include an ActRIIA fusion protein or variant, a buffer, a surfactant, a stabilizer, and optionally one or more antioxidants. In certain embodiments, the pharmaceutical formulations described herein include an ActRIIA fusion protein of SEQ ID NO: 32 or a variant of SEQ ID NO: 32 lacking a C-terminal lysine, a buffer, a surfactant, a stabilizer, and optionally one or more antioxidants.

[0013] In some embodiments, the pharmaceutical formulations provided herein contain 10 to 100 mg / mL of recombinant fusion protein comprising the extracellular domain (ECD) or derivative thereof of human activin receptor type IIA (ActRIIA) protein linked to a constant domain of an immunoglobulin such as a human IgG1 Fc domain. In other embodiments, the pharmaceutical formulations contain recombinant fusion protein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg / mL comprising the extracellular domain (ECD) or derivative thereof of human activin receptor type IIA (ActRIIA) protein linked to a constant domain of an immunoglobulin such as a human IgG1 Fc domain. In other embodiments, the pharmaceutical formulations contain 40 to 50 mg / mL of recombinant fusion protein comprising the extracellular domain (ECD) or derivative thereof of human activin receptor type IIA (ActRIIA) protein linked to a constant domain of an immunoglobulin such as a human IgG1 Fc domain. In other embodiments, the pharmaceutical formulation contains 50 mg / mL of a recombinant fusion protein comprising the extracellular domain (ECD) or a derivative thereof of a human activin receptor type IIA (ActRIIA) protein linked to a constant domain of an immunoglobulin such as the human IgG1 Fc domain.

[0014] In certain embodiments, the pharmaceutical preparation provided herein comprises 10 to 100 mg / mL of human ActRIIA fusion protein of SEQ ID NO: 32 or a variant of SEQ ID NO: 32 lacking a C-terminal lysine. In certain embodiments, the pharmaceutical preparation provided herein comprises 50 to 100 mg / mL of human ActRIIA fusion protein of SEQ ID NO: 32 or a variant of SEQ ID NO: 32 lacking a C-terminal lysine. In certain embodiments, the pharmaceutical preparation provided herein comprises 40 to 50 mg / mL of human ActRIIa fusion protein of SEQ ID NO: 32 or a variant of SEQ ID NO: 32 lacking a C-terminal lysine. In certain embodiments, the pharmaceutical preparation provided herein comprises 100 mg / mL of human ActRIIa fusion protein of SEQ ID NO: 32 or a variant of SEQ ID NO: 32 lacking a C-terminal lysine. In certain embodiments, the pharmaceutical preparation provided herein comprises 50 mg / mL of human ActRIIa fusion protein of SEQ ID NO: 32 or a variant of SEQ ID NO: 32 lacking a C-terminal lysine.

[0015] In certain embodiments, the pharmaceutical preparation provided herein comprises 10 to 100 mg / mL of human ActRIIa fusion protein of SEQ ID NO: 32 and / or SEQ ID NO: 41. In certain embodiments, the pharmaceutical preparation provided herein comprises 50 to 100 mg / mL of human ActRIIa fusion protein of SEQ ID NO: 32 and / or SEQ ID NO: 41. In certain embodiments, the pharmaceutical preparation provided herein comprises 40 to 50 mg / mL of human ActRIIa fusion protein of SEQ ID NO: 32 and / or SEQ ID NO: 41. In certain embodiments, the pharmaceutical preparation provided herein comprises 50 mg / mL of human ActRIIa fusion protein of SEQ ID NO: 32 and / or SEQ ID NO: 41. In certain embodiments, the pharmaceutical preparation provided herein comprises 100 mg / mL of human ActRIIa fusion protein of SEQ ID NO: 32 and / or SEQ ID NO: 41.

[0016] In certain embodiments, the pharmaceutical preparation provided herein comprises 10~100 mg / mL of a human ActRIIa fusion protein, a buffering agent, a surfactant, a stabilizer and optionally one or more antioxidants, wherein the buffering agent is not histidine.

[0017] In certain embodiments, the pharmaceutical preparation provided herein comprises 10~100 mg / mL of a human ActRIIa fusion protein of SEQ ID NO: 32 and / or SEQ ID NO: 41, a buffering agent, a surfactant, a stabilizer and optionally one or more antioxidants, wherein the buffering agent is not histidine.

[0018] In certain embodiments, the pharmaceutical preparation provided herein comprises 10~100 mg / mL of a human ActRIIa fusion protein of SEQ ID NO: 32, a buffering agent, a surfactant, a stabilizer and optionally one or more antioxidants, wherein the buffering agent is not histidine.

[0019] In certain embodiments, the pharmaceutical preparation provided herein comprises 50~100 mg / mL of a human ActRIIa fusion protein which is SEQ ID NO: 32 or a variant of SEQ ID NO: 32 lacking a C-terminal lysine, a buffering agent, a surfactant, a stabilizer and optionally one or more antioxidants, wherein the buffering agent is not histidine.

[0020] In some embodiments, the buffer comprises an organic acid, succinate, phosphate, acetate, citrate, citric acid, Tris, HEPES, glutamate, amino acids, MES (2-(N-morpholino)ethanesulfonic acid), lactate, or a mixture of amino acids. In some embodiments, the buffer comprises an organic acid, succinate, phosphate, acetate, citrate, citric acid, Tris, HEPES, glutamate, amino acids, or a mixture of amino acids. In some embodiments, the buffer comprises trisodium citrate dihydrate. In some embodiments, the buffer comprises succinate, phosphate, acetate, citrate, lactate, or glutamate. In some embodiments, the buffer comprises succinate, phosphate, acetate, citrate, or glutamate. In some embodiments, the buffer comprises trisodium citrate dihydrate. In some embodiments, the buffer comprises citric acid monohydrate. In some embodiments, the buffer comprises citrate. In some embodiments, the buffer comprises trisodium citrate dihydrate and citric acid monohydrate. In certain embodiments, if the protein has a negative charge due to the presence of glycans, the buffer is not histidine.

[0021] In some embodiments, the buffer is selected to be physiologically compatible and to maintain a pH higher than 4. In some embodiments, the buffer is selected to be physiologically compatible and to maintain a pH between 5 and 7. In some embodiments, the buffer is selected to be physiologically compatible and to maintain a pH of 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0. In some embodiments, the buffer is selected to be physiologically compatible and to maintain a pH of 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0. In some embodiments, the buffer is selected to be physiologically compatible and to maintain a pH of 5.8.

[0022] In certain embodiments, the pharmaceutical formulation described herein comprises a buffer, wherein the buffer is a phosphate buffer for maintaining the pH of the pharmaceutical formulation between 5 and 7. In certain embodiments, the pharmaceutical formulation described herein comprises a buffer, wherein the buffer is a citrate buffer for maintaining the pH of the pharmaceutical formulation between 4.5 and 7. In certain embodiments, the pharmaceutical formulation described herein comprises a buffer, wherein the buffer is an acetate buffer for maintaining the pH of the pharmaceutical formulation between 4.5 and 6. In certain embodiments, the pharmaceutical formulation described herein comprises a buffer, wherein the buffer is a succinate buffer for maintaining the pH of the pharmaceutical formulation between 4.5 and 5. In certain embodiments, the pharmaceutical formulation described herein comprises a buffer, wherein the buffer is a glutamate buffer for maintaining the pH of the pharmaceutical formulation between 5 and 7.

[0023] In some embodiments, the buffer is present at a concentration of at least 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 500 mM. In some embodiments, the buffer is present at a concentration of 10 to 50 mM. In some embodiments, the buffer is present at a concentration of at least 10 mM.

[0024] In some embodiments, the buffer is a 10 mM citrate buffer that maintains the pH of the pharmaceutical formulation at approximately pH 5.5 to approximately pH 6.5. In some embodiments, the buffer is a 10 mM succinate buffer that maintains the pH of the pharmaceutical formulation at approximately pH 5.5 to approximately pH 6.5. In some embodiments, the buffer is a 10 mM histidine buffer that maintains the pH of the pharmaceutical formulation at approximately pH 5.5 to approximately pH 6.0. In some embodiments, the buffer is a 10 mM citrate buffer that maintains the pH of the pharmaceutical formulation at approximately pH 5.8. In some embodiments, the buffer is a 10 mM succinate buffer that maintains the pH of the pharmaceutical formulation at approximately pH 5.8.

[0025] In some embodiments, the stabilizer is selected from the group consisting of: carboxymethylcellulose (CMC), dextrose, polyethylene glycol (PEG), albumin, kelptoose, proline, sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, genthiobiose, isomaltose, arabinose, glucosamine, fructose, mannitol, sorbitol, polyhydroxy compounds, polysaccharides, dextran, starch, hydroxyethyl starch, cyclodextrin, N-methylpyrrolidene, cellulose, and hyaluronic acid. In some embodiments, the stabilizer is selected from the group consisting of: sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, genthiobiose, isomaltose, arabinose, glucosamine, fructose, mannitol, sorbitol, polyhydroxy compounds, polysaccharides, dextran, starch, hydroxyethyl starch, cyclodextrin, N-methylpyrrolidone, cellulose, and hyaluronic acid. In some embodiments, the stabilizer is sucrose.

[0026] In some embodiments, the stabilizer is present in the formulation at a concentration of 2-16% (weight / volume). In some embodiments, the stabilizer is present at a concentration of about 6-10% (weight / volume). In some embodiments, the stabilizer is present at a concentration of at least 0.005% (weight / volume), 0.01% (weight / volume), 0.02% (weight / volume), 0.03% (weight / volume), 0.05% (weight / volume), 0.06% (weight / volume), 0.07% (weight / volume), 0.08% (weight / volume), 0.09% (weight / volume), 0.1% (weight / volume), 0.5% (weight / volume), 0.7% (weight / volume), 0.8% (weight / volume), 0.9% (weight / volume), 1.0% (weight / volume), 1.2% (weight / volume), 1.5% (weight / volume). It is present in concentrations of 1.7% (weight / volume), 2% (weight / volume), 3% (weight / volume), 4% (weight / volume), 5% (weight / volume), 6% (weight / volume), 7% (weight / volume), 8% (weight / volume), 9% (weight / volume), 10% (weight / volume), 11% (weight / volume), 12% (weight / volume), 13% (weight / volume), 14% (weight / volume), 15% (weight / volume), 16% (weight / volume), 17% (weight / volume), 18% (weight / volume), 19% (weight / volume), or 20% (weight / volume). In some embodiments, the stabilizer is present in a concentration of at least 8% (weight / volume).

[0027] In some embodiments, the surfactant is selected from the group consisting of: sodium lauryl sulfate, sodium dioctyl sulfosuccinate and sodium dioctyl sulfonate, chenodeoxycholic acid, sodium N-lauroyl sarcosinate, lithium dodecyl sulfate, sodium 1-octanesulfonate, sodium cholate hydrate, sodium deoxycholate and sodium glycodeoxycholate, benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride monohydrate, hexadecyltrimethylammonium bromide, CHAPS, CHAPSO, SB3-10, SB3-12, digitonin, Triton X-100, Triton X-114, TWEEN®-20, TWEEN®-80, Lauromacrogol 400, Polyoxyl Stearate 40, Polyoxyethylene Hydrogenated Castor Oil 10, 40, 50 and 60, Glycerol Monostearate, Polysorbate 20, Polysorbate 40, Polysorbate 60, Polysorbate 65, Polysorbate 80, Poloxamer 188, and Soy Lecithin. In certain embodiments, the surfactant is selected from the group consisting of Poloxamer 188, Sodium Dodecyl Sulfate (SDS), N-Dodecyl-β-D-Maltoside (DDM), Polysorbate 20, and Triton X. In certain embodiments, the surfactant is selected from the group consisting of Polysorbate 20, Polysorbate 80, Poloxamer 124, Poloxamer 127, Poloxamer 188, and Poloxamer 407.

[0028] In some embodiments, the surfactant is polysorbate 80. In some embodiments, the surfactant is polysorbate 20. In some embodiments, the surfactant is poloxamer 188.

[0029] In some embodiments, the surfactant is present in the formulation at a concentration of 0.02 to 2.0 mg / mL. In some embodiments, the surfactant is present in the formulation at concentrations of 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL, and 2.0 mg / mL. In some embodiments, the surfactant is present in the formulation at a concentration of 0.05 to 0.3 g / mL. In some embodiments, the surfactant is present at a concentration of at least 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mg / mL. In some embodiments, the pharmaceutical formulation contains a surfactant at a concentration of at least 0.2 mg / mL. In some embodiments, the pharmaceutical formulation contains a surfactant at a concentration of at least 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, or 0.5 mg / mL. In some embodiments, the pharmaceutical formulation contains a surfactant at a concentration of 0.05 to 0.5 mg / mL. In some embodiments, the pharmaceutical formulation contains a surfactant at a concentration of 0.1 to 0.5 mg / mL.

[0030] In some embodiments, one or more of the pharmaceutical additives and / or excipients are antioxidants. In some embodiments of the pharmaceutical formulations provided herein, the concentration of the antioxidant is 0.001 to 50 mM. In some embodiments of the pharmaceutical formulations provided herein, the concentration of the antioxidant is 7.5 to 50 mM. In some embodiments of the pharmaceutical formulations provided herein, the concentration of the antioxidant is 5 to 20 mM. In some embodiments of the pharmaceutical formulations provided herein, the concentration of the antioxidant is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, or 50 mM.

[0031] In one embodiment, the pharmaceutical formulation comprises an antioxidant selected from the group consisting of methionine (L-type or D-type), tryptophan (L-type or D-type), dimercaprol, and pyridoxine. In one embodiment, the pharmaceutical formulation comprises an antioxidant selected from the group consisting of methionine (L-type or D-type), tryptophan (L-type or D-type), and pyridoxine. In a specific embodiment, the formulation comprises L-methionine.

[0032] In certain embodiments, the formulations described herein contain 1 to 50 mM of an antioxidant. In certain embodiments, the formulations described herein contain 5 mM, 10 mM, or 50 mM of an antioxidant. In certain embodiments, the formulation contains 1 to 30 mM of an antioxidant. In certain embodiments, the formulation contains 1 to 20 mM of an antioxidant. In certain embodiments, the formulation contains 5 to 15 mM of an antioxidant. In certain embodiments, the formulation contains 5 to 10 mM of an antioxidant. In certain embodiments, the formulation contains 10 mM or at least 10 mM of an antioxidant.

[0033] In certain embodiments, the formulation contains 1 to 50 mM L-methionine. In certain embodiments, the formulation contains 1 to 30 mM L-methionine. In certain embodiments, the formulation contains 1 to 20 mM L-methionine. In certain embodiments, the formulation contains 5 to 15 mM L-methionine. In certain embodiments, the formulation contains 5 to 10 mM L-methionine. In certain embodiments, the formulation contains 10 mM or at least 10 mM L-methionine. In certain embodiments, the formulations described herein contain 5 mM, 10 mM, or 50 mM L-methionine.

