Formulations comprising ACTRIIA protein variants

Abstract

In certain aspects, the present disclosure provides stable liquid pharmaceutical formulations comprising a recombinant fusion protein and one or more pharmaceutical additives and / or excipients, the recombinant fusion protein comprises an extracellular domain (ECD) of a 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.

Description

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

[0002] Reference to electronically submitted sequence lists This application contains a sequence list, which is submitted electronically in XML format and incorporated herein by reference in its entirety. The XML file, created on March 6, 2024, is named 1848179-0002-170-WO1_Sequence_Listing.xml and is 51.2 KB in size. Technical Field

[0003] This article describes a stable liquid pharmaceutical formulation comprising a recombinant fusion protein and one or more pharmaceutical additives and / or excipients, wherein the recombinant fusion protein comprises an 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 the human IgG1 Fc domain. Background Technology

[0004] Pulmonary hypertension (PH) is a condition 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 ≥25 mm Hg (at rest) or ≥30 mm Hg (during exercise) (Hill et al., Respiratory Care 54(7):958-68 (2009)). The primary symptom of PH is shortness of breath or difficulty breathing, and other symptoms include fatigue, dizziness, fainting, peripheral edema (swelling of the feet, legs, or ankles), cyanosis of the lips and skin, chest pain, angina, dizziness during exercise, dry cough, rapid pulse, and palpitations. PH can be a serious condition leading to heart failure, one of the most common causes of death in people with pulmonary hypertension. Postoperative pulmonary hypertension can complicate many types of surgery or procedures and presents challenges associated with high mortality rates.

[0005] PH can be classified according to the different manifestations of the disease based on similarities in pathophysiology, clinical presentation, and treatment approach (Simonneau et al., JACC 54(1): S44-54 (2009)). The clinical classification of PH was first proposed in 1973, and the recently updated clinical classification was approved by the World Health Organization (WHO) in 2008. According to the updated clinical classification of PH, PH is primarily classified into five categories: (1) pulmonary arterial hypertension (PAH) characterized by PA wedge pressure < 15 mm Hg; (2) PH caused by left-sided heart disease (also known as pulmonary venous hypertension or congestive heart failure); (3) PH characterized by PA wedge pressure > 15 mm Hg; (4) PH caused by lung disease and / or hypoxia; chronic thromboembolic PH; and (5) PH with unclear 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), a sporadic disease without a family history of PAH and without clear risk factors; heritable PAH; PAH induced by drugs and toxins; PAH associated with connective tissue disease, HIV infection, portal hypertension, congenital heart disease, schistosomiasis, and chronic hemolytic anemia; and persistent PH of the newborn (Simonneau et al., JACC 54(1): S44-54 (2009)). Diagnosis of the various types of PH requires a series of tests.

[0006] In general, PH treatment depends on the cause or classification of the PH. Where the PH is caused by a known drug or medical condition, it is referred to as secondary PH, and its treatment typically involves the underlying disease. Treatment of pulmonary venous hypertension generally involves optimizing left ventricular function by administration of diuretics, beta blockers, and ACE inhibitors, or repair or replacement of the mitral or aortic valves. PAH therapy includes pulmonary vasodilators, digoxin, diuretics, anticoagulants, and oxygen therapy. Pulmonary vasodilators target different pathways, including the prostacyclin pathway (e.g., prostacyclin analogs, including intravenous epoprostenol, subcutaneous or intravenous treprostinil, and inhaled iloprost), the nitric oxide pathway (e.g., phosphodiesterase-5 inhibitors, including sildenafil and tadalafil), and the endothelin-1 pathway (e.g., endothelin receptor antagonists, including 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 therapies do not provide a cure for PH, and they do not directly treat the underlying vascular remodeling and vascular muscularization observed in many PH patients.

[0007] Accordingly, it is an object of the present disclosure to provide stable liquid pharmaceutical formulations comprising ActRIIa fusion proteins and corresponding methods of treating, preventing, or reducing the progression rate and / or severity of PH, in particular of treating, preventing, or reducing the progression rate and / or severity of one or more PH-related complications. SUMMARY

[0008] Provided herein are stable liquid pharmaceutical formulations comprising a recombinant fusion protein comprising an extracellular domain (ECD) of human Activin Receptor Type IIA (ActRIIa) or a variant thereof linked to a constant domain of an immunoglobulin, e.g., a human IgGl Fc domain, and one or more pharmaceutical additives and / or excipients.

[0009] In some embodiments, the ActRIIa protein comprises 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 starting at any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of SEQ ID NO: 9 and 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, 132, 133, 134, or 135 of SEQ ID NO: 9. In some embodiments, the ActRIIa protein comprises 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 comprises 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.

[0010] In some embodiments, the ActRIIa protein is an ActRIIa fusion protein comprising an ActRIIa extracellular domain and one or more protein domains that are heterologous to ActRIIa. In some embodiments, the ActRIIa protein is a fusion protein comprising an Fc domain of an immunoglobulin. In some embodiments, the Fc domain of an immunoglobulin is an Fc domain of an IgGl immunoglobulin. In some embodiments, the ActRIIa fusion protein further comprises a linker domain between the ActRIIa protein domain and the one or more heterologous domains (e.g., 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 consists of 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: 41. In some embodiments, the ActRIIa fusion protein consists of the amino acid sequence of SEQ ID NO: 32 or 41. In some embodiments, the ActRIIa fusion protein consists of a variant of the amino acid sequence set forth 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 comprises 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 obtainable by expression in Chinese hamster ovary cells.

[0011] In certain embodiments, the pharmaceutical formulations described herein comprise 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 comprise an ActRIIA fusion protein of SEQ ID NO: 32 or a variant of SEQ ID NO: 32 lacking the C-terminal lysine, a buffer, a surfactant, a stabilizer, and optionally one or more antioxidants.

[0012] In some embodiments, the pharmaceutical formulations provided herein comprise 10-100 mg / mL of a recombinant fusion protein comprising an extracellular domain (ECD) of a human activin receptor type IIA (ActRIIa) protein or derivative thereof linked to a constant domain of an immunoglobulin, e.g., a human IgGl Fc domain. In other embodiments, the pharmaceutical formulations comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg / mL of a recombinant fusion protein comprising an extracellular domain (ECD) of a human activin receptor type IIA (ActRIIa) protein or derivative thereof linked to a constant domain of an immunoglobulin, e.g., a human IgGl Fc domain. In other embodiments, the pharmaceutical formulations comprise 40-50 mg / mL of a recombinant fusion protein comprising an extracellular domain (ECD) of a human activin receptor type IIA (ActRIIa) protein or derivative thereof linked to a constant domain of an immunoglobulin, e.g., a human IgGl Fc domain. In other embodiments, the pharmaceutical formulations comprise 50 mg / mL of a recombinant fusion protein comprising an extracellular domain (ECD) of a human activin receptor type IIA (ActRIIa) protein or derivative thereof linked to a constant domain of an immunoglobulin, e.g., a human IgGl Fc domain.

[0013] In certain embodiments, the pharmaceutical formulations provided herein comprise 10-100 mg / mL of a human ActRIIa fusion protein of SEQ ID NO: 32 or a variant of SEQ ID NO: 32 lacking the C-terminal lysine. In certain embodiments, the pharmaceutical formulations provided herein comprise 50-100 mg / mL of a human ActRIIa fusion protein of SEQ ID NO: 32 or a variant of SEQ ID NO: 32 lacking the C-terminal lysine. In certain embodiments, the pharmaceutical formulations provided herein comprise 40-50 mg / mL of a human ActRIIa fusion protein of SEQ ID NO: 32 or a variant of SEQ ID NO: 32 lacking the C-terminal lysine. In certain embodiments, the pharmaceutical formulations provided herein comprise 100 mg / mL of a human ActRIIa fusion protein of SEQ ID NO: 32 or a variant of SEQ ID NO: 32 lacking the C-terminal lysine. In certain embodiments, the pharmaceutical formulations provided herein comprise 50 mg / mL of a human ActRIIa fusion protein of SEQ ID NO: 32 or a variant of SEQ ID NO: 32 lacking the C-terminal lysine.

[0014] In certain embodiments, the pharmaceutical formulations provided herein comprise 10-100 mg / mL of a human ActRIIa fusion protein of SEQ ID NO: 32 and / or SEQ ID NO: 41. In certain embodiments, the pharmaceutical formulations provided herein comprise 50-100 mg / mL of a human ActRIIa fusion protein of SEQ ID NO: 32 and / or SEQ ID NO: 41. In certain embodiments, the pharmaceutical formulations provided herein comprise 40-50 mg / mL of a human ActRIIa fusion protein of SEQ ID NO: 32 and / or SEQ ID NO: 41. In certain embodiments, the pharmaceutical formulations provided herein comprise 50 mg / mL of a human ActRIIa fusion protein of SEQ ID NO: 32 and / or SEQ ID NO: 41. In certain embodiments, the pharmaceutical formulations provided herein comprise 100 mg / mL of a human ActRIIa fusion protein of SEQ ID NO: 32 and / or SEQ ID NO: 41.

[0015] In certain embodiments, the pharmaceutical formulations provided herein comprise 10-100 mg / mL of a human ActRIIa fusion protein, a buffer, a surfactant, a stabilizer, and optionally one or more antioxidants, wherein the buffer is not histidine.

[0016] In certain embodiments, the pharmaceutical formulations provided herein comprise 10-100 mg / mL of a human ActRIIa fusion protein of SEQ ID NO: 32 and / or SEQ ID NO: 41, a buffer, a surfactant, a stabilizer, and optionally one or more antioxidants, wherein the buffer is not histidine.

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

[0018] In certain embodiments, the pharmaceutical formulations provided herein comprise 50-100 mg / mL of a human ActRIIa fusion protein of SEQ ID NO: 32 or a variant of SEQ ID NO: 32 lacking the C-terminal lysine, a buffer, a surfactant, a stabilizer, and optionally one or more antioxidants, wherein the buffer is not histidine.

[0019] In some embodiments, the buffer comprises an organic acid, succinate, phosphate, acetate, citrate, citric acid, Tris, HEPES, glutamate, an amino acid, 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, an amino acid, 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 where the protein has a negative charge due to the presence of glycans, the buffer is not histidine.

[0020] In some embodiments, the buffer is selected to be physiologically compatible and to maintain a pH of greater than 4. In some embodiments, the buffer is selected to be physiologically compatible and to maintain a pH between 5-7. 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.

[0021] In certain embodiments, the pharmaceutical formulations described herein include a buffer, wherein the buffer is a phosphate buffer to maintain a pH of the pharmaceutical formulation between 5-7. In certain embodiments, the pharmaceutical formulations described herein include a buffer, wherein the buffer is a citrate buffer to maintain a pH of the pharmaceutical formulation between pH 4.5-7. In certain embodiments, the pharmaceutical formulations described herein include a buffer, wherein the buffer is an acetate buffer to maintain a pH of the pharmaceutical formulation between pH 4.5-6. In certain embodiments, the pharmaceutical formulations described herein include a buffer, wherein the buffer is a succinate buffer to maintain a pH of the pharmaceutical formulation between pH 4.5-5. In certain embodiments, the pharmaceutical formulations described herein include a buffer, wherein the buffer is a glutamate buffer to maintain a pH of the pharmaceutical formulation between pH 5-7.

[0022] 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 10-50 mM. In some embodiments, the buffer is present at a concentration of at least 10 mM.

[0023] In some embodiments, the buffering agent is a citrate buffer that maintains the pH of the pharmaceutical formulation at about pH 5.5 to about pH 6.5. In some embodiments, the buffering agent is a succinate buffer that maintains the pH of the pharmaceutical formulation at about pH 5.5 to about pH 6.5. In some embodiments, the buffering agent is a histidine buffer that maintains the pH of the pharmaceutical formulation at about pH 5.5 to about pH 6.0. In some embodiments, the buffering agent is a citrate buffer that maintains the pH of the pharmaceutical formulation at about pH 5.8. In some embodiments, the buffering agent is a succinate buffer that maintains the pH of the pharmaceutical formulation at about pH 5.8.

[0024] In some embodiments, the stabilizer is selected from the group consisting of carboxymethylcellulose (CMC), glucose, polyethylene glycol (PEG), albumin, kelptose, proline, sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, gentiobiose, 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 selected from the group consisting of sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, gentiobiose, 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.

[0025] In some embodiments, the stabilizing agent is present in the formulation at a concentration of 2-16% weight / volume. In some embodiments, the stabilizing agent is present at a concentration of 6-10% weight / volume. In some embodiments, the stabilizing agent 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, 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 stabilizing agent is present at a concentration of at least 8% weight / volume.

[0026] In some embodiments, the surfactant is selected from the group consisting of sodium lauryl sulfate, dioctyl sodium sulfosuccinate and sodium dioctyl sulfonate, chenodeoxycholic acid, sodium N-lauroylsarcosinate, lithium dodecyl sulfate, sodium 1-octane sulfonate, sodium cholate hydrate, sodium deoxycholate and sodium glycocholate, benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride monohydrate, cetrimonium bromide, CHAPS, CHAPSO, SB3-10, SB3-12, digitonin, Triton X-100, Triton X-114, TWEEN-20, TWEEN-80, laureth 400, polyoxyl 40 stearate, polyoxyl hydrogenated castor oil 10, 40, 50 and 60, glycerol monostearate, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, poloxamer 188, and soybean lecithin. In certain embodiments, the surfactant is selected from the group consisting of poloxamer 188, sodium dodecyl sulfate (SDS), N-dodecyl-P-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.

