Formulations containing ActRIIa polypeptide variants

Abstract

In certain aspects, the present disclosure provides a lyophilized 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, and one or more pharmaceutical additives and / or excipients.

Description

[Technical Field]

[0001] Cross-reference to related applications This application claims the benefit of U.S. Provisional Application No. 63 / 451,198, filed March 9, 2023, the contents of which are incorporated herein by reference in their entirety.

[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 January 29, 2024, has the file name 1848179-0002-169-WO1_SL.XML, and is 37,058 in size.

[0003] Described herein is a lyophilized 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, and one or more pharmaceutical additives and / or excipients as defined herein. [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 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 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, wherein the formulation is lyophilized.

[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. In some embodiments, the ActRIIa protein is a fusion protein comprising an ActRIIa extracellular domain and one or more protein domains that are heterologous to ActRIIa. In some embodiments, the ActRIIa protein is a fusion protein comprising an 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 (eg, an Fc immunoglobulin domain).In some embodiments, the linker domain is selected from the group consisting of TGGG (SEQ ID NO: 23), TGGGG (SEQ ID NO: 21), SGGGG (SEQ ID NO: 22), GGGGS (SEQ ID NO: 25), GGG (SEQ ID NO: 19), GGGG (SEQ ID NO: 20), and SGGG (SEQ ID NO: 24). In some embodiments, the ActRIIa fusion protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 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 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 obtained by expression in Chinese hamster ovary cells.

[0011] The pharmaceutical formulations described herein comprise an ActRIIa fusion protein and one or more pharmaceutical additives and / or excipients. In certain embodiments, the pharmaceutical formulations described herein comprise a human ActRIIa fusion protein or a variant thereof, a buffer, a surfactant, and a stabilizer.

[0012] In some embodiments, one or more of the pharmaceutical additives and / or excipients is a buffering agent. In some embodiments, the buffering agent is selected to be physiologically compatible and to maintain a pH of greater than or equal to 2. In some embodiments, the buffering agent is selected to be physiologically compatible and to be able to maintain a pH of greater than or equal to 2 of the reconstituted lyophilized solutions described herein. In some embodiments, the buffering agent is selected to be physiologically compatible and to maintain the pH of the pharmaceutical formulation at 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 the pH of the pharmaceutical formulation between 5 and 7 (i.e., 5.0 and 7.0). In some embodiments, the buffering agent is selected to be physiologically compatible and to maintain the pH of the pharmaceutical formulation at 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 the pH of the pharmaceutical formulation at a pH of 5.5 to 6.5 (i.e., 5.5 to 6.5). In some embodiments, the buffering agent is selected to be physiologically compatible and to maintain the pH of the pharmaceutical formulation at 5.8. In some embodiments, the buffer comprises an organic acid, succinate, phosphate, acetate, citrate, citric acid, Tris, HEPES, an amino acid, or a mixture of amino acids. In certain embodiments, the buffer is selected from the group consisting of sodium citrate, succinate, and histidine. In some embodiments, the buffer comprises citrate. In certain embodiments, when the buffer is citrate, the citrate buffer comprises trisodium citrate dihydrate and citric acid monohydrate. In some embodiments, the buffer comprises trisodium citrate dihydrate. In some embodiments, the buffer comprises citric acid monohydrate. 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.

[0013] In some embodiments, the buffering agent is present in an amount of 4 mM to 50 mM. 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 pharmaceutical formulation comprises at least 10 mM buffering agent.

[0014] In some embodiments, the buffer is a citrate buffer that maintains the pH of the pharmaceutical formulation at about pH 5.5 to about pH 6.5. In some embodiments, the buffer is a succinate buffer that maintains the pH of the pharmaceutical formulation at about pH 5.5 to about pH 6.5. In some embodiments, the buffer is a histidine buffer that maintains the pH of the pharmaceutical formulation at about pH 5.5 to about pH 6.0. In some embodiments, the buffer is a citrate buffer that maintains the pH of the pharmaceutical formulation at about pH 5.8. In some embodiments, the buffer is a succinate buffer that maintains the pH of the pharmaceutical formulation at about pH 5.8.

[0015] 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.

[0016] In some embodiments, one or more of the pharmaceutical additives and / or excipients is a surfactant, which may be 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 sodium glycodeoxycholic acid 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 40, polysorbate 60, polysorbate 65, polysorbate 80, and soy lecithin. In some embodiments, the surfactant is polysorbate 80 or polysorbate 20. In some embodiments, the surfactant is polysorbate 80.

[0017] In some embodiments, the surfactant is present in an amount of 0.05-0.3 mg / mL. In some embodiments, the pharmaceutical formulation comprises 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) of the surfactant. In some embodiments, the pharmaceutical formulation comprises 0.01-0.05% (weight / volume) of the surfactant. In some embodiments, the pharmaceutical formulation comprises at least 0.02% (weight / volume) of the surfactant. In some embodiments, the pharmaceutical formulation comprises 0.02% (weight / volume) of the surfactant. In some embodiments, the pharmaceutical formulation comprises 0.02% polysorbate 80 or polysorbate 20.

[0018] In some embodiments, one or more of the pharmaceutical additives and / or excipients is a stabilizer. In some embodiments, the stabilizer is selected from the group consisting of sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, glucosamine, fructose, mannitol, sorbitol, polyhydroxy compounds, polysaccharides, dextran, starch, hydroxyethyl starch, cyclodextrin, N-methylpyrrolidone, cellulose, and hyaluronic acid. In some embodiments, the stabilizer is sucrose.

[0019] In some embodiments, the stabilizer is present in the pharmaceutical formulation at a concentration of 2 to 16% (w / v). In some embodiments, the pharmaceutical formulation comprises about 8% to about 10% (w / v) of the stabilizer. In some embodiments, the pharmaceutical formulation comprises at least 0.005% (w / v), 0.01% (w / v), 0.02% (w / v), 0.03% (w / v), 0.05% (w / v), 0.06% (w / v), 0.07% (w / v), 0.08% (w / v), 0.09% (w / v), 0.1% (w / v), 0.5% (w / v), 0.7% (w / v), 0.8% (w / v), 0.9% (w / v), 1.0% (w / v), 1.2% (w / v), 1.5% (w / v), or 2.0% (w / v). (wt / vol), 1.7% (wt / vol), 2% (wt / vol), 3% (wt / vol), 4% (wt / vol), 5% (wt / vol), 6% (wt / vol), 7% (wt / vol), 8% (wt / vol), 9% (wt / vol), 10% (wt / vol), 11% (wt / vol), 12% (wt / vol), 13% (wt / vol), 14% (wt / vol), 15% (wt / vol), 16% (wt / vol), 17% (wt / vol), 18% (wt / vol), 19% (wt / vol), or 20% (wt / vol) of stabilizer. In some embodiments, the stabilizer is present in a concentration of at least 8% (wt / vol).

[0020] In certain embodiments, the pharmaceutical formulations described herein do not include salts.

[0021] Also described herein are lyophilized pharmaceutical formulations prepared by lyophilizing the liquid formulations described herein. Also described herein are pharmaceutical formulations, wherein the formulations are reconstituted from the lyophilized formulations.

[0022] In some embodiments, the lyophilized pharmaceutical formulation is reconstituted with sterile water for injection. In some embodiments, the lyophilized pharmaceutical formulation is reconstituted with sterile water for injection to a final protein concentration of 10 to 100 mg / mL. In some embodiments, the lyophilized pharmaceutical formulation is reconstituted with sterile water for injection to a final protein concentration of 45 to 55 mg / mL. In some embodiments, the lyophilized pharmaceutical formulation is reconstituted with sterile water for injection to a final protein concentration of about 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 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, 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. In certain embodiments, the human ActRIIa fusion protein is present in an amount of about 75 mg / mL or less. In certain embodiments, the human ActRIIa fusion protein is present in an amount of about 50 mg / mL.

[0023] In some embodiments, the protein is provided in a lyophilized pharmaceutical formulation in a vial, hi some embodiments, the lyophilized formulation comprises an ActRIIa fusion protein as defined herein in an amount of 45 mg / vial or 60 mg / vial.

[0024] In some embodiments, the lyophilized pharmaceutical formulation is reconstituted with sterile water for injection. In some embodiments, the lyophilized pharmaceutical formulation is reconstituted with sterile water for injection to a final protein 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. In some embodiments, the lyophilized pharmaceutical formulation is reconstituted with sterile water for injection to a final protein concentration of about 50 mg / mL.

[0025] In certain embodiments, the lyophilized formulation is reconstituted with about 0.5 mL to 1.8 mL of sterile water for injection. In some embodiments, the lyophilized pharmaceutical formulation is reconstituted with about 0.5 mL, 0.56 mL, 0.58 mL, 0.6 mL, 0.62 mL, 0.64 mL, 0.66 mL, 0.68 mL, 0.70 mL, 0.72 mL, 0.74 mL, 0.76 mL, 0.78 mL, 0.8 mL, 0.82 mL, 0.84 mL, 0.86 mL, 0.88 mL, 0.9 mL, 0.92 mL, 0.94 mL, 0.96 mL, 0.98 mL, 1 mL, 1.1 mL, 1.15 mL, 1.2 mL, 1.25 mL, 1.3 mL, 1.35 mL, 1.4 mL, 1.45 mL, 1.5 mL, 1.55 mL, 1.6 mL, 1.65 mL, 1.7 mL, 1.75 mL, or 1.8 mL of sterile water for injection. In some embodiments, the lyophilized pharmaceutical formulation comprises an ActRIIa fusion protein provided in an amount of 45 mg / vial, and the formulation is reconstituted with 0.9 mL, 0.95 mL, 1 mL, or 1.1 mL of sterile water for injection. In some embodiments, the lyophilized pharmaceutical formulation comprises an ActRIIa fusion protein provided in an amount of 45 mg / vial, and the formulation is reconstituted with 1.1 mL of sterile water for injection. In some embodiments, the lyophilized pharmaceutical formulation comprises an ActRIIa fusion protein provided in an amount of 60 mg / vial, and the formulation is reconstituted with 0.90 mL, 0.95 mL, 1.00 mL, 1.05 mL, 1.10 mL, 1.15 mL, 1.20 mL, 1.25 mL, 1.30 mL, 1.35 mL, 1.40 mL, 1.45 mL, or 1.50 mL of sterile water for injection. In some embodiments, the lyophilized pharmaceutical formulation comprises an ActRIIa fusion protein provided in an amount of 60 mg / vial, and the formulation is reconstituted with 1.30 mL of sterile water for injection.

