Formulations containing ActRIIA protein variants

Abstract

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

Description

[Technical Field]

[0001] Cross-reference to related applications This application claims the benefit of priority from 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 foregoing applications are incorporated herein by reference in their entireties.

[0002] Reference to an electronically submitted sequence listing This application contains a Sequence Listing that has been submitted electronically in XML format, which is incorporated herein by reference in its entirety. The XML file was created on March 6, 2024, has the file name 1848179-0002-170-WO1_Sequence_Listing.xml, and is 51.2 KB in size.

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

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

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

[0006] Treatment of PH generally depends on the cause or classification of the condition. When PH is caused by a known drug or medical condition, it is known as secondary PH, and treatment is usually directed at the underlying condition. Treatment of pulmonary venous hypertension generally involves optimizing left ventricular function by administering diuretics, beta-blockers, and ACE inhibitors, or repair or replacement of the mitral or aortic valve. Treatments for PAH include pulmonary vasodilators, digoxin, diuretics, anticoagulants, and oxygen therapy. Pulmonary vasodilators target various pathways, including the prostacyclin pathway (e.g., prostacyclins, e.g., intravenous epoprostenol, subcutaneous or intravenous treprostinil, and inhaled iloprost), the nitric oxide pathway (e.g., phosphodiesterase-5 inhibitors, e.g., sildenafil and tadalafil), and the endothelin-1 pathway (e.g., endothelin receptor antagonists, e.g., oral bosentan and oral ambrisentan) (Humbert, M. Am. J. Respir. Crit. Care Med. 179:650-6 (2009); Hill et al., Respiratory Care 54(7):958-68 (2009)). However, current therapies do not reverse PH, nor do they directly address the underlying vascular remodeling and muscularization seen in many PH patients. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Hill et al., Respiratory Care 54(7):958-68 (2009) [Non-patent document 2] Simonneau et al., JACC 54(1):S44-54 (2009) [Non-patent document 3] Humbert, M. Am. J. Respir. Crit. Care Med. 179:650-6 (2009) Summary of the Invention [Problem to be solved by the invention]

[0008] Accordingly, it is an object of the present disclosure to provide stable liquid pharmaceutical formulations comprising ActRIIa fusion proteins and corresponding methods for treating, preventing or reducing the rate of progression and / or severity of PH, particularly for treating, preventing or reducing the rate of progression and / or severity of one or more PH-related complications. [Means for solving the problem]

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

[0010] In some embodiments, the ActRIIa protein begins with any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of SEQ ID NO:9 and includes 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, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 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.

[0011] In some embodiments, the ActRIIa protein is an ActRIIa fusion protein comprising an ActRIIa extracellular domain and one or more protein domains heterologous to ActRIIa. In some embodiments, the ActRIIa protein is a fusion protein comprising an immunoglobulin Fc domain. In some embodiments, the immunoglobulin Fc domain is an IgG1 immunoglobulin Fc domain. In some embodiments, the ActRIIa fusion protein further comprises a linker domain disposed between the ActRIIa protein domain and the one or more heterologous domains (e.g., Fc immunoglobulin domains). 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 that is achieved by expression in Chinese hamster ovary cells.

[0012] 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.

[0013] In some embodiments, the pharmaceutical formulations provided herein contain 10-100 mg / mL of a recombinant fusion protein comprising the extracellular domain (ECD) of human activin receptor type IIA (ActRIIA) protein or a derivative thereof linked to a constant domain of immunoglobulin, such as a human IgG1 Fc domain. In other embodiments, the pharmaceutical formulations contain 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 the extracellular domain (ECD) of human activin receptor type IIA (ActRIIA) protein or a derivative thereof linked to a constant domain of immunoglobulin, such as a human IgG1 Fc domain. In other embodiments, the pharmaceutical formulations contain 40-50 mg / mL of a recombinant fusion protein comprising the extracellular domain (ECD) of human activin receptor type IIA (ActRIIA) protein or a derivative thereof linked to a constant domain of immunoglobulin, such as a human IgG1 Fc domain. In other embodiments, the pharmaceutical formulation comprises 50 mg / mL of a recombinant fusion protein comprising the extracellular domain (ECD) of human activin receptor type IIA (ActRIIA) protein or a derivative thereof linked to a constant domain of an immunoglobulin, such as a human IgG1 Fc domain.

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

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

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

[0019] In certain embodiments, the pharmaceutical formulations provided herein comprise 50 to 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.

[0020] 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, if the protein has a negative charge due to the presence of glycans, the buffer is not histidine.

[0021] In some embodiments, the buffering agent is selected to be physiologically compatible and to maintain a pH above 4. In some embodiments, the buffering agent is selected to be physiologically compatible and to maintain a pH between 5 and 7. In some embodiments, the buffering agent 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 buffering agent 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 buffering agent is selected to be physiologically compatible and to maintain a pH of 5.8.

[0022] In certain embodiments, the pharmaceutical formulations described herein comprise a buffering agent, wherein the buffering agent is a phosphate buffer to maintain the pH of the pharmaceutical formulation between 5 and 7. In certain embodiments, the pharmaceutical formulations described herein comprise a buffering agent, wherein the buffering agent is a citrate buffer to maintain the pH of the pharmaceutical formulation between 4.5 and 7. In certain embodiments, the pharmaceutical formulations described herein comprise a buffering agent, wherein the buffering agent is an acetate buffer to maintain the pH of the pharmaceutical formulation between 4.5 and 6. In certain embodiments, the pharmaceutical formulations described herein comprise a buffering agent, wherein the buffering agent is a succinate buffer to maintain the pH of the pharmaceutical formulation between 4.5 and 5. In certain embodiments, the pharmaceutical formulations described herein comprise a buffering agent, wherein the buffering agent is a glutamate buffer to maintain the pH of the pharmaceutical formulation between 5 and 7.

[0023] 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 buffering agent is present at a concentration of 10-50 mM. In some embodiments, the buffering agent is present at a concentration of at least 10 mM.

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

[0025] In some embodiments, the stabilizer is selected from the group consisting of carboxymethylcellulose (CMC), dextrose, 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, hi some embodiments, the stabilizer is sucrose.

[0026] In some embodiments, the stabilizer is present in the formulation at a concentration of 2-16% (wt / vol). In some embodiments, the stabilizer is present at a concentration of about 6% to about 10% (wt / vol). In some embodiments, the stabilizer is present at a concentration of at least 0.005% (wt / vol), 0.01% (wt / vol), 0.02% (wt / vol), 0.03% (wt / vol), 0.05% (wt / vol), 0.06% (wt / vol), 0.07% (wt / vol), 0.08% (wt / vol), 0.09% (wt / vol), 0.1% (wt / vol), 0.5% (wt / vol), 0.7% (wt / vol), 0.8% (wt / vol), 0.9% (wt / vol), 1.0% (wt / vol), 1.2% (wt / vol), 1.5% (wt / vol). In some embodiments, the stabilizer is present in a concentration of at least 8% wt / vol. In some embodiments, the stabilizer is present in a concentration of at least 8% wt / vol.