[0034] In some embodiments of the pharmaceutical formulations described herein, the pharmaceutical formulations optionally include a chelating agent. In certain embodiments, the chelating agent is DTPA or EDTA. In certain embodiments, the chelating agent has a concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 μM. In certain embodiments, the liquid formulation contains DTPA or EDTA in concentrations of 1-100 μM, 1-30 μM, 1-20 μM, or 10-30 μM. In certain embodiments, the chelating agent has a concentration of 7.5-100 μM. In certain embodiments, the chelating agent has a concentration of 10 μM.

[0035] In some embodiments, the pharmaceutical formulations provided herein contain 10-100 mg / mL of human ActRIIa fusion protein of SEQ ID NO: 32, 10-50 mM citrate buffer, 2-16% (weight / volume) sucrose, 0.05-0.5 mg / mL polysorbate 80, polysorbate 20, or poloxamer 188, 0-50 mM L-methionine, and 0-100 μM DTPA or EDTA. In other embodiments, the pharmaceutical formulations provided herein contain 50 mg / mL of human ActRIIa fusion protein of SEQ ID NO: 32, 10 mM citrate buffer, 8% (weight / volume) sucrose, 0.2 mg / mL polysorbate 80, and 20 mM L-methionine. In some embodiments, the pharmaceutical formulations provided herein contain 10-100 mg / mL of human ActRIIa fusion protein of SEQ ID NO: 32 or a variant of SEQ ID NO: 32 lacking a C-terminal lysine, 10-50 mM / mL of citrate buffer, 2-16% (weight / volume) of sucrose, 0.05-0.5 mg / mL of polysorbate 80, polysorbate 20, or poloxamer 188, 0-50 mM of L-methionine, and 0-100 μM of DTPA or EDTA.

[0036] In other embodiments, the pharmaceutical formulations provided herein include 50 mg / mL of human ActRIIa fusion protein of SEQ ID NO: 32 or a variant of SEQ ID NO: 32 lacking a C-terminal lysine, 10 mM citrate buffer, 8% (weight / volume) sucrose, 0.2 mg / mL polysorbate 80, and 20 mM L-methionine.

[0037] In some embodiments, the pharmaceutical formulations provided herein contain 10-100 mg / mL of human ActRIIa fusion protein of SEQ ID NO: 32 and / or SEQ ID NO: 41, 10-50 mM citrate buffer, 2-16% (weight / volume) sucrose, 0.05-0.5 mg / mL polysorbate 80, polysorbate 20, or poloxamer 188, 0-50 mM L-methionine, and 0-100 μM DTPA or EDTA. In other embodiments, the pharmaceutical formulations provided herein contain 50 mg / mL of human ActRIIa fusion protein of SEQ ID NO: 32 and / or SEQ ID NO: 41, 10 mM citrate buffer, 8% (weight / volume) sucrose, 0.2 mg / mL polysorbate 80, and 20 mM L-methionine.

[0038] In some embodiments, the human ActRIIa fusion protein comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, the lyophilized formulation comprises a human ActRIIa fusion protein comprising the amino acid sequence of SEQ ID NO: 32 and a human ActRIIa fusion protein comprising the amino acid sequence of SEQ ID NO: 41. In some embodiments, the pharmaceutical formulation comprises sotatercept.

[0039] In certain embodiments, the pharmaceutical formulation described herein comprises a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 lacking a C-terminal lysine residue (SEQ ID NO: 41). In certain embodiments, the pharmaceutical formulation described herein comprises a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 lacking a C-terminal lysine residue (SEQ ID NO: 41), wherein the mixture is in the following proportions by weight: 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 6 Includes SEQ ID NO: 32 with values ​​of 1%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0040] In certain embodiments, the pharmaceutical formulations described herein comprise a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 (SEQ ID NO: 41) lacking a C-terminal lysine residue, wherein the mixture comprises, by weight, approximately 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, Includes SEQ ID NO: 32 with values ​​of 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0041] In certain embodiments, the pharmaceutical formulations described herein comprise a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 (SEQ ID NO: 41) lacking a C-terminal lysine residue, wherein the mixture comprises, by weight, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, and 6 Includes SEQ ID NO: 41 with values ​​of 1%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0042] In certain embodiments, the pharmaceutical formulations described herein comprise a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 (SEQ ID NO: 41) lacking a C-terminal lysine residue, wherein the mixture comprises, by weight, approximately 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, Includes SEQ ID NO: 41 with 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0043] In certain embodiments, the pharmaceutical formulation described herein includes 100% by weight of SEQ ID NO: 32. In certain embodiments, the pharmaceutical formulation described herein includes 100% by weight of SEQ ID NO: 41.

[0044] The pharmaceutical formulations provided herein can be used to treat pulmonary arterial hypertension in subjects requiring treatment for pulmonary arterial hypertension. In certain embodiments, the pharmaceutical formulations provided herein are liquids.

[0045] In certain embodiments, the pharmaceutical formulations provided herein are stable when stored at 2–8°C. In certain embodiments, the liquid pharmaceutical formulation is stored under refrigerated conditions (temperature range: typically about 2–8°C, but under certain circumstances, the aqueous formulation may exhibit stability at other temperatures, including about 25°C and about 40°C, for periods of up to about 3, 6, 9, or 12 months). In certain embodiments, the pharmaceutical formulation is administered via an autoinjector. In certain embodiments, the pharmaceutical formulation is administered by subcutaneous injection. In some embodiments, the pharmaceutical formulation is administered parenterally.

[0046] In certain embodiments, the formulations described herein are contained in an injection device, which is an autoinjector. In other embodiments, the formulations described herein are contained in a glass vial. [Brief explanation of the drawing]

[0047] [Figure 1] Figure 1 shows multiple sequence alignments of various vertebrate ActRIIA proteins and human ActRIIA (SEQ ID NOs: 61-68). [Figure 2A] Figures 2A and 2B show the purification of ActRIIA-hFc expressed in CHO cells. The protein is purified as a single, distinct peak visualized by a sizing column (upper panel) and Coomassie-stained SDS-PAGE (lower panel) (left lane: molecular weight standard; right lane: ActRIIA-hFc). See Example 1. [Figure 2B] Figures 2A and 2B show the purification of ActRIIA-hFc expressed in CHO cells. The protein is purified as a single, distinct peak visualized by a sizing column (upper panel) and Coomassie-stained SDS-PAGE (lower panel) (left lane: molecular weight standard; right lane: ActRIIA-hFc). See Example 1. [Figure 3A] Figures 3A and 3B show that the dissociation constant (KD) of ActRIIA-hFc bound to activin is 5 × 10⁻¹², and the KD of ActRIIA-hFc bound to GDF11 is 9.96 × 10⁻⁹. See Example 1. [Figure 3B] Figures 3A and 3B show that the dissociation constant (KD) of ActRIIA-hFc bound to activin is 5 × 10⁻¹², and the KD of ActRIIA-hFc bound to GDF11 is 9.96 × 10⁻⁹. See Example 1. [Figure 4] Figure 4 shows the results of the "Stability pH Screening Study: Effects of pH and Buffer at t=0". Please refer to "Example 3: Feasibility Study of pH and Buffer". [Figure 5] Figure 5 shows the results of the "Stability pH Screening Study: Effects of pH and Buffering Agent over 2 Weeks at 40°C." Please refer to "Example 3: Feasibility Study of pH and Buffering Agent." [Figure 6] Figure 6 shows the results of the "Stability pH Screening Study: Effects of pH and Buffering Agent at 40°C for 1 Month." Please refer to "Example 3: Feasibility Study of pH and Buffering Agent." [Figure 7] Figure 7 shows the results of the "Stability pH Screening Study: Effects of pH and Buffering Agent at 40°C for 2 Months." Please refer to "Example 3: Feasibility Study of pH and Buffering Agent." [Figure 8] Figure 8 shows the 1H NMR data for Sequence ID No. 32 in the presence and absence of DTPA. See "Example 3: Effect of Metal Chelating Agent on Sequence ID No. 32". [Figure 9] Figure 9 shows diffusion NMR data for Sequence ID No. 32 in the presence and absence of DTPA. See "Example 3: Effect of Metal Chelating Agent on Sequence ID No. 32". [Figure 10]Figure 10 shows the results of colloidal stability studies for Sequence ID No. 32 and PS80 conducted under stirring stress. See "Example 3: Colloidal Stability and Surfactant Screening". [Figure 11] Figure 11 shows the results of colloidal stability studies for Sequence ID No. 32 and PS80 conducted under stirring stress. See "Example 3: Colloidal Stability and Surfactant Screening". [Figure 12] Figure 12 shows the results of colloidal stability studies for Sequence ID No. 32 and PS80 conducted by freeze-thaw cycles. See "Example 3: Colloidal Stability and Surfactant Screening". [Figure 13] Figure 13 shows the results of colloidal stability studies for Sequence ID No. 32 and PS80 conducted by freeze-thaw cycles. See "Example 3: Colloidal Stability and Surfactant Screening". [Figure 14] Figure 14 shows the results of exposing formulations of Sequence ID No. 32, containing different levels of L-methionine (0 mM, 10 mM, 20 mM, 30 mM), to photostress in a photostable chamber in the presence of one of the following chelating agents: EDTA (0 μM, 7.5 μM, 15 μM, 30 μM, 60 μM). See "Example 3: Chelating Agent / L-methionine Variation Study". [Figure 15] Figure 15 shows the results of exposing formulations of Sequence ID No. 32, containing different levels of L-methionine (0 mM, 10 mM, 20 mM, 30 mM), to photostress in a photostable chamber in the presence of one of the following chelating agents: DTPA (0 μM, 7.5 μM, 15 μM, 30 μM, 60 μM). See "Example 3: Chelating Agent / L-methionine Variation Study". [Modes for carrying out the invention]

[0048] Detailed explanation definition The terms used herein generally have their common meanings in the art within the context of this disclosure and in the specific context in which each term is used. Certain terms are discussed below or elsewhere in this specification to provide additional guidance to those skilled in the art when describing the formulations and methods of this disclosure, as well as their manufacturing methods and uses. The scope or meaning of any term in its use will become apparent from the specific context in which it is used.

[0049] "About" and "approximately" generally refer to the degree of acceptable error in a measured quantity, taking into account the nature or precision of the measurement, which may occur, for example, through typical measurement, handling, and sampling procedures involved in the preparation, characterization, and / or use of a substance or composition, through careless errors in these procedures, or through differences in the manufacture, source, or purity of the components employed in the manufacture or use of the composition or the execution of the procedure. Typically, an exemplary degree of error is within 10%, more preferably within 5%, of a given value or range of values. In certain embodiments, "about" may mean a variation of ±0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%.

[0050] Alternatively, particularly in biological systems, the terms “about” and “approximately” may mean a value within a range of decimal places of a given value, preferably within five times, and more preferably within two times. Unless otherwise specified, the numerical quantities shown herein are approximate values; that is, the terms “about” and “approximately” mean that they can be estimated unless explicitly stated otherwise.

[0051] The terms “a” and “an” encompass multiple references unless the context in which the terms are used clearly indicates a different context. The terms “a” (or “an”) and “one or more” and “at least one” are interchangeable within this specification. Furthermore, “and / or” is considered a specific disclosure for each of two or more designated features or components, with or without the other, wherever it is used within this specification. Accordingly, within this specification, the term “and / or” as used in phrases such as “A and / or B” is intended to encompass “A and B,” “A or B,” “A (alone),” and “B (alone).” Similarly, the term “and / or” as used in phrases such as “A, B and / or C” is intended to encompass each of the following embodiments: 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).

[0052] Numerical ranges disclosed herein include the numerical values ​​that define the range. For example, when a range of pH values ​​is stated, such as "pH 5.5 to 6.0", the range is intended to include the stated values. For example, pH 5.0 to 7.0 includes values ​​between pH 5.0 and pH 7.0, and between 5.0 and 7.0. Where used herein, a formulation containing a citrate buffer at pH X refers to a solution at pH X containing the citrate buffer. That is, pH is intended to indicate the pH of the solution.

[0053] The proteins disclosed herein may include amino acid sequences that do not exist in nature. Such variants will inevitably have less than 100% sequence identity or similarity to the starting molecule. In certain embodiments, the variant has an amino acid sequence that has, for example, about 75% to less than 100% amino acid sequence identity or similarity, more preferably about 80% to less than 100%, even more preferably about 85% to less than 100%, even more preferably about 90% to less than 100% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), and most preferably about 95% to less than 100% amino acid sequence identity or similarity over the length of the variant molecule.

[0054] A “stable” formulation is one in which the proteins within it inherently retain their physical and / or chemical stability and / or biological activity during storage. Various analytical techniques for measuring protein stability are available in the art and are described in “Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991)” and “Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993).” Stability can be measured over a selected period and at a selected temperature.

[0055] A “stable” liquid formulation is a pharmaceutical formulation that shows no significant changes for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably 2 years at refrigerated temperatures (2–8°C). Furthermore, “stable” liquid formulations include formulations that exhibit desired characteristics for periods including 1 month, 3 months, 6 months, 12 months, and / or 24 months, and at temperatures including 25°C and 40°C. Typical acceptable criteria for stability are as follows: Typically, less than about 10%, preferably less than about 5%, of the protein is degraded, as measured by SEC-HPLC. The pharmaceutical formulation is colorless or transparent to slightly opaque upon visual analysis. The concentration, pH, and osmotic pressure of the formulation do not vary by more than ±10%. The potency is usually within the reference range of 50–150. Typically, less than about 10%, preferably less than about 5%, of aggregates are present in the formulation. The term "buffering agent" encompasses agents that maintain the solution pH of the formulation of the present invention within an acceptable range, or, in the case of the lyophilized formulation of the present invention, agents that provide an acceptable solution pH before lyophilization. The terms "lyophilization," "lyophilized," and "freeze-dried" refer to a process in which the material to be dried is first frozen, and then the ice or freezing solvent is removed by sublimation under a vacuum. Excipients may be included in the pre-lyophilized formulation to enhance the storage stability of the lyophilized product. The term "pharmaceutical formulation" refers to a preparation in which the active ingredient is in an effective form and which does not contain additional ingredients that are toxic to the person to whom the formulation is administered. The terms "formulation" and "pharmaceutical formulation" are used interchangeably throughout. "Pharmacologically acceptable" means that the excipients (vehicles, additives) and composition can be reasonably administered to a subject to provide an effective dose of the active ingredient used, and are "generally considered safe," for example, physiologically acceptable and do not typically cause allergic reactions or similar unpleasant reactions (e.g., stomach upset) when administered to humans.In another embodiment, this term refers to molecular entities and compositions approved by a federal or state regulatory agency, or listed in the United States Pharmacopeia or other generally accepted pharmacopoeias for use in animals (more specifically, humans). A “reconstituted” formulation is a formulation prepared by dissolving a lyophilized protein formulation in a diluent so that the protein is dispersed in the reconstituted formulation. Reconstituted formulations are suitable for administration (e.g., parenteral administration) and, in some cases, for subcutaneous administration.