[0027] In some embodiments, the surfactant is polysorbate 80. In some embodiments, the surfactant is polysorbate 20. In some embodiments, the surfactant is poloxamer 188.

[0028] In some embodiments, the surfactant is present in the formulation at a concentration of 0.02-2.0 mg / mL. In some embodiments, the surfactant is present in the formulation at a concentration 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 0.05-0.3 mg / 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 comprises a surfactant at a concentration of at least 0.2 mg / mL. In some embodiments, the pharmaceutical formulation comprises 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 comprises a surfactant at a concentration of 0.05-0.5 mg / mL. In some embodiments, the pharmaceutical formulation comprises a surfactant at a concentration of 0.1-0.5 mg / mL.

[0029] In some embodiments, one or more of the pharmaceutical additives and / or excipients is an antioxidant. In some embodiments of the pharmaceutical formulations provided herein, the antioxidant is at a concentration of 0.001-50 mM. In some embodiments of the pharmaceutical formulations provided herein, the antioxidant is at a concentration of 7.5-50 mM. In some embodiments of the pharmaceutical formulations provided herein, the antioxidant is at a concentration of 5-20 mM. In some embodiments of the pharmaceutical formulations provided herein, the antioxidant is at a concentration of 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.

[0030] In one embodiment, the pharmaceutical formulation comprises an antioxidant selected from the group consisting of methionine (L or D form), tryptophan (L or D form), dimercaprol, and pyridoxine. In one embodiment, the pharmaceutical formulation comprises an antioxidant selected from the group consisting of methionine (L or D form), tryptophan (L or D form), and pyridoxine. In certain embodiments, the formulation comprises L-methionine.

[0031] In certain embodiments, the formulations described herein comprise 1-50 mM of an antioxidant. In certain embodiments, the formulations described herein comprise 5 mM, 10 mM, or 50 mM of an antioxidant. In certain embodiments, the formulation comprises 1-30 mM of an antioxidant. In certain embodiments, the formulation comprises 1-20 mM of an antioxidant. In certain embodiments, the formulation comprises 5-15 mM of an antioxidant. In certain embodiments, the formulation comprises 5-10 mM of an antioxidant. In certain embodiments, the formulation comprises 10 mM or at least 10 mM of an antioxidant.

[0032] In certain embodiments, the formulation comprises 1-50 mM of L-methionine. In certain embodiments, the formulation comprises 1-30 mM of L-methionine. In certain embodiments, the formulation comprises 1-20 mM of L-methionine. In certain embodiments, the formulation comprises 5-15 mM of L-methionine. In certain embodiments, the formulation comprises 5-10 mM of L-methionine. In certain embodiments, the formulation comprises 10 mM or at least 10 mM of L-methionine. In certain embodiments, the formulations described herein comprise 5 mM, 10 mM, or 50 mM of L-methionine.

[0033] In some embodiments of the pharmaceutical formulations described herein, the pharmaceutical formulation optionally includes a chelator. In certain embodiments, the chelator is DTPA or EDTA. In certain embodiments, the chelator 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 μΜ. In one embodiment, the liquid formulation includes 1-100 μΜ, 1-30 μΜ, 1-20 μΜ, 10 μΜ-30 μΜ of DTPA or EDTA. In certain embodiments, the chelator has a concentration of 7.5-100 μΜ. In certain embodiments, the chelator has a concentration of 10 μΜ.

[0034] In some embodiments, the pharmaceutical formulations provided herein comprise 10-100 mg / mL of the human ActRIIa fusion protein of SEQ ID NO: 32, 10-50 mM of a citrate buffer, 2-16% weight / volume sucrose, 0.05-0.5 mg / mL of polysorbate 80, polysorbate 20, or poloxamer 188, 0-50 mM of L-methionine, and 0-100 μΜ of DTPA or EDTA. In other embodiments, the pharmaceutical formulations provided herein comprise 50 mg / mL of the human ActRIIa fusion protein of SEQ ID NO: 32, 10 mM of a citrate buffer, 8% weight / volume sucrose, 0.2 mg / mL of polysorbate 80, and 20 mM of L-methionine. In some embodiments, the pharmaceutical formulations provided herein comprise 10-100 mg / mL of the human ActRIIa fusion protein of SEQ ID NO: 32, or a variant of SEQ ID NO: 32 lacking the C-terminal lysine, 10-50 mM of a citrate buffer, 2-16% weight / volume sucrose, 0.05-0.5 mg / mL of polysorbate 80, polysorbate 20, or poloxamer 188, 0-50 mM of L-methionine, and 0-100 μΜ of DTPA or EDTA.

[0035] In other embodiments, the pharmaceutical formulations provided herein comprise 50 mg / mL of the human ActRIIa fusion protein of SEQ ID NO: 32, or a variant of SEQ ID NO: 32 lacking the C-terminal lysine, 10 mM of a citrate buffer, 8% weight / volume sucrose, 0.2 mg / mL of polysorbate 80, and 20 mM of L-methionine.

[0036] In some embodiments, the pharmaceutical formulations provided herein comprise 10-100 mg / mL of a human ActRIIa fusion protein of SEQ ID NO: 32 and / or SEQ ID NO: 41, 10-50 mM of a citrate buffer, 2-16% weight / volume sucrose, 0.05-0.5 mg / mL of polysorbate 80, polysorbate 20, or poloxamer 188, 0-50 mM of L-methionine, and 0-100 μΜ of DTPA or EDTA. In other embodiments, the pharmaceutical formulations provided herein comprise 50 mg / mL of a human ActRIIa fusion protein of SEQ ID NO: 32 and / or SEQ ID NO: 41, 10 mM of a citrate buffer, 8% weight / volume sucrose, 0.2 mg / mL of polysorbate 80, and 20 mM of L-methionine.

[0037] In some embodiments, the human ActRIIa fusion protein consists of the amino acid sequence of SEQ ID NO: 41. In some embodiments, the lyophilized formulation includes 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 solithromycin.

[0038] In certain embodiments, the pharmaceutical formulations described herein contain a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 that lacks the C-terminal lysine residue (SEQ ID NO: 41). In certain embodiments, the pharmaceutical formulations described herein contain a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 that lacks the C-terminal lysine residue (SEQ ID NO: 41), wherein the mixture contains 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%, 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% by weight of SEQ ID NO: 32.

[0039] In certain embodiments, the pharmaceutical formulations described herein contain a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 that lacks the C-terminal lysine residue (SEQ ID NO: 41), wherein the mixture contains about 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%, 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% by weight of SEQ ID NO: 32.

[0040] In certain embodiments, the pharmaceutical formulations described herein contain a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 lacking the C-terminal lysine residue (SEQ ID NO: 41), wherein the mixture contains 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%, 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% by weight of SEQ ID NO: 41.

[0041] In certain embodiments, the pharmaceutical formulations described herein contain a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 lacking the C-terminal lysine residue (SEQ ID NO: 41), wherein the mixture contains about 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%, 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% by weight of SEQ ID NO: 41.

[0042] In certain embodiments, the pharmaceutical formulations described herein contain 100% by weight of SEQ ID NO: 32. In certain embodiments, the pharmaceutical formulations described herein contain 100% by weight of SEQ ID NO: 41.

[0043] The pharmaceutical formulations provided herein can be used to treat pulmonary arterial hypertension in a subject in need thereof. In certain embodiments, the pharmaceutical formulations provided herein are liquids.

[0044] In certain embodiments, the pharmaceutical formulations provided herein are stable when stored at 2-8 °C. In certain embodiments, the liquid pharmaceutical formulations are stored under refrigerated conditions (temperature range: typically about 2-8 °C, but in certain cases, aqueous formulations can show stability at other temperatures, including at about 25 °C and about 40 °C, for a period of up to about 3, 6, 9, or 12 months). In certain embodiments, the pharmaceutical formulations are administered by an auto-injector. In certain embodiments, the pharmaceutical formulations are administered by subcutaneous injection. In some embodiments, the pharmaceutical formulations are administered parenterally.

[0045] In certain embodiments, the formulations described herein are contained in an injection device. In certain embodiments, the formulations described herein are contained in an injection device, wherein the injection device is an auto-injector. In other embodiments, the formulations described herein are contained in a glass vial. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 A multiple sequence alignment showing various vertebrate ActRIIA proteins and human ActRIIA (SEQ ID NO: 61-68).

[0047] Figure 2A A multiple sequence alignment showing various vertebrate ActRIIA proteins and human ActRIIA (SEQ ID NO: 61-68). 2B Purification of ActRIIA-hFc expressed in CHO cells is shown. The protein was purified as a single sharp peak as seen by size exclusion column (top panel) and Coomassie-stained SDS-PAGE (bottom panel) (left lane: molecular weight standards; right lane: ActRIIA-hFc). See Example 1.

[0048] Figure 3A A multiple sequence alignment showing various vertebrate ActRIIA proteins and human ActRIIA (SEQ ID NO: 61-68). 3B ActRIIA-hFc binds activin with a dissociation constant (K -12 ) of 5 x 10 D and binds GDF11 with a K -9 of 9.96 x 10 D . See Example 1.

[0049] Figure 4 Results of a stability pH screen study: effect of pH and buffer at t=0. See Example 3: pH and buffer feasibility study.

[0050] Figure 5 Results of a stability pH screen study: effect of pH and buffer at 2 weeks at 40°C. See Example 3: pH and buffer feasibility study.

[0051] Figure 6 Results of a stability pH screen study: effect of pH and buffer at 1 month at 40°C. See Example 3: pH and buffer feasibility study.

[0052] Figure 7 Results of a stability pH screen study: effect of pH and buffer at 2 months at 40°C. See Example 3: pH and buffer feasibility study.

[0053] Figure 8 1H NMR data for SEQ ID NO: 32 with and without DTPA is shown. See Example 3: Effect of metal chelators on SEQ ID NO: 32.

[0054] Figure 9 The diffusion NMR data for SEQ ID NO:32 are shown with and without DTPA. See Example 3: Effect of metal chelating agents on SEQ ID NO:32.

[0055] Figure 10 The results of a colloidal stability study of SEQ ID NO:32 and PS80 performed under stirring stress are shown. See Example 3: Colloidal Stability and Surfactant Screening.

[0056] Figure 11 The results of a colloidal stability study of SEQ ID NO:32 and PS80 performed under stirring stress are shown. See Example 3: Colloidal Stability and Surfactant Screening.

[0057] Figure 12 The results of a colloidal stability study of SEQ ID NO:32 and PS80 performed by freeze-thaw cycles are shown. See Example 3: Colloidal Stability and Surfactant Screening.

[0058] Figure 13 The results of a colloidal stability study of SEQ ID NO:32 and PS80 performed by freeze-thaw cycles are shown. See Example 3: Colloidal Stability and Surfactant Screening.

[0059] Figure 14 The formulation of L-methionine at different levels (0 mM, 10 mM, 20 mM, 30 mM) was exposed to photostress in a photostable chamber in the presence of different levels of any EDTA as a chelating agent (0 µM, 7.5 µM, 15 µM, 30 µM, 60 µM). See Example 3: Chelating Agent / L-Methionine Range Study.

[0060] Figure 15 The formulation of L-methionine at different levels (0 mM, 10 mM, 20 mM, 30 mM) was exposed to photostress in a photostable chamber in the presence of any DTPA as a chelating agent (0 µM, 7.5 µM, 15 µM, 30 µM, 60 µM). See Example 3: Chelating Agent / L-Methionine Range Study. Detailed Implementation

[0061] Definitions The terms used in this specification generally have their ordinary meanings in the art, within the context of the disclosure, and in the specific context wherein each term is used. Certain terms are discussed below or elsewhere in the specification to provide additional guidance to the practitioner in order to describe the formulations and processes of the present disclosure and how to make and use them. The scope and meaning of any use of a term will be apparent from the specific context in which each term is used.

[0062] "About" and "approximately" shall generally mean an acceptable degree of error for the quantity measured considering the measurement in question, e.g., measuring a quantity of a material or composition, the typical measurement, operation and sampling procedures used in preparing, characterizing and / or using the material or composition; through inadvertent error in the experiments; through variations in the manufacture, source, or purity of the ingredients used to make the composition or in the manufacture of the composition; etc. Generally, an exemplary degree of error is within 10% of and preferably within 5% of the stated value or range of values. In certain embodiments, "about" can mean ±0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10% variation.

[0063] Alternatively, and particularly in biological systems, the terms "about" and "approximately" can mean a value that is within one order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a given value. Unless otherwise indicated, numerical quantities given herein are approximate, meaning that the term "about" or "approximately" can be inferred when not expressly stated.

[0064] The terms "a" and "an" include both the singular and plural, unless the context clearly indicates otherwise. The terms "a" (or "an"), as well as the terms "one or more" and "at least one," can be used interchangeably in the present disclosure. Furthermore, "and / or" where used herein is to be taken as specific disclosure of each of the various terms and components in the list. For example, "A and / or B" is to be taken as specific disclosure of each of the following terms: A; B; and A and B. Likewise, "A, B, and / or C" is to be taken as specific disclosure of each of the following terms: A; B; C; A and B; A and C; B and C; and A, B, and C. As used in the present disclosure, the term "or" as used herein means any one member of a set or none. For example, in the phrase "A or B" A or B can be selected, but not both.