[0027] In some embodiments, the surfactant is selected from the group consisting of sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate, chenodeoxycholic acid, N-lauroylsarcosine sodium salt, lithium dodecyl sulfate, 1-octanesulfonic acid sodium salt, sodium cholate hydrate, sodium deoxycholate and glycodeoxycholic acid sodium salt, benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride monohydrate, hexadecyltrimethylammonium bromide, CHAPS, CHAPSO, SB3-10, SB3-12, digitonin, Triton X-100, Triton X-114, TWEEN®-20, TWEEN®-80, lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 40, 50, and 60, glycerol monostearate, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, poloxamer 188, and soy lecithin. In certain embodiments, the surfactant is selected from the group consisting of poloxamer 188, sodium dodecyl sulfate (SDS), N-dodecyl-β-D-maltoside (DDM), polysorbate 20, and Triton X. In certain embodiments, the surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, poloxamer 124, poloxamer 127, poloxamer 188, and poloxamer 407.

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

[0029] In some embodiments, the surfactant is present in the formulation at a concentration of 0.02-2.0 mg / mL, or 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, or 2.0 mg / mL. In some embodiments, the surfactant is present in the formulation at a concentration of 0.05-0.3 g / mL. In some embodiments, the surfactant is present at a concentration of at least 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mg / mL. In some embodiments, the pharmaceutical formulation 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.

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

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

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

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

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

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

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

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

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

[0039] In certain embodiments, the pharmaceutical formulations described herein comprise a mixture of SEQ ID NO:32 and a variant of SEQ ID NO:32 lacking a C-terminal lysine residue (SEQ ID NO:41), wherein the mixture is 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%, 64%, 65%, 65%, 66%, 67%, 68%, 69 ... 1%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of SEQ ID NO: 32.

[0040] In certain embodiments, the pharmaceutical formulations described herein comprise a mixture of SEQ ID NO:32 and a variant of SEQ ID NO:32 lacking a C-terminal lysine residue (SEQ ID NO:41), wherein the mixture is 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%, 64% or 65% by weight. 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% of SEQ ID NO: 32.

[0041] In certain embodiments, the pharmaceutical formulations described herein comprise a mixture of SEQ ID NO:32 and a variant of SEQ ID NO:32 lacking a C-terminal lysine residue (SEQ ID NO:41), wherein the mixture is 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%, 64%, 65%, 66%, 67%, 68%, 69%, 69%, 68 ... 1%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of SEQ ID NO: 41.

[0042] In certain embodiments, the pharmaceutical formulations described herein comprise a mixture of SEQ ID NO:32 and a variant of SEQ ID NO:32 lacking a C-terminal lysine residue (SEQ ID NO:41), wherein the mixture is 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%, 64% or 65% by weight. 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% of SEQ ID NO: 41.

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

[0044] 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.

[0045] 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., although under certain circumstances, the aqueous formulations may exhibit stability at other temperatures, including about 25° C. and about 40° C., for periods up to about 3, 6, 9, or 12 months). In certain embodiments, the pharmaceutical formulations are administered via an autoinjector. In certain embodiments, the pharmaceutical formulations are administered by subcutaneous injection. In some embodiments, the pharmaceutical formulations are administered parenterally.

[0046] 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 autoinjector. In other embodiments, the formulations described herein are contained in a glass vial. [Brief explanation of the drawings]

[0047] [Figure 1] Figure 1 shows a multiple sequence alignment of various vertebrate ActRIIA proteins and human ActRIIA (SEQ ID NOs: 61-68). [Figure 2A] Figures 2A and 2B show the purification of ActRIIA-hFc expressed in CHO cells. The protein is purified as a single, well-defined peak (left lane: molecular weight standard; right lane: ActRIIA-hFc), visualized by sizing column (top panel) and Coomassie-stained SDS-PAGE (bottom panel). See Example 1. [Figure 2B] Figures 2A and 2B show the purification of ActRIIA-hFc expressed in CHO cells. The protein is purified as a single, well-defined peak (left lane: molecular weight standard; right lane: ActRIIA-hFc), visualized by sizing column (top panel) and Coomassie-stained SDS-PAGE (bottom panel). See Example 1. [Figure 3A] Figures 3A and 3B show that the dissociation constant (KD) of ActRIIA-hFc bound to activin is 5 x 10-12 and the KD for binding to GDF11 is 9.96 x 10-9. See Example 1. [Figure 3B] Figures 3A and 3B show that the dissociation constant (KD) of ActRIIA-hFc bound to activin is 5 x 10-12 and the KD for binding to GDF11 is 9.96 x 10-9. See Example 1. [Figure 4] Figure 4 shows the results of "Stability pH Screening Study: Effect of pH and Buffer at t=0". See "Example 3: pH and Buffer Feasibility Study". [Figure 5] Figure 5 shows the results of the "Stability pH Screening Study: Effect of pH and Buffer at 40°C for 2 weeks." See "Example 3: pH and Buffer Feasibility Study." [Figure 6] Figure 6 shows the results of the "Stability pH Screening Study: Effect of pH and Buffer at 40°C for 1 month." See "Example 3: pH and Buffer Feasibility Study." [Figure 7] Figure 7 shows the results of the "Stability pH Screening Study: Effect of pH and Buffer at 40°C for 2 months." See "Example 3: pH and Buffer Feasibility Study." [Figure 8] Figure 8 shows the 1H NMR data for SEQ ID NO:32 in the presence and absence of DTPA. See "Example 3: Effect of Metal Chelators on SEQ ID NO:32." [Figure 9] Figure 9 shows diffusion NMR data for SEQ ID NO:32 in the presence and absence of DTPA. See "Example 3: Effect of Metal Chelators on SEQ ID NO:32." [Figure 10]Figure 10 shows the results of a colloidal stability study on SEQ ID NO:32 and PS80 performed by agitation stress. See "Example 3: Colloidal Stability and Surfactant Screening." [Figure 11] Figure 11 shows the results of a colloidal stability study on SEQ ID NO:32 and PS80 performed by agitation stress. See "Example 3: Colloidal Stability and Surfactant Screening." [Figure 12] Figure 12 shows the results of a colloidal stability study on SEQ ID NO:32 and PS80 performed by freeze-thaw cycling. See "Example 3: Colloidal Stability and Surfactant Screening." [Figure 13] Figure 13 shows the results of a colloidal stability study on SEQ ID NO:32 and PS80 performed by freeze-thaw cycling. See "Example 3: Colloidal Stability and Surfactant Screening." [Figure 14] Figure 14 shows the results of exposing formulations of SEQ ID NO:32 containing different levels of L-methionine (0 mM, 10 mM, 20 mM, 30 mM) to light stress in a photostability chamber in the presence of different levels of EDTA as a chelator (0 μM, 7.5 μM, 15 μM, 30 μM, 60 μM). See "Example 3: Chelator / L-methionine Variation Study." [Figure 15] Figure 15 shows the results of exposing a formulation of SEQ ID NO:32 containing different levels of L-methionine (0 mM, 10 mM, 20 mM, 30 mM) to light stress in a photostability chamber in the presence of different levels of DTPA as a chelator (0 μM, 7.5 μM, 15 μM, 30 μM, 60 μM). See "Example 3: Chelator / L-methionine Variation Study." DETAILED DESCRIPTION OF THE INVENTION

[0048] Detailed Description definition The terms used herein generally have their ordinary meaning in the art, within the context of this disclosure and in the specific context in which each term is used. Particular terms are discussed below or elsewhere in this specification to provide additional guidance to those skilled in the art in describing the formulations and methods of the present disclosure and how to make and use them. The scope or meaning of any use of a term will be apparent from the specific context in which the term is used.

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

[0050] Alternatively, particularly in biological systems, the terms "about" and "approximately" can mean values ​​within an order of magnitude of a given value, preferably within 5-fold, and more preferably within 2-fold. Numerical quantities given herein are approximations unless otherwise indicated, i.e., the terms "about" and "approximately" mean that they can be estimated unless expressly stated.