[0056] Pharmaceutical preparations Provided herein are pharmaceutical formulations comprising recombinant fusion proteins containing an extracellular domain (ECD) or derivative thereof of a human activin receptor type IIA (ActRIIA) protein ligated to a constant domain of an immunoglobulin, such as a human IgG1 Fc domain. In certain embodiments, the disclosure relates to stable liquid pharmaceutical formulations comprising an extracellular domain (ECD) or derivative thereof of a human ActRIIA protein ligated to a constant domain of an immunoglobulin, such as a human IgG1 Fc domain.

[0057] The liquid formulation of the present invention minimizes the formation of aggregates (high molecular weight species) and fine particles, improves colloidal stability, minimizes fragmentation (low molecular weight species), and ensures that the protein maintains its biological activity over a long period. In certain embodiments, the formulation is produced, for example, by extracting ActRIIA from an aqueous pharmaceutical formulation and exchanging it with a desired buffer as the final step in a purification process. The ActRIIA is then concentrated to a desired concentration. In this embodiment, there is no lyophilization step. Furthermore, excipients such as stabilizers and surfactants can be added to the ActRIIA formulation, and the formulation is diluted with an appropriate buffer until the final protein concentration is reached. The final formulation is filtered and filled into a final container (e.g., an auto-injector). Alternatively, the formulation can be stored in a vial and delivered through a syringe or container.

[0058] The pharmaceutical formulations provided herein contain an ActRIIa fusion protein. In certain embodiments, this disclosure provides a pharmaceutical formulation containing an ActRIIa fusion protein, wherein the protein is present in concentrations of approximately 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 8 mg / mL, 10 mg / mL, 15 mg / mL, approximately 17.5 mg / mL, approximately 20 mg / mL, approximately 22.5 mg / mL, approximately 25 mg / mL, approximately 27.5 mg / mL, approximately 30 mg / mL, approximately 32.5 mg / mL, approximately 35 mg / mL, approximately 37.5 mg / mL, approximately 40 mg / mL, and approximately 42 mg / mL. It is present in concentrations of 5 mg / mL, approximately 45 mg / mL, approximately 47.5 mg / mL, approximately 50 mg / mL, approximately 52.5 mg / mL, approximately 55 mg / mL, approximately 57.5 mg / mL, approximately 60 mg / mL, approximately 62.5 mg / mL, approximately 65 mg / mL, approximately 67.5 mg / mL, approximately 70 mg / mL, approximately 72.5 mg / mL, approximately 75 mg / mL, approximately 77.5 mg / mL, approximately 80 mg / mL, approximately 82.5 mg / mL, approximately 85 mg / mL, approximately 90 mg / mL, approximately 92.5 mg / mL, approximately 95 mg / mL, approximately 97.5 mg / mL, or approximately 100 mg / mL.

[0059] In certain embodiments, the ActRIIa fusion protein has concentrations of approximately 45 mg / mL, 46 mg / mL, 47 mg / mL, 48 mg / mL, 49 mg / mL, 50 mg / mL, 51 mg / mL, 52 mg / mL, 53 mg / mL, 54 mg / mL, or 55 mg / mL. In other embodiments, the ActRIIa fusion protein has concentrations of 45 mg / mL, 46 mg / mL, 47 mg / mL, 48 mg / mL, 49 mg / mL, 50 mg / mL, 51 mg / mL, 52 mg / mL, 53 mg / mL, 54 mg / mL, or 55 mg / mL. In some embodiments, the ActRIIa fusion protein has a concentration of approximately 50 mg / mL.

[0060] In some embodiments, the pharmaceutical formulations provided herein contain 10 to 100 mg / mL of recombinant fusion protein comprising the extracellular domain (ECD) or derivative thereof of human activin receptor type IIA (ActRIIA) protein linked to a constant domain of an immunoglobulin such as a human IgG1 Fc domain. In other embodiments, the pharmaceutical formulations contain recombinant fusion proteins 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg / mL of recombinant fusion protein comprising the extracellular domain (ECD) or derivative thereof of human activin receptor type IIA (ActRIIA) protein linked to a constant domain of an immunoglobulin such as a human IgG1 Fc domain. In other embodiments, the pharmaceutical formulations contain 40 to 50 mg / mL of recombinant fusion protein comprising the extracellular domain (ECD) or derivative thereof of human activin receptor type IIA (ActRIIA) protein linked to a constant domain of an immunoglobulin such as a human IgG1 Fc domain. In other embodiments, the pharmaceutical formulation contains 50 mg / mL of a recombinant fusion protein comprising the extracellular domain (ECD) or a derivative thereof of a human activin receptor type IIA (ActRIIA) protein linked to a constant domain of an immunoglobulin such as the human IgG1 Fc domain.

[0061] In some embodiments, the liquid pharmaceutical formulations of ActRIIa fusion proteins provided herein comprise the ActRIIa fusion protein and one or more pharmaceutical additives and / or excipients. In certain embodiments, the one or more pharmaceutical additives and / or excipients comprise buffers, stabilizers, surfactants, and optionally one or more antioxidants, which are described in further detail below. Buffers may be selected to maintain the pH of the formulation during processing. Surfactants may be selected based on their ability to function as emulsifiers, wetting agents, solubilizers, and / or dispersants.

[0062] Those skilled in the art will recognize that the concentrations of excipients described herein share interdependence within a particular formulation. For example, the concentration of a volume extender is, in one embodiment, lower, for instance, when the protein concentration is high. Excipients and other additives are added to impart or improve manufacturability and / or the quality of the final product, such as the stability and delivery of the drug product (e.g., protein). The formulations provided herein include appropriate excipients that enhance stability and safety.

[0063] cushioning agent Typically, the stability of pharmacologically active protein formulations is observed to be maximized within a narrow pH range. This pH range, which exhibits optimal stability, needs to be identified early in pre-formulation studies. Several approaches are useful in this endeavor, including accelerated stability studies and calorific screening studies (Remmele RL Jr., et al., Biochemistry, 38(16): 5241-7 (1999)). Once the formulation is finalized, the protein must be manufactured and maintained throughout its storage period. Therefore, buffers are almost always used to control the pH of the formulation.

[0064] When selecting a buffer, several factors must be considered. First and foremost, the type and concentration of the buffer must be determined based on the buffer's pKa and the desired pH of the formulation. Equally important is ensuring that the buffer is reliably compatible with the protein and other formulation excipients, and that it does not catalyze any degradation reactions. A third important aspect to consider is the tingling and irritation that the buffer may induce upon administration. The likelihood of tingling and irritation is greater for drugs administered via subcutaneous (SC) or intramuscular (IM) routes, where the drug solution remains at the administration site for a relatively longer period, than for drugs administered via IV routes, where the formulation is rapidly diluted into the bloodstream upon administration. For formulations administered by direct IV infusion, it is necessary to monitor the total amount of buffer (and any other formulation components).

[0065] In some embodiments, the buffer comprises an organic acid, succinate, phosphate, acetate, citrate, citric acid, Tris, HEPES, glutamate, amino acids, MES (2-(N-morpholino)ethanesulfonic acid), lactate, or a mixture of amino acids. In some embodiments, the buffer comprises an organic acid, succinate, phosphate, acetate, citrate, citric acid, Tris, HEPES, glutamate, amino acids, or a mixture of amino acids. In some embodiments, the buffer comprises trisodium citrate dihydrate. In some embodiments, the buffer comprises succinate, phosphate, acetate, citrate, lactate, or glutamate. In some embodiments, the buffer comprises succinate, phosphate, acetate, citrate, or glutamate. In some embodiments, the buffer comprises trisodium citrate dihydrate. In some embodiments, the buffer comprises citric acid monohydrate. In some embodiments, the buffer comprises citrate. In one embodiment, the buffer comprises trisodium citrate dihydrate and citric acid monohydrate. In another embodiment, the buffer is trisodium citrate dihydrate and citric acid monohydrate. In certain embodiments where the protein has a negative charge due to the presence of glycans, the buffer is not histidine.

[0066] In one embodiment, the buffer present in the formulation is selected to be physiologically compatible and to maintain a desired pH of the pharmaceutical formulation. In another embodiment, the pH of the formulation is 4 or higher. In yet another embodiment, the pH of the formulation is between pH 4.0 and pH 12.0. For example, in various embodiments, the pH of the reconstituted formulation is 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0. In some embodiments, the pH of the formulation is pH 5 to pH 7. In some embodiments, the pH of the stable liquid formulation is 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0. In one embodiment, the pH of the stable liquid formulation is 5.8.

[0067] In certain embodiments, the buffer is a phosphate buffer for maintaining the pH of the pharmaceutical formulation at 5 to 7. In certain embodiments, the buffer is a citrate buffer for maintaining the pH of the pharmaceutical formulation at 4.5 to 7. In certain embodiments, the buffer is an acetate buffer for maintaining the pH of the pharmaceutical formulation at 4.5 to 6. In certain embodiments, the buffer is a succinate buffer for maintaining the pH of the pharmaceutical formulation at 4.5 to 5. In certain embodiments, the buffer is a glutamate buffer for maintaining the pH of the pharmaceutical formulation at 5 to 7.

[0068] The pH buffer compound can be present in any amount suitable for maintaining the pH of the formulation at a predetermined level. Crystallization and pH shift can be avoided if a sufficiently low level of buffer is used. In one embodiment, the concentration of the buffer is 0.1 mM to 500 mM (1 M). For example, the buffer is intended to be at least 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 500 mM. In certain embodiments, the buffer is 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 500 mM. In certain embodiments, the buffer is 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mM. In certain embodiments, the concentration of the buffer is 10 mM.

[0069] In some embodiments, the buffer comprises trisodium citrate dihydrate. In some embodiments, the buffer comprises citric acid monohydrate. In some embodiments, the buffer comprises citrate. In some embodiments, the buffer comprises trisodium citrate dihydrate and citric acid monohydrate. In certain embodiments, the buffer is not histidine. In some embodiments, the buffer is present at concentrations of at least 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 500 mM. In some embodiments, the concentration of the pharmaceutical formulation is at least 10 mM.

[0070] Stabilizer In certain embodiments, the pharmaceutical formulations provided herein include stabilizers. These stabilizers may be classified based on the mechanisms by which they stabilize proteins against various chemical and physical stresses. Some stabilizers are used to mitigate the effects of specific stresses or to modulate the specific sensitivity of certain proteins. Other stabilizers have a more general effect on the physical and covalent stability of proteins. With consideration of the teachings and guidance provided herein, those skilled in the art will know what amounts or ranges of stabilizers may be included in any particular formulation to achieve the formulations of this disclosure that are likely to enhance the retention and stability of ActRIIa fusion proteins.

[0071] In some embodiments, stabilizers (or combinations of stabilizers) are added to the formulation to prevent or reduce aggregation and chemical degradation induced by storage. A cloudy or turbid solution upon reconstitution usually indicates that proteins have precipitated or at least aggregated. Stabilizers can prevent aggregation or chemical degradation (e.g., autolysis, deamidation, oxidation, etc.). Some stabilizers may also act as anticoagulants when the formulation is administered to a patient. In some embodiments, the stabilizer is selected from the group consisting of: CMC, dextrose, PEG, albumin, kelptoose, proline, sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, genthiobiose, isomaltose, arabinose, glucosamine, fructose, mannitol, sorbitol, polyhydroxy compounds, polysaccharides, dextran, starch, hydroxyethyl starch, cyclodextrin, N-methylpyrrolidone, cellulose, and hyaluronic acid. In certain embodiments, the pharmaceutical formulations provided herein include stabilizers, for example, sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, genthiobiose, isomaltose, arabinose, glucosamine, fructose, mannitol, sorbitol, polyhydroxy compounds, for example, polysaccharides, for example, dextran, starch, hydroxyethyl starch, cyclodextrin, N-methylpyrrolidone, cellulose, and hyaluronic acid (Carpenter et al., Develop. Biol. Standard 74:225, (1991)). In one embodiment of the present disclosure, sucrose is used as a stabilizer.

[0072] In certain embodiments, the formulation contains a stabilizer at a concentration of about 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 700, 900, or 1000 mM. Similarly, in certain embodiments of the present disclosure, the stabilizer is incorporated at a concentration of about 0.005, 0.01, 0.02, 0.03, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20% (weight / volume). In certain embodiments, the stabilizer has a concentration of 2–16% (weight / volume). In certain embodiments, the stabilizer has a concentration of 6–10% (weight / volume). In certain embodiments, the stabilizer has a concentration of at least 8% (weight / volume). In certain embodiments, the stabilizer is sucrose and has a concentration of 2-16% (weight / volume). In certain embodiments, the stabilizer is sucrose and has a concentration of 6-10% (weight / volume). In certain embodiments, the stabilizer is sucrose and has a concentration of at least 8% (weight / volume).

[0073] surfactants In certain embodiments, the pharmaceutical formulations provided herein may further include surfactants. Surfactants are commonly used in protein formulations to prevent surface-induced degradation. Surfactants are amphiphilic molecules that have the ability to compete with proteins for interfacial location (and / or to facilitate the proper refolding of structurally altered protein molecules). The hydrophobic portion of the surfactant molecule occupies the interfacial location (e.g., air / liquid), while the hydrophilic portion remains oriented toward the bulk solvent. At sufficient concentrations (typically near the critical micelle concentration of the surfactant), the surface layer of the surfactant molecule plays a role in preventing protein molecules from adsorbing to the interface. This minimizes surface-induced degradation. Surfactants considered herein include, but are not limited to, fatty acid esters of sorbitan polyethoxylate, i.e., polysorbate 20 and polysorbate 80. These two types differ only in the length of the aliphatic chains that confer hydrophobicity to the molecules, C-12 and C-18, respectively. Therefore, polysorbate 80 has higher surface activity and a lower critical micelle concentration than polysorbate 20.

[0074] Surfactants (detergents) also affect the thermodynamic structural stability of proteins. Nonionic surfactants are generally useful in stabilizing proteins. Ionic surfactants (detergents) typically destabilize proteins. Here again, the effect of a given surfactant excipient is specific to the protein. For example, polysorbates have been shown to decrease the stability of some proteins and increase the stability of others. Destabilization of proteins by surfactants can be reasonably explained in terms of the hydrophobic tails of surfactant molecules that may be involved in specific binding to proteins in a partially or completely unfolded state. This type of interaction may cause a conformational shift to a more expanded protein state (i.e., increasing the exposure of hydrophobic portions of the protein molecule that complement the binding with the polysorbate). Alternatively, if the native state of a protein exhibits some hydrophobic surfaces, a surfactant that binds to that native state may stabilize its conformation. Another aspect of polysorbates is that they are inherently susceptible to oxidative degradation. In many cases, the raw materials contain enough peroxides to cause oxidation of the side chains of protein residues (especially methionine). The potential for oxidative damage resulting from the addition of stabilizers highlights the need to use the lowest effective concentration of excipients in formulations. In the case of surfactants, the effective concentration for a given protein depends on the stabilization mechanism.