[0026] In certain embodiments, the lyophilized formulation is provided in an amount of 45 mg / vial, wherein the formulation is reconstituted with 0.65 mL to 0.75 mL of sterile water for injection. In certain embodiments, the lyophilized formulation is provided in an amount of 60 mg / vial, wherein the formulation is reconstituted with 0.65 mL to 1.75 mL of sterile water for injection.

[0027] In certain embodiments, the lyophilized pharmaceutical formulation comprises a human ActRIIa fusion protein of SEQ ID NO: 32 or a variant of SEQ ID NO: 32 lacking the C-terminal lysine (SEQ ID NO: 41), a buffer, a surfactant, and a stabilizer, wherein the buffer is selected to be physiologically compatible and to maintain a pH of 5.8 upon reconstitution with sterile water for injection. In certain embodiments, the buffer comprises citrate. In certain embodiments, the stabilizer is sucrose. In certain embodiments, the surfactant is polysorbate 20 or polysorbate 80. In certain embodiments, the surfactant is polysorbate 80. In certain embodiments, the lyophilized pharmaceutical formulation comprises sotatercept, a buffer, a surfactant, and a stabilizer, wherein the buffer is selected to be physiologically compatible and to maintain a pH of 5.8 upon reconstitution with sterile water for injection.

[0028] In certain embodiments, the lyophilized pharmaceutical formulation comprises a human ActRIIa fusion protein comprising the amino acid sequence of SEQ ID NO: 32 or comprising the amino acid sequence of SEQ ID NO: 41, citrate, polysorbate 80, and sucrose. 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 lyophilized pharmaceutical formulation comprises sotatercept.

[0029] In certain embodiments, the lyophilized pharmaceutical formulation comprises 55.0 mg of human ActRIIa fusion protein comprising the amino acid sequence of SEQ ID NO: 32 or a variant of SEQ ID NO: 32 lacking the C-terminal lysine residue (e.g., comprising the amino acid sequence of SEQ ID NO: 41), 0.48 mg of citric acid monohydrate, 2.56 mg of trisodium citrate dihydrate, 0.22 mg of polysorbate 80, and 88.0 mg of sucrose. 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 lyophilized pharmaceutical formulation comprises sotatercept.

[0030] In certain embodiments, the lyophilized pharmaceutical formulation comprises 72.5 mg of a human ActRIIa fusion protein comprising SEQ ID NO: 32 or a variant of SEQ ID NO: 32 lacking the C-terminal lysine residue (SEQ ID NO: 41), 0.64 mg of citric acid monohydrate, 3.37 mg of trisodium citrate dihydrate, 0.29 mg of polysorbate 80, and 116.0 mg of sucrose. 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 lyophilized pharmaceutical formulation comprises sotatercept.

[0031] In certain embodiments, the reconstituted pharmaceutical formulation comprises 50 mg / mL of human ActRIIa protein comprising the amino acid sequence of SEQ ID NO: 32 or human ActRIIa protein comprising the amino acid sequence of SEQ ID NO: 41, 10 mM citrate, 0.2 mg / mL polysorbate 80, and 80 mg / mL sucrose (pH 5.8). In some embodiments, the human ActRIIa fusion protein consists of the amino acid sequence of SEQ ID NO: 41. In some embodiments, the reconstituted 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 reconstituted pharmaceutical formulation comprises sotatercept.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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. 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.

[0036] In some embodiments, the pharmaceutical formulation is administered parenterally (e.g., by intravenous infusion). In some embodiments, the pharmaceutical formulation is administered by subcutaneous injection. In certain embodiments, the formulation is contained in a glass vial or injection device.

[0037] Also described herein are methods for treating pulmonary arterial hypertension (PAH), comprising administering to a patient in need thereof a pharmaceutical formulation described herein.

[0038] Also described herein is the use of the pharmaceutical formulations for treating pulmonary arterial hypertension (PAH), wherein the use comprises administering to a patient in need of such treatment the pharmaceutical formulations described herein.

[0039] In some embodiments, the present disclosure provides a lyophilized pharmaceutical formulation for treating pulmonary arterial hypertension in a subject in need thereof, wherein the lyophilized pharmaceutical formulation comprises a human ActRIIa fusion protein of SEQ ID NO: 32, citrate, polysorbate 80, and sucrose, wherein the formulation is lyophilized. In some embodiments, the vial contains a lyophilized pharmaceutical formulation comprising a human ActRIIa fusion protein of SEQ ID NO: 32, citric acid monohydrate, trisodium citrate dihydrate, polysorbate 80, and sucrose, wherein the formulation is lyophilized. In some embodiments, the vial contains a lyophilized pharmaceutical formulation comprising 55 mg of protein (i.e., the ActRIIa fusion protein), 0.48 mg of citric acid monohydrate, 2.56 mg of trisodium citrate dihydrate, 0.22 mg of polysorbate 80, and 88.0 mg of sucrose, wherein the formulation is lyophilized. In some embodiments, the vial contains a lyophilized pharmaceutical formulation comprising 72.5 mg of ActRIIa fusion protein, 0.64 mg of citric acid monohydrate, 3.37 mg of trisodium citrate dihydrate, 0.29 mg of polysorbate 80, and 116.0 mg of sucrose, wherein the formulation is lyophilized. In some embodiments, the human ActRIIa fusion protein is a variant of SEQ ID NO: 32 lacking the C-terminal lysine. In some embodiments, the human ActRIIa fusion protein comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, the human ActRIIa fusion protein is sotatercept.

[0040] In some embodiments, once reconstituted with sterile water, the vial contains 50 mg / mL of ActRIIa fusion protein comprising the amino acid sequence of SEQ ID NO: 32, 10 mM citrate, 0.2 mg / mL polysorbate 80, and 8% by weight of sucrose. In some embodiments, the human ActRIIa fusion protein comprises or consists of the amino acid sequence of SEQ ID NO: 41, 10 mM citrate, 0.2 mg / mL polysorbate 80, and 8% by weight of sucrose. In some embodiments, once reconstituted with sterile water, the vial contains 50 mg / mL of sotatercept, 10 mM citrate, 0.2 mg / mL polysorbate 80, and 8% by weight of sucrose.

[0041] In some embodiments, the reconstituted pharmaceutical formulation comprises 50 mg / mL of ActRIIa fusion protein comprising the amino acid sequence of SEQ ID NO: 32, 10 mM citrate, 0.02 mg / mL of polysorbate 80, and 80 mg / mL of sucrose at pH 5.8. In some embodiments, the human ActRIIa protein comprises the amino acid sequence of SEQ ID NO: 41, 10 mM citrate, 0.02 mg / mL of polysorbate 80, and 80 mg / mL of sucrose at pH 5.8. In some embodiments, the reconstituted pharmaceutical formulation comprises 50 mg / mL of sotatercept, 10 mM citrate, 0.02 mg / mL of polysorbate 80, and 80 mg / mL of sucrose at pH 5.8. [Brief explanation of the drawings]

[0042] [Figure 1]FIG. 1 shows the components of the kit, including a lyophilized pharmaceutical formulation containing an ActRIIa fusion protein and an injection device. Vial (1) contains the lyophilized pharmaceutical formulation containing the ActRIIa fusion protein, a reconstituted sterile injectable solution, or a sterile injectable solution. Prefilled syringe (2) containing the reconstituted solution is used to reconstitute the lyophilized pharmaceutical formulation containing the ActRIIa fusion protein from (1) into a sterile injectable solution. Vial adapter (3) connects vial (1) to prefilled syringe (2) by attaching one end to the vial and the other end to the prefilled syringe. Syringe (4) and injection needle (5) are provided for administering the sterile injectable solution. Swab wipes (6) are provided for sterilizing the individual components of the kit. [Figure 2] FIG. 2 shows a multiple sequence alignment of ActRIIa proteins from various vertebrates with human ActRIIa (SEQ ID NOs: 62-68). [Figure 3A] Figures 3A and 3B 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). [Figure 3B] Figures 3A and 3B 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). [Figure 4A] Figures 4A and 4B show the binding of ActRIIa-hFc to activin (top panel) and GDF-11 (bottom panel b) as measured by Biacore™ assay. [Figure 4B] Figures 4A and 4B show the binding of ActRIIa-hFc to activin (top panel) and GDF-11 (bottom panel b) as measured by Biacore™ assay. [Figure 5] Figure 5 shows the effect of pH and buffer on the percentage of monomeric species by HP-SEC after 6 M at 25°C. [Figure 6] Figure 6 shows the effect of pH and buffer on Biacore™ binding activity after stabilization at 6M and 25°C. [Figure 7] FIG. 7 shows the effect of buffer and pH on % HMW, monomeric, and % LMW species by HP-SEC after 3 months based on stability. [Figure 8] Figure 8 shows the effect of pH and buffer on % minor peaks and % major peaks by CE-SDS NR after 1 month stability. [Figure 9] FIG. 9 shows the effect of pH and buffer on % acidic species, % basic species, and % total major species by desialylated icIEF after 3 months of stability. [Figure 10] FIG. 10 shows the effect of concentration on % monomer species by HP-SEC. [Figure 11] FIG. 11 shows the effect of buffer and concentration on % monomer species by HP-SEC based on stability. [Figure 12] FIG. 12 shows the effect of protein and sucrose concentration on purity by HP-SEC based on stability. DETAILED DESCRIPTION OF THE INVENTION

[0043] 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.

[0044] "About" and "approximately" generally refer to an acceptable degree of error for the measured quantity given the nature or precision of the measurement. Typically, exemplary degrees of error are within 20%, preferably within 10%, and more preferably within 5% of a given value or range of values.

[0045] 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.

[0046] 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.

[0047] Numerical ranges disclosed herein are inclusive of the numbers defining the range, for example, a pH of 5.0 to 7.0 includes pH 5.0 and pH 7.0 and values ​​between 5.0 and 7.0.

[0048] 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.

[0049] Pharmaceutical preparations Provided herein is a pharmaceutical formulation comprising a recombinant fusion protein comprising the extracellular domain (ECD) of a 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, wherein the pharmaceutical formulation is lyophilized. In a particular aspect, the present disclosure relates to a pharmaceutical formulation comprising the extracellular domain (ECD) of a human ActRIIa protein or a derivative thereof linked to a constant domain of an immunoglobulin, such as a human IgG1 Fc domain, wherein the pharmaceutical formulation is reconstituted from a lyophilized formulation into a sterile solution for injection.