[0051] The terms "a" and "an" include plural references unless the context in which the term is used clearly dictates otherwise. The terms "a" (or "an") and "one or more" and "at least one" can be used interchangeably herein. Furthermore, "and / or," when used herein, is considered a specific disclosure of two or more specified features or components, each with or without the other. Thus, herein, the term "and / or" used in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A alone," and "B alone." Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone.

[0052] Numerical ranges disclosed herein are inclusive of the values ​​defining the range. For example, when a range of pH values ​​is described, such as "pH 5.5 to 6.0," the range is intended to include the described values. For example, pH 5.0 to 7.0 includes pH 5.0 and pH 7.0, as well as values ​​between 5.0 and 7.0. As used herein, a formulation containing "a citrate buffer of pH X" refers to a solution of pH X containing a citrate buffer. That is, pH is intended to refer to the pH of the solution.

[0053] The proteins disclosed herein can include amino acid sequences that do not occur in nature. Such variants necessarily have less than 100% sequence identity or similarity with the starting molecule. In certain embodiments, the variants have an amino acid sequence that has less than about 75% to 100% amino acid sequence identity or similarity, more preferably less than about 80% to 100%, even more preferably less than about 85% to 100%, even more preferably less than about 90% to 100% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), and most preferably less than about 95% to 100% amino acid sequence identity or similarity with the amino acid sequence of the starting protein (e.g., a naturally occurring or wild-type protein), e.g., over the length of the variant molecule.

[0054] 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 for measuring protein stability are available in the art and are described in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). Stability can be measured for a selected period of time at a selected temperature.

[0055] A "stable" liquid formulation is a pharmaceutical formulation in which no significant changes are observed at refrigerated temperatures (2-8°C) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably 2 years. Furthermore, "stable" liquid formulations include formulations that exhibit desired characteristics at temperatures including 25°C and 40°C for periods including 1 month, 3 months, 6 months, 12 months, and / or 24 months. Typical acceptance criteria for stability are as follows: Typically, less than about 10%, preferably less than about 5%, of the protein degrades as measured by SEC-HPLC; the pharmaceutical formulation is colorless or clear to slightly opaque upon visual analysis; the concentration, pH, and osmolality of the formulation do not vary by more than ±10%; and the potency is usually within 50-150% of the nominal value. Typically, the formulation contains less than about 10%, preferably less than about 5%, of aggregates. The term "buffering agent" includes an agent that maintains the solution pH of a formulation of the invention within an acceptable range, or, for a lyophilized formulation of the invention, an agent that provides an acceptable solution pH prior to lyophilization. The terms "lyophilization," "lyophilized," and "freeze-dried" refer to a process in which the material to be dried is first frozen, and then the ice or freezing solvent is removed by sublimation under a vacuum. Excipients can be included in the pre-lyophilized formulation to enhance the storage stability of the lyophilized product. The term "pharmaceutical formulation" refers to a preparation in a form that allows the active ingredient to be effective and that does not contain additional ingredients that are toxic to the subject to which the formulation is administered. The terms "formulation" and "pharmaceutical formulation" are used interchangeably throughout. "Pharmaceutically acceptable" refers to excipients (vehicles, additives) and compositions that can reasonably be administered to a subject to provide an effective dose of the active ingredient employed, and that are "generally regarded as safe," e.g., physiologically tolerable and do not normally produce an allergic reaction or similar unpleasant reaction (e.g., stomach upset, etc.) when administered to humans.In another embodiment, the term refers to molecular entities and compositions approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals (more particularly, humans). A "reconstituted" formulation is one 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 optionally, for subcutaneous administration.

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

[0057] The liquid formulations of the present invention minimize the formation of aggregates (high molecular weight species) and particulates, improve colloidal stability, minimize fragmentation (low molecular weight species), and ensure that the protein maintains its biological activity over time. In certain embodiments, the formulations are produced, for example, by collecting ActRIIA in an aqueous pharmaceutical formulation and buffer-exchanging it into a desired buffer as the final step of the purification process. The ActRIIA is then concentrated to the desired concentration. In this embodiment, there is no lyophilization step. Further, excipients such as stabilizers and surfactants can be added to the ActRIIA formulation, and the formulation is diluted with an appropriate buffer to the final protein concentration. The final formulation is filtered and filled into a final container (e.g., an autoinjector). Alternatively, the formulation can be stored in a vial and delivered via a syringe or container.

[0058] Pharmaceutical formulations provided herein comprise an ActRIIa fusion protein. In certain embodiments, the present disclosure provides pharmaceutical formulations comprising an ActRIIa fusion protein, wherein the protein is 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. It is present in a concentration of about 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.

[0059] 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.

[0060] In some embodiments, the pharmaceutical formulations provided herein contain 10-100 mg / mL of a recombinant fusion protein comprising the extracellular domain (ECD) of human activin receptor type IIA (ActRIIA) protein or a derivative thereof linked to a constant domain of an immunoglobulin, such as a human IgG1 Fc domain. In other embodiments, the pharmaceutical formulations contain 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 the extracellular domain (ECD) of human activin receptor type IIA (ActRIIA) protein or a derivative thereof linked to a constant domain of an immunoglobulin, such as a human IgG1 Fc domain. In other embodiments, the pharmaceutical formulations contain 40-50 mg / mL of a recombinant fusion protein comprising the extracellular domain (ECD) of human activin receptor type IIA (ActRIIA) protein or a derivative thereof linked to a constant domain of an immunoglobulin, such as a human IgG1 Fc domain. In other embodiments, the pharmaceutical formulation comprises 50 mg / mL of a recombinant fusion protein comprising the extracellular domain (ECD) of human activin receptor type IIA (ActRIIA) protein or a derivative thereof linked to a constant domain of an immunoglobulin, such as a human IgG1 Fc domain.

[0061] In some embodiments, the liquid pharmaceutical formulations of 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 include a buffer, a stabilizer, a surfactant, and optionally one or more antioxidants, which are described in further detail below. Buffers can be selected to maintain the pH of the formulation during processing. Surfactants can be selected based on their ability to function as emulsifiers, wetting agents, solubilizers, and / or dispersants.

[0062] Those skilled in the art will recognize that the concentrations of excipients described herein share interdependencies within a particular formulation. As an example, the concentration of a bulking agent may be reduced in one embodiment, for example, when the protein concentration is high. Excipients and other additives are added to confer or improve manufacturability and / or final product quality, such as stability and delivery of a drug product (e.g., protein). The formulations provided herein include appropriate excipients that enhance stability and safety.

[0063] buffer Typically, the stability of pharmacologically active protein formulations is observed to be maximized within a narrow pH range. This pH range exhibiting optimal stability must be identified early in preformulation studies. Several approaches, such as accelerated stability studies and calorimetric screening studies, are useful in this endeavor (Remmele RL Jr., et al., Biochemistry, 38(16): 5241-7 (1999)). Once the formulation is finalized, the protein must be manufactured and maintained throughout its shelf life. Therefore, buffers are often used to control the pH of the formulation.

[0064] Several factors must be considered when selecting a buffer. First and foremost, the type and concentration of buffer must be determined based on the buffer's pKa and the desired formulation pH. Equally important is ensuring 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 tingling or irritation that the buffer may induce upon administration. The potential for tingling or irritation is greater for drugs administered via subcutaneous (SC) or intramuscular (IM) routes, where the drug solution remains at the administration site for a relatively long time, than for drugs administered via IV routes, where the formulation is rapidly diluted into the blood upon administration. For formulations administered via direct IV infusion, it is necessary to monitor the total amount of buffer (and any other formulation components).