[0075] Surfactants are also added in appropriate amounts to prevent surface-related aggregation (Chang, B, J. Pharm. Sci. 85:1325, (1996)). Therefore, exemplary surfactants include, but are not limited to, anionic, cationic, nonionic, zwitterionic, and amphoteric surfactants, which include surfactants derived from naturally occurring amino acids. Anionic surfactants include, but are not limited to, sodium lauryl sulfate, sodium dioctyl sulfosuccinate and sodium dioctyl sulfonate, chenodeoxycholic acid, sodium N-lauroyl sarcosinate, lithium dodecyl sulfate, sodium 1-octanesulfonate, sodium cholate hydrate, sodium deoxycholate, and sodium glycodeoxycholate. Cationic surfactants include, but are not limited to, benzalkonium chloride or benzethonium chloride, cetylpyridinium chloride monohydrate, and hexadecyltrimethylammonium bromide. Examples of zwitterionic surfactants include, but are not limited to, CHAPS, CHAPSO, SB3-10, and SB3-12. Examples of nonionic surfactants include, but are not limited to, digitonin, Triton X-100, Triton X-114, TWEEN(registered trademark)-20, and TWEEN(registered trademark)-80. Surfactants include, but are not limited to, the following: lauromacrogol 400, polyoxyl stearate 40, polyoxyethylene hydrogenated castor oil 10, 40, 50 and 60, glycerol monostearate, polysorbate 40, polysorbate 60, polysorbate 65 and polysorbate 80, soy lecithin and other phospholipids, such as dioleylphosphatidylcholine (DOPC), dimyristoylphosphatidylglycerol (DMPG), dimyristoylphosphatidylcholine (DMPC) and (dioleylphosphatidylglycerol)DOPG; sucrose fatty acid esters, methylcellulose and carboxymethylcellulose.In certain embodiments, the surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, poloxamer 124, poloxamer 127, poloxamer 188, and poloxamer 407. In certain embodiments, the surfactant is polysorbate 80. In certain embodiments, the surfactant is polysorbate 20. In certain embodiments, the surfactant is poloxamer 188.

[0076] In some embodiments, the surfactant is selected from the group consisting of: sodium lauryl sulfate, sodium dioctyl sulfosuccinate and sodium dioctyl sulfonate, chenodeoxycholic acid, sodium N-lauroyl sarcosinate, lithium dodecyl sulfate, sodium 1-octanesulfonate, sodium cholate hydrate, sodium deoxycholate, sodium glycodeoxycholate, benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride monohydrate, hexadecyltrimethylammonium bromide, CHAPS, CHAPSO, SB3-10, SB3-12, digitonin, Triton X-100, Triton X-114, TWEEN®-20, TWEEN®-80, Lauromacrogol 400, Polyoxyl Stearate 40, Polyoxyethylene Hydrogenated Castor Oil 10, 40, 50 and 60, Glycerol Monostearate, Polysorbate 20, Polysorbate 40, Polysorbate 60, Polysorbate 65, Polysorbate 80, Poloxamer 188, and Soy Lecithin. In certain embodiments, the surfactant is selected from the group consisting of Poloxamer 188, Sodium Dodecyl Sulfate (SDS), N-Dodecyl-β-D-Maltoside (DDM), Polysorbate 20, and Triton X. In certain embodiments, the surfactant is selected from the group consisting of Polysorbate 20, Polysorbate 80, Poloxamer 124, Poloxamer 127, Poloxamer 188, and Poloxamer 407.

[0077] Accordingly, formulations containing these surfactants alone or in mixtures of various ratios are further provided. In the formulations of the present invention, the surfactant is incorporated at a concentration of about 0.01 to about 0.5 mg / mL. In the formulations of the present invention, the surfactant is incorporated at a concentration of about 0.05 to about 0.5 mg / mL. In the formulations of the present invention, the surfactant is incorporated at a concentration of about 0.1 to about 0.5 mg / mL. In various embodiments of the pharmaceutical formulations provided herein, the surfactant concentration is 0.005, 0.01, 0.02, 0.03, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mg / mL. In the formulations of the present invention, the surfactant is incorporated at a concentration of 0.2 mg / mL. Similarly, in certain embodiments of the present disclosure, the surfactant is incorporated at a concentration of about 0.001, 0.002, 0.003, 0.004, 0.005, 0.01, 0.02, 0.03, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 0.8, 0.9, or 1.0% (weight / volume).

[0078] In some embodiments, the surfactant is polysorbate 80. In some embodiments, the surfactant is present in the formulation at a concentration of 0.05 to 0.3 mg / mL. In some embodiments, the surfactant is present at a concentration of 0.2 mg / mL. In some embodiments, the surfactant is present at a concentration of at least 0.001, 0.002, 0.003, 0.004, 0.005, 0.01, 0.02, 0.03, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0% (weight / volume). In some embodiments, the surfactant is present at a concentration of at least 0.02% (weight / volume).

[0079] Antioxidants In certain embodiments, the pharmaceutical formulations described herein include one or more antioxidants. Oxidation of protein residues can result from many different causes. In addition to the addition of specific antioxidants, preventing oxidative protein damage requires careful control of many factors throughout the manufacturing process and product storage, including atmospheric oxygen, temperature, light exposure, and chemical contamination.

[0080] Accordingly, this disclosure intends to describe the use of pharmaceutical antioxidants, including reducing agents, oxygen / free radical scavengers, or chelating agents, but is not limited thereto. In one embodiment, antioxidants in therapeutic protein formulations are water-soluble and maintain their activity throughout the product's shelf life. Reducing agents and oxygen / free radical scavengers work by removing reactive oxygen species from solution. In some embodiments of the pharmaceutical formulations described herein, the antioxidant concentration is 0.005, 0.01, 0.02, 0.03, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mg / mL.

[0081] In one embodiment, the pharmaceutical formulation described herein comprises methionine (L-form or D-form), tryptophan (L-form or D-form), dimercaprol, or pyridoxine. In one embodiment, the pharmaceutical formulation described herein comprises methionine (L-form or D-form), tryptophan (L-form or D-form), or pyridoxine. In one embodiment, the pharmaceutical formulation described herein comprises methionine (L-form or D-form). In one embodiment, the pharmaceutical formulation described herein comprises L-methionine. In one embodiment, the pharmaceutical formulation described herein comprises D-methionine.

[0082] In certain embodiments, the pharmaceutical formulations described herein may further contain 1 to 50 mM of antioxidants. In certain embodiments, the pharmaceutical formulations described herein may also contain 1 to 30 mM of antioxidants. In one embodiment, the pharmaceutical formulations described herein may also contain 1 to 20 mM of antioxidants. The pharmaceutical formulations described herein may further contain 5 to 15 mM of antioxidants. The pharmaceutical formulations described herein may further contain 5 to 10 mM of antioxidants. In certain embodiments, the pharmaceutical formulations described herein contain 5 mM, 10 mM, or 50 mM of antioxidants. The pharmaceutical formulations described herein may further contain 10 mM of antioxidants or at least 10 mM of antioxidants.

[0083] In certain embodiments, the pharmaceutical formulations described herein may further contain 1 to 50 mM L-methionine. In certain embodiments, the pharmaceutical formulations described herein may also contain 1 to 30 mM L-methionine. In one embodiment, the pharmaceutical formulations described herein may also contain 1 to 20 mM L-methionine. The pharmaceutical formulations described herein may also contain 5 to 15 mM L-methionine. The pharmaceutical formulations described herein may also contain 5 to 10 mM L-methionine. In certain embodiments, the pharmaceutical formulations described herein may also contain 5 mM, 10 mM, or 50 mM L-methionine. The pharmaceutical formulations described herein may further contain 10 mM L-methionine or at least 10 mM L-methionine.

[0084] Chelating agents In certain embodiments, the pharmaceutical formulations described herein may optionally contain one or more chelating agents. In certain embodiments, the chelating agent is DTPA or EDTA. The pharmaceutical formulations described herein may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 μM of a chelating agent. In certain embodiments, the stable liquid formulation contains 1 to 100 μM, 1 to 30 μM, 1 to 20 μM, or 10 to 30 μM of DTPA or EDTA. In certain embodiments, the stable liquid formulation contains 7.5 to 100 μM of DTPA or EDTA.

[0085] In certain embodiments, the stable liquid formulation contains 10 μM of DTPA or EDTA.

[0086] Actriia Protein Preparation Some embodiments of the pharmaceutical formulations provided herein contain 10–100 mg / mL of human ActRIIa fusion protein, 10–50 mM citrate buffer, 2–16% (weight / volume) of sucrose, 0.05–0.5 mg / mL of polysorbate 80, polysorbate 20, or poloxamer 188, 0–50 mM of L-methionine, and 0–100 μM of DTPA or EDTA. Other embodiments of the pharmaceutical formulations provided herein contain 50 mg / mL of human ActRIIa fusion protein of SEQ ID NO: 32 or a variant of SEQ ID NO: 32 lacking C-terminal lysine, 10 mM citrate buffer, 8% (weight / volume) of sucrose, 0.2 mg / mL of polysorbate 80, and 20 mM of L-methionine. Other embodiments of the pharmaceutical formulations provided herein include 50 mg / mL of human ActRIIa fusion protein of SEQ ID NO: 32, 10 mM citrate buffer, 8% (weight / volume) sucrose, 0.2 mg / mL of polysorbate 80, and 20 mM L-methionine.

[0087] Some embodiments of the pharmaceutical formulations provided herein contain 10–100 mg / mL of human ActRIIa fusion protein, 10–50 mM citrate buffer, 2–16% (weight / volume) of sucrose, 0.05–0.5 mg / mL of polysorbate 80, polysorbate 20, or poloxamer 188, 0–50 mM of L-methionine, and 0–100 μM of DTPA or EDTA. Other embodiments of the pharmaceutical formulations provided herein contain 50 mg / mL of human ActRIIa fusion protein of SEQ ID NO: 41, 10 mM citrate buffer, 8% (weight / volume) of sucrose, 0.2 mg / mL of polysorbate 80, and 20 mM of L-methionine.

[0088] Some embodiments of the pharmaceutical formulations provided herein contain 10–100 mg / mL of human ActRIIa fusion protein, 10–50 mM citrate buffer, 2–16% (weight / volume) of sucrose, 0.05–0.5 mg / mL of polysorbate 80, polysorbate 20, or poloxamer 188, 0–50 mM of L-methionine, and 0–100 μM of DTPA or EDTA. Other embodiments of the pharmaceutical formulations provided herein contain 50 mg / mL of human ActRIIa fusion protein in a mixture of SEQ ID NO: 32 and SEQ ID NO: 41, 10 mM citrate buffer, 8% (weight / volume) of sucrose, 0.2 mg / mL of polysorbate 80, and 20 mM of L-methionine.

[0089] In certain embodiments, the dose is administered parenterally. In some embodiments, the dose is administered by subcutaneous injection. In some embodiments, the dose is administered by intradermal injection. In some embodiments, the dose is administered by intramuscular injection. In some embodiments, the dose is administered by intravenous injection. In some embodiments, the dose is self-administered.

[0090] stability In certain embodiments, the liquid pharmaceutical formulation is stored under refrigerated conditions (temperature range: typically about 2–8°C, but under certain circumstances, aqueous formulations may exhibit stability at other temperatures, including about 25°C and about 40°C, for periods of up to about 3, 6, 9, or 12 months). In certain embodiments, the pharmaceutical formulation provided herein is stable when stored at 2–8°C. In certain embodiments, the pharmaceutical formulation provided herein is stable when stored at 2–8°C for 1 month, 3 months, 6 months, or 12 months. In certain embodiments, the pharmaceutical formulation provided herein is stable when stored at 5°C. In certain embodiments, the pharmaceutical formulation provided herein is stable when stored at 2–8°C for 1 month, 3 months, 6 months, or 12 months. In certain embodiments, the pharmaceutical formulation provided herein is stable when stored at 5°C. In certain embodiments, the pharmaceutical formulation provided herein is stable when stored at 25°C for 1 month, 3 months, 6 months, or 12 months. In certain embodiments, the pharmaceutical formulations provided herein are stable when stored at 5°C. In certain embodiments, the pharmaceutical formulations provided herein are stable when stored at 40°C for 1 month, 3 months, 6 months, or 12 months.

[0091] kit This disclosure provides kits comprising pharmaceutical formulations and injection devices provided herein. In certain embodiments, the pharmaceutical formulation comprises an ActRIIA protein or ActRIIA fusion protein (for example, a protein that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9, SEQ ID NO: 32, or SEQ ID NO: 41) or a fragment, functional variant, or modified form thereof. In certain embodiments, the protein binds to one or more ligands selected from the group consisting of activin A, activin B, and GDF11. In certain such embodiments, the protein further binds to one or more ligands selected from the group consisting of BMP10, GDF8, and BMP6. In certain embodiments, the protein binds to activin and / or GDF11.

[0092] In some embodiments, the pharmaceutical formulation includes a protein comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9, SEQ ID NO: 32, or SEQ ID NO: 41, a protein consisting essentially of that amino acid sequence, or a protein consisting of that amino acid sequence. In certain such embodiments, the protein comprises an amino acid sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 9, SEQ ID NO: 32, or SEQ ID NO: 41, wherein the protein binds to activin and / or GDF11. In certain embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 32, or SEQ ID NO: 41. In other embodiments, the protein consists of the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 32, or SEQ ID NO: 41.

[0093] In some embodiments, the pharmaceutical formulation contains an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 41. In certain embodiments, the protein consists essentially of the amino acid sequence of SEQ ID NO: 41. In other embodiments, the protein consists of the amino acid sequence of SEQ ID NO: 41.

[0094] In some embodiments, the pharmaceutical formulation contains an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 9. In certain embodiments, the protein consists essentially of the amino acid sequence of SEQ ID NO: 9. In other embodiments, the protein consists of the amino acid sequence of SEQ ID NO: 9.

[0095] In some embodiments, the pharmaceutical formulation contains an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 32. In certain embodiments, the protein consists essentially of the amino acid sequence of SEQ ID NO: 32. In other embodiments, the protein consists of the amino acid sequence of SEQ ID NO: 32.

[0096] In the particular embodiments described above, the protein comprises a fusion protein further comprising the Fc domain of an immunoglobulin. In certain such embodiments, the Fc domain of the immunoglobulin is the Fc domain of IgG1 immunoglobulin. In other embodiments, the fusion protein further comprises a linker domain positioned between the protein domain and the Fc domain of the immunoglobulin. In certain embodiments, the linker domain is a polyglycine linker.

[0097] In certain embodiments, the ActRIIA fusion protein is part of a homodimeric protein complex.

[0098] In certain embodiments, the ActRIIA fusion protein is glycosylated.

[0099] This disclosure provides a kit comprising a pharmaceutical formulation and an injection device as described herein. In certain embodiments, the kit comprises a pharmaceutical formulation and an injection device as described herein. In some embodiments of the kits disclosed herein, the pharmaceutical formulation comprising the protein is pre-filled in one or more containers, such as auto-injectors.

[0100] In certain embodiments, the pH range of the protein-containing pharmaceutical formulation is 5 to 7. In some embodiments, the protein-containing pharmaceutical formulation further includes a buffer. In some embodiments, the buffer is added in an amount of at least 10 mM. In some embodiments, the buffer is added in an amount ranging from about 10 mM to about 200 mM. In some embodiments, the buffer includes a citrate.