[0050] In a specific aspect, the present disclosure relates to a lyophilized pharmaceutical formulation comprising the extracellular domain (ECD) of the human ActRIIa protein or a derivative thereof linked to an immunoglobulin constant domain, such as the human IgG1 Fc domain, for reconstitution into a sterile solution for injection. During lyophilization, the liquid formulation is converted from an aqueous phase to an amorphous solid phase, which is believed to protect the protein from chemical and / or conformational instability. Lyophilization is performed using techniques common in the art, and the lyophilized formulation is optimized for stability, shelf life, and to reduce the level of high molecular weight (HMW) species and aggregates. Tang et al., Pharm Res. 21:191-200, (2004) and Chang et al., Pharm Res. 13:243-9 (1996)). The pharmaceutical formulation helped stabilize the protein against the stresses of manufacturing, transportation, and storage. The excipients and additives used in the lyophilized formulation are essential components of the formulation and therefore must be safe and well tolerated by patients. For protein pharmaceuticals, the choice of excipients and additives is particularly important because it can affect both the efficacy and immunogenicity of the pharmaceutical. Excipients and additives are also useful in reducing the viscosity of these formulations, enabling the delivery of highly concentrated protein formulations and enhancing patient convenience. The formulation excipients and additives disclosed herein provide stability against these stresses. Common excipients are known in the art and can be found in Powell et al., Compendium of Excipients for Parenteral Formulations (1998), PDA J. Pharm. Sci. Technology, 52:238-311.

[0051] In another aspect, provided herein is a pharmaceutical formulation comprising an ActRIIa fusion protein. In some embodiments, the formulation is lyophilized. In further embodiments, the formulation is reconstituted from a lyophilized formulation. In certain embodiments, the present disclosure provides a pharmaceutical formulation comprising an ActRIIa protein, wherein the pharmaceutical formulation is in lyophilized form in a vial. In certain embodiments, the pharmaceutical formulation comprises 15 mg to 100 mg of an ActRIIa fusion protein. In certain embodiments, the pharmaceutical formulation comprises 20 mg to 80 mg of an ActRIIa fusion protein. In certain embodiments, the pharmaceutical formulation comprises 40 mg to 70 mg of an ActRIIa fusion protein. In certain embodiments, the pharmaceutical formulation comprises about 15 mg, about 17.5 mg, about 20 mg, about 22.5 mg, about 25 mg, about 27.5 mg, about 30 mg, about 32.5 mg, about 35 mg, about 37.5 mg, about 40 mg, about 42.5 mg, about 45 mg, about 47.5 mg, about 50 mg, about 52.5 mg, about 55 mg, about 57.5 mg, about 60 mg, about 62.5 mg, about 65 mg, about 67.5 mg, about 70 mg, about 72.5 mg, about 75 mg, about 77.5 mg, about 80 mg, about 82.5 mg, about 85 mg, about 90 mg, about 92.5 mg, about 95 mg, about 97.5 mg or about 100 mg of the ActRIIa fusion protein.

[0052] In certain embodiments, the lyophilized pharmaceutical formulation comprising an ActRIIa fusion protein is provided as a lyophilized powder or cake in a vial. In certain embodiments, the lyophilized pharmaceutical formulation comprises an amount of ActRIIa fusion protein of 45 mg / vial or 60 mg / vial. In more specific embodiments, the 45 mg / vial or the 60 mg / vial are each reconstituted with sterile water for injection, each containing a final concentration of 45 to 55 mg / mL of reconstituted ActRIIa fusion protein. In even more specific embodiments, the 45 mg / vial or the 60 mg / vial are each reconstituted with sterile water for injection, each containing a final concentration of 50 mg / mL of reconstituted ActRIIa fusion protein (active pharmaceutical ingredient).

[0053] In certain embodiments, the lyophilized pharmaceutical formulation comprises an ActRIIa fusion protein in an amount of 45 mg / vial or 60 mg / vial and is reconstituted with sterile water for injection. In more specific embodiments, the lyophilized pharmaceutical formulation provided in each of the 45 mg / vial or 60 mg / vial amounts is reconstituted with sterile water for injection to a final 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 reconstituted ActRIIa fusion protein. In more specific embodiments, the lyophilized pharmaceutical formulation provided in each of the 45 mg / vial or the 60 mg / vial is reconstituted with sterile water for injection to a final 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 reconstituted ActRIIa fusion protein. In some embodiments, the lyophilized pharmaceutical formulation provided in each of the 45 mg / vial or the 60 mg / vial is reconstituted with sterile water for injection to a final concentration of about 50 mg / mL of reconstituted ActRIIa fusion protein. In some embodiments, the lyophilized pharmaceutical formulation provided in each of the 45 mg / vial or the 60 mg / vial is reconstituted with sterile water for injection to a final concentration of 50 mg / mL of reconstituted ActRIIa fusion protein.

[0054] In some embodiments, the 45 mg / vial or 60 mg / vial lyophilized pharmaceutical formulation is reconstituted with 0.5 to 2 mL of sterile water for injection. In some embodiments, the 45 mg / vial or 60 mg / vial lyophilized pharmaceutical formulation is reconstituted with approximately 0.5 mL, 0.56 mL, 0.58 mL, 0.6 mL, 0.62 mL, 0.64 mL, 0.66 mL, 0.68 mL, 0.70 mL, 0.72 mL, 0.74 mL, 0.76 mL, 0.78 mL, 0.8 mL, 0.82 mL, 0.84 mL, 0.86 mL, 0.88 mL, 0.89 mL, 0.90 mL, 0.91 mL, 0.92 mL, 0.94 mL, 0.96 mL, 0.98 mL, 0.99 mL, 10 ... Reconstituted with 0.88mL, 0.9mL, 0.92mL, 0.94mL, 0.96mL, 0.98mL, 1mL, 1.1mL, 1.15mL, 1.2mL, 1.25mL, 1.3mL, 1.35mL, 1.4mL, 1.45mL, 1.5mL, 1.55mL, 1.6mL, 1.65mL, 1.7mL, 1.75mL or 1.8mL of sterile water for injection. In some embodiments, the lyophilized pharmaceutical formulation provided in each of the 45 mg / vial or the 60 mg / vial is 0.5 mL, 0.55 mL, 0.56 mL, 0.57 mL, 0.58 mL, 0.59 mL, 0.6 mL, 0.65 mL, 0.66 mL, 0.67 mL, 0.68 mL, 0.69 mL, 0.70 mL, 0.71 mL, 0.72 mL, 0.73 mL, 0.74 mL, 0.75 mL, 0.76 mL, 0.77 mL, 0.78 mL, 0.79 mL, 0.8 mL, 0.85 mL, 0.9 mL, 0.95 mL, 1 mL, 1.1 mL, 1.15 mL, 1.2 mL, 1.25 mL, 1.3 mL, 1.4 mL, 1.5 mL, 1.6 mL, 1.7 mL, 1.8 mL, 1.9 mL, 2.0 mL, 2.1 mL, 2.2 mL, 2.3 mL, 2.4 mL, 2.5 mL, 2.6 mL, 2.7 mL, 2.8 mL, 2.9 mL, 3.0 mL, 3.1 mL, 3.2 mL, 3.3 mL, 3.4 mL, 3.5 mL, 3.6 mL, 3.7 mL, 3.8 mL, 3.9 mL, 4.0 mL, 4.1 mL, 4.2 mL, 4.3 mL, 4.4 mL, 4.5 mL, 4.6 mL, 4.7 mL, 4.8 mL, 4.9 mL, 5.0 mL, 5.1 mL, 5.1 mL, 5.2 mL, 5.3 mL, 5.4 mL, 5.5 mL, 5.6 mL, Reconstituted with 1.6 mL, 1.3 mL, 1.35 mL, 1.4 mL, 1.45 mL, 1.5 mL, 1.51 mL, 1.52 mL, 1.53 mL, 1.54 mL, 1.55 mL, 1.56 mL, 1.57 mL, 1.58 mL, 1.59 mL, 1.6 mL, 1.61 mL, 1.62 mL, 1.63 mL, 1.64 mL, 1.65 mL, 1.66 mL, 1.67 mL, 1.68 mL, 1.69 mL, 1.7 mL, 1.71 mL, 1.72 mL, 1.73 mL, 1.74 mL, 1.75 mL, 1.76 mL, 1.77 mL, 1.78 mL, 1.79 mL or 1.8 mL of sterile water for injection.In some embodiments, the lyophilized pharmaceutical formulation provided at 45 mg / vial is reconstituted with 0.8 mL, 0.85 mL, 0.9 mL, 0.95 mL, 1 mL, 1.1 mL, 1.15 mL, 1.2 mL, 1.25 mL, 1.3 mL, 1.35 mL, or 1.4 mL or 0.75 mL of sterile water for injection. In some embodiments, the lyophilized pharmaceutical formulation provided at 45 mg / vial is reconstituted with 0.9 mL, 0.95 mL, 1 mL, or 1.1 mL of sterile water for injection. In some embodiments, the lyophilized pharmaceutical formulation provided at 45 mg / vial is reconstituted with 1.1 mL of sterile water for injection. In some embodiments, the lyophilized pharmaceutical formulation provided at 60 mg / vial is reconstituted with 0.8 mL, 0.85 mL, 0.9 mL, 0.95 mL, 1 mL, 1.1 mL, 1.15 mL, 1.2 mL, 1.25 mL, 1.3 mL, 1.35 mL, or 1.4 mL or 0.75 mL of sterile water for injection. In some embodiments, the lyophilized pharmaceutical formulation provided at 60 mg / vial is reconstituted with 1 mL, 1.1 mL, 1.2 mL, or 1.3 mL of sterile water for injection. In some embodiments, the lyophilized pharmaceutical formulation provided at 60 mg / vial is reconstituted with 1.3 mL of sterile water for injection.

[0055] In some embodiments, the lyophilized pharmaceutical formulations 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, and a surfactant. The buffer may be selected to maintain the pH of the formulation during processing and upon reconstitution. The stabilizer may include cryoprotectants and lyoprotectants, such as polyols, sugars, and polysaccharides. The stabilizer may be selected to protect the formulation from freeze / thaw cycle stress and stabilize the formulation in the lyophilized state. The surfactant may be selected based on its ability to function as an emulsifier, wetting agent, solubilizer, and / or dispersant.

[0056] The formulations provided herein include buffers, surfactants, and sugars, which are described in more detail below.