[0065] In some embodiments, the buffer comprises an organic acid, succinate, phosphate, acetate, citrate, citric acid, Tris, HEPES, glutamate, 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.

[0066] 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 or equal to 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.

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

[0068] The pH buffering compound can be present in any amount suitable for maintaining the pH of the formulation at a predetermined level. When an appropriately low level of buffering agent is used, crystallization and pH shifts can be avoided. 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 may be at least 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 500 mM. In certain embodiments, the 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, 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.

[0069] 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 pharmaceutical formulation is at least 10 mM.

[0070] stabilizers In certain embodiments, the pharmaceutical formulations provided herein include stabilizers. These stabilizers can be classified based on the mechanism by which they stabilize proteins against various chemical and physical stresses. Some stabilizers are used to mitigate the effects of specific stresses or to regulate the specific 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 what amount or range of stabilizer can be included in any particular formulation to achieve a formulation of the present disclosure that is likely to promote the retention and stability of an ActRIIa fusion protein.

[0071] In some embodiments, a stabilizer (or a 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 typically indicates that the protein has precipitated or at least aggregated. The stabilizer can prevent aggregation or chemical degradation (e.g., autolysis, deamidation, oxidation, etc.). Some stabilizers can also act as anticoagulants when the formulation is administered to a patient. In some embodiments, the stabilizer is selected from the group consisting of CMC, dextrose, PEG, albumin, kerptose, 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 certain embodiments, the pharmaceutical formulations provided herein include a stabilizer, such as, but not limited to, sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, glucosamine, fructose, mannitol, sorbitol, polyhydroxy compounds, such as polysaccharides, e.g., dextran, starch, hydroxyethyl starch, cyclodextrin, N-methylpyrrolidone, cellulose, and hyaluronic acid (Carpenter et al., Develop. Biol. Standard 74:225, (1991)). In one embodiment of the present disclosure, sucrose is used as a stabilizer.

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

[0073] surfactants In certain embodiments, the pharmaceutical formulations provided herein can further comprise a surfactant. Surfactants are commonly used in protein formulations to prevent surface-induced degradation. Surfactants are amphipathic molecules that have the ability to compete with proteins for interfacial positions (and / or promote the proper refolding of conformationally altered protein molecules). The hydrophobic portion of the surfactant molecule occupies the interfacial position (e.g., air / liquid), while the hydrophilic portion of the surfactant molecule remains oriented toward the bulk solvent. At sufficient concentrations (usually near the surfactant's critical micelle concentration), the surface layer of surfactant molecules serves to prevent protein molecules from adsorbing to the interface, thereby minimizing surface-induced degradation. Surfactants contemplated herein include, but are not limited to, fatty acid esters of sorbitan polyethoxylate, i.e., polysorbate 20 and polysorbate 80. These two types differ only in the length of the aliphatic chain, C-12 and C-18, respectively, which confers hydrophobicity to the molecule. Thus, polysorbate 80 has a higher surface activity and a lower critical micelle concentration than polysorbate 20.

[0074] Detergents also affect the thermodynamic structural stability of proteins. Nonionic surfactants are generally useful in stabilizing proteins. Ionic surfactants (detergents) typically destabilize proteins. Again, the effect of a given surfactant excipient is protein-specific. For example, polysorbates have been shown to decrease the stability of some proteins and increase the stability of others. Detergent-induced protein destabilization can be rationalized in terms of the hydrophobic tails of the surfactant molecules, which may be involved in specific binding with proteins in their partially or fully unfolded states. This type of interaction may cause a shift in conformational equilibrium toward a more expanded protein state (i.e., increasing the exposure of hydrophobic portions of the protein molecule that complement binding with polysorbate). Alternatively, if the native state of a protein exhibits several hydrophobic surfaces, surfactant binding to that native state may stabilize that conformation. Another aspect of polysorbates is that they are inherently susceptible to oxidative degradation. As raw materials, they often contain sufficient amounts of peroxide to cause oxidation of the side chains of protein residues, especially methionine. The potential for oxidative damage resulting from the addition of stabilizers emphasizes the need to use the lowest effective concentration of excipients in formulations. In the case of surfactants, the effective concentration for a given protein depends on the mechanism of stabilization.

[0075] Surfactants are also added in an appropriate amount to prevent surface-related aggregation phenomena (Chang, B, J. Pharm. Sci. 85:1325, (1996)). Accordingly, exemplary surfactants include, but are not limited to, anionic, cationic, nonionic, 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, N-lauroyl sarcosine sodium salt, lithium dodecyl sulfate, 1-octanesulfonic acid sodium salt, sodium cholate hydrate, sodium deoxycholate, and sodium glycodeoxycholic acid salt. Cationic surfactants include, but are not limited to, benzalkonium chloride or benzethonium chloride, cetylpyridinium chloride monohydrate, and hexadecyltrimethylammonium bromide. Zwitterionic detergents include, but are not limited to, CHAPS, CHAPSO, SB3-10, and SB3-12. Non-ionic detergents include, but are not limited to, digitonin, Triton X-100, Triton X-114, TWEEN®-20, and TWEEN®-80. Surfactants include, but are not limited to, lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 40, 50 and 60, glycerol monostearate, polysorbate 40, polysorbate 60, polysorbate 65 and polysorbate 80, soybean lecithin and other phospholipids such as dioleylphosphatidylcholine (DOPC), dimyristoylphosphatidylglycerol (DMPG), dimyristoylphosphatidylcholine (DMPC) and (dioleylphosphatidylglycerol) DOPG; sucrose fatty acid esters, methylcellulose and carboxymethylcellulose.In certain embodiments, the surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, poloxamer 124, poloxamer 127, poloxamer 188, and poloxamer 407. In certain embodiments, the surfactant is polysorbate 80. In certain embodiments, the surfactant is polysorbate 20. In certain embodiments, the surfactant is poloxamer 188.

[0076] In some embodiments, the surfactant is selected from the group consisting of sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate, chenodeoxycholic acid, N-lauroylsarcosine sodium salt, lithium dodecyl sulfate, 1-octanesulfonic acid sodium salt, sodium cholate hydrate, sodium deoxycholate, glycodeoxycholic acid sodium salt, benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride monohydrate, hexadecyltrimethylammonium bromide, CHAPS, CHAPSO, SB3-10, SB3-12, digitonin, Triton X-100, Triton X-114, TWEEN®-20, TWEEN®-80, lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 40, 50, and 60, glycerol monostearate, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, poloxamer 188, and soy lecithin. In certain embodiments, the surfactant is selected from the group consisting of poloxamer 188, sodium dodecyl sulfate (SDS), N-dodecyl-β-D-maltoside (DDM), polysorbate 20, and Triton X. In certain embodiments, the surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, poloxamer 124, poloxamer 127, poloxamer 188, and poloxamer 407.

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

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

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

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

[0081] In one embodiment, the pharmaceutical 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.

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

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

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

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

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

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

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

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

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

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

[0092] In some embodiments, the pharmaceutical formulation comprises a protein comprising, consisting essentially of, or consisting of an amino acid sequence 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 at least 90%, 95%, or 99% identical to SEQ ID NO:9, SEQ ID NO:32, or SEQ ID NO:41, wherein the protein binds to activin and / or GDF11. In certain embodiments, the protein comprises the amino acid sequence of SEQ ID NO:9, SEQ ID NO:32, or SEQ ID NO:41. In other embodiments, the protein consists of the amino acid sequence of SEQ ID NO:9, SEQ ID NO:32, or SEQ ID NO:41.