[0101] In some embodiments, the pharmaceutical formulation containing a protein further comprises a surfactant. In some embodiments, the surfactant comprises a polysorbate. In some embodiments, the surfactant comprises polysorbate 80 or polysorbate 20.

[0102] In some embodiments, the pharmaceutical formulation containing protein further contains sugars such as disaccharides (e.g., sucrose). In some embodiments, the stable liquid pharmaceutical formulation containing protein contains sucrose, trehalose, mannitol, polyvinylpyrrolidone (PVP), dextrose, and / or glycine. In some embodiments, the pharmaceutical formulation containing protein contains sucrose. In some embodiments, the pharmaceutical formulation contains protein and sugar in a weight ratio of at least 1:1 protein to sugar. In some embodiments, the pharmaceutical formulation contains protein and sugar in a weight ratio of 1:1 to 1:10 protein to sugar. In some embodiments, the pharmaceutical formulation contains protein and sugar in a weight ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10 protein to sugar. In some embodiments, the pharmaceutical formulation contains protein and sugar in a weight ratio of 1:6 protein to sugar. In the particular embodiment described above, the pharmaceutical formulation contains a protein and a sugar in an amount sufficient to stabilize the protein.

[0103] In certain embodiments of the kits disclosed herein, the injection device includes a syringe. In certain such embodiments, the syringe is pre-filled with a stable liquid formulation.

[0104] In certain embodiments of the kits disclosed herein, the kit further includes an injectable device for use in parenterally administering a sterile injection. In some embodiments, the sterile injection is administered by subcutaneous injection. In some embodiments, the sterile injection is administered by intradermal injection. In some embodiments, the sterile injection is administered by intramuscular injection. In some embodiments, the sterile injection is administered by intravenous injection.

[0105] In certain embodiments of the kits disclosed herein, the kit further includes a self-injector for use in administering a sterile injection solution. In some embodiments, the sterile injection solution is self-administered. In some embodiments, the sterile injection solution contains a therapeutically effective dose. In some embodiments, the therapeutically effective dose contains a weight-based dose.

[0106] Actriia protein In certain embodiments, this disclosure relates to ActRIIA proteins. Where used herein, the term “ActRIIA” refers to the activin receptor type IIA (ActRIIA) protein family derived from any species, and variants derived from such ActRIIA proteins by mutagenesis or other modifications. References to ActRIIA herein are understood to refer to any one of the currently identified forms. Members of the ActRIIA family are generally transmembrane proteins comprising a ligand-binding extracellular domain containing a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain in which serine / threonine kinase activity is expected.

[0107] The term “ActRIIA protein” encompasses any naturally occurring protein of an ActRIIA family member and any variant thereof that retains useful activity (e.g., mutants, fragments, fusions, and peptide-mimicking forms). Examples of such variant ActRIIA proteins are provided throughout this disclosure and in International Patent Application Publications WO2006 / 012627 and WO2007 / 062188 (which are incorporated herein by reference in their entirety). The amino acid numbering for all ActRIIA-related proteins described herein is based on the numbering of the human ActRIIA precursor protein sequence (SEQ ID NO: 9) provided below, unless otherwise specifically designated.

[0108] The canonical sequence of the human ActRIIA precursor protein is as follows: [Table 1]

[0109] Signal peptides are single underlineThe extracellular domain is shown in bold; and the potential endogenous N-linked glycosylation site is double underline This is shown.

[0110] The sequence of the processed (mature) extracellular human ActRIIA protein is as follows: [Table 2]

[0111] The C-terminal "tail" of the extracellular domain is single underline As shown above, the sequences from which the "tail" has been removed (Δ15 sequences) are as follows: [Table 3]

[0112] The nucleic acid sequence encoding the human ActRIIA precursor protein is shown below (SEQ ID NO: 12), followed by nucleotides 159-1700 of Genbank Reference Sequence NM_001616.4. The signal sequence is: underline It has been deducted. [Table 4]

[0113] TIFF0007918343000005.tif220161

[0114] The nucleic acid sequence encoding the processed soluble (extracellular) human ActRIIA protein is as follows: [Table 5]

[0115] ActRIIA is well-conserved among vertebrates, and most of its extracellular domain is completely conserved. For example, Figure 1 shows a multi-sequence alignment of the human ActRIIA extracellular domain compared to various ActRIIA orthologues. Many of the ligands that bind to ActRIIA are also highly conserved. Therefore, from these alignments, it is possible to predict the key amino acid positions within the ligand-binding domain that are important for normal ActRIIA-ligand-binding activity, and also to predict amino acid positions that are likely to be resistant to substitution without significantly altering normal ActRIIA-ligand-binding activity. Thus, a useful active human ActRIIA mutant protein according to the currently disclosed method may contain one or more amino acids at corresponding positions in the sequences of other vertebrate ActRIIA, or may contain residues similar to those in the human or other vertebrate sequences.

[0116] While not limiting, the following examples illustrate this approach to defining active ActRIIA variants. As shown in Figure 1, F13 in the human extracellular domain corresponds to Y in ActRIIA of sheep (Ovis aries) (SEQ ID NO: 62), red junglefowl (Gallus) (SEQ ID NO: 65), cattle (Bos Taurus) (SEQ ID NO: 66), barn owl (Tyto alba) (SEQ ID NO: 67), and bat (Myotis davidii) (SEQ ID NO: 68), indicating that aromatic residues containing F, W, and Y are permissible at this position. Q24 in the human extracellular domain corresponds to R in Bos Taurus ActRIIA, indicating that charged residues containing D, R, K, H, and E are permissible at this position. S95 in the human extracellular domain is F in the ActRIIA of the red junglefowl (Gallus gallus) and barn owl (Tyto alba), indicating that this site can tolerate a wide variety of changes, including polar residues such as E, D, K, R, H, S, T, P, G, and Y, and possibly hydrophobic residues such as L, I, or F. E52 in the human extracellular domain is D in the sheep (Ovis aries) ActRIIA, indicating that acidic residues including D and E are tolerated at this position. P29 in the human extracellular domain is relatively poorly conserved, appearing as S in the sheep (Ovis aries) ActRIIA and as L in the bat (Myotis davidii) ActRIIA. Therefore, essentially any amino acid should be tolerated at this position.

[0117] Furthermore, as discussed above, ActRII proteins have been characterized in the art in terms of their structural and functional properties, particularly with respect to ligand binding (Attisano et al. (1992) Cell 68(1):97-108; Greenwald et al. (1999) Nature Structural Biology 6(1): 18-22; Allendorph et al. (2006) PNAS 103(20: 7643-7648; Thompson et al. (2003); and U.S. Patents 7,709,605, 7,612,041 and 7,842,663). In addition to the teachings herein, these references provide ample guidance on how to generate ActRII variants that retain one or more desired activities (e.g., ligand-binding activity).

[0118] For example, a distinctive structural motif known as the three-finger toxin fold is crucial for ligand binding by type I and type II receptors and is formed by conserved cysteine ​​residues located at various positions within the extracellular domain of each monomeric receptor ("Greenwald et al. (1999) Nat Struct Biol 6:18-22" and "Hinck (2012) FEBS Lett 586:1860-1870"). Therefore, the core ligand-binding domain of human ActRIIA, defined by the outermost of these conserved cysteines, corresponds to positions 30-110 of SEQ ID NO: 9 (ActRIIA precursor). Therefore, structurally disordered amino acids adjacent to the core sequences defined by these cysteines can cleave approximately residues 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 at the N-terminus and approximately residues 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 at the C-terminus, without necessarily altering ligand binding. Exemplary cleavage of the ActRIIA extracellular domain includes SEQ ID NOs: 10 and 11.

[0119] Therefore, the general formula for the active region of ActRIIA (e.g., ligand binding of ActRIIA) is either a protein containing amino acids 30-110 of SEQ ID NO: 9, or a protein consisting essentially of amino acids 30-110, or a protein consisting of amino acids 30-110. Thus, an ActRIIA protein begins with a residue corresponding to any one of amino acids 21-30 of SEQ ID NO: 9 (e.g., any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) and ends at a position corresponding to any one of amino acids 110-135 of SEQ ID NO: 9 (e.g., amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123 It may contain, or may consist of, or may consist of, a portion of ActRIIA (ending in any one of 24, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135) an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or may consist of, or may consist of, such amino acid sequences. Other examples include starting at a position selected from 21-30 of SEQ ID NO: 9 (for example, starting with any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30), or starting at a position selected from 22-30 (for example, starting with any one of amino acids 22, 23, 24, 25, 26, 27, 28, 29, or 30), or starting at a position selected from 23-30 (for example, starting with any one of amino acids 23, 24, 25, 26, 27, 28, 29, or 30). ), or starting at a position selected from 24-30 (for example, starting with any one of amino acids 24, 25, 26, 27, 28, 29, or 30), and ending at a position selected from 111-135 of SEQ ID NO: 9 (for example, ending with any one of amino acids 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135), or,It ends at a position selected from 112-135 (for example, ending with any one of amino acids 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134 or 135), or it ends at a position selected from 113-135 (for example, amino acids 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 1 (Ends with any one of 32, 133, 134, or 135) or ends at a position selected from 120-135 (e.g., ending with any one of amino acids 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135) or ends at a position selected from 130-135 (e.g., ending with any one of amino acids 130, 131, 132, 133, 134, or 135) or ends at a position selected from 111-134 (e.g., ending with any one of amino acids 110, 111, 1 (ending in any one of 12, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133 or 134), or (ending in any one of 111-133 (for example, ending in any one of amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132 or 133), or , or end at a position selected from 111-132 (for example, ending at any one of amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 or 132), or end at a position selected from 111-131 (for example, amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,There are constructs, etc., ending in either 130 or 131. Variants within these ranges are also intended, in particular, variants containing amino acid sequences that have at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the corresponding portion of SEQ ID NO: 9, variants consisting essentially of those amino acid sequences, or variants consisting of those amino acid sequences. Accordingly, in some embodiments, the ActRIIA protein may include, essentially consist of, or be composed of, proteins that are at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 30-110 of SEQ ID NO: 9. In some cases, ActRIIA proteins are at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 30-110 of SEQ ID NO: 9, and include proteins containing 1 or fewer, 2 or fewer, 5 or fewer, 10 or fewer, or 15 or fewer conservative amino acid changes in the ligand-binding pocket.

[0120] In certain embodiments, this disclosure relates to GDF / BMP antagonists (inhibitors) comprising ActRIIA proteins (which include their fragments, functional variants and modified forms), and their uses (e.g., to enhance the immune response in patients who require it, and to treat cancer). Preferably, the ActRIIA protein is soluble (e.g., the extracellular domain of ActRIIA). In some embodiments, the ActRIIA protein inhibits (e.g., Smad signaling) one or more GDF / BMP ligands (e.g., GDF11, GDF8, activin (activin A, activin B, activin AB, activin C, activin E), BMP6, GDF3, BMP15, and / or BMP10). In some embodiments, the ActRIIA protein binds to one or more GDF / BMP ligands (e.g., GDF11, GDF8, activin (activin A, activin B, activin AB, activin C, activin E), BMP6, GDF3, BMP15, and / or BMP10). In some embodiments, the ActRIIA protein of this disclosure begins at a residue corresponding to amino acids 21-30 of SEQ ID NO: 9 (e.g., any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) and ends at a position corresponding to any one of amino acids 110-135 of SEQ ID NO: 9 (e.g., amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123) It contains, essentially consists of, or consists of amino acid sequences that are at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the portion of ActRIIA (ending in any one of 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135).In some embodiments, the ActRIIA protein contains, consists of, or essentially consists of amino acid sequences that are at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 30-110 of SEQ ID NO: 9. In certain embodiments, the ActRIIA protein contains, consists of, or essentially consists of amino acid sequences that are at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 21-135 of SEQ ID NO: 9. In some embodiments, the ActRIIA protein or ActRIIA fusion protein contains, consists of, or essentially consists of amino acid sequences that are at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to any one of the amino acid sequences of SEQ ID NOs: 9, 10, 11, 32, 36, 39, and 41.

[0121] In some embodiments, the ActRIIa fusion protein contains an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 32. In some embodiments, the ActRIIa fusion protein contains the amino acid sequence of SEQ ID NO: 32. In some embodiments, the ActRIIa fusion protein consists of the amino acid sequence of SEQ ID NO: 32. In some embodiments, the ActRIIa fusion protein is part of a homodimeric protein complex. In some embodiments, the ActRIIa fusion protein is glycosylated. In some embodiments, the ActRIIa fusion protein has a glycosylation pattern obtained by expression in Chinese hamster ovary cells.

[0122] In some alternative embodiments, the ActRII protein (e.g., SEQ ID NO: 32) lacks a C-terminal lysine. In some embodiments, the ActRII protein lacking a C-terminal lysine is SEQ ID NO: 41. In some embodiments, the formulation comprises a mixture of SEQ ID NO: 32 and SEQ ID NO: 41.

[0123] In certain embodiments, the pharmaceutical formulation described herein comprises a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 lacking a C-terminal lysine residue (SEQ ID NO: 41). In certain embodiments, the pharmaceutical formulation described herein comprises a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 lacking a C-terminal lysine residue (SEQ ID NO: 41), wherein the mixture is in the following proportions by weight: 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, Includes SEQ ID NO: 32 with values ​​of 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0124] In certain embodiments, the pharmaceutical formulations described herein comprise a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 lacking a C-terminal lysine residue (SEQ ID NO: 41), wherein the mixture comprises, by weight, approximately 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, Includes SEQ ID NO: 32 with values ​​of 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0125] In certain embodiments, the pharmaceutical formulations described herein comprise a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 lacking a C-terminal lysine residue (SEQ ID NO: 41), wherein the mixture comprises, by weight, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, Includes SEQ ID NO: 41 with 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0126] In certain embodiments, the pharmaceutical formulations described herein comprise a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 lacking a C-terminal lysine residue (SEQ ID NO: 41), wherein the mixture comprises, by weight, approximately 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, Includes SEQ ID NO: 41 with 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0127] In certain embodiments, the pharmaceutical formulation described herein includes 100% by weight of SEQ ID NO: 32. In certain embodiments, the pharmaceutical formulation described herein includes 100% by weight of SEQ ID NO: 41.

[0128] In certain embodiments, this disclosure relates to GDF trap proteins (also referred to as “GDF traps”). In some embodiments, the GDF traps of this disclosure are mutant ActRII proteins (e.g., ActRIIA) comprising one or more mutations (e.g., addition, deletion, substitution, and combination thereof) in the extracellular domain (also referred to as the ligand-binding domain) of an ActRIIA protein (e.g., “wild-type” or unmodified ActRIIA protein) such that the mutant ActRIIA protein has one or more altered ligand-binding activities compared to the corresponding wild-type ActRIIA protein. In some embodiments, the GDF trap proteins of this disclosure retain at least one similar activity to the corresponding wild-type ActRIIA protein. For example, a preferred GDF trap binds to GDF11 and / or GDF8 and inhibits their function (e.g., antagonizes them). In some embodiments, the GDF traps of this disclosure further bind to one or more ligands of GDF / BMP and inhibit them. Thus, this disclosure provides GDF trap proteins having altered binding specificity to one or more ActRII ligands.