[0057] 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.

[0058] 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.

[0059] 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).

[0060] Buffers for lyophilized formulations require further consideration. For example, certain buffers, such as sodium phosphate, tend to crystallize from the amorphous phase of the protein during freezing, resulting in a pH shift. In certain embodiments, exemplary buffers used to buffer the pharmaceutical formulations described herein include, but are not limited to, organic acids, succinates, phosphates, acetates, citrates, Tris, HEPES, and amino acids or mixtures of amino acids, including, but not limited to, aspartates, arginine, and glycine. 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. In some embodiments, the buffer comprises citrate, succinate, or histidine.

[0061] In certain embodiments, the amount of buffering agent in the lyophilized formulation is 0.3 mg to 5 mg. In certain embodiments, the amount of buffering agent in the lyophilized formulation is 0.3 mg, 0.4 mg, 0.5 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, 3.5 mg, 4.0 mg, 4.5 mg, or 5.0 mg. In certain embodiments, the amount of buffering agent in the lyophilized formulation is about 0.5 mg, about 1.0 mg, about 1.5 mg, about 2.0 mg, about 2.5 mg, about 3.0 mg, about 3.5 mg, about 4.0 mg, about 4.5 mg, or about 5.0 mg.

[0062] In certain embodiments, the buffering agent comprises citric acid monohydrate and trisodium citrate dihydrate, wherein the amount of citric acid monohydrate is 0.1 mg to 1.0 mg. In certain embodiments, the amount of citric acid monohydrate is 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, or 1.0 mg. In certain embodiments, the amount of citric acid monohydrate is about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, or about 1.0 mg. In certain embodiments, the amount of citric acid monohydrate is 0.48 mg. In certain embodiments, the amount of citric acid monohydrate is 0.48 mg. In a particular embodiment, the amount of citric acid monohydrate is 0.64 mg.

[0063] In certain embodiments, the buffering agent comprises citric acid monohydrate and trisodium citrate dihydrate, wherein the amount of trisodium citrate dihydrate is 1.0 mg to 5.0 mg. In certain embodiments, the amount of trisodium citrate dihydrate is 1.0 mg, 2.0 mg, 2.5 mg, 3.0 mg, 4.0 mg, or 5.0 mg. In certain embodiments, the amount of trisodium citrate dihydrate is about 1.0 mg, about 2.0 mg, about 2.5 mg, about 3.0 mg, about 4.0 mg, or about 5.0 mg. In certain embodiments, the amount of trisodium citrate dihydrate is 2.56 mg. In certain embodiments, the amount of trisodium citrate dihydrate is 3.37 mg.

[0064] 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 upon reconstitution with sterile water for injection. In another embodiment, the pH of the solution is greater than 2. In another embodiment, the pH of the solution is between pH 2.0 and pH 12.0. For example, in various embodiments, the pH of the reconstituted solution 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, when reconstituted in solution, the pH of the solution is between pH 5 and pH 7. In some embodiments, the pH of the reconstituted solution 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 reconstituted solution is ≦6.5. In one embodiment, the pH of the reconstituted solution is about 5.5 to about 6.5. In one embodiment, the pH of the reconstituted solution is about 5.3 to about 6.3. In one embodiment, the pH of the reconstituted solution is ≦6.5. In one embodiment, the pH of the reconstituted solution is about 6.0 to about 6.5. In one embodiment, the pH of the reconstituted solution is 5.8.

[0065] In some embodiments, the buffer comprises citrate and the reconstituted solution has a pH of about 5.5 to about 6.5. In some embodiments, the buffer comprises citrate and the reconstituted solution has a pH of about 5.3 to about 6.3. In some embodiments, the buffer comprises citrate and the reconstituted solution has a pH of about 5.8.

[0066] In some embodiments, the buffer comprises succinate and the pH of the reconstituted solution is about 5.5 to about 6.5. In some embodiments, the buffer comprises succinate and the pH of the reconstituted solution is about 5.3 to about 6.3. In some embodiments, the buffer comprises succinate and the pH of the reconstituted solution is about 5.8.

[0067] 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 buffering agent has a concentration of 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 one embodiment, the buffering agent has a concentration of 0.1 mM to 20 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 buffering agent is present in an amount of 10 mM.

[0068] 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.

[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 protein has a negative charge due to the presence of glycans, and the buffering agent is not histidine. In some embodiments, the buffering agent is present in the pharmaceutical formulation 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 pharmaceutical formulation comprises at least 10 mM of a buffering agent.

[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 ActRIIa protein.

[0071] In some embodiments, a stabilizer (or combination of stabilizers) is added to the formulation to prevent or reduce storage-induced aggregation and chemical degradation. A hazy or cloudy solution upon reconstitution 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 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 lyophilized formulations described herein contain a stabilizer in an amount between 50 mg and 150 mg. In certain embodiments, the lyophilized formulations described herein contain a stabilizer in an amount of 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, or 150 mg. In certain embodiments, the lyophilized formulations described herein contain a stabilizer in an amount of about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, or about 150 mg.

[0073] In certain embodiments, the stabilizer is sucrose in an amount of 88.0 mg. In certain embodiments, the stabilizer is sucrose in an amount of 116.0 mg.

[0074] In certain embodiments, the reconstituted 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).

[0075] In some embodiments, the formulation comprises about 8% to about 10% (weight / volume) sucrose. In some embodiments, the formulation comprises 8-10% (weight / volume) sucrose. In some embodiments, the formulation comprises 8%, 9%, or 10% (weight / volume) sucrose. In some embodiments, the formulation comprises 8% (weight / volume) sucrose.

[0076] 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.

[0077] Surfactants 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.

[0078] Surfactants are also added in an appropriate amount to prevent surface-related aggregation phenomena during freezing and drying (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. Thus, formulations containing these surfactants individually or as mixtures of these surfactants in various ratios are further provided.In one embodiment of the present disclosure, the surfactant is polysorbate 80 or polysorbate 20. In one embodiment of the present disclosure, the surfactant is polysorbate 80.

[0079] In certain embodiments, the surfactant is polysorbate 80 or polysorbate 20, and the amount of polysorbate 80 or polysorbate 20 is 0.1 mg to 1.0 mg. In certain embodiments, the amount of polysorbate 80 or polysorbate 20 is 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, or 1.0 mg. In certain embodiments, the amount of polysorbate 80 or polysorbate 20 is about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, or about 1.0 mg. In certain embodiments, the amount of polysorbate 80 is 0.22 mg. In certain embodiments, the amount of polysorbate 80 is 0.29 mg. In certain embodiments, the amount of polysorbate 20 is 0.22 mg. In certain embodiments, the amount of polysorbate 20 is 0.29 mg.

[0080] In the reconstituted formulation, the surfactant is present at a concentration of about 0.01 to about 0.5 g / L. 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 g / L. 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). In certain embodiments of the present disclosure, the surfactant is incorporated at a concentration of about 0.01 to about 0.05% (weight / volume). In certain embodiments, the surfactant is incorporated at a concentration of about 0.02% (weight / volume).

[0081] In some embodiments, the surfactant is polysorbate 20 or polysorbate 80. In some embodiments, the surfactant is polysorbate 80. In some embodiments, the pharmaceutical formulation comprises 0.05-0.3 mg / mL of surfactant. In some embodiments, the pharmaceutical formulation comprises 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) of surfactant. In some embodiments, the surfactant is present in the pharmaceutical formulation at a concentration of at least 0.02% (weight / volume). In certain embodiments, polysorbate 80 or polysorbate 20 is incorporated at a concentration of about 0.01 to about 0.05% (weight / volume). In certain embodiments, polysorbate 80 or polysorbate 20 is incorporated at a concentration of about 0.02% (weight / volume).

[0082] ActRIIa preparations In some embodiments of the pharmaceutical formulations provided herein, a vial of an ActRIIa fusion protein provided herein comprises an ActRIIa fusion protein and one or more pharmaceutical additives and / or excipients. In some embodiments, the pharmaceutical formulation is lyophilized. In some embodiments, the formulation is reconstituted from a lyophilized formulation. In one embodiment of the pharmaceutical formulations provided herein, a vial of the lyophilized pharmaceutical formulation contains 55 mg of ActRIIa fusion protein; 0.48 mg of citric acid monohydrate, 2.56 mg of trisodium citrate dehydrate, 0.22 mg of polysorbate 80, and 88 mg of sucrose. In another embodiment, the vial is rehydrated with 1.0 mL of liquid (e.g., sterile water for injection). In another embodiment of the pharmaceutical formulation provided herein, a vial contains 72.5 mg of ActRIIa fusion protein; 0.64 mg of citric acid monohydrate, 3.37 mg of trisodium citrate dehydrate, 0.29 mg of polysorbate 80, and 116 mg of sucrose, wherein the formulation is lyophilized. In another embodiment, the vial is rehydrated with 1.3 mL of liquid (e.g., sterile water for injection). In a further embodiment, the vial contains one, two, or all three of the following: citrate, e.g., 10 mM citrate; sucrose, e.g., 8% (wt / vol) sucrose; and / or polysorbate 80 (e.g., pH 5.0-7.0), e.g., 0.02% (wt / vol) polysorbate 80 (pH 5.8).

[0083] In some embodiments, once reconstituted with sterile water, the vial contains 50 mg / mL of the ActRIIa fusion protein of SEQ ID NO: 32 or SEQ ID NO: 41, 10 mM citrate, 0.2 mg / mL of polysorbate 80, and 8% by weight of sucrose. In some embodiments, once reconstituted with sterile water, the vial contains 50 mg / mL of sotatercept, 10 mM citrate, 0.2 mg / mL of polysorbate 80, and 8% by weight of sucrose.

[0084] In some embodiments, the reconstituted pharmaceutical formulation comprises 50 mg / mL of SEQ ID NO:32 or SEQ ID NO:41, 10 mM citrate, 0.2 mg / mL of polysorbate 80, and 80 mg / mL of sucrose at pH 5.8. In some embodiments, the reconstituted pharmaceutical formulation comprises 50 mg / mL of sotatercept, 10 mM citrate, 0.2 mg / mL of polysorbate 80, and 80 mg / mL of sucrose at pH 5.8.

[0085] 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.