[0093] In some embodiments, the pharmaceutical formulation 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.

[0094] 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: 9. In certain embodiments, the protein consists essentially of the amino acid sequence of SEQ ID NO: 9. In other embodiments, the protein consists of the amino acid sequence of SEQ ID NO: 9.

[0095] In some embodiments, the pharmaceutical formulation 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.

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

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

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

[0099] The present disclosure provides kits comprising the pharmaceutical formulations and injection devices described herein. In certain embodiments, the kits comprise the pharmaceutical formulations and injection devices described herein. In some embodiments of the kits disclosed herein, the pharmaceutical formulations comprising the proteins are pre-filled into one or more containers, such as an autoinjector.

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

[0101] 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.

[0102] In some embodiments, the pharmaceutical formulation comprising a protein further comprises a sugar, such as a disaccharide (e.g., sucrose). In some embodiments, the stable liquid pharmaceutical formulation comprising a protein comprises sucrose, trehalose, mannitol, polyvinylpyrrolidone (PVP), dextrose, 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 protein-to-sugar weight ratio of at least 1:1. In some embodiments, the pharmaceutical formulation comprises a protein and a sugar in a protein-to-sugar weight ratio of 1:1 to 1:10. In some embodiments, the pharmaceutical formulation comprises a protein and a sugar in a protein-to-sugar weight ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In some embodiments, the pharmaceutical formulation comprises a protein and a sugar in a protein-to-sugar weight ratio of 1:6. In certain of the foregoing embodiments, the pharmaceutical formulation comprises a protein and a sugar in an amount sufficient to stabilize the protein.

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

[0104] In certain embodiments of the kits disclosed herein, the kit further comprises an injectable device for use in parenterally administering a sterile injectable solution. In some embodiments, the sterile injectable solution is administered subcutaneously. In some embodiments, the sterile injectable solution is administered intradermally. In some embodiments, the sterile injectable solution is administered intramuscularly. In some embodiments, the sterile injectable solution is administered intravenously.

[0105] In certain embodiments of the kits disclosed herein, the kit further comprises an autoinjector for use in 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.

[0106] ActRIIA protein In certain embodiments, the present disclosure relates to an ActRIIA protein. 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 modifications. Reference to ActRIIA herein is understood to refer to any one of the currently identified forms. Members of the ActRIIA family are generally transmembrane proteins composed of a ligand-binding extracellular domain containing a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain predicted to exhibit serine / threonine kinase activity.

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

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

[0109] The signal peptide is single underlinethe extracellular domain is shown in bold font; and potential endogenous N-linked glycosylation sites are shown in bold font. double underline is shown.

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

[0111] The C-terminal "tail" of the extracellular domain is single underline The sequence with the "tail" deleted (Δ15 sequence) is as follows: [Table 3]

[0112] The nucleic acid sequence encoding the human ActRIIA precursor protein is shown below (SEQ ID NO: 12) and follows nucleotides 159-1700 of Genbank Reference Sequence NM_001616.4. The signal sequence includes: underline is drawn. [Table 4]

[0113] TIFF2026500069000006.tif220161

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

[0115] ActRIIA is highly conserved among vertebrates, with most of the extracellular domain being completely conserved. For example, Figure 1 shows a multi-sequence alignment of the human ActRIIA extracellular domain compared with various ActRIIA orthologs. Many of the ligands that bind to ActRIIA are also highly conserved. Therefore, from 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 are likely to tolerate substitutions without significantly altering normal ActRIIA-ligand binding activity. Thus, active human ActRIIA mutant proteins useful according to the presently disclosed methods may contain one or more amino acids at corresponding positions from other vertebrate ActRIIA sequences, or may contain residues similar to residues in human or other vertebrate sequences.

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

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

[0118] For example, a distinctive structural motif known as the three-finger toxin fold is important for ligand binding by type I and type II receptors and is formed by conserved cysteine ​​residues at various positions within the extracellular domain of each monomeric receptor (Greenwald et al. (1999) Nat Struct Biol 6:18-22; and Hinck (2012) FEBS Lett 586:1860-1870). 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, structurally unordered amino acids adjacent to the core sequence defined by these cysteines can be truncated at 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 at the N-terminus and 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 at the C-terminus without necessarily altering ligand binding. Exemplary ActRIIA extracellular domain truncations include SEQ ID NOs: 10 and 11.

[0119] Thus, a general formula for an active portion of ActRIIA (e.g., ligand binding of ActRIIA) is a protein that includes, consists essentially of, or consists of amino acids 30-110 of SEQ ID NO:9. Thus, an ActRIIA protein can be, for example, an ActRIIA protein beginning at a residue corresponding to any one of amino acids 21-30 of SEQ ID NO:9 (e.g., beginning at any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) and ending at a position corresponding to any one of amino acids 110-135 of SEQ ID NO:9 (e.g., amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135). Other examples include those starting at a position selected from 21-30 of SEQ ID NO: 9 (e.g., starting at any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30), or those starting at a position selected from 22-30 (e.g., starting at any one of amino acids 22, 23, 24, 25, 26, 27, 28, 29, or 30), or those starting at a position selected from 23-30 (e.g., starting at any one of amino acids 23, 24, 25, 26, 27, 28, 29, or 30). ), or beginning at a position selected from 24-30 (e.g., beginning at any one of amino acids 24, 25, 26, 27, 28, 29, or 30) and ending at a position selected from 111-135 of SEQ ID NO:9 (e.g., ending at any one of amino acids 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135); orterminating at a position selected from 112-135 (e.g., terminating at any one of amino acids 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135), or terminating at a position selected from 113-135 (e.g., terminating at any one of amino acids 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135). or terminating at a position selected from 120-135 (e.g., terminating at any one of amino acids 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135), or terminating at a position selected from 130-135 (e.g., terminating at any one of amino acids 130, 131, 132, 133, 134, or 135), or terminating at a position selected from 111-134 (e.g., terminating at any one of amino acids 110, 111, 1 12, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134), or ending at a position selected from 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), or , 111-132 (e.g., terminating 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 terminating at a position selected from 111-131 (e.g., terminating 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, 129,9, 100%, 130, or 131). Variants within these ranges are also contemplated, particularly variants that comprise, consist essentially of, or consist of an amino acid sequence 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 the corresponding portion of SEQ ID NO:9. Thus, in some embodiments, an ActRIIA protein may comprise, consist essentially of, or consist of a protein that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 30-110 of SEQ ID NO:9. Optionally, the ActRIIA protein includes a protein that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 30-110 of SEQ ID NO:9 and contains no more than 1, no more than 2, no more than 5, no more than 10 or no more than 15 conservative amino acid changes in the ligand binding pocket.

[0120] In certain embodiments, the present disclosure relates to GDF / BMP antagonists (inhibitors) comprising ActRIIA proteins (including fragments, functional variants, and modified forms thereof) and their uses (e.g., enhancing immune responses and treating cancer in patients in need thereof). Preferably, the ActRIIA proteins are soluble (e.g., the extracellular domain of ActRIIA). In some embodiments, the ActRIIA proteins inhibit (e.g., Smad signaling) one or more GDF / BMP ligands (e.g., GDF11, GDF8, activin (activin A, activin B, activin AB, activin C, activin E), BMP6, GDF3, BMP15, and / or BMP10). In some embodiments, an ActRIIA protein binds to one or more GDF / BMP ligands (e.g., GDF11, GDF8, activin (activin A, activin B, activin AB, activin C, activin E), BMP6, GDF3, BMP15, and / or BMP10). In some embodiments, an ActRIIA protein of the disclosure begins at a residue corresponding to amino acids 21-30 of SEQ ID NO:9 (e.g., beginning 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., 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, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 1 , 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134 or 135).In some 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 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.