[0129] For example, one or more mutations may be selected that increase the selectivity of the modified ligand-binding domain for GDF11 and / or GDF8 compared to one or more ActRIIA-binding ligands (e.g., activin, in particular activin A). In some cases, the modified ligand-binding domain may have a K ratio for GDF11 and / or GDF8 binding that is at least 2, 5, 10, 20, 50, 100, or 1000 times greater than the ratio for the wild-type ligand-binding domain. d K related to activin binding d The ratio is such that, in some cases, the modified ligand-binding domain is at least 2 times, 5 times, 10 times, 20 times, 50 times, 100 times, or even 1000 times larger than the wild-type ligand-binding domain in terms of IC for inhibiting GDF11 and / or GDF8. 50 IC to inhibit activin 50It has the ratio of . In some cases, the modified ligand-binding domain is used to inhibit IC for activin 50 ICs that are at least 2 times, 5 times, 10 times, 20 times, 50 times, and 100 times smaller than the others. 50 And, or even, an IC that is 1000 times smaller. 50 This inhibits GDF11 and / or GDF8.

[0130] How to use In certain embodiments, the Disclosure provides a method for treating pulmonary arterial hypertension (PAH), the method comprising administering a pharmaceutical formulation described herein to a patient in need of treatment for pulmonary arterial hypertension.

[0131] In certain embodiments, the Disclosure provides a pharmaceutical formulation for treating PAH in subjects requiring treatment of PAH, comprising administering a lyophilized human ActRIIA fusion protein ligated to a constant domain of immunoglobulin, wherein the administration regimen comprises (1) administering an initial dose of 0.3 mg / kg; (2) administering a subsequent dose of 0.7 mg / kg three weeks after the initial dose; and (3) monitoring the subject's response; and (4) modifying the subsequent dose, wherein the subject receives the subsequent dose every three weeks. In some embodiments, the subsequent dose is modified based on the subject's response.

[0132] In certain embodiments, the Disclosure provides a method for treating pulmonary arterial hypertension (PAH), wherein the method comprises administering a pharmaceutical formulation described herein to a patient in need of treatment for pulmonary arterial hypertension, wherein the administration of the pharmaceutical formulation results in a change in one or more of the following hemodynamic or functional parameters: a reduction in pulmonary vascular resistance (PVR); an increase in the 6-minute walk distance (6MWD); a decrease in N-terminal pro-B natriuretic peptide (NT-proBNP) levels; prevention or reduction of progression of the World Health Organization (WHO) recognized functional classification of pulmonary hypertension; promotion or increase of regression of the WHO recognized functional classification of pulmonary hypertension; improvement of right ventricular function; improvement of pulmonary artery pressure; and / or improvement of mean right atrial pressure.

[0133] In certain embodiments, the Disclosure provides a method for treating pulmonary arterial hypertension (PAH), the method comprising administering a pharmaceutical formulation described herein to a patient in need of treatment for pulmonary arterial hypertension, wherein the administration of the pharmaceutical formulation results in increased exercise capacity, provides clinical improvement, improves WHO functional class (FC), and slows disease progression (this includes reducing the risk of death and hospitalization related to PAH).

[0134] In certain embodiments, the Disclosure provides a method for treating, preventing or reducing the rate of progression and / or severity of one or more complications of pulmonary arterial hypertension, wherein the method comprises administering a pharmaceutical formulation described herein to a patient in need of treatment, prevention or reduction of the rate of progression and / or severity of one or more complications of pulmonary arterial hypertension, wherein the administration of the protein results in one or more changes in the following hemodynamic or functional parameters: a reduction in pulmonary vascular resistance (PVR); an increase in the 6-minute walk distance (6MWD); a decrease in N-terminal pro-B natriuretic peptide (NT-proBNP) levels; prevention or reduction of progression of the World Health Organization (WHO) functional classification of pulmonary hypertension; promotion or increase of regression of the WHO functional classification of pulmonary hypertension; improvement of right ventricular function; improvement of pulmonary artery pressure; and / or improvement of mean right atrial pressure. In some embodiments, the one or more complications of pulmonary arterial hypertension described above are selected from the group consisting of proliferation of smooth muscle and / or endothelial cells in the pulmonary arteries, angiogenesis in the pulmonary arteries, dyspnea, chest pain, pulmonary vascular remodeling, right ventricular hypertrophy, and pulmonary fibrosis.

[0135] In some embodiments, administration of the pharmaceutical formulations described herein reduces the patient's PVR. In some embodiments, administration of the pharmaceutical formulations described herein reduces the patient's PVR by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%). In some embodiments, administration of the pharmaceutical formulations described herein reduces the patient's PVR by at least 20%. In some embodiments, the reduction in PVR is a result of a decrease in mean pulmonary artery pressure. In some embodiments, administration of the pharmaceutical formulations described herein increases the patient's 6-minute walking distance. In some embodiments, administration of the pharmaceutical formulations described herein increases the patient's 6-minute walking distance by at least 10 meters (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300 meters, or 400 meters or more). In some embodiments, administration of the pharmaceutical formulation described herein increases a patient's 6-minute walking distance by at least 30 meters. In some embodiments, administration of the pharmaceutical formulation described herein lowers a patient's NT-proBNP levels. In some embodiments, administration of the pharmaceutical formulation described herein lowers a patient's NT-proBNP levels by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least 80%). In some embodiments, administration of the pharmaceutical formulation described herein lowers a patient's NT-proBNP levels by at least 30%. In some embodiments, administration of the pharmaceutical formulation described herein lowers NT-proBNP levels to normal levels. In some embodiments, a normal level of NT-proBNP is <100 pg / mL.

[0136] In some embodiments, administration of the pharmaceutical formulations described herein prevents or reduces the progression of pulmonary hypertension functional classification as recognized by the WHO. In some embodiments, administration of the pharmaceutical formulations described herein prevents or reduces the progression of pulmonary hypertension functional classification from functional class I to class II pulmonary hypertension as recognized by the WHO. In some embodiments, administration of the pharmaceutical formulations described herein prevents or reduces the progression of pulmonary hypertension functional classification from functional class II to class III pulmonary hypertension as recognized by the WHO. In some embodiments, administration of the pharmaceutical formulations described herein prevents or reduces the progression of pulmonary hypertension functional classification from functional class III to class IV pulmonary hypertension as recognized by the WHO. In some embodiments, administration of the pharmaceutical formulations described herein promotes or increases the regression of pulmonary hypertension functional classification as recognized by the WHO. In some embodiments, administration of the pharmaceutical formulations described herein promotes or increases the regression of pulmonary hypertension functional classification from class IV to class III pulmonary hypertension as recognized by the WHO. In some embodiments, administration of the pharmaceutical formulations described herein promotes or increases the regression of the functional classification of pulmonary hypertension from Class III to Class II pulmonary hypertension as recognized by the WHO. In some embodiments, administration of the pharmaceutical formulations described herein promotes or increases the regression of the functional classification of pulmonary hypertension from Class II to Class I pulmonary hypertension as recognized by the WHO.

[0137] In some embodiments, administration of the pharmaceutical formulations described herein improves the right ventricular function of a patient. In some embodiments, the improvement in right ventricular function is due to an increase in the change in right ventricular volume. In some embodiments, the improvement in right ventricular function is due to a decrease in right ventricular hypertrophy. In some embodiments, the improvement in right ventricular function is due to an increase in ejection fraction. In some embodiments, the improvement in right ventricular function is due to an increase in both the change in right ventricular volume and the ejection fraction.

[0138] In some embodiments, administration of the pharmaceutical formulations described herein improves the patient's pulmonary artery pressure. In some embodiments, the improvement in pulmonary artery pressure is a decrease in mean pulmonary artery pressure (mPAP). In some embodiments, administration of the pharmaceutical formulations described herein reduces the patient's mPAP by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%). In some embodiments, administration of the pharmaceutical formulations described herein reduces the patient's mPAP by at least 3 mmHg (e.g., at least 3, 5, 7, 10, 12, 15, 20, or 25 mmHg). In some embodiments, administration of the pharmaceutical formulations described herein improves the patient's mean right atrial pressure (mRAP). In some embodiments, the improvement in mRAP is a decrease in mRAP. In some embodiments, administration of the pharmaceutical formulations described herein reduces a patient's mRAP by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%). In some embodiments, administration of the pharmaceutical formulations described herein reduces a patient's mRAP by at least 1 mmHg (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mmHg).

[0139] In some embodiments, the patient has a pulmonary vascular resistance (PVR) of 3 Wood units or more. In some embodiments, the patient has a 6-minute walking distance of 150 to 550 meters. In some embodiments, the patient has elevated NT-proBNP levels compared to healthy patients. In some embodiments, the patient has an NT-proBNP level of at least 100 pg / mL (e.g., 100, 150, 200, 300, 400, 500, 1000, 3000, 5000, 10000, 15000, or 20000 pg / mL). In some embodiments, the patient has elevated brain natriuretic peptide (BNP) levels compared to healthy patients. In some embodiments, the patient has a BNP level of at least 100 pg / mL (e.g., 100, 150, 200, 300, 400, 500, 1000, 3000, 5000, 10000, 15000, or 20000 pg / mL). In some embodiments, administration of the pharmaceutical formulation described herein reduces the patient's BNP level by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least 80%). In some embodiments, administration of the pharmaceutical formulation described herein reduces the BNP level to a normal level (i.e., <100 pg / mL). In some embodiments, the patient has a mean pulmonary artery pressure (mPAP) selected from the group consisting of at least 20 mmHg mPAP; at least 25 mmHg mPAP; at least 30 mmHg mPAP; at least 35 mmHg mPAP; at least 40 mmHg mPAP; at least 45 mmHg mPAP; and at least 50 mmHg mPAP. In some embodiments, the patient has a mean right atrial pressure (mRAP) selected from the group consisting of at least 5 mmHg mRAP; at least 6 mmHg mRAP; at least 8 mmHg mRAP; at least 10 mmHg mRAP; at least 12 mmHg mRAP; at least 14 mmHg mRAP; and at least 16 mmHg mRAP.

[0140] In some embodiments, PAH is idiopathic pulmonary arterial hypertension (PAH). In some embodiments, the PAH is hereditary PAH. In some embodiments, the PAH is drug-induced or toxin-induced PAH. In some embodiments, the PAH is PAH associated with a simple congenital systemic-pulmonary shunt at least one year after shunt repair. In some embodiments, the patient has functional class II or III pulmonary hypertension according to the World Health Organization's functional classification system for pulmonary hypertension. In some embodiments, the patient has functional class I, class II, class III or class IV pulmonary hypertension as recognized by the World Health Organization. In some embodiments, the patient has functional class I, class II, class III or class IV pulmonary hypertension according to the World Health Organization's functional classification system for pulmonary hypertension. In some embodiments, the patient has functional class IV pulmonary hypertension according to the World Health Organization's functional classification system for pulmonary hypertension. In some embodiments, administration of the pharmaceutical formulations described herein increases the transplant-free survival in the patient. In some embodiments, administration of the pharmaceutical formulations described herein increases the transplant-free survival rate in patients by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%). In some embodiments, administration of the pharmaceutical formulations described herein reduces right ventricular hypertrophy in patients. In some embodiments, administration of the pharmaceutical formulations described herein reduces right ventricular hypertrophy in patients by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%). In some embodiments, administration of the pharmaceutical formulations described herein reduces smooth muscle hypertrophy in patients. In some embodiments, administration of the pharmaceutical formulations described herein reduces smooth muscle hypertrophy in patients by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%). In some embodiments, administration of the pharmaceutical formulations described herein reduces pulmonary arteriole muscularity in patients.In some embodiments, administration of the pharmaceutical formulations described herein reduces pulmonary arteriole muscle hypertrophy in a patient by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%).

[0141] In some embodiments, administration of the pharmaceutical formulations described herein improves the patient's exercise capacity. In some embodiments, administration of the pharmaceutical formulations described herein reduces the patient's Borg dyspnea index (BDI). In some embodiments, administration of the pharmaceutical formulations described herein reduces the patient's BDI by at least 0.5 index points (e.g., at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 index points). In some embodiments, the patient has impaired renal function. In some embodiments, administration of the pharmaceutical formulations described herein further improves renal function. In some embodiments, administration of the pharmaceutical formulations described herein delays the clinical progression of pulmonary arterial hypertension. In some embodiments, administration of the pharmaceutical formulations described herein delays the clinical progression of pulmonary arterial hypertension according to the World Health Organization's functional classification system for pulmonary hypertension. In some embodiments, administration of the pharmaceutical formulations described herein reduces the risk of hospitalization for one or more complications associated with pulmonary arterial hypertension. In some embodiments, administration of the pharmaceutical formulations described herein reduces the risk of morbidity for one or more complications associated with pulmonary arterial hypertension. In some embodiments, the morbidity includes changes in one or more of the following: increased need for lung transplantation and / or heart transplantation; need to initiate salvage therapy with known treatments for PAH; need to increase prostacyclin by at least 10%; need for atrial septal resection; PAH-specific hospitalization for at least 24 hours; and worsening of PAH. In some embodiments, worsening of PAH includes worsening of WHO functional class and a decrease of at least 15% in 6MWD. In some embodiments, administration of the pharmaceutical formulations described herein reduces the risk of death associated with pulmonary arterial hypertension. In some embodiments, administration of the pharmaceutical formulations described herein reduces the risk of death associated with pulmonary arterial hypertension by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%).In some embodiments, the patient has hemoglobin levels of >8 g / dL and <15 g / dL. In some embodiments, the patient's hemoglobin level is <18 g / dL.

[0142] In certain embodiments, the patient treated according to the method described herein is female. In certain embodiments, the patient treated according to the method described herein is male. In certain embodiments, the patient treated according to the method described herein is of any age. In certain embodiments, the patient treated according to the method described herein is under 18 years of age. In certain embodiments, the patient treated according to the method described herein is under 13 years of age. In another particular embodiment, the patient treated according to the method described herein is under 12 years of age, under 11 years of age, under 10 years of age, under 9 years of age, under 8 years of age, under 7 years of age, under 6 years of age, or under 5 years of age. In another particular embodiment, the patient treated according to the method described herein is between 1 and 3 years of age, 3 and 5 years of age, 5 and 7 years of age, 7 and 9 years of age, 9 and 11 years of age, 11 and 13 years of age, 13 and 15 years of age, 15 and 20 years of age, 20 and 25 years of age, 25 and 30 years of age, or 30 years of age or older. In another specific embodiment, the patients treated according to the method described herein are 30–35 years old, 35–40 years old, 40–45 years old, 45–50 years old, 50–55 years old, 55–60 years old, or 60 years old or older. In another specific embodiment, the patients treated according to the method described herein are 18–64 years old, 65–74 years old, or 75 years old or older.

[0143] In certain embodiments, the hemoglobin level of a patient treated according to the dosage forms and methods provided herein is less than 10 g / dL, less than 9 g / dL, less than 8 g / dL, or less than 7 g / dL. In certain embodiments, the hemoglobin level in a patient treated according to the dosage forms and methods provided herein is 7 g / dL to 7.5 g / dL, 7.5 g / dL to 8 g / dL, 8 g / dL to 8.5 g / dL, 8.5 g / dL to 9.0 g / dL, 9.0 g / dL to 9.5 g / dL, or 9.5 g / dL to 10.0 g / dL.