[0086] kit The present disclosure provides a kit comprising a lyophilized pharmaceutical formulation and an injection device. In certain embodiments, the lyophilized pharmaceutical formulation comprises an ActRIIa 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 or SEQ ID NO:32) 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. In some embodiments, the lyophilized 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 or SEQ ID NO:32. In certain such embodiments, the protein comprises an amino acid sequence at least 90%, 95%, or 99% identical to SEQ ID NO:9 or SEQ ID NO:32, wherein the protein binds to activin and / or GDF11. In certain embodiments, the protein comprises the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:32. In other embodiments, the protein consists of the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:32. In certain embodiments, the protein comprises 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.

[0087] In some embodiments, the lyophilized pharmaceutical formulation comprises a protein comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to 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. In other embodiments, the protein consists of a variant of SEQ ID NO: 32 lacking the C-terminal lysine residue (i.e., SEQ ID NO: 41).

[0088] In certain of the foregoing embodiments, the lyophilized pharmaceutical formulation comprises a fusion protein further comprising an Fc domain of an immunoglobulin. In certain such embodiments, the Fc domain of the immunoglobulin is the Fc domain of an IgG1 immunoglobulin. In other embodiments, the fusion protein further comprises a linker domain disposed between the protein domain and the Fc domain of the immunoglobulin. In certain embodiments, the linker domain is a polyglycine linker.

[0089] In certain embodiments, the protein is part of a homodimeric protein complex.

[0090] In certain embodiments, the protein is glycosylated.

[0091] The present disclosure further provides kits comprising a sterile, lyophilized pharmaceutical formulation comprising a protein disclosed herein and an injection device. In some embodiments of the kits disclosed herein, the sterile, lyophilized pharmaceutical formulation comprising the protein is pre-filled into one or more containers (e.g., one or more vials) (FIG. 1).

[0092] In certain embodiments, the sterile, lyophilized pharmaceutical formulation comprising an ActRIIa fusion protein has a pH range of 5 to 7. In certain embodiments, the sterile, lyophilized pharmaceutical formulation comprising an ActRIIa fusion protein 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 anions.

[0093] In some embodiments, the pharmaceutical formulation further comprises a surfactant. In some embodiments, the surfactant comprises a polysorbate. In some embodiments, the surfactant comprises polysorbate 20 or polysorbate 80. In some embodiments, the surfactant comprises polysorbate 80.

[0094] In some embodiments, the lyophilized pharmaceutical formulation further comprises a lyoprotectant. In some embodiments, the lyoprotectant comprises a sugar such as a disaccharide (e.g., sucrose). In some embodiments, the lyoprotectant comprises sucrose, trehalose, mannitol, polyvinylpyrrolidone (PVP), dextrose, and / or glycine. In some embodiments, the lyoprotectant comprises sucrose. In some embodiments, the lyophilized pharmaceutical formulation comprises a lyoprotectant and a protein in a weight ratio of protein to lyoprotectant of at least 1:1. In some embodiments, the lyophilized pharmaceutical formulation comprises a lyoprotectant and a protein in a weight ratio of protein to lyoprotectant of 1:1 to 1:10. In some embodiments, the lyophilized pharmaceutical formulation comprises a lyoprotectant and a protein in a weight ratio of protein to lyoprotectant 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 lyophilized pharmaceutical formulation comprises a lyoprotectant and a protein in a weight ratio of protein to lyoprotectant of 1:6. In certain of the foregoing embodiments, the lyophilized pharmaceutical formulation comprises an amount of lyoprotectant sufficient to stabilize the protein.

[0095] In certain embodiments of the kits disclosed herein, the injection device includes a syringe, as shown in FIG. 1 . In certain such embodiments, the syringe is prefilled with a reconstitution solution. In some embodiments, the reconstitution solution includes a pharmaceutically acceptable carrier and / or excipient. In some embodiments, the pharmaceutically acceptable carrier includes an aqueous solution, such as water, physiologically buffered saline, or other solvent or vehicle (e.g., glycol, glycerol, oil, or injectable organic ester). In some embodiments, the pharmaceutically acceptable excipient includes a pharmaceutically acceptable excipient selected from calcium phosphate, calcium carbonate, calcium sulfate, rock salt, metal oxides, sugars, sugar alcohols, starches, glycols, povidone, mineral hydrocarbons, acrylic polymers, fatty alcohols, mineral stearates, glycerin, and / or lipids. In certain embodiments, the reconstitution solution includes a pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solution, dispersion, suspension, or emulsion. In certain such embodiments, the reconstitution solution comprises an antioxidant, a buffer, a bacteriostat, and / or a solute that renders the formulation isotonic with the blood of the intended recipient, hi other embodiments, the reconstitution solution comprises a suspending agent or a thickening agent.

[0096] In certain embodiments of the kits disclosed herein, the kit further comprises a vial adapter, as shown in FIG. 1 . In some embodiments, a vial (1) prefilled with a lyophilized pharmaceutical formulation of the present invention is attached to one end of the vial adapter (3). In some embodiments, a syringe (2) prefilled with a reconstitution solution disclosed herein is attached to one end of the vial adapter (3). In some embodiments, the syringe (2) prefilled with a reconstitution solution disclosed herein and the vial (1) prefilled with the lyophilized pharmaceutical formulation are attached to opposite ends of the vial adapter (3). In some embodiments, the reconstitution solution is transferred from the prefilled syringe to the vial. In some embodiments, the lyophilized formulation is reconstituted into a sterile injectable solution by transferring the reconstitution solution to the vial prefilled with the lyophilized pharmaceutical formulation. In some embodiments, the lyophilized formulation is reconstituted into a sterile injectable solution. In some embodiments, the lyophilized formulation is reconstituted into a sterile injectable solution prior to use.

[0097] In other embodiments of the kits disclosed herein, the kit further comprises a pump device. In certain embodiments, the pump device comprises an electromechanical pump assembly. In certain embodiments, the pump device comprises a reservoir for holding a sterile injectable fluid. In certain embodiments, the reservoir holds 1 mL of sterile injectable fluid. In certain embodiments, the pump device comprises one or more vials or cartridges containing sterile injectable fluid. In certain embodiments, the vials or cartridges are prefilled with sterile injectable fluid. In certain embodiments, the vials or cartridges contain sterile injectable fluid reconstituted from a lyophilized formulation. In certain embodiments, the reservoir is coupled to the vial or cartridge. In certain embodiments, the vial or cartridge holds 1-20 mL of sterile injectable fluid. In certain embodiments, the electromechanical pump assembly comprises a pump chamber. In certain embodiments, the electromechanical pump assembly is coupled to a reservoir. In certain embodiments, the sterile injectable fluid is received from the reservoir into the pump chamber. In some embodiments, the electromechanical pump assembly includes a plunger positioned such that the sterile injectable fluid in the pump chamber is in direct contact with the plunger. In certain embodiments, the sterile injectable fluid is received into the pump chamber from a reservoir during a first pumping stage and delivered from the pump chamber to the subject during a second pumping stage. In certain embodiments, the electromechanical pump assembly includes a control circuit. In certain embodiments, the control circuit drives the plunger to (a) draw the sterile injectable fluid into the pump chamber during the first pumping stage and (b) deliver the sterile injectable fluid from the pump chamber with multiple discrete strokes of the plunger during the second pumping stage, thereby delivering the therapeutic substance to the subject in multiple controlled, discrete doses throughout the second pumping stage. In certain embodiments, the alternating cycle of the first and second pumping stages is repeated until the desired dose is administered. In certain embodiments, the pump device is coupled to a wearable patch. In certain embodiments, the pump device is a wearable pump device.

[0098] The present disclosure provides kits used to reconstitute lyophilized pharmaceutical formulations into sterile injectable solutions. In certain embodiments, the resulting sterile injectable solutions are useful in the methods disclosed herein.

[0099] In certain embodiments of the kits disclosed herein, the kit further comprises an injectable device for use in parenterally administering the sterile injectable solution. In some embodiments, the sterile injectable solution is administered by subcutaneous injection. In some embodiments, the sterile injectable solution is administered by intradermal injection. In some embodiments, the sterile injectable solution is administered by intramuscular injection. In some embodiments, the sterile injectable solution is administered by intravenous injection. 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 dose based on body weight.

[0100] ActRIIa polypeptides In certain embodiments, the present disclosure relates to ActRIIa fusion proteins. As used herein, the term "ActRIIa" refers to the activin receptor type IIA (ActRIIa) protein family from any species, and variants derived from such ActRIIa fusion proteins by mutagenesis or other modifications. Reference to ActRIIa herein is understood to refer to any one of its 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.

[0101] The term "ActRIIa fusion protein" encompasses proteins comprising 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 fusion proteins are provided throughout this disclosure and in International Patent Application Publication Nos. WO2006 / 012627 and WO2007 / 062188, which are incorporated 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.

[0102] The canonical sequence of the human ActRIIa precursor protein is as follows: [Table 1]

[0103] The signal peptide is single underline the extracellular domain is shown in bold font; and potential endogenous N-linked glycosylation sites are shown in bold font. double underline is shown.

[0104] The sequence of the processed (mature) extracellular human ActRIIa protein is as follows: ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM EVTQPTSNPVTPKPP (SEQ ID NO: 10).

[0105] The C-terminal "tail" of the extracellular domain is single underline The sequence with the "tail" deleted (Δ15 sequence) is as follows: ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM (SEQ ID NO: 11).

[0106] 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.

[0107] [Table 2]

[0108] The nucleic acid sequence encoding the processed soluble (extracellular) human ActRIIa protein is as follows: [Table 3]

[0109] ActRIIa is highly conserved among vertebrates, with most of the extracellular domain being completely conserved. For example, Figure 2 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.

[0110] While not meant to be limiting, the following example illustrates this approach to defining active ActRIIa variants. As shown in Figure 2, F13 of the human extracellular domain is Y in ActRIIa of sheep (Ovis aries) (SEQ ID NO: 62), red junglefowl (Gallus 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.

[0111] Furthermore, as discussed above, ActRIIa 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 ActRIIa variants that retain one or more desired activities (e.g., ligand binding activity).

[0112] For example, a 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.

[0113] 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, 290, 291, 292, 293, 294, 295, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 124, 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, the 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.

[0114] 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 present 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 , 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.

[0115] In certain embodiments, the extracellular domain (ECD) of a human activin receptor type IIA (ActRIIa) protein or a derivative thereof is linked to a constant domain of an immunoglobulin, such as a human IgG1 Fc domain. In some embodiments, the ActRIIa protein is a fusion protein comprising an ActRIIa 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 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%, 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 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.

[0116] In some embodiments, the ActRIIa fusion protein comprises, consists of, or essentially consists 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 and 39.