[0065] The numerical ranges disclosed herein include the numbers defining the range. For example, when a range of pH values is recited, for example, "a pH between 5.5 and 6.0," the range is intended to include the recited values. For example, a pH between 5.0 and 7.0 includes pH 5.0 and pH 7.0 and values in between 5.0 and 7.0. As used herein, a formulation that includes "a citrate buffer at pH X" refers to a solution at pH X and including a citrate buffer, i.e., the pH is intended to refer to the pH of the solution.

[0066] The proteins disclosed herein can comprise an amino acid sequence that is not naturally occurring. Such variants necessarily have less than 100% sequence identity or similarity to the starting molecule. In certain embodiments, the variant has an amino acid sequence that has about 75% to less than 100% amino acid sequence identity or similarity to the amino acid sequence of the starting (e.g., naturally occurring or wild type) protein, more preferably about 80% to less than 100%, more preferably about 85% to less than 100%, 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%, e.g., over the entire length of the variant molecule.

[0067] A "stable" formulation is one in which the protein therein essentially retains its physical stability and / or chemical stability and / or biological activity upon storage. Various analytical techniques are available to measure protein stability, and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). Stability can be measured at a selected temperature for a selected time.

[0068] A "stable" liquid formulation is a pharmaceutical formulation that shows no significant change at refrigerated temperatures (2-8°C) for at least 3 months, preferably 6 months, and more preferably 1 year, and even more preferably up to 2 years. In addition, a "stable" liquid formulation includes a liquid formulation that shows the desired characteristics for a period of time including 1 month, 3 months, 6 months, 12 months, and / or 24 months at temperatures including 25°C and 40°C. Typical acceptable criteria for stability are as follows. Typically, no more than about 10%, preferably about 5%, of the protein is degraded, as measured by SEC-HPLC. The pharmaceutical formulation is colorless or clear to slightly opalescent by visual analysis. The concentration, pH, and osmolality of the formulation have no more than + / - 10% change. Potency is typically within 50-150 of reference. Typically, no more than about 10%, preferably about 5%, of the protein is aggregated in the formulation. The term "buffer" includes an agent that maintains the pH of the solution of the formulation of the application within an acceptable range, or for the lyophilized formulations of the application, provides an acceptable solution pH prior to lyophilization. The terms "lyophilization", "lyophilized", and "freeze-dried" refer to a process in which a material to be dried is first frozen and then the ice or frozen solvent is removed by sublimation in a vacuum environment. Excipients can be included in the pre-lyophilization formulation to improve the stability of the lyophilized product upon storage. The term "pharmaceutical formulation" refers to a preparation that is in a form acceptable for effective administration to a subject and does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered. The terms "formulation" and "pharmaceutical formulation" are used interchangeably throughout. "Pharmaceutically acceptable" means an excipient (vehicle, additive) and composition that is reasonably administered to a subject to provide an effective dose of the active ingredient used, and is "generally regarded as safe", e.g., is physiologically tolerable and does not typically produce an allergic or similar untoward reaction, such as gastric upset, etc., when administered to a human. In another embodiment, the term refers to molecular entities and compositions approved by a regulatory structure of the federal or state governments or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. A "reconstituted" formulation is a formulation that has been prepared by dissolving a lyophilized protein formulation in a diluent such that the protein is dispersed in the reconstituted formulation. The reconstituted formulation is suitable for administration, e.g., parenteral administration, and can optionally be suitable for subcutaneous administration.

[0069] Pharmaceutical Formulations Provided herein are pharmaceutical formulations comprising a recombinant fusion protein comprising an extracellular domain (ECD) of human Activin Receptor IIA Type II (ActRIIA) protein or a derivative thereof linked to a constant domain of an immunoglobulin, e.g., a human IgGl Fc domain. In certain aspects, the disclosure relates to stable, liquid pharmaceutical formulations or injectables comprising an extracellular domain (ECD) of human ActRIIA protein or a derivative thereof linked to a constant domain of an immunoglobulin, e.g., a human IgGl Fc domain.

[0070] The liquid formulations of the present application minimize the formation of aggregates (high molecular weight species) and microparticles, improve colloidal stability, minimize fragmentation (low molecular weight species), and ensure that the protein maintains its biological activity over time. In certain embodiments, the formulation is prepared by taking ActRIIA, e.g., in an aqueous pharmaceutical formulation, and exchanging its buffer into the desired buffer as the last step in a purification process. The ActRIIA is then concentrated to the desired concentration. There is no lyophilization step in this embodiment. Additionally, excipients such as stabilizers and surfactants can be added to the ActRIIA formulation, which is diluted to the final protein concentration using the appropriate buffer. The final formulation is filtered and filled into the final container, e.g., an auto-injector. Alternatively, the formulation can be stored in a vial and delivered by an injection device or container.

[0071] The pharmaceutical formulations provided herein comprise an ActRIIa fusion protein. In certain embodiments, the present disclosure provides a pharmaceutical formulation comprising an ActRIIa fusion protein, wherein the protein is present at a concentration of about 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, about 17.5 mg / mL, about 20 mg / mL, about 22.5 mg / mL, about 25 mg / mL, about 27.5 mg / mL, about 30 mg / mL, about 32.5 mg / mL, about 35 mg / mL, about 37.5 mg / mL, about 40 mg / mL, about 42.5 mg / mL, about 45 mg / mL, about 47.5 mg / mL, about 50 mg / mL, about 52.5 mg / mL, about 55 mg / mL, about 57.5 mg / mL, about 60 mg / mL, about 62.5 mg / mL, about 65 mg / mL, about 67.5 mg / mL, about 70 mg / mL, about 72.5 mg / mL, about 75 mg / mL, about 77.5 mg / mL, about 80 mg / mL, about 82.5 mg / mL, about 85 mg / mL, about 90 mg / mL, about 92.5 mg / mL, about 95 mg / mL, about 97.5 mg / mL, or about 100 mg / mL.

[0072] In certain embodiments, the ActRIIa fusion protein has a concentration of about 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 of ActRIIa fusion protein. In other embodiments, the ActRIIa fusion protein has a concentration 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 of ActRIIa fusion protein. In some embodiments, the ActRIIa fusion protein has a concentration of about 50 mg / mL.

[0073] In some embodiments, the pharmaceutical formulations provided herein comprise 10-100 mg / mL of a recombinant fusion protein comprising an extracellular domain (ECD) of a human Activin Receptor Type IIA (ActRIIa) protein or a derivative thereof linked to a constant domain of an immunoglobulin, e.g., a human IgGl Fc domain. In other embodiments, the pharmaceutical formulations comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg / mL of a recombinant fusion protein comprising an extracellular domain (ECD) of a human Activin Receptor Type IIA (ActRIIa) protein or a derivative thereof linked to a constant domain of an immunoglobulin, e.g., a human IgGl Fc domain. In other embodiments, the pharmaceutical formulations comprise 40-50 mg / mL of a recombinant fusion protein comprising an extracellular domain (ECD) of a human Activin Receptor Type IIA (ActRIIa) protein or a derivative thereof linked to a constant domain of an immunoglobulin, e.g., a human IgGl Fc domain. In other embodiments, the pharmaceutical formulations comprise 50 mg / mL of a recombinant fusion protein comprising an extracellular domain (ECD) of a human Activin Receptor Type IIA (ActRIIa) protein or a derivative thereof linked to a constant domain of an immunoglobulin, e.g., a human IgGl Fc domain.

[0074] In some embodiments, the liquid pharmaceutical formulations of the ActRIIa fusion proteins provided herein comprise an 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 a buffer, a stabilizer, a surfactant, and optionally one or more antioxidants, which are described in more detail below. The buffer can be selected to maintain the pH of the formulation during processing. The surfactants can be selected based on their ability to act as emulsifying agents, wetting agents, solubilizing agents, and / or dispersing agents.

[0075] One of ordinary skill in the art will recognize that the concentrations of the excipients described herein have dependencies within a particular formulation. For example, the concentration of a bulking agent is lower in one aspect, e.g., where there is a high protein concentration. The excipients and other additives are added to provide or improve manufacturability and / or final product quality, e.g., stability and delivery of a pharmaceutical product (e.g., a protein). The formulations provided herein comprise suitable excipients that improve stability and safety.

[0076] Buffering Agents Generally, the stability of a pharmaceutically active protein formulation is observed to be greatest within a narrow pH range. This pH range of optimal stability needs to be identified early in the pre-formulation phase of development. Several methods, such as accelerated stability and calorimetric screening studies, can be used in this endeavor (Remmele R. L. Jr., et al., Biochemistry, 38(16): 5241-7 (1999)). Once the formulation is finalized, the protein must be manufactured and maintained throughout its shelf life. Therefore, a buffer is almost always used to control the pH of the formulation.

[0077] Several factors must be considered when selecting a buffer. First and foremost, the buffer species and its concentration must be defined according to its pKa and the desired formulation pH. It is also important to ensure that the buffer is compatible with the protein and other formulation excipients, and does not catalyze any degradation reactions. A third important aspect to consider is the potential for stinging and irritation that the buffer can induce upon administration. For drugs administered by subcutaneous (SC) or intramuscular (IM) routes, where the drug solution remains at the site for a relatively longer period of time, the potential for stinging and irritation is greater than for drugs administered by IV routes, where the formulation is rapidly diluted into the bloodstream upon administration. For formulations administered by direct IV infusion, the total amount of buffer (as well as any other formulation components) needs to be monitored.

[0078] In some embodiments, the buffer comprises an organic acid, succinate, phosphate, acetate, citrate, citric acid, Tris, HEPES, glutamate, an amino acid, 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, an amino acid, 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.

[0079] In one embodiment, the buffer present in the formulation is selected to be physiologically compatible and to maintain the desired pH of the pharmaceutical formulation. In another embodiment, the pH of the formulation is greater than 4. In 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 between pH 5 and 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.

[0080] In certain embodiments, the buffer is a phosphate buffer to maintain the pH of the pharmaceutical formulation between 5-7. In certain embodiments, the buffer is a citrate buffer to maintain the pH of the pharmaceutical formulation between 4.5-7. In certain embodiments, the buffer is an acetate buffer to maintain the pH of the pharmaceutical formulation between 4.5-6. In certain embodiments, the buffer is a succinate buffer to maintain the pH of the pharmaceutical formulation between 4.5-5. In certain embodiments, the buffer is a glutamate buffer to maintain the pH of the pharmaceutical formulation between 5-7.

[0081] The pH buffering compound can be present in any amount suitable to maintain the pH of the formulation at a predetermined level. Crystallization and pH shift can be avoided when using a suitably low level of buffering agent. In one embodiment, the concentration of the buffering agent is 0.1 mM to 500 mM (1 M). For example, it is contemplated that the buffering agent is 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 buffering agent 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 buffering agent is 10 mM.

[0082] In some embodiments, the buffering agent comprises trisodium citrate dihydrate. In some embodiments, the buffering agent comprises citric acid monohydrate. In some embodiments, the buffering agent comprises citrate. In some embodiments, the buffering agent comprises trisodium citrate dihydrate and citric acid monohydrate. In certain embodiments, the buffering agent is not histidine. In some embodiments, the buffering agent 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 concentration of the buffering agent is at least 10 mM.

[0083] Stabilizing Agents In certain embodiments, the pharmaceutical formulations provided herein comprise a stabilizer. These stabilizers can be categorized according to their mechanism of stabilizing proteins against various chemical and physical stresses. Some stabilizers act to mitigate the effects of a particular stress or modulate a particular susceptibility of a particular protein. Other stabilizers have a more general effect on the physical and covalent stability of proteins. Given the teachings and guidance provided herein, one of skill in the art will know the amount or range of stabilizers that can be included in any particular formulation to achieve the stability of the formulations of the disclosure that likely promote the preservation and stability of the ActRIIa fusion proteins.

[0084] In some embodiments, a stabilizer (or combination of stabilizers) is added to the formulation to prevent or reduce storage-induced aggregation and chemical degradation. A hazy or cloudy solution upon reconstitution often indicates that the protein has precipitated or at least aggregated. Stabilizers are capable of preventing aggregation or chemical degradation (e.g., autolysis, deamidation, oxidation, etc.). Some stabilizers are also capable of acting as an anti-coagulant when the formulation is administered to a patient. In some embodiments, the stabilizer is selected from the group consisting of: CMC, glucose, PEG, albumin, kelptose, proline, sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, glucosamine, fructose, mannitol, sorbitol, polyhydroxyl compounds, polysaccharides, dextran, starch, hydroxyethyl starch, cyclodextrin, N-methylpyrrolidone, cellulose, and hyaluronic acid. In certain embodiments, the pharmaceutical formulations provided herein include a stabilizer, including but not limited to sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, glucosamine, fructose, mannitol, sorbitol, polyhydroxyl compounds, including polysaccharides such as 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 disclosure, sucrose is used as a stabilizer.

[0085] In certain embodiments, the formulation comprises 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. Also, in certain embodiments of the 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.

[0086] Surfactants In certain embodiments, the pharmaceutical formulations provided herein can additionally include a surfactant. Surfactants are commonly used in protein formulations to prevent surface-induced degradation. Surfactants are amphiphilic molecules that have the ability to outcompete proteins (and / or facilitate the proper refolding of structurally altered protein molecules) for interfacial sites. The hydrophobic portion of the surfactant molecule occupies the interfacial site (e.g., air / liquid), while the hydrophilic portion of the molecule remains oriented toward the bulk solvent. At sufficient concentrations (typically near the critical micelle concentration of the detergent), a surface layer of surfactant molecules serves to prevent protein molecules from adsorbing at the interface. In this way, surface-induced degradation is minimized. The surfactants contemplated herein include, but are not limited to, fatty acid esters of sorbitan polyoxyethylene ethers, i.e., polysorbate 20 and polysorbate 80. The only difference between the two is the length of the aliphatic chain that imparts the hydrophobic character to the molecule, C-12 and C-18, respectively. Thus, polysorbate 80 has a higher surface activity and a lower critical micelle concentration than polysorbate 20.