[0121] 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 obtained by expression in Chinese hamster ovary cells.

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

[0123] In certain embodiments, the pharmaceutical formulations described herein comprise a mixture of SEQ ID NO:32 and a variant of SEQ ID NO:32 lacking a C-terminal lysine residue (SEQ ID NO:41), wherein the mixture is 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%, or 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% of SEQ ID NO: 32.

[0124] In certain embodiments, the pharmaceutical formulations described herein comprise a mixture of SEQ ID NO:32 and a variant of SEQ ID NO:32 lacking a C-terminal lysine residue (SEQ ID NO:41), wherein the mixture is 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% of SEQ ID NO: 32.

[0125] In certain embodiments, the pharmaceutical formulations described herein comprise a mixture of SEQ ID NO:32 and a variant of SEQ ID NO:32 lacking a C-terminal lysine residue (SEQ ID NO:41), wherein the mixture is 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% of SEQ ID NO: 41.

[0126] In certain embodiments, the pharmaceutical formulations described herein comprise a mixture of SEQ ID NO:32 and a variant of SEQ ID NO:32 lacking a C-terminal lysine residue (SEQ ID NO:41), wherein the mixture is 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% of SEQ ID NO: 41.

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

[0128] In certain aspects, the present disclosure relates to GDF trap proteins (also referred to as "GDF traps"). In some embodiments, the GDF traps of the present disclosure are mutant ActRII proteins (e.g., ActRIIA) that include 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 mutant ActRIIA protein has one or more altered ligand-binding activities compared to the corresponding wild-type ActRIIA protein. In some embodiments, the GDF trap proteins of the present disclosure retain at least one activity similar to that of the corresponding wild-type ActRIIA protein. For example, preferred GDF traps bind to GDF11 and / or GDF8 and inhibit (e.g., antagonize) their function. In some embodiments, the GDF traps of the present disclosure further bind to and inhibit one or more GDF / BMP ligands. Thus, the present disclosure provides GDF trap proteins with altered binding specificity for one or more ActRII ligands.

[0129] For example, one or more mutations can be selected that increase the selectivity of the modified ligand-binding domain for GDF11 and / or GDF8 over one or more ActRIIA-binding ligands (e.g., activin, particularly activin A). Optionally, the modified ligand-binding domain has a K for GDF11 and / or GDF8 binding that is at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or 1000-fold greater than the ratio for the wild-type ligand-binding domain. d K for activin binding to d Optionally, the modified ligand-binding domain has an IC for inhibiting GDF11 and / or GDF8 that is at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold greater, or even 1000-fold greater than the wild-type ligand-binding domain. 50 IC for inhibiting activin against 50Optionally, the modified ligand-binding domain has an IC 50 IC at least 2x, 5x, 10x, 20x, 50x, 100x smaller than 50 or even 1000 times smaller IC 50 and inhibits GDF11 and / or GDF8.

[0130] How to use In certain aspects, the present disclosure provides methods of treating pulmonary arterial hypertension (PAH), wherein the method comprises administering to a patient in need of treatment for pulmonary arterial hypertension a pharmaceutical formulation described herein.

[0131] In certain aspects, the present disclosure provides a pharmaceutical formulation for treating PAH in a subject in need thereof, comprising administering a lyophilized human ActRIIA fusion protein linked to an immunoglobulin constant domain, 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's response; and (3) modifying the subsequent dose; wherein the subject is administered the subsequent dose every three weeks. In some embodiments, the subsequent dose is modified based on the subject's response.

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

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

[0134] In certain aspects, the present disclosure provides methods for treating, preventing, or reducing the rate of progression and / or severity of one or more complications of pulmonary arterial hypertension, wherein the methods comprise administering a pharmaceutical formulation described herein to a patient in need of treatment, prevention, or reduction in the rate of progression and / or severity of one or more complications of pulmonary arterial hypertension, wherein administration of the protein results in a change in one or more of the following hemodynamic or functional parameters: a reduction in pulmonary vascular resistance (PVR); an increase in 6-minute walk distance (6MWD); a reduction in N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels; prevention or reduction of progression of World Health Organization (WHO) pulmonary hypertension functional class; promotion or increase in regression of WHO pulmonary hypertension functional class; improvement in right ventricular function; improvement in pulmonary artery pressure; and / or 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.

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

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

[0137] 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 fractional area 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 right ventricular fractional area change and an increase in ejection fraction.

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

[0139] In some embodiments, the patient has a pulmonary vascular resistance (PVR) of 3 Wood units or greater. In some embodiments, the patient has a 6-minute walk distance of 150 to 550 meters. In some embodiments, the patient has elevated NT-proBNP levels compared to healthy patients. In some embodiments, the patient has an NT-proBNP level of at least 100 pg / mL (e.g., 100, 150, 200, 300, 400, 500, 1000, 3000, 5000, 10,000, 15,000, or 20,000 pg / mL). In some embodiments, the patient has elevated brain natriuretic peptide (BNP) levels compared to healthy patients. In some embodiments, the patient has a BNP level of at least 100 pg / mL (e.g., 100, 150, 200, 300, 400, 500, 1000, 3000, 5000, 10000, 15000, or 20000 pg / mL). In some embodiments, administration of a 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 a 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 artery pressure (mPAP) selected from the group consisting of at least 20 mmHg mPAP, at least 25 mmHg mPAP, at least 30 mmHg mPAP, at least 35 mmHg mPAP, at least 40 mmHg mPAP, at least 45 mmHg mPAP, and at least 50 mmHg mPAP. In some embodiments, the patient has a mean right atrial pressure (mRAP) selected from the group consisting of at least 5 mmHg mRAP, at least 6 mmHg mRAP, at least 8 mmHg mRAP, at least 10 mmHg mRAP, at least 12 mmHg mRAP, at least 14 mmHg mRAP, and at least 16 mmHg mRAP.

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

[0141] In some embodiments, administration of a pharmaceutical formulation described herein improves a patient's exercise capacity. In some embodiments, administration of a pharmaceutical formulation described herein reduces a patient's Borg Dyspnea Index (BDI). In some embodiments, administration of a pharmaceutical formulation 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 renal function. In some embodiments, administration of a pharmaceutical formulation described herein further improves renal function. In some embodiments, administration of a pharmaceutical formulation described herein delays clinical worsening of pulmonary arterial hypertension. In some embodiments, administration of a pharmaceutical formulation described herein delays clinical worsening of pulmonary arterial hypertension according to the World Health Organization Functional Classification System of Pulmonary Hypertension. In some embodiments, administration of the pharmaceutical formulations described herein reduces the risk of hospitalization for one or more complications associated with pulmonary arterial hypertension. In some embodiments, administration of the pharmaceutical formulations described herein reduces the risk of morbidity for one or more complications associated with pulmonary arterial hypertension. In some embodiments, the morbidity includes a change in one or more of the following: an increased need for lung transplant and / or heart transplant; the need to initiate rescue therapy with known treatments for PAH; the need to increase prostacyclin by at least 10%; the need for atrial septal resection; PAH-specific hospitalization for at least 24 hours; and worsening of PAH. In some embodiments, worsening of PAH includes a worsening of WHO functional class and a decrease in 6MWD by 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 g / dL and <15 g / dL, hi some embodiments, the patient's hemoglobin level is <18 g / dL.