[0117] 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.

[0118] In some alternative embodiments, the ActRIIa fusion protein (e.g., SEQ ID NO: 32) may lack a C-terminal lysine. In some embodiments, the ActRIIa fusion protein lacking a C-terminal lysine is SEQ ID NO: 41. For example, in certain embodiments of the pharmaceutical preparations described herein, the ActRIIa fusion protein may consist of 1 to 100% of SEQ ID NO: 32. For example, in certain embodiments of the pharmaceutical preparations described herein, the ActRIIa fusion protein consists of 1 to 100% of SEQ ID NO: 41.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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 ActRIIa proteins (e.g., ActRIIa) comprising one or more mutations (e.g., amino acid additions, deletions, substitutions, and combinations thereof) in the extracellular domain (also referred to as the ligand-binding domain) of an ActRIIa protein (e.g., a "wild-type" or unmodified ActRIIa protein) such that the mutant ActRIIa protein has one or more altered ligand-binding activities compared to the corresponding wild-type ActRIIa protein. In certain 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 specificities for one or more ActRIIa ligands.

[0125] 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.

[0126] 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.

[0127] In certain aspects, the present disclosure provides a method of treating pulmonary arterial hypertension (PAH) in a patient in need thereof, wherein the method comprises reconstituting a lyophilized pharmaceutical formulation described herein to produce a reconstituted formulation, and administering the reconstituted formulation to the patient.

[0128] In certain aspects, the present disclosure provides a pharmaceutical formulation for treating PAH in a subject in need thereof, comprising reconstituting a lyophilized pharmaceutical formulation comprising a human ActRIIa fusion protein linked to an immunoglobulin constant domain and administering the reconstituted formulation to the subject, wherein the dosing regimen comprises: (1) administering an initial dose of 0.3 mg / kg; (2) monitoring the subject's response; and (3) administering subsequent doses of 0.7 mg / kg; and wherein the subject receives the subsequent doses every three weeks. In some embodiments, the subsequent doses are modified based on the subject's response.

[0129] In certain aspects, the present disclosure provides methods of treating pulmonary arterial hypertension (PAH), the methods comprising administering to a patient in need thereof a pharmaceutical formulation described herein, 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 a World Health Organization (WHO) pulmonary hypertension functional class; promotion or increase in regression of a WHO pulmonary hypertension functional class; improvement in right ventricular function; improvement in pulmonary artery pressure; and / or improvement in mean right atrial pressure.

[0130] 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).

[0131] In certain aspects, the present disclosure provides methods of treating, preventing, or reducing the rate of progression and / or severity of one or more complications of pulmonary arterial hypertension, comprising administering to a patient in need thereof a pharmaceutical formulation described herein, wherein administration of the 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 a World Health Organization (WHO) pulmonary hypertension functional class; promotion or increase in regression of a 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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).

[0136] 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.

[0137] 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%).

[0138] 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.

[0139] 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 can be 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 other specific embodiments, 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 other specific embodiments, 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.

[0140] 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. [Example]

[0141] 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.

[0142] Having generally described the invention, 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 invention and are not intended to limit the invention.

[0143] 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, designated ActRIIa-hFc and ActRIIa-mFc, respectively.

[0144] ActRIIa-hFc shown below is sotatercept purified from a CHO cell line (SEQ ID NO: 32): ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPP TGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPI EKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK .

[0145] 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).

[0146] The form selected employs a TPA leader and has the following raw amino acid sequence: MDAMKRGLCCVLLLCGAVFVSPGAAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 36).

[0147] This protein is encoded by the following nucleic acid sequence:

[0148] Both ActRIIa-hFc and ActRIIa-mFc were highly suitable for recombinant expression. As shown in Figures 3A and 3B, 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 chromatography, Q Sepharose chromatography, phenyl Sepharose chromatography, size exclusion chromatography, 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.

[0149] 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 protein and ActRIIa-mFc protein were immobilized on a CM5 chip. The ActRIIa-hFc protein and ActRIIa-mFc protein were loaded into 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 4A and 4B. 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.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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).

[0154] Example 3 Preparation of lyophilized ActRIIa-hFc fusion protein formulation Initially, the ActRIIa-hFc fusion protein SEQ ID NO:32 (sotatercept) was first formulated as a frozen solution in phosphate-buffered saline. Based on subsequent development work, a lyophilized citrate buffer formulation of the ActRIIa-hFc fusion protein SEQ ID NO:32 containing sucrose and polysorbate 80 was developed, allowing for a stable formulation with sufficient shelf life for commercialization.

[0155] Two formulations of ActRIIa-hFc fusion protein SEQ ID NO:32 were developed: 45 mg / vial and 60 mg / vial. The 45 mg / mL formulation contained 55.0 mg of SEQ ID NO:32 or a variant of SEQ ID NO:32 lacking the C-terminal lysine residue, 0.48 mg of citric acid monohydrate, 2.56 mg of trisodium citrate dihydrate, 0.22 mg of polysorbate 80, and 88.0 mg of sucrose. The 60 mg / mL formulation contained 72.5 mg of SEQ ID NO:32 or a variant of SEQ ID NO:32 lacking the C-terminal lysine residue, 0.64 mg of citric acid monohydrate, 3.37 mg of trisodium citrate dihydrate, 0.29 mg of polysorbate 80, and 116.0 mg of sucrose. The formulations were identical in composition before lyophilization and upon reconstitution with sWFI. They differ only in their fill volume: the 45 mg / vial has a fill volume of 1.1 mL, whereas the 60 mg / vial has a fill volume of 1.45 mL. Both formulations are lyophilized and reconstituted prior to subcutaneous administration: the 45 mg / vial is reconstituted with 1.0 mL of sWFI, and the 60 mg / vial is reconstituted with 1.3 mL of sWFI.

[0156] Formulation development of the ActRIIa-hFc fusion protein SEQ ID NO: 32 was guided by the results of various screening studies, described below. These studies focused on the selection of optimal pH, buffer system, protein concentration, and excipients for stabilization of the ActRIIa-hFc fusion protein SEQ ID NO: 32. The screening studies were conducted under accelerated or stressed conditions to observe differences in the stability of the ActRIIa-hFc fusion protein SEQ ID NO: 32 in test samples.

[0157] Preliminary pH Screening Studies: Early evaluation and identification of suitable buffers and pH ranges for ActRIIa-hFc fusion protein SEQ ID NO:32 for optimal biophysical stability and binding capacity To understand the effect of pH and buffer system on the stability of the ActRIIa-hFc fusion protein SEQ ID NO:32, an initial buffer and pH screen was performed. In this study, the ActRIIa-hFc fusion protein SEQ ID NO:32 was formulated at a concentration of 5 mg / mL in 50 mM acetate, citrate, histidine, phosphate, succinate, and Tris buffers at pH levels ranging from 4 to 8. These formulations were filled into Type I glass vials, subjected to short-term stability tests at 5°C, 25°C, and 45°C for up to 6 months, and analyzed for purity by high-performance size-exclusion chromatography (HP-SEC), thermal stability using differential scanning calorimetry (DSC), and binding activity using surface plasmon resonance (Biacore). Data from these analyses are summarized in Table 1.

[0158] [Table 4] TIFF2026501040000005.tif115170

[0159] Observation of changes in monomer species % (purity) by HP-SEC revealed no significant change in purity after 6 months at 5°C, with all formulations retaining >99.0% monomer. As shown in Figure 5, after 6 months at 25°C, formulations with pH ≥ 6.5 showed a significant decrease in monomer % but no significant change in formulations with pH < 6.5. After 2 months at 45°C, formulations with pH ≥ 6.5 showed a similar decrease in monomer % but similarly correlated with a lower pH < 5.5.

[0160] Based on these observations under accelerated (25°C) and stressed (45°C) conditions, the ActRIIa-hFc fusion protein SEQ ID NO:32 showed the greatest stabilizing effect in citrate and succinate buffers at pH 5.5-6.5. Histidine at pH 5.5-6.0 also showed similar stability to citrate and succinate at low pH, but decreased stability at high pH.

[0161] Binding affinity is usually measured by the equilibrium dissociation constant (Kd), which is used to evaluate and rank the strength of bimolecular interactions. In this case, the interaction or binding affinity of ActRIIa-hFc fusion protein SEQ ID NO:32 with the target activin by Biacore was used as a measure of product stability. The smaller the Kd value, the higher the binding affinity of the ligand to the target.

[0162] There was no significant change in the equilibrium dissociation constant (Kd) of SEQ ID NO:32 after 6 months at 5° C. For formulations at pH<5.5, there was a significant decrease in binding after 6 months at 25° C., as shown by the sharp increase in Kd values ​​in Figure 6. For formulations at pH<6.0, there was a decrease in binding after 2 months at 45° C.

[0163] Based on these observations, the ActRIIa-hFc fusion protein SEQ ID NO:32 is able to retain binding affinity at pH ≥ 5.5 in many different buffer systems.

[0164] The unfolding temperature of the ActRIIa-hFc fusion protein SEQ ID NO:32 increased consistently with pH, ​​leveling off around 67-70°C at pH ≥ 5.5. All buffers at comparable pH values ​​performed similarly, except for the histidine and Tris buffers, which had significantly lower melting points.

[0165] Overall, the results of this experiment suggest that a pH range of 5.5 to 6.5 is the optimal range for SEQ ID NO:32 in terms of physical stability as monitored by HP-SEC and retention of binding affinity by Biacore. Furthermore, citrate and succinate buffer systems performed better than histidine at the higher end of this range. Furthermore, acetate and phosphate buffers were not pursued further in development due to the potential for pH shifts due to precipitation during freezing or lyophilization.

[0166] Identifying the optimal buffer concentration: Evaluating the effect of buffer concentration on the conformational and colloidal stability of the ActRIIa-hFc fusion protein SEQ ID NO:32 using citrate, one of the lead buffer systems. In initial pH screening studies, various buffer systems were evaluated at 50 mM concentration to provide high buffering capacity. However, this concentration may not be optimal for stabilizing the ActRIIa-hFc fusion protein SEQ ID NO:32 and is not ideal for subcutaneous injection, where high ionic strength buffers (especially citrate) are known to cause injection pain. In this study, the ActRIIa-hFc fusion protein SEQ ID NO:32 was formulated in a range of concentrations of citrate buffer, one of the lead buffer systems from previous studies. 50 mg / mL ActRIIa-hFc fusion protein SEQ ID NO:32 in pH 5.8 citrate buffer (5-50 mM) containing 8% (wt / vol) sucrose was evaluated using DSC to assess the effect of buffer strength on thermal stability and DLS to assess the diffusion interaction parameter (kD), a measure of the molecule's tendency to self-associate. The results are summarized in Table 2.