[0144] Method of administration Described herein is a method of administering the liquid formulation described herein. The liquid formulation described herein can be administered to a patient via a parenteral route, for example, by injection (e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, etc.), or via transdermal administration, transmucosal administration, nasal administration, pulmonary administration or oral administration. In certain embodiments, the liquid formulation is administered subcutaneously.

[0145] Also described herein is a method of administering any of the liquid formulations described herein to a patient, wherein the method comprises administering any of the liquid formulations described herein to the patient using a delivery device such as a reusable pen or an auto-injector delivery device. In certain embodiments, the liquid formulation is administered using a reusable pen or an auto-injector delivery device. In certain embodiments, the liquid formulation described herein is administered using an auto-injector delivery device.

[0146] A number of reusable pen or auto-injector delivery devices can be used for subcutaneous delivery of the pharmaceutical formulation of the present invention. Examples include, but are not limited to: Molly TM , and / or DAI TM , and / or PSDI TM (SHL Medical, Zug, Switzerland), and Autopen® (Owen Mumford, Inc., Woodstock, UK) from other manufacturers, Disetronic Pen (Disetronic Medical Systems, Bergdorf, Switzerland), Humalog® Mix75 / 25 TMpen, Humalog® pen, Humulin® 70 / 30 pen (Eli Lilly and Co., Indianapolis, Ind.), NovoPen® I, II and III (Novo Nordisk, Copenhagen, Denmark), NovoPen® Junior (Novo Nordisk, Copenhagen, Denmark), BD TM OptiPen (Becton Dickinson, Franklin Lakes, NJ), OptiPen®, OptiPen Pro®, OptiPen Starlet TM , and OptiClik® (Sanofi-Aventis, Frankfurt, Germany). Examples of disposable pen or auto-injector delivery devices applicable to the subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, the following: SoloSTAR® pen (Sanofi-Aventis), FlexPen® (Novo Nordisk), and KwikPen. TM (Eli Lilly), SureClick TM Autoinjector (Amgen, Thousand Oaks, Calif.), Penlet® (Haselmeier, Stuttgart, Germany), EpiPen® (Dey, LP), and Humira® Pen (Abbott Labs, Abbott Park, Ill.).

[0147] In certain embodiments, the liquid formulations described herein are administered using a reusable pen or auto-injector delivery device, wherein the reusable pen or auto-injector delivery device includes a pre-filled syringe. In certain embodiments, the volume of the pre-filled syringe is selected from syringes of 0.5 mL to 10 mL. In certain embodiments, the pre-filled syringe volumes are 0.5 mL, 0.75 mL, 1.0 mL, 1.25 mL, 1.5 mL, 1.75 mL, 2.0 mL, 2.25 mL, 2.5 mL, 2.75 mL, 3.0 mL, 3.25 mL, 3.5 mL, 3.75 mL, 4.0 mL, 4.25 mL, 4.5 mL, 4.75 mL, 5.0 mL, 5.25 mL, 5.5 mL, 5.75 mL, 6.0 mL, 6.25 mL, 6.5 mL, 6.75 mL, 7.0 mL, 7.25 mL, 7.5 mL, 7.75 mL, 8.0 mL, 8.25 mL, 8.5 mL, 8.75 mL, 9.0 mL, 9.25 mL, 9.5 mL, 9.75 mL, and 10.0 mL, each pre-filled with one of the liquid formulations described herein.

[0148] In certain embodiments, the pre-filled syringe contains one of the formulations described herein, wherein the concentration of ActRIIa protein is 5 mg / mL to 100 mg / mL. In certain embodiments, the concentration of ActRIIa protein is 5 mg / mL to 50 mg / mL. In certain embodiments, the concentration of ActRIIa protein is 8.3 mg / mL to 50 mg / mL. In certain embodiments, the concentration of ActRIIa protein is 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, 95 mg / mL, or 100 mg / mL.

[0149] In one embodiment, the reusable pen or auto-injector delivery device includes a needle having a gauge of 27G or less in diameter. In one embodiment, the needle gauge is in the range of 25G to 33G (this includes the intermediate range, e.g., 25sG, 26, 26sG, 27G, 28G, 29G, 30G, 31G, 32G, and 33G). In one embodiment, the minimum needle diameter and appropriate length are selected according to the viscosity characteristics of the formulation and the device used to deliver the formulation of the present invention.

[0150] In certain embodiments, the liquid formulations described herein are administered using a reusable pen or auto-injector delivery device, where the delivery volume of any of the liquid formulations described herein is 1 mL to 2 mL. In certain embodiments, the delivery volume of any of the liquid formulations described herein is 1.0 mL, 1.1 mL, 1.2 mL, 1.3 mL, 1.4 mL, 1.5 mL, 1.6 mL, 1.7 mL, 1.8 mL, 1.9 mL, or 2.0 mL. In certain embodiments, the delivery volume of any of the liquid formulations described herein is 1.8 mL.

[0151] In certain embodiments, the reusable pen or auto-injector delivery device is useful for injecting any of the liquid formulations described herein within a range of 1 to 5 seconds. In certain embodiments, the reusable pen or auto-injector delivery device is useful for injecting any of the liquid formulations described herein within a range of 1 to 4 seconds. In certain embodiments, the reusable pen or auto-injector delivery device is useful for injecting any of the liquid formulations described herein within a range of 1 to 3 seconds. In certain embodiments, the reusable pen or auto-injector delivery device enables the injection of any of the liquid formulations described herein within a range of 2 to 3 seconds. In certain embodiments, the reusable pen or auto-injector delivery device enables the injection of any of the liquid formulations described herein within a range of 2 to 4 seconds. In certain embodiments, the reusable pen or auto-injector delivery device enables the injection of any of the liquid formulations described herein within a range of 1, 2, 3, 4, or 5 seconds.

[0152] In certain embodiments, the reusable pen or auto-injector delivery device is useful for injecting any of the liquid formulations described herein using a spring force of 10N to 100N. In certain embodiments, the reusable pen or auto-injector delivery device is useful for injecting any of the liquid formulations described herein using a spring force of 10N to 50N. In certain embodiments, the reusable pen or auto-injector delivery device is useful for injecting any of the liquid formulations described herein using a spring force of 10N, 20N, 30N, 40N, 50N, 60N, 70N, 80N, 90N, or 100N. In certain embodiments, the reusable pen or auto-injector delivery device is useful for injecting any of the liquid formulations described herein using a spring force of approximately 10N, 20N, 30N, 40N, 50N, 60N, 70N, 80N, 90N, or 100N. In certain embodiments, the reusable pen or auto-injector delivery device is useful for injecting any of the liquid formulations described herein, using a spring force of about 50 N.

[0153] In certain embodiments, the reusable pen or auto-injector delivery device enables the injection of any of the liquid formulations described herein using a press force of 1N to 10N. In certain embodiments, the reusable pen or auto-injector delivery device enables the injection of any of the liquid formulations described herein using a press force of 1N, 2N, 3N, 4N, 5N, 6N, 7N, 8N, 9N, or 10N. In certain embodiments, the reusable pen or auto-injector delivery device enables the injection of any of the liquid formulations described herein using a press force of about 1N, 2N, 3N, 4N, 5N, 6N, 7N, 8N, 9N, or 10N. In certain embodiments, the reusable pen or auto-injector delivery device enables the injection of any of the liquid formulations described herein using a press force of about 6N. [Examples]

[0154] Examples This disclosure is generally described, but will be more readily understood by referring to the following examples. These examples are included solely for the purpose of illustrating specific embodiments of this disclosure and are not intended to limit this disclosure. [Table 6]

[0155] Example 1: ActRIIA-Fc Fusion Protein Soluble ActRIIA fusion proteins were constructed by fusing the extracellular domain of human ActRIIA to a human or mouse Fc domain and adding a minimal linker between them. These constructs are referred to as ActRIIA-hFc and ActRIIA-mFc, respectively.

[0156] ActRIIA-hFc, shown below, is also known as sotatercept and was purified from the CHO cell line (SEQ ID NO: 32): [Table 7]

[0157] ActRIIA-hFc and ActRIIA-mFc proteins were expressed in CHO cell lines. Three different leader sequences were investigated: (i) Honeybee melittin (HBML): MKFLVNVALVFMVVYISYIYA (Sequence ID: 33) (ii) Tissue plasminogen activator (TPA): MDAMKRGLCCVLLLCGAVFVSP (Sequence ID: 34) (iii) Native: MGAAAKLAFAVFLISCSSGA (Sequence ID: 35).

[0158] The selected form employs a TPA reader and has the following raw amino acid sequence: [Table 8]

[0159] This protein is encoded by the following nucleic acid sequence: [Table 9]

[0160] Both ActRIIA-hFc and ActRIIA-mFc were highly suitable for recombinant expression. As shown in Figures 2A and 2B, the protein was purified as a single, distinct peak. N-terminal sequencing revealed the single sequence -ILGRSETQE (SEQ ID NO: 38). Purification can be achieved by a series of column chromatography steps, for example, comprising three or more of the following in any order: protein A chromatography, Q Sepharose chromatography, phenyl Sepharose chromatography, size exclusion chromatography, and cation exchange chromatography. The purification can be completed by viral filtration and buffer exchange. ActRIIA-hFc proteins were purified to >98% purity as measured by size exclusion chromatography and to >95% purity as measured by SDS-PAGE.

[0161] ActRIIA-hFc and ActRIIA-mFc showed high affinity for their ligands. GDF11 or activin A was used with Biacore using a standard amine coupling method. TM The proteins were immobilized on a CM5 chip. ActRIIA-hFc and ActRIIA-mFc proteins were loaded into the system and their binding was measured. ActRIIA-hFc was immobilized on a CM5 chip. -12 The dissociation constant (K D ) binds to activin, 9.96 × 10 -9 K D ActRIIA-hFc bound to GDF11. See Figures 3A and 3B. Using a similar binding assay, ActRIIA-hFc was found to have high to moderate affinity for other TGF-β superfamily ligands, including activin B, GDF8, BMP6, and BMP10. ActRIIA-mFc exhibited similar behavior.

[0162] ActRIIA-hFc was very stable in pharmacokinetic studies. Rats were administered 1 mg / kg, 3 mg / kg, or 10 mg / kg of ActRIIA-hFc protein, and plasma concentrations of the protein were measured at 24, 48, 72, 144, and 168 hours. In another study, rats were administered 1 mg / kg, 10 mg / kg, or 30 mg / kg. In rats, the serum half-life of ActRIIA-hFc was 11–14 days, and the circulating level of the drug was quite high after two weeks (11 μg / mL, 110 μg / mL, or 304 μg / mL for the initial dose of 1 mg / kg, 10 mg / kg, or 30 mg / kg, respectively). In cynomolgus monkeys, the plasma half-life was substantially more than 14 days, and the circulating levels of the drug were 25 μg / mL, 304 μg / mL, and 1440 μg / mL, respectively, after initial doses of 1 mg / kg, 10 mg / kg, and 30 mg / kg.

[0163] Example 2: Characterization of ActRIIA-hFc protein The ActRIIA-hFc fusion protein was expressed in CHO-DUKX B11 cells stably transfected from a pAID4 vector (SV40 original / enhancer, CMV promoter) using the tissue plasminogen reader sequence of SEQ ID NO: 34. The purified protein in Example 1, as described above, had the sequence of SEQ ID NO: 32. Its Fc portion is a human IgG1 Fc sequence, as shown in SEQ ID NO: 32. Protein analysis revealed that the ActRIIA-hFc fusion protein is formed as a homodimer with a disulfide bond.

[0164] The CHO cell expression exhibited higher affinity for activin B ligand than that reported for ActRIIA-hFc fusion proteins expressed in human 293 cells (see "del Re et al. (2004) J Biol Chem. 279(51):53126-53135"). Furthermore, the use of the TPA reader sequence resulted in higher yields than other reader sequences and provided a purer N-terminal sequence, unlike ActRIIA-Fc expressed with the native reader. The use of the native reader sequence resulted in two major species of ActRIIA-Fc, each with a different N-terminal sequence.

[0165] Further ActRIIA ligand traps (ActRIIA-Fc fusion proteins modified to reduce the ratio of activin A binding to myostatin or GDF11) are described in International Patent Application Publications WO2006 / 012627 and WO2007 / 062188 (these are incorporated herein by reference).

[0166] Example 3: Preparation of a liquid ActRIIA-hFc fusion protein formulation Initially, the ActRIIA-hFc fusion protein sotatercept (SEQ ID NO: 32) was formulated as a frozen solution in phosphate-buffered saline. Based on further developmental studies, a lyophilized citrate-buffered formulation containing sucrose and polysorbate 80 of ActRIIA-hFc fusion protein sotatercept was developed, enabling a stable formulation with sufficient shelf life for commercialization. This lyophilized formulation consists of a lyophilized cake of sotatercept (45 mg / vial or 60 mg / vial) as part of an injection kit that also includes a vial adapter, a pre-filled syringe of sterile water for injection, an injection syringe, a needle, and an alcohol swab. However, users disliked the need to reconstitute the lyophilized cake before administration. To improve the user experience, a sotatercept-containing liquid formulation suitable for combinations with pre-filled syringes and / or auto-injector devices was formulated, taking the following experiments into consideration.

[0167] Feasibility study of concentrated liquid To evaluate the short-term biophysical stability of sotatercept (SEQ ID NO: 32) at various concentrations, concentration stability studies were conducted. Formulations containing 100 mg / mL and 50 mg / mL of sotatercept in 10 mM citrate buffer and 8% (weight / volume) sucrose were prepared at pH 5.8. Table 1 shows the stability of the formulation containing 100 mg / mL of sotatercept. The formulation containing 100 mg / mL of sotatercept was segmented at various temperatures (5°C, 25°C, and 40°C), and selected time points were analyzed. The formulations in citrate buffer and sucrose showed minimal changes in high molecular weight formation (measured by UPSEC) and charge distribution (measured by cIEF) at 5°C and 25°C.

[0168] [Table 10]

[0169] Tables 2 and 3 show the stability of formulations containing 50 mg / mL sotatercept. Formulations containing 50 mg / mL sotatercept were segmented at various temperatures (5°C, 25°C, and 40°C), and selected time points were analyzed. It was shown that formulations in citrate buffer, sucrose, and PS80, both in and out of DTPA, showed minimal changes in subvisible particle formation (measured by MFI), high molecular weight formation (measured by UPSEC), charge distribution (measured by cIEF), and fragmentation (measured by CE-SDS) at 5°C and 25°C.