[0142] In certain embodiments, the patient treated according to the methods described herein is female. In certain embodiments, the patient treated according to the methods described herein is 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 under 18 years of age. In certain embodiments, the patient treated according to the methods described herein is under 13 years of age. In another specific embodiment, the patient treated according to the methods described herein is under 12 years of age, under 11 years of age, under 10 years of age, under 9 years of age, under 8 years of age, under 7 years of age, under 6 years of age, or under 5 years of age. In another specific 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 30 years of age or older. In another specific embodiment, the patient treated according to the methods described herein is 30-35 years old, 35-40 years old, 40-45 years old, 45-50 years old, 50-55 years old, 55-60 years old, or over 60 years old. In another specific embodiment, the patient treated according to the methods described herein is 18-64 years old, 65-74 years old, or over 75 years old.

[0143] In certain embodiments, patients treated in accordance with the dosage forms and methods provided herein have hemoglobin levels of less than 10 g / dL, less than 9 g / dL, less than 8 g / dL, or less than 7 g / dL. In certain embodiments, patients treated in accordance with the dosage forms and methods provided herein have hemoglobin levels of between 7 g / dL and 7.5 g / dL, between 7.5 g / dL and 8 g / dL, between 8 g / dL and 8.5 g / dL, between 8.5 g / dL and 9.0 g / dL, between 9.0 g / dL and 9.5 g / dL, or between 9.5 g / dL and 10.0 g / dL.

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

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

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

[0147] In certain embodiments, the liquid formulations described herein are administered using a reusable pen or autoinjector delivery device, wherein the reusable pen or autoinjector delivery device comprises a pre-filled syringe. In certain embodiments, the volume of the pre-filled syringe is selected from a 0.5 mL to 10 mL syringe. In certain embodiments, the volume of the pre-filled syringe is 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 pre-filled with any of the liquid formulations described herein.

[0148] In certain embodiments, a 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.

[0149] In one embodiment, the reusable pen or autoinjector delivery device includes a needle having a gauge of 27 G or less in diameter. In one embodiment, the needle gauge ranges in size from 25 G to 33 G (including intermediate ranges, e.g., 25 G, 26, 26 G, 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 being used to deliver the formulation of the invention.

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

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

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

[0153] In certain embodiments, the reusable pen or autoinjector delivery device allows for injection of any of the liquid formulations described herein with an extrusion force of 1N to 10N. In certain embodiments, the reusable pen or autoinjector delivery device allows for injection of any of the liquid formulations described herein with an extrusion force of 1N, 2N, 3N, 4N, 5N, 6N, 7N, 8N, 9N, or 10N. In certain embodiments, the reusable pen or autoinjector delivery device allows for injection of any of the liquid formulations described herein with an extrusion force of about 1N, 2N, 3N, 4N, 5N, 6N, 7N, 8N, 9N, or 10N. In certain embodiments, the reusable pen or autoinjector delivery device allows for injection of any of the liquid formulations described herein with an extrusion force of about 6N. [Example]

[0154] Example Having generally described the present disclosure, it will be more readily understood by reference to the following examples, which are included solely for the purpose of illustrating particular embodiments of the disclosure and are not intended to limit the disclosure. [Table 6]

[0155] Example 1 ActRIIA-Fc fusion protein Soluble ActRIIA fusion proteins were constructed by fusing the extracellular domain of human ActRIIA to the human or mouse Fc domain with a minimal linker between them, and the constructs were designated ActRIIA-hFc and ActRIIA-mFc, respectively.

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

[0157] ActRIIA-hFc and ActRIIA-mFc proteins were expressed in a CHO cell line. Three different leader sequences were investigated: (i) Honeybee melittin (HBML): MKFLVNVALVFMVVYISYIYA (SEQ ID NO: 33) (ii) Tissue plasminogen activator (TPA): MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO: 34) (iii) Native: MGAAAKLAFAVFLISCSSGA (SEQ ID NO: 35).

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

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

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

[0161] ActRIIA-hFc and ActRIIA-mFc showed high affinity for their ligands. GDF11 or activin A were coupled to Biacore using standard amine coupling methods. TM ActRIIA-hFc and ActRIIA-mFc proteins were immobilized on a CM5 chip. The ActRIIA-hFc and ActRIIA-mFc proteins were loaded onto the system and binding was measured. ActRIIA-hFc was 5×10 -12 Dissociation constant (K D ) binds to activin and 9.96 × 10 -9 K D ActRIIA-hFc bound to GDF11 at a binding affinity of 1000 kJ / s. See Figures 3A and 3B. Using similar binding assays, ActRIIA-hFc was confirmed to have high to moderate affinity for other TGF-β superfamily ligands, including activin B, GDF8, BMP6, and BMP10. ActRIIA-mFc also exhibited similar behavior.

[0162] ActRIIA-hFc was very stable in pharmacokinetic studies. Rats were administered 1 mg / kg, 3 mg / kg, or 10 mg / kg of ActRIIA-hFc protein, and plasma concentrations of the protein were measured at 24, 48, 72, 144, and 168 hours. In another study, rats were administered 1 mg / kg, 10 mg / kg, or 30 mg / kg. In rats, the serum half-life of ActRIIA-hFc was 11–14 days, and circulating levels of the drug after 2 weeks were quite high (11 μg / mL, 110 μg / mL, or 304 μg / mL after initial administration of 1 mg / kg, 10 mg / kg, or 30 mg / kg, respectively). In cynomolgus monkeys, the plasma half-life was substantially greater than 14 days, and circulating levels of the drug were 25 μg / mL, 304 μg / mL, or 1440 μg / mL following initial doses of 1 mg / kg, 10 mg / kg, or 30 mg / kg, respectively.

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

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

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

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

[0167] Concentrate feasibility study A concentration stability study was conducted to evaluate the short-term biophysical stability of sotatercept (SEQ ID NO: 32) at various concentrations. Formulations containing 100 mg / mL and 50 mg / mL sotatercept at pH 5.8 were prepared in 10 mM citrate buffer and 8% (wt / vol) sucrose. Table 1 shows the stability of the formulation containing 100 mg / mL sotatercept. Formulations containing 100 mg / mL sotatercept were staged at various temperatures (5°C, 25°C, and 40°C) and analyzed at selected time points. The formulation in citrate buffer and sucrose showed minimal changes in polymer formation (measured by UPSEC) and charge distribution (measured by cIEF) at 5°C and 25°C.

[0168] [Table 10]

[0169] Tables 2 and 3 show the stability of formulations containing 50 mg / mL sotatercept. Formulations containing 50 mg / mL sotatercept were staged at various temperatures (5°C, 25°C, and 40°C) and analyzed at selected time points, demonstrating that formulations in citrate buffer, sucrose, and PS80, with or without DTPA, showed minimal changes in subvisible particle formation (measured by MFI), high molecular weight formation (measured by UPSEC), charge distribution (measured by cIEF), and fragmentation (measured by CE-SDS) at 5°C and 25°C.

[0170] [Table 11] [Table 12]

[0171] pH and buffer feasibility studies The behavior of sotatercept (SEQ ID NO: 32) was analyzed in six buffers at various pHs and 8% sucrose. The buffers tested were: histidine at pH 6.5 and 7; phosphate at pH 5, 5.5, 6, 6.5, and 7; citrate at pH 4.5, 5, 5.5, 6, 6.5, and 7; acetate at pH 4.5, 5, 5.5, and 6; succinate at pH 4.5 and 5; and glutamate at pH 4.5 and 5. The results are shown in Tables 4-13. Formulations were subjected to heat stress at 40°C for up to two months and tested for biophysical stability (turbidity), subvisible particle formation (measured by MFI), high molecular weight formation (measured by UPSEC), fragmentation (measured by R-CESDS and NR-CESDS), and charge distribution (measured by cIEF).