[0167] [Table 5]

[0168] The results of this study showed that the dissociation constant (kD) of the ActRIIa-hFc fusion protein SEQ ID NO:32 was highest at 10 mM (+6.7 mL / g) and decreased to a negative kD (-4.5 mL / g) at higher concentrations up to 50 mM. A more positive kD value indicates stronger intermolecular repulsion and higher colloidal stability, while a negative kD value indicates a tendency toward self-association, which can lead to aggregation. Furthermore, the melting temperature of the ActRIIa-hFc fusion protein SEQ ID NO:32 was nearly constant and sufficiently high at all buffer concentrations. Based on this data, a buffer concentration of 10 mM was selected for subsequent formulation development.

[0169] Selecting the pH for optimal stability Short-term stability studies were performed in which the ActRIIa-hFc fusion protein SEQ ID NO:32 was formulated at a concentration of 5 mg / mL in 10 mM succinate, citrate, and histidine (at pH levels ranging from 5 to 7). These formulations were filled into Type I glass vials and subjected to short-term stability at 5°C, 25°C, and 40°C for up to 3 months. These formulations were analyzed by biophysical methods for purity by high-performance size-exclusion chromatography (HP-SEC), thermal stability by differential scanning calorimetry (DSC), fragmentation by CE-SDS Non-Reduced, chemical degradation by desialylated iCIEF, and pH. The data from these analyses are summarized in Tables 3, 4, and 5, and yielded the following results:

[0170] [Table 6]

[0171] [Table 7]

[0172] [Table 8]

[0173] As shown in Figure 7, the results of this study indicated that citrate and succinate had comparable performance for HP-SEC between pH 5.3 and 6.3 (measured pH) for best thermal stability based on stability results at 25 °C. Higher fragment amounts (LMW%) were observed by HP-SEC at 40 °C in citrate and succinate buffer systems, but this was attributed to the nature of the stress and method integration challenges. The HP-SEC observations were consistent with the non-reducing CE-SDS data shown in Figure 8. Characterization of charge profiles by desialylated iCIEF, shown in Figure 9, also confirmed the optimal stability pH range of 5.3–6.3. For formulations in histidine buffer, a slightly higher pH range of 6.0–6.5 was required to achieve comparable performance to citrate and succinate; however, this buffer system also had lower Tm1, indicating poorer biophysical stability.

[0174] The results of this study demonstrated that the ActRIIa-hFc fusion protein SEQ ID NO:32 was biophysically and chemically stable in both citrate and succinate buffers at measured pH values ​​between 5.3 and 6.3. Furthermore, this range offered the possibility of reducing the rate of potential deamidation by selecting a less basic pH, as commonly observed with biologics. Therefore, a target pH of 5.8 was selected for further product development using citrate as the buffer system.

[0175] Physical stability of the ActRIIa-hFc fusion protein of SEQ ID NO:32 To evaluate the physical stability of the ActRIIa-hFc fusion protein of SEQ ID NO:32, a worst-case scenario was tested in the absence of excipients in 10 mM citrate, one of the lead buffer systems, under physical stress due to agitation. The ActRIIa-hFc fusion protein of SEQ ID NO:32 was diluted to 5 mg / mL and 50 mg / mL concentrations in 10 mM citrate (pH 5.8). These two formulations were agitated alongside unagitated "control" samples, in which 4 mL samples were placed in 10 mL Type I glass vials with an analog agitator for up to 4 days and analyzed for monomer loss by HP-SEC, as summarized in Table 6.

[0176] [Table 9]

[0177] Regardless of protein concentration, no significant changes in monomer content were observed between control and stressed samples even after stirring for up to 4 days. The results of this study suggest that the ActRIIa-hFc fusion protein of SEQ ID NO:32 is biophysically stable to shear and interfacial stress, even in the absence of added sugars and surfactants.

[0178] Concentration ranging study of ActRIIa-hFc fusion protein of SEQ ID NO:32 A study was conducted to evaluate the short-term biophysical stability of the ActRIIa-hFc fusion protein of SEQ ID NO: 32 at various concentrations. The ActRIIa-hFc fusion protein of SEQ ID NO: 32 was prepared from 20 mg / mL to 200 mg / mL in 10 mM citrate buffer (pH 5.8) without other excipients, filled into Type I glass vials, and subjected to short-term stability at 5°C and 25°C for up to 7 days. The purity of the formulations was determined by HP-SEC analysis throughout the study and is summarized in Table 7.

[0179] [Table 10]

[0180] None of the formulations showed significant changes in purity upon storage at 5°C for up to 7 days. However, at concentrations above 75 mg / mL, significant monomer loss was observed during short-term storage at 25°C, and this trend continued with increasing concentrations up to 200 mg / mL, as shown in Figure 10. The results of this study indicated that protein concentrations below 75 mg / mL did not significantly affect the biophysical stability of the ActRIIa-hFc fusion protein of SEQ ID NO:32. Therefore, the target concentration of the ActRIIa-hFc fusion protein of SEQ ID NO:32 was selected to be ≦75 mg / mL based on agitation and concentration range studies.

[0181] Assessment of the long-term stability of lead buffers supporting the composition of drug substances A long-term stability study was performed to compare the performance of two buffer systems at two different concentrations (based on early understanding of clinical administration needs). The ActRIIa-hFc fusion protein of SEQ ID NO:32 was formulated at 75 mg / mL and 50 mg / mL in 10 mM succinate and 10 mM citrate at pH 5.8, filled into Type I glass vials, and stored at -80°C, -20°C, 5°C, and 25°C for up to 30 months at each storage condition.

[0182] The stability data from HP-SEC analysis is shown in Table 8, and the following results were obtained:

[0183] [Table 11]

[0184] After 24 months at -80°C, there was no significant change in purity by HP-SEC, and all formulations retained 97.8% or more of the monomer. After 24 months at 5°C, the ActRIIa-hFc fusion protein of SEQ ID NO:32 had a loss of monomer to 94-95% in the citrate formulation and 93-94% in the succinate formulation. These results demonstrate that the ActRIIa-hFc fusion protein of SEQ ID NO:32 is biophysically stable in solution, even in the absence of detergents or stabilizing sugars, as no formulation showed a percent loss of monomer below 94% monomer even after 2 years at 2-8°C. After 12 months at 25°C, the ActRIIa-hFc fusion protein of SEQ ID NO:32 had a loss of monomer to 91-93% in the citrate formulation and 90-92% in the succinate formulation. In both the 5°C and 25°C storage data, the 75 mg / mL drug substance degraded slightly more than the 50 mg / mL in each buffer system, as expected, and these trends are shown in Figure 11.

[0185] In conclusion, the data from this study demonstrate that the ActRIIa-hFc fusion protein of SEQ ID NO:32 in citrate buffer has slightly better long-term stability than succinate buffer when stored at 5° C. and 25° C., but is identical at the recommended storage condition of −80° C. Therefore, 10 mM citrate at pH 5.8 was selected as the target formulation for SEQ ID NO:32 DS under frozen conditions.

[0186] Salt concentration range study The ActRIIa-hFc fusion protein of SEQ ID NO: 32 was prepared at 50 mg / mL in 10 mM citrate (pH 5.8) with 8% (wt / vol) sucrose and sodium chloride concentrations ranging from 0 to 150 mM to assess the effect of buffer strength on its biophysical properties. These formulations were evaluated for thermal stability using DSC and diffusion interaction parameter (kD) by DLS. Results are summarized in Table 9.

[0187] [Table 12]

[0188] The results of this study showed that the kD of the ActRIIa-hFc fusion protein of SEQ ID NO: 32 was highest (16.2 mL / g) in the absence of sodium chloride, decreased to approximately 5 mL / g with increasing concentration, and plateaued at concentrations above 50 mM. A more positive kD value of a molecule indicates stronger intermolecular repulsion, indicating better colloidal stability. This suggests that the ActRIIa-hFc fusion protein of SEQ ID NO: 32 has the best colloidal stability in the absence of sodium chloride. Furthermore, the melting point of the ActRIIa-hFc fusion protein of SEQ ID NO: 32 was nearly identical and sufficiently high at all sodium chloride concentrations. Based on this data, sodium chloride was not considered suitable for further development work in stabilizing the ActRIIa-hFc fusion protein of SEQ ID NO: 32.

[0189] Excipient Screening Studies A screening study was designed to evaluate a series of excipients with the aim of increasing the biophysical stability of the ActRIIa-hFc fusion protein of SEQ ID NO: 32 in solution. In this excipient screen, the ActRIIa-hFc fusion protein of SEQ ID NO: 32 was formulated at a concentration of 2 mg / mL with various excipients (salt (sodium chloride), sugars (sucrose, mannitol), and amino acids (arginine, histidine)). These solutions, summarized in Table 10, were subjected to stability tests at 5°C, 25°C, and 45°C for up to 12 months, 9 months, and 3 months, respectively, and then tested for purity (% monomer content) by HP-SEC.

[0190] [Table 13]

[0191] All formulations were biophysically stable at storage temperatures of 5°C and 25°C, retaining >99% of monomer after 12 months at 5°C and 9 months at 25°C. The excipients evaluated, with the exception of histidine, exhibited comparable biophysical stability after 3 months under stress conditions (45°C), retaining 97–99% of monomer. Increasing histidine concentrations decreased biophysical stability, with up to 2% (wt / vol) histidine resulting in a 6% reduction in monomer compared to the 0.1% (wt / vol) histidine formulation.

[0192] In conclusion, the ActRIIa-hFc fusion protein of SEQ ID NO: 32 was confirmed to be biophysically stable in all prototypes tested.

[0193] Surfactant screening research Surfactants are widely used to stabilize proteins from shear and interfacial stresses encountered during manufacturing, shipping, and handling. Therefore, a series of experiments were conducted to evaluate polysorbate 80 (PS80) as a surfactant for stabilizing SEQ ID NO:32 under various stress conditions.