[0087] Detergents can also affect the thermodynamic conformational stability of proteins. Non-ionic surfactants are generally used for protein stabilization. Ionic surfactants (detergents) generally destabilize proteins. Again, it is emphasized that the effects of the provided detergent excipients are protein specific. For example, polysorbate has been shown to decrease the stability of some proteins and increase the stability of others. The detergent destabilization of proteins can be reasonably explained in terms of the hydrophobic tail of the detergent molecule being able to participate in specific binding to partially or completely unfolded protein states. These types of interactions can result in a shift in the conformational equilibrium toward more extended protein states (i.e., increasing the exposure of the hydrophobic portion of the protein molecule to aid in the binding of polysorbate). Alternatively, if the native state of the protein displays some hydrophobic surface, binding of the detergent to the native state can stabilize that conformation. Another aspect of polysorbates is that they are inherently susceptible to oxidative degradation. Generally, as a raw material, they contain sufficient amounts of peroxide to cause oxidation of protein residue side chains, particularly methionine. The potential for oxidative damage due to the addition of stabilizers emphasizes the point that the minimum effective concentration of excipients should be used in formulations. For surfactants, the effective concentration for a given protein will depend on the stabilization mechanism.

[0088] A surfactant is also added in an appropriate amount to prevent surface-related aggregation phenomena (Chang, B, J. Pharm. Sci. 85: 1325, (1996)). Thus, exemplary surfactants include, but are not limited to, anionic, cationic, non-ionic, zwitterionic, and amphoteric surfactants, including surfactants derived from naturally occurring amino acids. Anionic surfactants include, but are not limited to, sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate, chenodeoxycholic acid, sodium N-lauroylsarcosinate, lithium dodecyl sulfate, sodium 1-octanesulfonate, sodium cholate hydrate, sodium deoxycholate, and sodium glycocholate. Cationic surfactants include, but are not limited to, benzalkonium chloride or benzethonium chloride, cetylpyridinium chloride monohydrate, and cetyltrimethylammonium bromide. Zwitterionic surfactants include, but are not limited to, CHAPS, CHAPSO, SB3-10, and SB3-12. Non-ionic surfactants include, but are not limited to, digitonin, Triton X-100, Triton X-114, TWEEN-20, and TWEEN-80. Surfactants also include, but are not limited to, laureth 400, polyoxyethylene 40 stearate, polyoxyethylene hydrogenated castor oil 10, 40, 50, and 60, glyceryl monostearate, polysorbate 40, polysorbate 60, polysorbate 65, and polysorbate 80, soybean lecithin, and other phospholipids, such as dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylglycerol (DMPG), dimyristoylphosphatidylcholine (DMPC), and (dioleoylphosphatidylglycerol) DOPG; sucrose fatty acid ester, 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.

[0089] In some embodiments, the surfactant is selected from the group consisting of: sodium lauryl sulfate, dioctyl sodium sulfosuccinate and sodium dioctyl sulfonate, chenodeoxycholic acid, sodium N-lauroylsarcosinate, lithium dodecyl sulfate, sodium 1-octane sulfonate, sodium cholate hydrate, sodium deoxycholate and sodium glycocholate, benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride monohydrate, cetrimonium bromide, CHAPS, CHAPSO, SB3-10, SB3-12, digitonin, Triton X-100, Triton X-114, TWEEN-20, TWEEN-80, laureth 400, polyoxyl 40 stearate, polyoxyl hydrogenated castor oil 10, 40, 50 and 60, glycerol monostearate, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, poloxamer 188, and soybean lecithin. In certain embodiments, the surfactant is selected from the group consisting of poloxamer 188, sodium dodecyl sulfate (SDS), N-dodecyl-P-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.

[0090] Further provided, therefore, are formulations comprising these surfactants, either alone or as mixtures in different ratios. In the formulations of the present application, the surfactant is incorporated at a concentration of about 0.01 to about 0.5 mg / ml. In the formulations of the present application, the surfactant is incorporated at a concentration of about 0.05 to about 0.5 mg / ml. In the formulations of the present application, the surfactant is incorporated at a concentration of about 0.1 to about 0.5 mg / ml. In each of the 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 application, the surfactant is incorporated at a concentration of 0.2 mg / ml. Likewise, 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.

[0091] In some embodiments, the surfactant is polysorbate 80. In some embodiments, the surfactant is present in the formulation at a concentration of 0.05-0.3 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.

[0092] Antioxidants In certain embodiments, the pharmaceutical formulations described herein include one or more antioxidants. Oxidation of protein residues arises from many different sources. In addition to the addition of specific antioxidants, prevention of oxidative protein damage includes careful control of many factors throughout the manufacturing process and storage of the product, such as oxygen in the atmosphere, temperature, light exposure, and chemical contamination.

[0093] Accordingly, the present disclosure contemplates the use of pharmaceutical antioxidants, including but not limited to reducing agents, oxygen / free radical scavengers, or chelators. In one aspect, the antioxidant in the therapeutic protein formulation is water soluble and remains active throughout the product shelf life. Reducing agents and oxygen / free radical scavengers act by abrogating reactive oxygen species in the 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.

[0094] In one embodiment, the pharmaceutical formulations described herein comprise methionine (L or D form), tryptophan (L or D form), dimercaprol, or pyridoxine. In one embodiment, the pharmaceutical formulations described herein comprise methionine (L or D form), tryptophan (L or D form), or pyridoxine. In one embodiment, the pharmaceutical formulations described herein comprise methionine (L or D form). In one embodiment, the pharmaceutical formulations described herein comprise L-methionine. In one embodiment, the pharmaceutical formulations described herein comprise D-methionine.

[0095] In certain embodiments, the pharmaceutical formulations described herein can also include 1-50 mM of an antioxidant. In certain embodiments, the pharmaceutical formulations described herein can also include 1-30 mM of an antioxidant. In one embodiment, the pharmaceutical formulations described herein can also include 1-20 mM of an antioxidant. The pharmaceutical formulations described herein can also include 5-15 mM of an antioxidant. The pharmaceutical formulations described herein can also include 5-10 mM of an antioxidant. In certain embodiments, the pharmaceutical formulations described herein include 5 mM, 10 mM, or 50 mM of an antioxidant. The pharmaceutical formulations described herein can also include 10 mM or at least 10 mM of an antioxidant.

[0096] In certain embodiments, the pharmaceutical formulations described herein can also include 1-50 mM L-methionine. In certain embodiments, the pharmaceutical formulations described herein can also include 1-30 mM L-methionine. In one embodiment, the pharmaceutical formulations described herein can also include 1-20 mM L-methionine. The pharmaceutical formulations described herein can also include 5-15 mM L-methionine. The pharmaceutical formulations described herein can also include 5-10 mM L-methionine. In certain embodiments, the pharmaceutical formulations described herein include 5 mM, 10 mM, or 50 mM of L-methionine. The pharmaceutical formulations described herein can also include 10 mM or at least 10 mM of L-methionine.

[0097] Chelating Agents In certain embodiments, the pharmaceutical formulations described herein can optionally include one or more chelators. In certain embodiments, the chelator is DTPA or EDTA. The pharmaceutical formulations described herein can include 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 chelator. In certain embodiments, the stable liquid formulation includes 1-100 µM, 1-30 µM, 1-20 µM, 10 µM-30 µM of DTPA or EDTA. In certain embodiments, the stable liquid formulation includes 7.5-100 µM of DTPA or EDTA.

[0098] In certain embodiments, the stable liquid formulation includes 10 µM of DTPA or EDTA.

[0099] ActRIIA Protein Formulations In some embodiments of the pharmaceutical formulations provided herein, 10-100 mg / mL of a human ActRIIa fusion protein, 10-50 mM of a citrate buffer, 2-16% w / v sucrose, 0.05-0.5 mg / mL of polysorbate 80, polysorbate 20, or poloxamer 188, 0-50 mM of L-methionine, and 0-100 μΜ of DTPA or EDTA are included. In other embodiments of the pharmaceutical formulations provided herein, 50 mg / mL of a human ActRIIa fusion protein of SEQ ID NO: 32, or a variant of SEQ ID NO: 32 lacking the C-terminal lysine, 10 mM of a citrate buffer, 8% w / v sucrose, 0.2 mg / mL of polysorbate 80, and 20 mM of L-methionine are included. In other embodiments of the pharmaceutical formulations provided herein, 50 mg / mL of a human ActRIIa fusion protein of SEQ ID NO: 32, 10 mM of a citrate buffer, 8% w / v sucrose, 0.2 mg / mL of polysorbate 80, and 20 mM of L-methionine are included.

[0100] In some embodiments of the pharmaceutical formulations provided herein, 10-100 mg / mL of a human ActRIIa fusion protein, 10-50 mM of a citrate buffer, 2-16% w / v sucrose, 0.05-0.5 mg / mL of polysorbate 80, polysorbate 20, or poloxamer 188, 0-50 mM of L-methionine, and 0-100 μΜ of DTPA or EDTA are included. In other embodiments of the pharmaceutical formulations provided herein, 50 mg / mL of a human ActRIIa fusion protein of SEQ ID NO: 41, 10 mM of a citrate buffer, 8% w / v sucrose, 0.2 mg / mL of polysorbate 80, and 20 mM of L-methionine are included.

[0101] In some embodiments of the pharmaceutical formulations provided herein, 10-100 mg / mL of a human ActRIIa fusion protein, 10-50 mM of a citrate buffer, 2-16% w / v sucrose, 0.05-0.5 mg / mL of polysorbate 80, polysorbate 20, or poloxamer 188, 0-50 mM of L-methionine, and 0-100 μΜ of DTPA or EDTA are included. In other embodiments of the pharmaceutical formulations provided herein, 50 mg / mL of a human ActRIIa fusion protein of SEQ ID NO: 32 and SEQ ID NO: 41 in a mixture, 10 mM of a citrate buffer, 8% w / v sucrose, 0.2 mg / mL of polysorbate 80, and 20 mM of L-methionine are included.

[0102] 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.

[0103] Stability In certain embodiments, the liquid pharmaceutical formulation is stored under refrigerated conditions (temperature range: typically about 2-8°C, although in certain cases the aqueous formulation can show stability at other temperatures, including at about 25°C and about 40°C, for a period of up to about 3, 6, 9, or 12 months). In certain embodiments, the pharmaceutical formulations provided herein are stable when stored at 2-8°C. In certain embodiments, the pharmaceutical formulations provided herein are stable when stored at 2-8°C for 1, 3, 6, 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 2-8°C for 1, 3, 6, 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 25°C for 1, 3, 6, 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, 3, 6, or 12 months.

[0104] Kits The present disclosure provides kits comprising a pharmaceutical formulation provided herein and an injection device. In certain embodiments, the pharmaceutical formulation comprises an ActRIIA protein or ActRIIa fusion protein, e.g., a protein having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity 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 one or more ligands selected from the group consisting of Activin A, Activin B, and GDF11. In certain such embodiments, the protein further binds one or more ligands selected from the group consisting of BMP10, GDF8, and BMP6. In certain embodiments, the protein binds Activin and / or GDF11.

[0105] In some embodiments, the pharmaceutical preparation comprises a protein comprising, consisting essentially of, or consisting of 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. 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 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.

[0106] In some embodiments, the pharmaceutical preparation 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%, 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.

[0107] In some embodiments, the pharmaceutical preparation 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%, 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.

[0108] In some embodiments, the pharmaceutical formulation 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%, 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.

[0109] In certain embodiments of the foregoing, the protein comprises a fusion protein further comprising an Fc domain of an immunoglobulin. In certain such embodiments, the Fc domain of an immunoglobulin is an Fc domain of an IgGl immunoglobulin. In other embodiments, the fusion protein further comprises a linker domain positioned between the protein domain and the Fc domain of an immunoglobulin. In certain embodiments, the linker domain is a polyglycine linker.

[0110] In certain embodiments, the ActRIIA fusion protein is part of a homodimeric protein complex.

[0111] In certain embodiments, the ActRIIa protein is glycosylated.

[0112] The present disclosure provides kits comprising the pharmaceutical formulations described herein and an injection device. In certain embodiments, the kits comprise a pharmaceutical formulation described herein and an injection device. In some embodiments of the kits disclosed herein, the pharmaceutical formulation comprising the protein is pre-filled in one or more containers, such as an auto-injector.

[0113] In certain embodiments, the pH of the pharmaceutical formulation comprising the protein ranges from 5-7. In some embodiments, the pharmaceutical formulation comprising the protein further comprises 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 comprises citrate.

[0114] In some embodiments, the pharmaceutical formulation comprising the protein further comprises a surfactant. In some embodiments, the surfactant comprises a polysorbate. In some embodiments, the surfactant comprises polysorbate 80 or polysorbate 20.