[0170] [Table 11] [Table 12]

[0171] Feasibility study on pH and buffering agents The behavior of sotatercept (SEQ ID NO: 32) was analyzed in six different buffers at various pH levels and in 8% sucrose. The buffers tested were: histidine at pH 6.5 and 7; phosphates at pH 5, 5.5, 6, 6.5 and 7; citrates at pH 4.5, 5, 5.5, 6, 6.5 and 7; acetates at pH 4.5, 5, 5.5 and 6; succinates at pH 4.5 and 5; and glutamates at pH 4.5 and 5. The results are shown in Tables 4-13. The formulations were subjected to heat stress at 40°C for up to two months, and their biophysical stability (turbidity), sub-visible particle formation (measured by MFI), high molecular weight formation (measured by UPSEC), fragmentation (measured by R-CESDS and NR-CESDS), and charge distribution (measured by cIEF) were tested.

[0172] [Table 13] [Table 14]

[0173] [Table 15] [Table 16]

[0174] [Table 17] [Table 18]

[0175] [Table 19] [Table 20]

[0176] [Table 21] [Table 22]

[0177] As shown in Tables 4-13 and Figures 4-7 above, all formulations exhibited reasonable stability at high temperatures, suggesting that all tested buffering systems are feasible for liquid formulations. Of these, histidine was the least stable, while the remaining buffers exhibited similar behavior.

[0178] The behavior of sotatercept was analyzed in six different buffers (pH 3, pH 5.8, pH 8) and 8% sucrose. The buffers tested were citrate, histidine, succinate, lactate, MES, and TRIS. The results are shown in Tables 14-30. The formulation was tested for biophysical stability (turbidity), high molecular weight formation (by UPSEC), aggregation (by UPSEC), fragmentation (by R-ESD and NR-ESDSUPSEC), and charge distribution (by cIEF) on days 0 (T0), 15, and 30. [Table 23] [Table 24]

[0179] [Table 25] [Table 26]

[0180] [Table 27] [Table 28]

[0181] [Table 29] [Table 30]

[0182] [Table 31] [Table 32]

[0183] [Table 33] [Table 34]

[0184] [Table 35] [Table 36] [Table 37]

[0185] [Table 38] [Table 39]

[0186] Based on the data shown in Tables 14-30, the tested buffers exhibited comparable stability across their entire strength at pH 5.8 and pH 8.0. However, at lower pH levels, stability significantly decreased for all tested buffers. While all buffers performed similarly, citrate and succinate buffers were slightly better than the others.

[0187] Effects of metal chelating agents on sotatercept The effect of the metal chelating agent DTPA on sotatercept (SEQ ID NO: 32) formulated in citrate buffer (pH 5.8) was investigated by 1H NMR profiling, 2D NMR fingerprinting, and NMR measurements of protein translational diffusion. The results of the 1D profiling and protein diffusion are shown in Figures 8 and 9. Identical behavior was observed in the protein 1H NMR spectra of sotatercept in the presence and absence of DTPA (Figure 8), indicating that this technique cannot detect direct effects due to binding or other interactions. Furthermore, data from protein detection diffusion measurements in the presence and absence of DTPA (Figure 9) showed no difference in behavior, indicating that the addition of DTPA does not affect the behavior of sotatercept.

[0188] Colloidal stability and surfactant screening Colloidal stability studies of sotatercept (SEQ ID NO: 32) were conducted by agitation stress by shaking vials containing sotatercept in 10 mM citrate buffer and 8% sucrose in the presence of various levels of PS80, and by freeze-thaw cycles (1 cycle: vial frozen at -80°C and thawed at room temperature). The results are shown in Figures 10-13. Figure 10 shows the percentage of high molecular weight species (%HMW) detected after agitation for 3 and 7 days in vials containing sotatercept in 10 mM citrate buffer and 8% sucrose in the presence of 0%, 0.005%, 0.01%, 0.02%, 0.03%, and 0.05% PS80. Figure 11 shows the %HMW detected in vials containing sotatercept in 10 mM citrate buffer and 8% sucrose in the presence of 0%, 0.005%, 0.01%, 0.02%, 0.03%, and 0.05% PS80, after 3, 5, and 8 freeze-thaw cycles. Figures 12 and 13 show the % monomer (Figure 12) and %HMW (Figure 13) detected in vials containing sotatercept in 10 mM citrate buffer and 8% sucrose in the presence of 0%, 0.01%, 0.02%, and 0.03% PS80, after 3, 5, and 8 freeze-thaw cycles. No changes were observed in %HMW or sub-visible particle formation after 7 days of stirring or 8 freeze-thaw cycles. Furthermore, a direct comparison of polysorbate 80 and poloxamer 188 was also performed (1-Mo / 25℃). The results showed that the molecules were stable under stirring stress, with no difference between PS80 and P188, exhibiting similar behavior. In terms of prototype stability, all analytical attributes were equivalent after 1-Mo stress at 25°C.

[0189] The results of additional surfactant stability studies are shown in Tables 31 to 35. The stability studies shown in Tables 31 to 35 were performed by agitation stress via shaking vials containing sotatercept in 10 mM citrate buffer and 8% sucrose in the presence of various levels of surfactants. The surfactants screened were poloxamer 188 at 0.02 mg / mL, 0.2 mg / mL and 2 mg / mL, sodium dodecyl sulfate (SDS), N-dodecyl-β-D-maltoside (DDM), polysorbate 20 and Triton X at 4 days and 7 days. Table 36 shows the results of a stability study on control sotatercept in 10 mM citrate buffer and 8% sucrose (no surfactant added). [Table 40] [Table 41]

[0190] [Table 42] [Table 43]

[0191] [Table 44] [Table 45]

[0192] The above results show that the preparation is stable against agitation stress in citrate buffer, and there was no significant difference among the tested surfactants when compared in terms of charged species, monomer species, aggregates or fragmentation.

[0193] Tables 37 to 41 show surfactant stability studies on sotatercept, which were conducted via agitation stress caused by shaking vials containing sotatercept in 10 mM histidine buffer and 8% sucrose in the presence of various levels of surfactants. The screened surfactants were 0.02 mg / mL, 0.2 mg / mL and 2 mg / mL of poloxamer 188, sodium dodecyl sulfate (SDS), N-dodecyl-β-D-maltoside (DDM), polysorbate 20 and Triton X, evaluated at 4 days and 7 days. Table 42 shows the results of a surfactant stability study for a control containing sotatercept in 10 mM histidine buffer and 8% sucrose (no surfactant added). [Table 46] [Table 47]

[0194] [Table 48] [Table 49]

[0195] [Table 50] [Table 51]

[0196] The above results show that sotatercept is stable against agitation stress when formulated with the tested surfactants in histidine buffer. A slightly higher level of high-molecular-weight species was detected at high SDS concentrations. When observing charge species after agitation stress, there was no significant difference among the tested surfactants.

[0197] Research on chelating agents Formulations containing sotatercept (SEQ ID NO: 32) in 10 mM citrate buffer, 8% sucrose, and 0.02% polysorbate 80 at pH 5.8 in the presence of various levels (0 mM, 10 mM, 20 mM, 30 mM) of L-methionine were exposed to photostress in a photostable chamber in the presence of various levels (0 μM, 7.5 μM, 15 μM, 30 μM, 60 μM) of DTPA or EDTA as chelating agents. High molecular weight formation was analyzed (using UPSEC) of the stressed samples together with a dark control, showing that %HMW formation decreased with the addition of L-methionine and that there were no adverse effects from the addition of chelating agents. The results are shown in Figures 14 and 15. Therefore, any of the chelating agents can be used in the formulation.

[0198] Furthermore, sotatercept was formulated at pH 5.8 in the presence of various levels (0 mM, 5 mM, 10 mM, 50 mM) of dimercaprol, using 10 mM citrate buffer or 10 mM histidine buffer, 8% sucrose, and 0.02% polysorbate 80. These samples were exposed to photostress in a photostability chamber. The stressed samples were analyzed for high molecular weight formation (using UPSEC) together with a dark control, indicating the presence of dimercaprol. The results of several tests on formulations containing sotatercept, 10 mM citrate buffer, 8% sucrose, and 0.02% polysorbate 80 at pH 5.8 in the presence of various levels (0 mM, 5 mM, 10 mM, 50 mM) of dimercaprol are shown in Table 43, and the results for the dark control are shown in Table 44. Table 45 shows the results of several tests conducted at pH 5.8 in the presence of various levels (0 mM, 5 mM, 10 mM, 50 mM) of dimercaprol on a formulation containing sotatercept, 10 mM histidine buffer, 8% sucrose, and 0.02% polysorbate 80, while the results of a dark control are shown in Table 46.

[0199] [Table 52] [Table 53]

[0200]

Table 54

Table 55

[0201] The above results show that including a low concentration of dimercaprol as an antioxidant can provide protection comparable to the control sample under light stress in both citrate buffer and histidine buffer. When dimercaprol is contained at a high concentration, the charged species increase and the loss of monomer content becomes greater upon exposure to light stress. This indicates that formulations containing high concentrations of dimercaprol as an antioxidant are not suitable.

[0202] Stabilizer screening The behavior of sotatercept (SEQ ID NO: 32) was analyzed among various stabilizers. The tested stabilizers were carboxymethyl cellulose (CMC), dextrose, polyethylene glycol (PEG), albumin, kelptose, proline, trehalose, mannitol and dextran. Tables 47 to 56 show the results of sotatercept formulations comprising 10 mM citrate, 20 mM L-methionine, 0.2 mg / mL and polysorbate 80 at pH 5.8 in the presence of various surfactants. Table 57 shows the results of a citrate control formulation without surfactant. Tables 58 to 67 show the results of sotatercept formulations comprising 10 mM histidine, 20 mM L-methionine, 0.2 mg / mL and polysorbate 80 at pH 5.8 in the presence of various surfactants. Table 68 shows the results of a control formulation without surfactant. The formulations were subjected to heat stress at 50°C for up to 2 weeks, and tested for biophysical stability (turbidity), subvisible particle formation (measured by Aura), high molecular weight species formation (measured by UPSEC), aggregation (measured by UPSEC) and fragmentation (measured by UPSEC).

[0203] Table 56 Table 57

[0204] Table 58 Table 59

[0205] Table 60 Table 61

[0206] Table 62

[0207] Table 63 Table 64

[0208] Table 65

[0209] Table 66

[0210] The above results indicate that, under high thermal stress, including the tested stabilizer in the formulation resulted in the same level of stability for the sotatercept molecule in the citrate buffer. Each test formulation maintained a high monomer content, and compared to the sample without the stabilizer, the high molecular weight species increased to a similar extent over time, and the levels of charged species were also similar. [Table 67] [Table 68]

[0211] [Table 69] [Table 70]

[0212] [Table 71] [Table 72]

[0213] [Table 73] [Table 74]

[0214] [Table 75] [Table 76]

[0215] [Table 77]

[0216] The above results indicate that, under high thermal stress, including the tested stabilizer in the formulation resulted in the same level of stability for the sotatercept molecule in the histidine buffer. Each test formulation maintained a high monomer content, and the high molecular weight species increased to a similar extent over time, as did the level of charged species.

[0217] Sotatercept preparations Tables 69-71 show the liquid pharmaceutical formulations prepared, containing sotatercept (SEQ ID NO: 32, SEQ ID NO: 41, or a mixture of both SEQ ID NO: 32 and SEQ ID NO: 41). [Table 78] [Table 79]

[0218] [Table 80]

[0219] array [Table 81] TIFF0007918343000083.tif243163TIFF0007918343000084.tif83162

[0220] While exemplary methods and materials are described herein, similar or equivalent methods and materials may also be used in carrying out or testing the methods and formulations currently disclosed. All publications, patent applications, patents, and other references mentioned herein are incorporated in their entirety by reference.

Claims

1. A liquid pharmaceutical preparation comprising 50 to 100 mg / mL in total amount of one or both of a human ActRIIaFc fusion protein containing the amino acid sequence of SEQ ID NO: 32 or a human ActRIIaFc fusion protein containing the amino acid sequence of SEQ ID NO: 41, a 10 to 100 mM citrate buffer, 0.02 to 2.0 mg / mL of polysorbate 80, 2 to 16% (weight / volume) of sucrose, and 1 to 30 mM L-methionine, wherein the liquid pharmaceutical preparation has a pH of 5.5 to 6.

5.

2. A liquid pharmaceutical formulation according to claim 1, comprising a total amount of 100 mg / mL of one or both of the human ActRIIaFc fusion proteins.

3. A liquid pharmaceutical formulation according to claim 1, comprising a total amount of 50 mg / mL of one or both of the human ActRIIaFc fusion proteins.

4. The liquid pharmaceutical preparation according to claim 1, wherein the liquid pharmaceutical preparation has a pH of 5.

8.

5. The liquid pharmaceutical formulation according to claim 1, wherein the liquid pharmaceutical formulation contains a citrate buffer in a concentration of 10 to 50 mM.

6. The liquid pharmaceutical formulation according to claim 5, wherein the liquid pharmaceutical formulation contains a 10 mM citrate buffer.

7. The liquid pharmaceutical preparation according to claim 1, wherein the citrate buffer comprises trisodium citrate dihydrate or citric acid monohydrate, or a combination thereof.

8. The liquid pharmaceutical preparation according to claim 1, wherein the liquid pharmaceutical preparation contains 6 to 10% (by weight / volume) of sucrose.

9. The liquid pharmaceutical preparation according to claim 8, wherein the liquid pharmaceutical preparation contains 8% (by weight / volume) sucrose.

10. The liquid pharmaceutical preparation according to claim 1, wherein the liquid pharmaceutical preparation contains 0.1 to 0.5 mg / mL of polysorbate 80.

11. The liquid pharmaceutical formulation according to claim 10, wherein the liquid pharmaceutical formulation contains 0.2 mg / mL of polysorbate 80.

12. The liquid pharmaceutical preparation according to claim 1, wherein the liquid pharmaceutical preparation contains 20 mM L-methionine.

13. A liquid pharmaceutical formulation comprising a total of 50 mg / mL of one or both of the human ActRIIaFc fusion protein having the amino acid sequence of SEQ ID NO: 32 or the human ActRIIaFc fusion protein having the amino acid sequence of SEQ ID NO: 41, a 10 mM citrate buffer, 0.2 mg / mL of polysorbate 80, 8% (weight / volume) of sucrose, and 20 mM L-methionine, wherein the liquid pharmaceutical formulation has a pH of 5.

8.

14. The liquid pharmaceutical preparation according to any one of claims 1 to 13, wherein the liquid pharmaceutical preparation is suitable for administration via an auto-injector.

15. The liquid pharmaceutical preparation according to any one of claims 1 to 13, wherein the liquid pharmaceutical preparation is suitable for subcutaneous administration.

16. The liquid pharmaceutical preparation according to any one of claims 1 to 13, wherein the liquid pharmaceutical preparation is stable when stored at 2 to 8°C for at least one month.

17. The liquid pharmaceutical formulation according to any one of claims 1 to 13, wherein the liquid pharmaceutical formulation is contained in an injection device.

18. The liquid pharmaceutical preparation according to claim 17, wherein the injection device is an auto-injector.

19. The liquid pharmaceutical preparation according to any one of claims 1 to 13, wherein the liquid pharmaceutical preparation is contained in a vial.

20. Use of a liquid pharmaceutical preparation according to any one of claims 1 to 13 in the preparation of a pharmaceutical for the treatment of pulmonary arterial hypertension (PAH).

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