[0172] [Table 13] [Table 14]

[0173] [Table 15] [Table 16]

[0174] [Table 17] [Table 18]

[0175] [Table 19] [Table 20]

[0176] [Table 21] [Table 22]

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

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

[0179] [Table 25] [Table 26]

[0180] [Table 27] [Table 28]

[0181] [Table 29] [Table 30]

[0182] [Table 31] [Table 32]

[0183] [Table 33] [Table 34]

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

[0185] [Table 38] [Table 39]

[0186] Based on the data presented in Tables 14-30, the buffers tested showed comparable stability across the buffer strength range at pH 5.8 and pH 8.0. However, at lower pH, stability decreased significantly for all buffers tested. All buffers performed comparable, with citrate and succinate performing slightly better than the other buffers.

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

[0188] Colloidal stability and surfactant screening Colloidal stability studies on sotatercept (SEQ ID NO: 32) were conducted by agitation stress vials containing sotatercept in 10 mM citrate buffer and 8% sucrose in the presence of various levels of PS80, as well as by freeze-thaw cycling (one cycle: vial frozen at -80°C and thawed at room temperature). The results are shown in Figures 10-13. Figure 10 shows the percentage of high molecular weight species (% HMW) detected in vials containing sotatercept in 10 mM citrate buffer and 8% sucrose in the presence of 0%, 0.005%, 0.01%, 0.02%, 0.03%, and 0.05% PS80 after 3 and 7 days of agitation. Figure 11 shows the % HMW detected in vials containing sotatercept in 10 mM citrate buffer and 8% sucrose in the presence of 0%, 0.005%, 0.01%, 0.02%, 0.03%, and 0.05% PS80 after three, five, and eight freeze-thaw cycles. Figures 12 and 13 show the % monomer (Figure 12) and % HMW (Figure 13) detected in vials containing sotatercept in 10 mM citrate buffer and 8% sucrose in the presence of 0%, 0.01%, 0.02%, and 0.03% PS80 after three, five, and eight freeze-thaw cycles. No changes in % HMW or subvisible particle formation were observed after 7 days of agitation or eight freeze-thaw cycles. Furthermore, a direct comparison of polysorbate 80 and poloxamer 188 was also performed (1-Mo / 25°C). The results showed that the molecules were stable to agitation stress and there was no difference between PS80 and P188, both of which behaved similarly. All analytical attributes were comparable for the prototype stability after 1-Mo stress at 25°C.

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

[0190] [Table 42] [Table 43]

[0191] [Table 44] [Table 45]

[0192] The results above show that the surfactants are stable to agitation stress in citrate buffer and there were no significant differences between the surfactants tested when comparing charge species, monomer species, aggregates, or fragmentation.

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

[0194] [Table 48] [Table 49]

[0195] [Table 50] [Table 51]

[0196] These results indicate that sotatercept is stable to agitation stress when formulated with the surfactants tested in a histidine buffer. At higher SDS concentrations, slightly higher molecular weight species were detected. There were no significant differences in charge species after agitation stress among the surfactants tested.

[0197] Chelating Agent Research Formulations containing sotatercept (SEQ ID NO: 32) in 10 mM citrate buffer, 8% sucrose, and 0.02% polysorbate 80 at pH 5.8 in the presence of various levels (0 mM, 10 mM, 20 mM, and 30 mM) of L-methionine were exposed to light stress in a photostability chamber in the presence of various levels (0 μM, 7.5 μM, 15 μM, 30 μM, and 60 μM) of DTPA or EDTA as chelators. The stressed samples, along with dark controls, were analyzed for high molecular weight formation (using UPSEC) and showed that the addition of L-methionine reduced % HMW formation and that there was no adverse effect from the addition of the chelator. The results are shown in Figures 14 and 15. Therefore, either chelator can be used in the formulation.

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

[0199] [Table 52] [Table 53]

[0200] [Table 54] [Table 55]

[0201] The results showed that low concentrations of dimercaprol as an antioxidant could provide comparable protection to the control samples under light stress in both citrate and histidine buffers. Higher concentrations of dimercaprol resulted in increased charge species and greater loss of monomer content upon light stress, indicating that formulations containing high concentrations of dimercaprol as an antioxidant are not suitable.

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

[0203] Table 56 Table 57

[0204] Table 58 Table 59

[0205] Table 60 Table 61

[0206] Table 62

[0207] Table 63 Table 64

[0208] Table 65

[0209] Table 66

[0210] These results demonstrate that the inclusion of the tested stabilizers in the formulations resulted in similar levels of stability for the sotatercept molecule in citrate buffer under high heat stress. Each test formulation maintained a high monomer content, showed a similar increase in high molecular weight species over time, and similar levels of charged species compared to samples without stabilizers. [Table 67] [Table 68]

[0211] [Table 69] [Table 70]

[0212] [Table 71] [Table 72]

[0213] [Table 73] [Table 74]

[0214] [Table 75] [Table 76]

[0215] [Table 77]

[0216] These results demonstrate that the inclusion of the tested stabilizers in the formulations resulted in similar levels of stability for the sotatercept molecule in histidine buffer under high heat stress. Each tested formulation maintained a high monomer content, showed a similar increase in high molecular weight species over time, and had similar levels of charged species.

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

[0218] [Table 80]

[0219] array [Table 81] TIFF2026500069000084.tif243163TIFF2026500069000085.tif83162

[0220] Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the presently disclosed methods and formulations. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

Claims

1. A pharmaceutical formulation comprising 50 to 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. 10. The pharmaceutical formulation of claim 1, wherein the fusion protein has a concentration of 100 mg / mL.

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

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

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

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

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

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

9. 4. The pharmaceutical formulation of claim 1, wherein the buffer is a glutamate buffer that maintains the pH of the formulation at 5-7.

10. 10. The pharmaceutical formulation of any one of claims 1 to 9, wherein the buffering agent maintains the pH of the formulation at 5.

8.

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

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

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

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

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

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

17. 17. The pharmaceutical formulation of any one of claims 1 to 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. 18. The pharmaceutical formulation of claim 17, wherein the surfactant is polysorbate 80.

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

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

21. 21. The pharmaceutical formulation of any one of claims 1 to 20, wherein the surfactant is present in a concentration of 0.05 to 0.5 mg / mL.

22. 21. The pharmaceutical formulation of any one of claims 1 to 20, wherein the surfactant is present in a concentration of 0.1 to 0.5 mg / mL.

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

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

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

26. 26. The pharmaceutical formulation of claim 25, wherein the chelating agent is present at a concentration of 7.5 to 100 μM.

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

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

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

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

31. 30. The pharmaceutical formulation of any one of claims 28 to 29, wherein the antioxidant is present in a concentration of 5 to 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 citrate buffer, 2-16% (weight / volume) sucrose, 0.05-0.5 mg / mL of polysorbate 80, polysorbate 20 or poloxamer 188, 0-50 mM L-methionine and 0-100 μM DTPA or EDTA.

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

34. 34. The pharmaceutical formulation of any one of claims 1 to 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 to 34, wherein the pharmaceutical formulation is administered via an autoinjector.

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

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

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

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

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

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

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

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