[0115] In some embodiments, the pharmaceutical formulation comprising a protein further comprises a sugar, e.g., a disaccharide (e.g., sucrose). In some embodiments, the stable liquid pharmaceutical formulation comprising a protein comprises sucrose, trehalose, mannitol, polyvinylpyrrolidone (PVP), glucose, and / or glycine. In some embodiments, the pharmaceutical formulation comprising a protein comprises sucrose. In some embodiments, the pharmaceutical formulation comprises a protein and a sugar in a weight ratio of at least 1 : 1 protein:sugar. In some embodiments, the pharmaceutical formulation comprises a protein and a sugar in a weight ratio of 1 : 1 to 1 : 10 protein:sugar. In some embodiments, the pharmaceutical formulation comprises a protein and a 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:sugar. In some embodiments, the pharmaceutical formulation comprises a protein and a sugar in a weight ratio of 1 :6 protein:sugar. In certain of the foregoing embodiments, the pharmaceutical formulation comprises a protein and a sugar in an amount sufficient to stabilize the protein.

[0116] In certain embodiments of the kits disclosed herein, the injection device comprises a syringe. In certain such embodiments, the syringe is pre-filled with the stable liquid formulation.

[0117] In certain embodiments of the kits disclosed herein, the kit further comprises an injectable device for administering the sterile injectable solution parenterally. In some embodiments, the sterile injectable solution is administered by subcutaneous injection. In some embodiments, the sterile injectable solution is administered by intradermal injection. In some embodiments, the sterile injectable solution is administered by intramuscular injection. In some embodiments, the sterile injectable solution is administered by intravenous injection.

[0118] In certain embodiments of the kits disclosed herein, the kit further comprises an auto-injector for administering the sterile injectable solution. In some embodiments, the sterile injectable solution is self-administered. In some embodiments, the sterile injectable solution comprises a therapeutically effective dose. In some embodiments, the therapeutically effective dose comprises a weight-based dose.

[0119] ActRIIA Proteins In certain embodiments, the disclosure relates to ActRIIA proteins. As used herein, the term "ActRIIA" refers to the Activin Receptor Type IIA (ActRIIA) protein family from any species and variants derived from such ActRIIA proteins by mutagenesis or other modification. Reference to ActRIIA herein is understood to refer to any one of the presently identified forms. Members of the ActRIIA family are generally transmembrane proteins, which consist of a ligand-binding extracellular domain containing a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine / threonine kinase activity.

[0120] The term "ActRIIA protein" includes any naturally occurring protein comprising an ActRIIA family member and any variant thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retains useful activity. Examples of such variant ActRIIA proteins are provided throughout the disclosure and in International Patent Application Publication Nos. WO 2006 / 012627 and WO 2007 / 062188, which are incorporated by reference herein in their entireties. Amino acid numbering for all ActRIIA-related proteins described herein is based on the numbering of the human ActRIIA precursor protein sequence provided below (SEQ ID NO: 9), unless explicitly indicated otherwise.

[0121] The standard human ActRIIA precursor protein sequence is as follows: The signal peptide is indicated by underlining; the extracellular domain is indicated in bold; and potential endogenous N-linked glycosylation sites are indicated by Single Underline .

[0122] The processed (mature) extracellular human ActRIIA protein sequence is as follows: The C-terminal "tail" of the extracellular domain is indicated by underlining. The sequence with the tail deleted (the Δ15 sequence) is as follows: Single Underline The nucleic acid sequence encoding the human ActRIIA precursor protein is shown below (SEQ ID NO: 12), corresponding to nucleotides 159-1700 of Genbank Reference Sequence NM_001616.4. The signal sequence is indicated by underlining. Underline

[0123] The nucleic acid sequence encoding the processed soluble (extracellular) human ActRIIA protein is as follows: ​​​ActRIIA is highly conserved in vertebrates, with large segments of its extracellular domains being completely conserved. For example, Figure 1 Multiple sequence alignments of the human ActRIIA extracellular domain with various ActRIIA orthologs are described. Many ActRIIA-binding ligands are also highly conserved. Therefore, based on these alignments, it is possible to predict key amino acid positions within the ligand-binding domain that are important for normal ActRIIA-ligand binding activity, as well as amino acid positions that may tolerate substitutions without significantly altering normal ActRIIA-ligand binding activity. Therefore, active human ActRIIA variant proteins usable according to the methods disclosed herein may include one or more amino acids at corresponding positions from sequences of ActRIIA from another vertebrate, or may include residues similar to residues in human or other vertebrate sequences.

[0124] This is not intended to be restrictive; the following examples illustrate this method of defining active ActRIIA variants. For example... Figure 1 As explained in the text, F13 in the extracellular domain of human cells is present in sheep (…). Ovis aries (SEQ ID NO: 62), Red Junglefowl ( Gallus (SEQ ID NO: 65), scalpers ( Bos Taurus ) (SEQ ID NO: 66), Barn Owl ( Tyto alba (SEQ ID NO: 67) and David's mouse-eared bat ( Myotis davidii (SEQ ID NO: 68) ActRIIA is Y, indicating that aromatic residues at this position are tolerant, including F, W, and Y. Q24 in the human extracellular domain is R in the cattle ActRIIA, indicating that charged residues at this position are tolerant, including D, R, K, H, and E. S95 in the human extracellular domain is F in the junglefowl and barn owl ActRIIA, indicating that this site is tolerant to a wide variety of variations, including polar residues such as E, D, K, R, H, S, T, P, G, Y, and possibly hydrophobic residues such as L, I, or F. E52 in the human extracellular domain is D in the sheep ActRIIA, indicating that acidic residues at this position are tolerant, including D and E. P29 in the human extracellular domain has relatively poor conservation, appearing as S in the sheep ActRIIA and as L in the David's mouse eared bat ActRIIA, therefore essentially any amino acid will be tolerant at this position.

[0125] In addition, as discussed above, ActRII proteins have been characterized in the art according to structural / functional features, 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) The EMBO Journal 22(7): 1555-1566; and U.S. Patent Nos. 7,709,605, 7,612,041, and 7,842,663). These references provide ample guidance on how to generate ActRII variants that retain one or more desired activities (e.g., ligand-binding activity) in addition to the teachings herein.

[0126] For example, a defining structural motif known as the three-finger toxin fold is important for ligand binding by type I and type II receptors, and is formed by conserved cysteine residues located at different 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). Thus, 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). Thus, the structurally more disordered amino acids flanking this cysteine-defined core sequence can be truncated at the N-terminus by about 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 residues and at the C-terminus by about 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 residues without necessarily altering ligand binding. Exemplary ActRIIA extracellular domain truncations include SEQ ID NOs: 10 and 11.

[0127] Therefore, the general formula for the active portion of ActRIIA (e.g., ligand binding) is a protein comprising or substantially composed of amino acids 30-110 of SEQ ID NO: 9. Thus, an ActRIIA protein may, for example, comprise residues starting at any of amino acids 21-30 corresponding to SEQ ID NO: 9 (e.g., starting at any of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) and corresponding to SEQ ID NO: The ActRIIA portion of amino acid 9 ending at any one of amino acids 110-135 (e.g., 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, 132, 133, 134, or 135) has 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, substantially composed of, or composed of. Other examples include starting at positions selected from SEQ ID NO: 9, 21-30 (e.g., starting at any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30), 22-30 (e.g., starting at any one of amino acids 22, 23, 24, 25, 26, 27, 28, 29, or 30), 23-30 (e.g., starting at any one of amino acids 23, 24, 25, 26, 27, 28, 29, or 30), and 24-30 (e.g., starting at any one of amino acids 24, 25, 26, 27, 28, 29, or 30), and at positions selected from SEQ ID NO: 9, 111-135. (e.g., ending at any 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), 112-135 (e.g., ending at any 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), 113-135 (e.g.,constructs 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, 132, 133, 134, or 135. Variants within these ranges are also contemplated, particularly those comprising, consisting essentially of, or consisting of 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 corresponding portion of SEQ ID NO: 9. Thus, in some embodiments, the amino acid sequence of the C-terminal portion of the polypeptide is selected from the group consisting of: 110-135 (e.g., 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, 132, 133, 134, or 135), 115-135 (e.g., ending at any one of amino acids 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135), 120-135 (e.g., ending at any one of amino acids 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135), 130-135 (e.g., ending at any one of amino acids 130, 131, 132, 133, 134, or 135), 111-134 (e.g., 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, 132, 133, or 134), 111-133 (e.g., 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, 132, or 133), 111-132 (e.g., 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 111-131 (e.g., 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, or 131).An ActRIIA protein can comprise, consist essentially of, or consist of amino acids 30-110 of SEQ ID NO: 9 having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto. Optionally, the ActRIIA protein comprises amino acids 30-110 of SEQ ID NO: 9 having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto and comprises no more than 1, 2, 5, 10, or 15 conservative amino acid changes in the ligand-binding pocket.

[0128] In certain embodiments, the disclosure relates to GDF / BMP antagonists (inhibitors) comprising ActRIIA proteins (including fragments, functional variants, and modified forms thereof) and uses thereof (e.g., increasing immune response and treating cancer in a patient in need thereof). Preferably, the ActRIIA proteins are soluble (e.g., the extracellular domain of ActRIIA). In some embodiments, the ActRIIA proteins inhibit one or more GDF / BMP ligands (e.g., Smad signaling) (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 proteins bind 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 proteins of the disclosure comprise 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, consists essentially of, or consists of an ActRIIA portion that begins at a residue corresponding to amino acid 21-30 of SEQ ID NO: 9 (e.g., begins at 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., ends 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, 132, 133, 134, or 135). In some embodiments, the ActRIIA proteins comprise, consist essentially of, or consist of 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 amino acids 30-110 of SEQ ID NO: 9.In certain embodiments, the ActRIIA protein comprises, consists of, or consists essentially of 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 amino acids 21-135 of SEQ ID NO: 9. In some embodiments, the ActRIIA protein or ActRIIA fusion protein comprises, consists of, or consists essentially of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 9, 10, 11, 32, 36, 39, and 41.

[0129] 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%, 99%, or 100% 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 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 obtainable by expression in Chinese hamster ovary cells.

[0130] In some alternative embodiments, the ActRII protein (e.g., SEQ ID NO: 32) lacks the C-terminal lysine. In some embodiments, the ActRII protein lacking the 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.

[0131] In certain embodiments, the pharmaceutical formulations described herein contain a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 that lacks the C-terminal lysine residue (SEQ ID NO: 41). In certain embodiments, the pharmaceutical formulations described herein contain a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 that lacks the C-terminal lysine residue (SEQ ID NO: 41), wherein the mixture contains 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%, 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% by weight of SEQ ID NO: 32.

[0132] In certain embodiments, the pharmaceutical formulations described herein contain a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 that lacks the C-terminal lysine residue (SEQ ID NO: 41), wherein the mixture contains about 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%, 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% by weight of SEQ ID NO: 32.

[0133] In certain embodiments, the pharmaceutical formulations described herein contain a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 lacking the C-terminal lysine residue (SEQ ID NO: 41), wherein the mixture contains 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%, 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% by weight of SEQ ID NO: 41.

[0134] In certain embodiments, the pharmaceutical formulations described herein contain a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 lacking the C-terminal lysine residue (SEQ ID NO: 41), wherein the mixture contains about 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%, 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% by weight of SEQ ID NO: 41.

[0135] In certain embodiments, the pharmaceutical formulations described herein contain 100% by weight of SEQ ID NO: 32. In certain embodiments, the pharmaceutical formulations described herein contain 100% by weight of SEQ ID NO: 41.

[0136] In certain aspects, the present disclosure relates to GDF-trapping proteins (also referred to as "GDF-trap"). In some embodiments, the GDF-trap of the present disclosure is a variant ActRII protein (e.g., ActRIIA) comprising one or more mutations (e.g., amino acid additions, deletions, substitutions, and combinations thereof) in the extracellular domain (also referred to as the ligand-binding domain) of an ActRIIA protein (e.g., a "wild-type" or unmodified ActRIIA protein) such that the variant ActRIIA protein has one or more altered ligand-binding activities as compared to the corresponding wild-type ActRIIA protein. In some embodiments, the GDF-trap of the present disclosure retains at least one activity similar to the corresponding wild-type ActRIIA protein. For example, preferred GDF-traps bind GDF11 and / or GDF8 and inhibit (e.g., antagonize) their function. In some embodiments, the GDF-trap of the present disclosure further binds and inhibits one or more GDF / BMP ligands. Thus, the present disclosure provides GDF-trap proteins having altered binding specificity for one or more ActRII ligands.

[0137] To illustrate, one or more mutations that increase the selectivity of the altered ligand-binding domain for GDF11 and / or GDF8 over one or more ActRIIA-binding ligands, e.g., activin, particularly activin A, can be selected. Optionally, the altered ligand-binding domain has an activin binding K d d50-fold of the ratio of the wild-type ligand-binding domain. Optionally, the altered ligand-binding domain has an IC d d50-fold of the ratio of the wild-type ligand-binding domain. Optionally, the altered ligand-binding domain has an IC 50 d50-fold of the ratio of the wild-type ligand-binding domain. Optionally, the altered ligand-binding domain has an IC 50 d50-fold of the ratio of the wild-type ligand-binding domain. Optionally, the altered ligand-binding domain has an IC 50 d50-fold of the ratio of the wild-type ligand-binding domain. Optionally, the altered ligand-binding domain has an IC 50 d50-fold of the ratio of the wild-type ligand-binding domain. Optionally, the altered ligand-binding domain has an IC

[0138] Methods of Use In certain aspects, the present disclosure provides a method of treating pulmonary arterial hypertension (PAH) comprising administering to a patient in need thereof a pharmaceutical formulation described herein.

[0139] In certain aspects, the disclosure provides a pharmaceutical formulation for treating PAH in a subject in need thereof comprising administering a lyophilized human ActRIIA protein linked to a constant domain of an immunoglobulin, wherein the dosing 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 for response; and 3) modifying the subsequent dose; and wherein the subsequent dose is administered to the subject every three weeks. In some embodiments, the subsequent dose is modified according to the response of the subject.

[0140] In certain aspects, the disclosure provides a method of treating pulmonary arterial hypertension (PAH) comprising administering to a patient in need thereof a pharmaceutical formulation described herein, wherein the administration of the pharmaceutical formulation results in a change in one or more of the following hemodynamic or functional parameters: a decrease in pulmonary vascular resistance (PVR); an increase in 6-minute walk distance (6MWD); a decrease in N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels; a prevention or reduction in progression of World Health Organization (WHO) functional classification of pulmonary hypertension; a promotion or increase in progression of WHO functional classification of pulmonary hypertension; an improvement in right ventricular function; an improvement in pulmonary arterial pressure; and / or an improvement in mean right atrial pressure.

[0141] In certain aspects, the disclosure provides a method of treating pulmonary arterial hypertension (PAH) comprising administering to a patient in need thereof a pharmaceutical formulation described herein, wherein the administration of the pharmaceutical formulation results in an increase in exercise capacity, provides a clinical improvement, improves WHO functional classification (FC), and delays disease progression, including a reduction in the risk of death and hospitalization from PAH.

[0142] In certain aspects, the disclosure provides a method of treating, preventing, or reducing the rate of progression and / or severity of one or more complications of pulmonary arterial hypertension comprising administering to a patient in need thereof a pharmaceutical formulation described herein, wherein the administration of the protein results in a change in one or more of the following hemodynamic or functional parameters: a decrease in pulmonary vascular resistance (PVR); an increase in 6-minute walk distance (6MWD); a decrease in N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels; a prevention or reduction in progression of World Health Organization (WHO) functional classification of pulmonary hypertension; a promotion or increase in progression of WHO functional classification of pulmonary hypertension; an improvement in right ventricular function; an improvement in pulmonary arterial pressure; and / or an improvement in mean right atrial pressure. In some embodiments, the one or more complications of pulmonary arterial hypertension are selected from the group consisting of: smooth muscle and / or endothelial cell proliferation in the pulmonary arteries, angiogenesis in the pulmonary arteries, dyspnea, chest pain, pulmonary vascular remodeling, right ventricular hypertrophy, and pulmonary fibrosis.

[0143] In some embodiments, administration of the pharmaceutical formulations described herein reduces PVR in a patient. In some embodiments, administration of the pharmaceutical formulations described herein reduces PVR in a patient 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 PVR in a patient by at least 20%. In some embodiments, the reduction in PVR is due to a reduction in mean pulmonary arterial pressure. In some embodiments, administration of the pharmaceutical formulations described herein increases the 6-minute walk distance in a patient. In some embodiments, administration of the pharmaceutical formulations described herein increases the 6-minute walk distance in a patient 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, or more than 400 meters). In some embodiments, administration of the pharmaceutical formulations described herein increases the 6-minute walk distance in a patient by at least 30 meters. In some embodiments, administration of the pharmaceutical formulations described herein reduces NT-proBNP levels in a patient. In some embodiments, administration of the pharmaceutical formulations described herein reduces NT-proBNP levels in a patient 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 formulations described herein reduces NT-proBNP levels in a patient by at least 30%. In some embodiments, administration of the pharmaceutical formulations described herein reduces NT-proBNP levels to normal levels. In some embodiments, normal levels of NT-proBNP are < 100 pg / ml.

[0144] In some embodiments, administration of the pharmaceutical formulations described herein prevents or reduces WHO-recognized functional classification progression of pulmonary hypertension. In some embodiments, administration of the pharmaceutical formulations described herein prevents or reduces WHO-recognized functional classification progression of pulmonary hypertension from functional Class I to Class II. In some embodiments, administration of the pharmaceutical formulations described herein prevents or reduces WHO-recognized functional classification progression of pulmonary hypertension from functional Class II to Class III. In some embodiments, administration of the pharmaceutical formulations described herein prevents or reduces WHO-recognized functional classification progression of pulmonary hypertension from functional Class III to Class IV. In some embodiments, administration of the pharmaceutical formulations described herein promotes or increases WHO-recognized functional classification progression of pulmonary hypertension. In some embodiments, administration of the pharmaceutical formulations described herein promotes or increases WHO-recognized functional classification progression of pulmonary hypertension from Class IV to Class III. In some embodiments, administration of the pharmaceutical formulations described herein promotes or increases WHO-recognized functional classification progression of pulmonary hypertension from Class III to Class II. In some embodiments, administration of the pharmaceutical formulations described herein promotes or increases WHO-recognized functional classification progression of pulmonary hypertension from Class II to Class I.

[0145] In some embodiments, administration of the pharmaceutical formulations described herein improves right ventricular function in a patient. In some embodiments, the improvement in right ventricular function is due to an increase in right ventricular area fraction change. 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 right ventricular area fraction change and ejection fraction.

[0146] In some embodiments, administration of the pharmaceutical formulations described herein improves pulmonary arterial pressure in a patient. In some embodiments, the improvement in pulmonary arterial pressure is a reduction in mean pulmonary arterial pressure (mPAP). In some embodiments, administration of the pharmaceutical formulations described herein reduces mPAP in a patient 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 mPAP in a patient 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 mean right atrial pressure (mRAP) in a patient. In some embodiments, the improvement in mRAP is a reduction in mRAP. In some embodiments, administration of the pharmaceutical formulations described herein reduces mRAP in a patient 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 mRAP in a patient 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).

[0147] In some embodiments, the patient has a pulmonary vascular resistance (PVR) greater than or equal to 3 Wood units. In some embodiments, the patient has a 6-minute walk distance of 150-550 meters. In some embodiments, the patient has an elevated NT-proBNP level compared to a healthy patient. 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, 10,000, 15,000, or 20,000 pg / mL). In some embodiments, the patient has an elevated brain natriuretic peptide (BNP) level compared to a healthy patient. 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, 10,000, 15,000, or 20,000 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 normal levels (i.e., < 100 pg / ml). In some embodiments, the patient has a mean pulmonary arterial pressure (mPAP) selected from the group consisting of: an mPAP of at least 20 mmHg; an mPAP of at least 25 mmHg; an mPAP of at least 30 mmHg; an mPAP of at least 35 mmHg; an mPAP of at least 40 mmHg; an mPAP of at least 45 mmHg; and an mPAP of at least 50 mmHg. In some embodiments, the patient has a mean right atrial pressure (mRAP) selected from the group consisting of: an mRAP of at least 5 mmHg; an mRAP of at least 6 mmHg; an mRAP of at least 8 mmHg; an mRAP of at least 10 mmHg; an mRAP of at least 12 mmHg; an mRAP of at least 14 mmHg; and an mRAP of at least 16 mmHg.

[0148] In some embodiments, the PAH is idiopathic pulmonary arterial hypertension (PAH). In some embodiments, the PAH is heritable PAH. In some embodiments, the PAH is drug or toxin-induced PAH. In some embodiments, the PAH is PAH associated with simple congenital systemic-pulmonary shunts at least 1 year after shunt repair. In some embodiments, the patient has functional Class II or III pulmonary hypertension according to the World Health Organization functional classification system for pulmonary hypertension. In some embodiments, the patient has World Health Organization-recognized functional Class I, II, III, or IV pulmonary hypertension. In some embodiments, the patient has functional Class I, II, III, or IV pulmonary hypertension according to the World Health Organization functional classification system for pulmonary hypertension. In some embodiments, the patient has functional Class IV pulmonary hypertension according to the World Health Organization functional classification system for pulmonary hypertension. In some embodiments, administration of the pharmaceutical formulation described herein increases the transplant-free survival of the patient. In some embodiments, administration of the pharmaceutical formulation described herein increases the transplant-free survival of the patient 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 formulation described herein decreases the right ventricular hypertrophy of the patient. In some embodiments, administration of the pharmaceutical formulation described herein decreases the right ventricular hypertrophy of the patient 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 formulation described herein decreases the smooth muscle hypertrophy of the patient. In some embodiments, administration of the pharmaceutical formulation described herein decreases the smooth muscle hypertrophy of the patient 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 formulation described herein decreases the muscularization of the pulmonary arterioles of the patient. In some embodiments, administration of the pharmaceutical formulation described herein decreases the muscularization of the pulmonary arterioles of the patient by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%).

[0149] In some embodiments, administration of the pharmaceutical formulations described herein improves a patient’s exercise capacity. In some embodiments, administration of the pharmaceutical formulations described herein reduces a patient’s Borg Dyspnea Index (BDI). In some embodiments, administration of the pharmaceutical formulations described herein reduces a 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 reduced kidney function. In some embodiments, administration of the pharmaceutical formulations described herein further improves kidney function. In some embodiments, administration of the pharmaceutical formulations described herein delays clinical worsening of pulmonary arterial hypertension. In some embodiments, administration of the pharmaceutical formulations described herein delays clinical worsening 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 comprises one or more of the following changes: increased need for lung and / or heart transplant; need for rescue therapy with initiation of a known PAH therapy; need for an increase in prostacyclin by at least 10%; need for atrial septostomy; PAH-specific hospitalization for at least 24 hours; and PAH worsening. In some embodiments, PAH worsening comprises WHO functional class worsening and a 6MWD decrease of at least 15%. 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 a hemoglobin level of >8 and <15 g / dl. In some embodiments, the patient’s hemoglobin level is <18 g / dl.

[0150] In certain embodiments, the patient treated according to the methods described herein is a female. In certain embodiments, the patient treated according to the methods described herein is a male. In certain embodiments, the patient treated according to the methods described herein is of any age. In certain embodiments, the patient treated according to the methods described herein is less than 18 years of age. In a particular embodiment, the patient treated according to the methods described herein is less than 13 years of age. In another particular embodiment, the patient treated according to the methods described herein is less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, or less than 5 years of age. In another particular embodiment, the patient treated according to the methods described herein is 1-3 years of age, 3-5 years of age, 5-7 years of age, 7-9 years of age, 9-11 years of age, 11-13 years of age, 13-15 years of age, 15-20 years of age, 20-25 years of age, 25-30 years of age, or greater than 30 years of age. In another particular embodiment, the patient treated according to the methods described herein is 30-35 years of age, 35-40 years of age, 40-45 years of age, 45-50 years of age, 50-55 years of age, 55-60 years of age, or greater than 60 years of age. In another particular embodiment, the patient treated according to the methods described herein is 18-64 years of age, 65-74 years of age, or greater than 75 years of age.

[0151] In certain embodiments, the patient treated according to the dosage forms and methods provided herein has a hemoglobin level of less than 10 g / dL, 9 g / dL, 8 g / dL, or 7 g / dL. In certain embodiments, the patient treated according to the dosage forms and methods provided herein has a hemoglobin level of 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.

[0152] Methods of administration Described herein are methods of administering the liquid formulations described herein. The liquid formulations described herein can be administered to a patient by parenteral routes, e.g., injection (e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, etc.) or transdermal, mucosal, nasal, pulmonary, or oral administration. In certain embodiments, the liquid formulation is administered subcutaneously.

[0153] Further described herein are methods of administering any one of the liquid formulations described herein to a patient, including administering any one of the liquid formulations described herein with a delivery device to a patient, e.g., a reusable pen or autoinjector delivery device. In certain embodiments, the liquid formulation is administered using a reusable pen or autoinjector delivery device. In certain embodiments, the liquid formulations described herein are administered using an autoinjector delivery device.

[0154] A number of reusable pen or auto-injector delivery devices can be used for subcutaneous delivery of the pharmaceutical formulations of the present application. Examples include, but are not limited to, the Molly™ and / or DAI™ and / or PSDI™ (SHL Medical, Zug, Switzerland) and other manufacturers Autopen® (Owen Mumford, Inc., Woodstock, UK), Disetronic Pen (Disetronic Medical Systems, Bergdorf, Switzerland), Humalog® Mix 75 / 25™ Pen, 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™ Pen (Becton Dickinson, Franklin Lakes, N.J.), OptiPen®, OptiPen Pro®, OptiPen Starlet™, and OptiClik® (Sanofi-Aventis, Frankfurt, Germany). Examples of disposable pen or auto-injector delivery devices with application for subcutaneous delivery of the pharmaceutical compositions of the present application include, but are not limited to, the SoloSTAR® pen (Sanofi-Aventis), FlexPen® (Novo Nordisk), and KwikPen™ (Eli Lilly), SureClick™ auto-injector (Amgen, Thousand Oaks, Calif.), Penlet® (Haselmeier, Stuttgart, Germany), EpiPen® (Dey, L. P.), and Humira® pen (Abbott Labs, Abbott Park, Ill.).

[0155] 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 comprises a pre-filled syringe. In certain embodiments, the capacity of the pre-filled syringe is selected from the group consisting of 0.5 ml to 10 ml syringes. In certain embodiments, the capacity of the pre-filled syringe is a 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, 10.0 ml syringe prefilled with any of the liquid formulations described herein.

[0156] In certain embodiments, the pre-filled syringe contains any of the formulations described herein, wherein the concentration of the ActRIIa protein is 5 mg / ml to 100 mg / ml. In certain embodiments, the concentration of the ActRIIa protein is 5 mg / ml to 50 mg / ml. In certain embodiments, the concentration of the ActRIIa protein is 8.3 mg / ml to 50 mg / ml. In certain embodiments, the concentration of the 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.

[0157] In one embodiment, the reusable pen or auto-injector delivery device contains a needle having a gauge of 27 G or smaller in diameter. In one embodiment, the needle gauge size ranges from 25 G to 33 G (including ranges therein, such as 25 sG, 26, 26 sG, 27 G, 28 G, 29 G, 30 G, 31 G, 32 G, and 33 G). In one embodiment, the smallest 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 application.

[0158] In certain embodiments, the liquid formulations described herein are administered using a reusable pen or auto-injector delivery device, wherein 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.

[0159] In certain embodiments, the reusable pen or auto-injector delivery device can be used to inject any of the liquid formulations described herein in the range of 1-5 seconds. In certain embodiments, the reusable pen or auto-injector delivery device can be used to inject any of the liquid formulations described herein in the range of 1-4 seconds. In certain embodiments, the reusable pen or auto-injector delivery device can be used to inject any of the liquid formulations described herein in the range of 1-3 seconds. In certain embodiments, the reusable pen or auto-injector delivery device allows for injection of any of the liquid formulations described herein in the range of 2-3 seconds. In certain embodiments, the reusable pen or auto-injector delivery device allows for injection of any of the liquid formulations described herein in the range of 2-4 seconds. In certain embodiments, the reusable pen or auto-injector delivery device allows for injection of any of the liquid formulations described herein in 1, 2, 3, 4, or 5 seconds.

[0160] In certain embodiments, the reusable pen or auto-injector delivery device can be used to inject any of the liquid formulations described herein with a spring force of 10 N to 100 N. In certain embodiments, the reusable pen or auto-injector delivery device can be used to inject any of the liquid formulations described herein with a spring force of 10 N to 50 N. In certain embodiments, the reusable pen or auto-injector delivery device can be used to inject any of the liquid formulations described herein with a spring force of 10 N, 20 N, 30 N, 40 N, 50 N, 60 N, 70 N, 80 N, 90 N, or 100 N. In certain embodiments, the reusable pen or auto-injector delivery device can be used to inject any of the liquid formulations described herein with a spring force of about 10 N, 20 N, 30 N, 40 N, 50 N, 60 N, 70 N, 80 N, 90 N, or 100 N. In certain embodiments, the reusable pen or auto-injector delivery device can be used to inject any of the liquid formulations described herein with a spring force of about 50 N.

[0161] In certain embodiments, the reusable pen or auto-injector delivery device allows for injection of any of the liquid formulations described herein with a squeeze force of 1 N - 10 N. In certain embodiments, the reusable pen or auto-injector delivery device allows for injection of any of the liquid formulations described herein with a squeeze force of 1 N, 2 N, 3 N, 4 N, 5 N, 6 N, 7 N, 8 N, 9 N, or 10 N. In certain embodiments, the reusable pen or auto-injector delivery device allows for injection of any of the liquid formulations described herein with a squeeze force of about 1 N, 2 N, 3 N, 4 N, 5 N, 6 N, 7 N, 8 N, 9 N, or 10 N. In certain embodiments, the reusable pen or auto-injector delivery device allows for injection of any of the liquid formulations described herein with a squeeze force of about 6 N. EMBODIMENTS

[0162] The presently generally described disclosure will be more readily understood by reference to the following examples, which are included for the purpose of illustration of certain embodiments of the present disclosure, and are not intended to limit the present disclosure.

[0163] The meaning of the abbreviations in the examples is shown below.

[0164] CHO = Chinese hamster ovary SDS PAGE = sodium dodecyl sulfate - polyacrylamide gel electrophoresis HMW = high molecular weight species LMW = low molecular weight species PS80 = polysorbate 80 DTPA = diethylene triamine pentaacetate EDTA = ethylenediaminetetraacetic acid MFI = mean fluorescence intensity UPSEC = ultra-performance size exclusion chromatography cIEF = capillary isoelectric focusing CE-SDS = capillary electrophoresis sodium dodecyl sulfate Fc = fragment crystallizable region IgG = immunoglobulin G P188 = poloxamer 188 Example 1: ActRIIA-Fc Fusion Proteins Soluble ActRIIA fusion proteins were constructed with the extracellular domain of human ActRIIA fused to a human or mouse Fc domain via a minimal linker. The constructs are referred to as ActRIIA-hFc and ActRIIA-mFc, respectively.

[0165] The ActRIIA-hFc shown below is also known as sotacept and was purified from the CHO cell line (SEQ ID NO:32): ActRIIA-hFc and ActRIIA-mFc proteins are expressed in CHO cell lines. Consider three different leader sequences: (i) Bee melittin (HBML): MKFLVNVALVFMVVYISYIYA (SEQ ID NO: 33) (ii) Tissue plasminogen activator (TPA): MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO: 34) (iii) Natural: MGAAAKLAFAVFLISCSSGA (SEQ ID NO: 35).

[0166] The chosen form uses a TPA precursor and has the following unprocessed amino acid sequence: This protein is encoded by the following nucleic acid sequence: Both ActRIIA-hFc and ActRIIA-mFc are highly suitable for recombination expression. For example... Figure 2A and 2B The protein is shown to be purified as a single, clear peak. N-terminal sequencing revealed a single sequence of –ILGRSETQE (SEQ ID NO: 38). Purification can be achieved via a series of column chromatography steps, including, for example, three or more steps in any order: protein A chromatography, Q agarose chromatography, phenyl agarose chromatography, size exclusion chromatography, and cation exchange chromatography. Purification can be accomplished via viral filtration and buffer exchange. ActRIIA-hFc protein is purified to >98% purity, as determined by size exclusion chromatography, and >95% purity, as determined by SDS-PAGE.

[0167] ActRIIA-hFc and ActRIIA-mFc exhibited high affinity for their ligands. GDF11 or activin A was immobilized on a Biacore™ CM5 chip using a standard amine conjugation procedure. ActRIIA-hFc and ActRIIA-mFc proteins were loaded onto the system and binding was measured. ActRIIA-hFc was loaded at 5 x 10⁻⁶ ppm. -12 dissociation constant (K) D ) binds to activin, and at 9.96 x 10 -9 KD Binding to GDF11. See Figure 3A and 3B Using similar binding assays, ActRIIA-hFc was determined to have high to moderate affinity for other TGF-beta superfamily ligands, including, for example, Activin B, GDF8, BMP6, and BMP10. ActRIIA-mFc has similar behavior.

[0168] ActRIIA-hFc is very stable in pharmacokinetic studies. Rats were given 1 mg / kg, 3 mg / kg, or 10 mg / kg of ActRIIA-hFc protein and the plasma levels of the protein were measured at 24, 48, 72, 144, and 168 hours. In another study, rats were given 1 mg / kg, 10 mg / kg, or 30 mg / kg. In rats, ActRIIA-hFc has a serum half-life of 11-14 days, and circulating levels of the drug are very high after two weeks (11 μg / ml, 110 μg / ml, or 304 μg / ml for initial administration of 1 mg / kg, 10 mg / kg, or 30 mg / kg, respectively). In cynomolgus monkeys, the plasma half-life is significantly greater than 14 days, and circulating levels of the drug are 25 μg / ml, 304 μg / ml, or 1440 μg / ml for initial administration of 1 mg / kg, 10 mg / kg, or 30 mg / kg, respectively.

[0169] Example 2: Characterization of ActRIIA-hFc Proteins The ActRIIA-hFc fusion protein was expressed from the pAID4 vector (SV40 ori / enhancer, CMV promoter) in stably transfected CHO-DUKX B11 cells using the tissue plasminogen leader sequence of SEQ ID NO: 34. The purified protein, as described above in Example 1, has the sequence of SEQ ID NO: 32. The Fc portion is the human IgGl Fc sequence as shown in SEQ ID NO: 32. Protein analysis revealed that the ActRIIA-hFc fusion protein forms as a homodimer held together by disulfide bonds.

[0170] CHO cell-expressed material has higher affinity for activin B ligand than has been reported for ActRIIA-hFc fusion proteins expressed in human 293 cells (see del Re et al. (2004) J Biol Chem. 279(51):53126-53135). In addition, use of the TPA leader sequence provides greater yield than other leaders, and provides a higher purity N-terminal sequence than ActRIIA-Fc expressed by the native leader. Use of the native leader sequence results in two major ActRIIA-Fc species, each with a different N-terminal sequence.

[0171] Additional ActRIIA ligand trap (ActRIIA-Fc fusion proteins modified to reduce activin A binding relative to myostatin or GDF11) are described in International Patent Application Publication Nos. WO 2006 / 012627 and WO 2007 / 062188, which are incorporated herein by reference.

[0172] Example 3: Preparation of Liquid ActRIIA-hFc Fusion Protein Formulations At the outset, ActRIIA-hFc fusion protein soliastercept (SEQ ID NO: 32) was formulated as a lyophilized solution in phosphate buffered saline. In further development work, a lyophilized citrate buffer formulation of ActRIIA-hFc fusion protein soliastercept containing sucrose and polysorbate 80 was developed to enable a stable formulation with a shelf life sufficient for commercialization. The lyophilized formulation consisted of a lyophilized cake of soliastercept (45 mg / vial or 60 mg / vial) as part of an injection kit that also contained a vial adapter, a pre-filled syringe of sterile water for injection, a syringe, a needle, and an alcohol swab. However, users did not like the need to reconstitute the lyophilized cake prior to administration. To improve the user experience, a liquid formulation containing soliastercept suitable for a pre-filled syringe and / or auto-injector device combination was formulated according to the following experiments.

[0173] Concentration Liquid Feasibility Studies Concentration stability studies were performed to evaluate the short term biophysical stability of secukinumab (SEQ ID NO: 32) at various concentrations. Formulations containing 100 mg / mL and 50 mg / mL secukinumab in 10 mM citrate buffer and 8% (w / v) sucrose pH 5.8 were prepared. Table 1 shows the stability of the formulation containing 100 mg / mL secukinumab. Formulations containing 100 mg / mL secukinumab were placed at different temperatures (5°C, 25°C, and 40°C) and selected time points were analyzed to show that the formulation in citrate buffer and sucrose had minimal changes in high molecular weight formation (as measured by UPSEC) and charge distribution (as measured by cIEF) at 5°C and 25°C.

[0174] Tables 2 and 3 show the stability of the formulation containing 50 mg / mL secukinumab. Formulations containing 50 mg / mL secukinumab were placed at different temperatures (5°C, 25°C, and 40°C) and selected time points were analyzed to show that the formulation in citrate buffer, sucrose, PS80, and with or without DTPA had minimal changes in sub-visible particle formation (as measured by MFI), high molecular weight formation (as measured by UPSEC), charge distribution (as measured by cIEF), and fragmentation (as measured by CE-SDS) at 5°C and 25°C.

[0175] pH and Buffer Feasibility Studies The behavior of secukinumab (SEQ ID NO: 32) was analyzed in 6 buffers at different pH and 8% sucrose. The buffers tested were histidine pH 6.5 and 7; phosphate pH 5, 5.5, 6, 6.5, and 7; citrate pH 4.5, 5, 5.5, 6, 6.5, and 7; acetate pH 4.5, 5, 5.5, and 6; succinate 4.5 and 5; and glutamate pH 4.5 and 5. The results are shown in Tables 4-13. The formulations were heat stressed at 40°C for up to 2 months and tested for biophysical stability (turbidity), sub-visible particle formation (as measured by MFI), high molecular weight formation (by UPSEC), fragmentation (by R- and NR-CE-SDS), and charge distribution (by cIEF).

[0176] As shown in Tables 4-13 and Figures 4-7 All formulations showed reasonable stability at elevated temperatures, suggesting that all of the buffer systems tested are viable for liquid formulations. Of these, histidine was the least stable, and the other buffers behaved identically.

[0177] The behavior of solithromycin in 6 buffers at pH 3, 5.8, and 8 and 8% sucrose was also analyzed. The buffers tested were citrate, histidine, succinate, lactate, MES, and TRIS. The results are shown in Tables 14-30. The biophysical stability (turbidity), high molecular weight formation (by UPSEC), aggregation (by UPSEC), and fragmentation (by R-CESDS and NR-CESDS UPSEC), and charge distribution (by cIEF) of the formulations were tested at time 0 (TO), 15 days, and 30 days.

[0178] Based on the data presented in Tables 14-30, the tested buffers had comparable stability between buffer concentrations at pH 5.8 and pH 8.0. However, at lower pH, the stability was significantly reduced for all buffers tested. Although all buffers were comparable, citrate and succinate performed slightly better than the others.

[0179] Effect of Metal Chelators on Solithicimab The effect of the metal chelator DTPA on solithromycin (SEQ ID NO: 32) formulated in citrate buffer pH 5.8 was investigated by 1H NMR spectra, 2D NMR fingerprinting, and NMR measurements of protein translational diffusion. The results of the 1D spectra and protein diffusion are shown in Figure 8 and 9 The same behavior was observed in the protein 1H NMR spectra of solithromycin with and without DTPA Figure 8 ), indicating that no direct effect from binding or other interactions was detected by this technique. Additionally, the data from the protein detection diffusion measurements Figure 9 ) did not show a difference in behavior, indicating that the addition of DTPA had no effect on solithromycin behavior.

[0180] Colloidal Stability and Surfactant Screening A colloidal stability study of solithromycin (SEQ ID NO: 32) was performed by agitation stress by shaking bottles containing solithromycin in 10 mM citrate buffer, 8% sucrose, and different levels of PS80, and by freeze-thaw cycling (one cycle - freezing the bottle at -80°C and thawing at room temperature). The results are shown in Figures 10-13 Figure 10 The percentage of high molecular weight species (%HMW) detected in bottles of solithromycin containing 10 mM citrate buffer, 8% sucrose, and 0%, 0.005%, 0.01%, 0.02%, 0.03%, and 0.05% PS80, after 3 and 7 days of agitation are shown. Figure 11 The %HMW detected in bottles of solithromycin containing 10 mM citrate buffer, 8% sucrose, and 0%, 0.005%, 0.01%, 0.02%, 0.03%, and 0.05% PS80, that had undergone three, five, and eight freeze-thaw cycles are shown.​Figure 12 and 13 % monomer (%M) and % HMW (%HMW) detected in vials of solithromycin containing 10 mM citrate buffer, 8% sucrose, and 0%, 0.01%, 0.02%, and 0.03% PS80 that had undergone three, five, and eight freeze-thaw cycles. Figure 12 ) and % HMW ( Figure 13 ) were not observed after 7 days of agitation or 8 freeze / thaw cycles. In addition, a positive comparison of polysorbate 80 and poloxamer 188 was performed, 1-Mo / 25°C. The results showed that the molecule was stable to agitation stress, and there was no difference between PS80 or P188, and they both behaved similarly. In terms of prototype stability, all analytical attributes were comparable after 1-Mo stress at 25°C.

[0181] Results of additional surfactant stability studies are shown in Tables 31-35. The stability studies shown in Tables 31-35 were performed by subjecting vials of solithromycin containing 10 mM citrate buffer and 8% sucrose and different levels of surfactant to agitation stress by shaking. The surfactants screened were poloxamer 188, sodium dodecyl sulfate (SDS), N-dodecyl- -D-maltoside (DDM), polysorbate 20, and triton X at 0.02 mg / mL, 0.2 mg / mL, and 2 mg / mL for 4 days and 7 days. Table 36 shows the results of a stability study of control solithromycin in 10 mM citrate buffer and 8% sucrose without surfactant.

[0182] The results show that solithromycin is stable to agitation stress in citrate buffer, and there is no significant difference between the surfactants tested when comparing charged species, monomeric species, aggregates, or fragmentation.

[0183] Tables 37-41 show surfactant stability studies of solithromycin performed by shake stress with bottles containing 10 mM histidine buffer, 8% sucrose, and different levels of surfactants in solithromycin. The surfactants screened were 0.02 mg / mL, 0.2 mg / mL, and 2 mg / mL of poloxamer 188, sodium dodecyl sulfate (SDS), N-dodecyl- -D-maltoside (DDM), polysorbate 20, and triton X for 4 days and 7 days. Table 42 shows a control surfactant stability study of solithromycin containing 10 mM histidine buffer, 8% sucrose, and no surfactant.

[0184] The results show that solithromycin is stable to shake stress when formulated with the tested surfactants in histidine buffer. At high SDS concentrations, slightly higher high molecular weight species were detected. When looking at charge species after shake stress, there were no significant differences between the tested surfactants.

[0185] Chelator Studies Formulations containing solithromycin (SEQ ID NO: 32) in 10 mM citrate buffer, 8% sucrose, and 0.02% polysorbate 80, pH 5.8, and different levels (0 mM, 10 mM, 20 mM, 30 mM) of L-methionine were exposed to light stress in a photostability chamber in the presence of different levels of DTPA or EDTA as chelators (0 µM, 7.5 µM, 15 µM, 30 µM, 60 µM). Along with the dark control, the stressed samples were analyzed for high molecular weight formation (using UPSEC) and showed that the addition of L-methionine reduced %HMW formation and the addition of chelators had no negative impact. The results are shown in Tables 33-36. Thus, either chelator can be used in the formulation. Figure 14 and 15

[0186] ​In addition, siltuximab was formulated with 10 mM citrate buffer or 10 mM histidine buffer, 8% sucrose, and 0.02% polysorbate 80, pH 5.8, and different levels (0 mM, 5 mM, 10 mM, 50 mM) of dimercaprol. The samples were exposed to light stress in a photostability chamber. The stressed samples were analyzed for high molecular weight formation (using UPSEC) along with a dark control and showed the addition of dimercaprol. The results of several tests of formulations containing siltuximab, 10 mM citrate buffer, 8% sucrose, and 0.02% polysorbate 80, pH 5.8, and different levels (0 mM, 5 mM, 10 mM, 50 mM) of dimercaprol are shown in Table 43, and the dark control results are shown in Table 44. The results of several tests of formulations containing siltuximab, 10 mM histidine buffer, 8% sucrose, and 0.02% polysorbate 80, pH 5.8, and different levels (0 mM, 5 mM, 10 mM, 50 mM) of dimercaprol are shown in Table 45, and the dark control results are shown in Table 46.

[0187] The results show that in both citrate and histidine buffers, the inclusion of low concentrations of dimercaprol as an antioxidant was able to provide comparable protection under light stress to the control samples. The inclusion of higher amounts of dimercaprol, as well as light stress, resulted in increased charge species and greater loss of monomer content, indicating that the formulation was not compatible with higher dimercaprol content as an antioxidant.

[0188] Stabilizer Screening The behavior of secukinumab (SEQ ID NO: 32) was analyzed in various stabilizers. The stabilizers tested were carboxymethylcellulose (CMC), glucose, polyethylene glycol (PEG), albumin, trehalose, proline, mannitol, and dextran. Tables 47-56 show the results for secukinumab formulations comprising 10 mM citrate, 20 mM L-methionine, 0.2 mg / mL and polysorbate 80, pH 5.8, and various surfactants. Table 57 shows the results for a citrate control formulation without surfactant. Tables 58-67 show the results for secukinumab formulations comprising 10 mM histidine, 20 mM L-methionine, 0.2 mg / mL and polysorbate 80, pH 5.8, and various surfactants. Table 68 shows the results for a control formulation without surfactant. The formulations were heat stressed at 50°C for up to two weeks and tested for biophysical stability (turbidity), sub-visible particle formation (measured by Aura), high molecular weight formation (by UPSEC), aggregation (by UPSEC), and fragmentation (by UPSEC).

[0189] The results show that the inclusion of the tested stabilizers in the formulation provided similar levels of stability to secukinumab molecules in citrate buffer at elevated heat stress. Each of the tested formulations maintained high monomer content and comparable high molecular weight species increase over time, as well as comparable levels of charged species, compared to samples without stabilizers.

[0190] The results show that the inclusion of the tested stabilizers in the formulation provided similar levels of stability to the solithromycin molecules in the histidine buffer under elevated heat stress. Each of the tested formulations maintained high monomer content and comparable high molecular weight species increase over time, as well as comparable levels of charged species.

[0191] Solithicimab Formulations Tables 69-71 show liquid pharmaceutical formulations containing solithromycin (SEQ ID NO: 32, SEQ ID NO: 41, or a mixture of both SEQ ID NO: 32 and SEQ ID NO: 41) prepared: Table 69 Human ActRIIA Fusion Protein SEQ ID NO: 32 50 mg / mL Citrate Buffer 10 mM Sucrose 8% weight / volume Polysorbate 80 0.2 mg / mL L-methionine 20 mM Table 70 Human ActRIIA Fusion Protein SEQ ID NO: 41 50 mg / mL Citrate Buffer 10 mM Sucrose 8% weight / volume Polysorbate 80 0.2 mg / mL L-methionine 20 mM Table 71 Human ActRIIA Fusion Proteins SEQ ID NO: 32 and SEQ ID NO: 41 50 mg / mL Citrate Buffer 10 mM Sucrose 8% weight / volume Polysorbate 80 0.2 mg / mL L-methionine 20 mM Human ActRIIA Fusion Proteins SEQ ID NO: 32 and SEQ ID NO: 41 50 mg / mL Citrate Buffer 10 mM Sucrose 8% weight / volume Polysorbate 80 0.2 mg / mL L-methionine 20 mM Sequence Table 72: Sequence Exemplary methods and materials are described herein, but methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the methods and formulations disclosed herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

Claims

1. A pharmaceutical formulation comprising 50-100 mg / mL of a human ActRIIa fusion protein comprising the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 41, a buffer, a surfactant, a stabilizer, and optionally one or more antioxidants; wherein the buffer is not histidine.

2. The pharmaceutical formulation of claim 1, wherein the fusion protein has a concentration of 100 mg / mL.

3. The pharmaceutical formulation of claim 1, wherein the fusion protein has a concentration of 50 mg / mL.

4. The pharmaceutical formulation of any one of claims 1-3, wherein the buffer is selected from the group consisting of succinate, phosphate, acetate, citrate, and glutamate.

5. The pharmaceutical formulation of claim 4, wherein the buffer is a phosphate buffer that maintains the pH of the formulation between 5-7.

6. The pharmaceutical formulation of claim 4, wherein the buffer is a citrate buffer that maintains the pH of the formulation between 4.5-7.

7. The pharmaceutical formulation of claim 4, wherein the buffer is an acetate buffer that maintains the pH of the formulation between 4.5-6.

8. The pharmaceutical formulation of claim 4, wherein the buffer is a succinate buffer that maintains the pH of the formulation between 4.5-5.

9. The pharmaceutical formulation of any one of claims 1-3, wherein the buffer is a glutamate buffer that maintains the pH of the formulation between 5-7.

10. The pharmaceutical formulation of any one of claims 1-9, wherein the buffer maintains the pH of the formulation at 5.

8.

11. The pharmaceutical formulation of any one of claims 1-10, wherein the buffer is present at a concentration of 10-50 mM.

12. The pharmaceutical formulation of any one of claims 1-10, wherein the buffer is present at a concentration of at least 10 mM.

13. The pharmaceutical formulation of claim 12, wherein the stabilizer is sucrose.

14. The pharmaceutical formulation of any one of claims 1-13, wherein the stabilizer is present at a concentration of 2-16% weight / volume.

15. The pharmaceutical formulation of any one of claims 1-14, wherein the stabilizer is present at a concentration of 6-10% weight / volume.

16. The pharmaceutical formulation of any one of claims 1-15, wherein the stabilizer is present at a concentration of at least 8% weight / volume.

17. The pharmaceutical formulation of any one of claims 1-16, wherein the surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, poloxamer 124, poloxamer 127, poloxamer 188, and poloxamer 407.

18. The pharmaceutical formulation of claim 17, wherein the surfactant is polysorbate 80.

19. The pharmaceutical formulation of claim 17, wherein the surfactant is polysorbate 20.

20. The pharmaceutical formulation of claim 17, wherein the surfactant is poloxamer 188.

21. The pharmaceutical formulation of any one of claims 1-20, wherein the surfactant is present at a concentration of 0.05-0.5 mg / ml.

22. The pharmaceutical formulation of any one of claims 1-20, wherein the surfactant is present at a concentration of 0.1-0.5 mg / ml.

23. The pharmaceutical formulation of any one of claims 1-22, wherein the surfactant is present at a concentration of at least 0.2 mg / ml.

24. The pharmaceutical formulation of any one of claims 1-23, wherein the pharmaceutical formulation further comprises an antioxidant.

25. The pharmaceutical formulation of any one of claims 1-24, wherein the pharmaceutical formulation further comprises a chelator, wherein the chelator is DTPA or EDTA.

26. The pharmaceutical formulation of claim 25, wherein the chelator is present at a concentration of 7.5-100 µM.

27. The pharmaceutical formulation of any one of claims 25-26, wherein the chelator is present at a concentration of 10 µM.

28. The pharmaceutical formulation of claim 24, wherein the antioxidant is methionine.

29. The pharmaceutical formulation of claim 28, wherein the antioxidant is L-methionine.

30. The pharmaceutical formulation of any one of claims 28-29, wherein the antioxidant is present at a concentration of 7.5-50 mM.

31. The pharmaceutical formulation of any one of claims 28-29, wherein the antioxidant is present at a concentration of 5-20 mM.

32. A liquid pharmaceutical formulation comprising 50-100 mg / mL of a human ActRIIa fusion protein comprising the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO:41, 10-50 mM of a citrate buffer, 2-16% weight / volume 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.

33. A liquid pharmaceutical formulation comprising 50 mg / mL of a human ActRIIa fusion protein comprising the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO:41, 10 mM of a citrate buffer, 8% weight / volume sucrose, 0.2 mg / mL of polysorbate 80, and 20 mM of L-methionine.

34. The pharmaceutical formulation of any one of claims 1-33, wherein the pharmaceutical formulation is for treating pulmonary arterial hypertension in a subject in need thereof.

35. The pharmaceutical formulation of any one of claims 1-34, wherein the pharmaceutical formulation is administered by an auto-injector.

36. The pharmaceutical formulation of any one of claims 1-35, wherein the pharmaceutical formulation is administered by subcutaneous injection.

37. The pharmaceutical formulation of any one of claims 1-35, wherein the pharmaceutical formulation is a liquid formulation.

38. The pharmaceutical formulation of any one of claims 1-35, wherein the pharmaceutical formulation is stable when stored at 2-8 °C for at least 1 month.

39. The pharmaceutical formulation of any one of claims 1-38, wherein the formulation is contained in an injection device.

40. The pharmaceutical formulation of claim 39, wherein the injection device is an autoinjector.

41. The pharmaceutical formulation of any one of claims 1-38, wherein the formulation is contained in a glass bottle.

42. A method of treating pulmonary arterial hypertension (PAH) in a human patient in need thereof, comprising administering to the patient the pharmaceutical formulation of any one of claims 1-41.

43. Use of the pharmaceutical formulation of any one of claims 1-41 for the treatment of pulmonary arterial hypertension (PAH) in a patient in need thereof.

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