[0194] Study 1: Screening surfactants by thermal stability A PS80 concentration-ranging stability study was conducted to evaluate the effect of PS80 on the long-term stability of the ActRIIa-hFc fusion protein of SEQ ID NO:32. In this study, the ActRIIa-hFc fusion protein of SEQ ID NO:32 was formulated at a concentration of 50 mg / mL with various levels of PS80 ranging from 0.001% to 0.05% in 10 mM citrate, pH 5.8, containing 8% (wt / vol) sucrose. These formulations were filled into Type I glass vials and subjected to stability tests at 5°C and 25°C for up to 24 months and at 45°C for up to 3 months. To monitor the physical stability of these formulations, the purity of the formulations was measured by HP-SEC throughout the study period. The HP-SEC stability data are shown in Table 11.

[0195] [Table 14]

[0196] No significant differences in biophysical stability profiles were observed between various levels of PS80, with all formulations having >99% monomer after 24 months at 5°C, >96% monomer after 24 months at 25°C, and >93% monomer after 3 months at 45°C. The results of this study showed that concentrations of PS80 between 0.001% and 0.05% were comparable in terms of biophysical stability under heat stress.

[0197] Study 2: Screening of surfactants by stirring and freeze-thawing To further evaluate the effect of PS80 on stabilizing the ActRIIa-hFc fusion protein of SEQ ID NO:32 against agitation and freeze-thaw stresses, an additional PS80 concentration ranging study was conducted. In this study, the ActRIIa-hFc fusion protein of SEQ ID NO:32 was formulated at a concentration of 50 mg / mL with various levels of PS80 ranging from 0% to 0.05% in 10 mM citrate, pH 5.8, containing 8% (wt / vol) sucrose. These formulations were filled into Type I glass vials and agitated at 300 RPM for up to 7 days. Separately, the formulations were subjected to up to eight freeze-thaw cycles at temperatures ranging from -80°C to 25°C. The biophysical and chemical stability of SEQ ID NO:32 was monitored throughout the study by measuring the purity of the formulations by UP-SEC, the microparticles by HIAC and MFI, and the charge variants by desialylated iCIEF. Stability data are shown in Tables 12-19.

[0198] [Table 15]

[0199] [Table 16]

[0200] [Table 17]

[0201] [Table 18]

[0202] [Table 19]

[0203] [Table 20]

[0204] [Table 21]

[0205] [Table 22]

[0206] There were no significant differences in purity of any of the tested formulations compared to the control sample for HMW content, monomer content, and LMW content by UP-SEC. No discernible trends were observed for the tested formulation prototypes by CE-SDS or charge variants by desialylated iCIEF when compared to the control. Starting at least at 0.01% PS80, a slight decrease in 2 μm and 5 μm fine particles was observed with freeze-thaw stress compared to 0% PS80.

[0207] The results of this study showed that there were no significant differences between samples containing 0.01-0.05% PS-80 after exposure to agitation or freeze / thaw stress. These studies did not distinguish between various concentrations of PS80; however, 0.02% (weight / volume) PS80 was selected to ensure product quality and minimize stability risks while maintaining safety to support emerging supply chains.

[0208] Sucrose concentration range study To ensure that injections of the ActRIIa-hFc fusion protein of SEQ ID NO:32 had an appropriate osmolality (260-380 mOsm / kg) for parenteral administration, the range of sucrose concentrations outlined in Table 20 were prepared in 10 mM citrate at pH 5.8 and the osmolality measured. Based on these preparations, 8-10% (wt / vol) was determined to be appropriate for the formulation optimization studies described below.

[0209] [Table 23]

[0210] A formulation containing 50 mg / mL of ActRIIa-hFc fusion protein of SEQ ID NO: 32, 8%-10% (wt / vol) sucrose in 10 mM citrate, pH 5.8, containing 0.02% (wt / vol) PS80 was prepared and purity was assessed by HP-SEC to monitor physical stability under different conditions. For thermal stability in this study, the formulation was subjected to stability at 5°C and 25°C for up to 24 months and at 45°C for up to 3 months. Stability data from HP-SEC analysis are shown in Table 21.

[0211] [Table 24]

[0212] For agitation in this study, formulations were filled into 3 mL Type I glass vials and shaken at 250 rpm on a plate shaker for up to 7 days. The agitation data from HP-SEC analysis are shown in Table 22.

[0213] [Table 25]

[0214] Regarding photostability in this study, another set of vials was filled with 1 mL of each formulation and divided into two sets for incubation in a photostability chamber: one set exposed to 25 kLux visible light for 48 hours (1.2 mLuxhr), and the other set exposed to 20 W / m 2 of UV rays for 10 hours (200Wh / m 2 ) exposure. Two control vials were also prepared for each formulation: one was protected from light with aluminum foil in a light stability chamber and the other was kept in a refrigerator at 5°C. Photostability data from HP-SEC analysis are shown in Table 23.

[0215] [Table 26]

[0216] There were no significant differences in stability profiles among the various levels of sucrose, with each formulation retaining 99% monomer after 24 months at 5°C, 98% monomer after 24 months at 25°C, and 92% monomer after 3 months at 45°C. After 7 days of stirring, no clear trends in purity were observed for any of the formulations, with all formulations retaining >98% monomer. Regarding photostability in this study, each formulation lost approximately 16-17% of its monomer percentage after visible light exposure and approximately 9-10% after UV exposure, revealing potential degradation pathways.

[0217] In conclusion, sucrose concentrations of 8% to 10% (wt / vol) did not significantly affect the long-term stability profile of the ActRIIa-hFc fusion protein of SEQ ID NO: 32 when protected from light or under physical stress due to aggregation. It was also observed that the ActRIIa-hFc fusion protein of SEQ ID NO: 32 was prone to photoinstability, a mechanism that is too complex to properly outline. Therefore, a lyophilized formulation was developed to reduce the potential for light-induced chemical instability. A sucrose concentration of 8% (wt / vol) was considered sufficient for the development of the lyophilized formulation.

[0218] Protein concentration range study To assess the effect of increasing protein concentration on the long-term stability profile, a protein concentration range stability study was conducted. In this study, the ActRIIa-hFc fusion protein of SEQ ID NO: 32 was formulated in 10 mM citrate, 8% (wt / vol) sucrose, and 0.02% (wt / vol) PS80 at concentrations ranging from 50 mg / mL to 100 mg / mL. These formulations were filled into Type I glass vials and subjected to stability tests at 5°C for 24 months and at 45°C for 3 months. To monitor physical stability, the purity of the formulations was measured by HP-SEC analysis throughout the study and is summarized in Table 24.

[0219] [Table 27]

[0220] There were no significant differences in the 5°C stability profiles between the different protein concentrations, with all formulations retaining 99% of the monomer after 24 months at 5°C. However, the 100 mg / mL concentration showed slightly more degradation (88% monomer content) after 3 months under stress conditions (45°C) than the lower concentration, which retained 91%.

[0221] The results of this study showed that SEQ ID NO:32 can be concentrated up to 75 mg / mL without significantly affecting long-term stability, and this initial physical stability data suggests that concentrations as high as 100 mg / mL may have sufficient long-term stability based on data at 5°C.

[0222] Composition of the formulation Based on the formulation development work summarized above and the potential for charge instability during long-term storage at the recommended storage conditions for the ready-to-use solution, a lyophilized formulation with an optimized composition was developed. The target compositions of the ActRIIa-hFc fusion protein of SEQ ID NO:32 before and after lyophilization are outlined below.

[0223] [Table 28]

[0224] The compositions are further provided in two vial strengths, and when reconstituted with the specified amount of sterile water for injection (SWFI), each composition contains 50 mg / mL of ActRIIa ligand trap (active pharmaceutical ingredient) and the following excipients: 10 mM citrate, 8% (weight / volume) sucrose, 0.02% (weight / volume) polysorbate 80 (pH 5.8).

[0225] The components present in the lyophilized ActRIIa-hFc fusion protein composition are as follows: [Table 29]

[0226] For administration, the 45 mg vial lyophilisate is reconstituted with 1.0 mL of sterile water for injection (sWFI), and the 60 mg vial lyophilisate is reconstituted with 1.3 mL of sWFI, both resulting in an ActRIIa ligand trap solution of at least 50 mg / mL.

[0227] pH robustness of freeze-dried formulations A study was conducted to assess the robustness of key components in the formulation. This initial study was conducted to evaluate the effect of pH on the stability of lyophilized formulations. Formulations ranging from pH 5.3 to 6.3 were prepared at 50 mg protein / mL in 10 mM citrate buffer at pH 5.8 containing 8% (wt / vol) sucrose and 0.02% (wt / vol) polysorbate 80. These formulations (2 mL) were filled into 5 mL Type I glass vials, lyophilized using an early-stage lyophilization cycle, and subjected to stability conditions at 25°C, 45°C, and 60°C. To monitor physical stability, the purity of the formulations was measured by HP-SEC throughout the study and is summarized in Table 26.

[0228] [Table 30]

[0229] There was no significant effect of pH on the stability profile of SEQ ID NO:32 at either 25°C or 45°C.

[0230] The results of this study demonstrated that the pH range of 5.3 to 6.3 in the formulation composition did not significantly affect the biophysical stability of the ActRIIa-hFc fusion protein of SEQ ID NO: 32 under lyophilization conditions. Additionally, previous development studies demonstrated that variation in PS80 did not affect biophysical stability in solution form. Therefore, pH and PS80 were excluded as parameters in the formulation robustness study under lyophilization conditions.

[0231] Formulation robustness studies A formulation robustness study was performed to confirm the formulation's robustness by varying the protein and sucrose concentrations from their target levels. These two components were chosen for evaluation because they were shown to have the greatest impact on determining the stability of the ActRIIa-hFc fusion protein of SEQ ID NO:32 as a lyophilized product. Additionally, this laboratory-scale study utilized the primary packaging and lyophilization cycle parameters for manufacturing.

[0232] Formulations with protein concentrations ranging from 45 mg / mL (Lo) to 55 mg / mL (Hi) and sucrose concentrations ranging from 7% (wt / vol) (Lo) to 9% (wt / vol) (Hi), as outlined in Table 27, were prepared in 10 mM citrate buffer containing 0.02% (wt / vol) polysorbate 80. 60 mg / vial containing 1.45 mL of each formulation was filled into 2R Type I glass vials and lyophilized using the targeted commercial lyophilization cycle.

[0233] [Table 31]

[0234] All formulation prototypes were placed as lyophilized cakes under stability conditions of 5°C, 25°C, and 40°C for up to 18 months, 6 months, and 3 months, respectively. The prototypes were analyzed for purity by HP-SEC, fragmentation by CE-SDS NR, charge variants by desialylated iCIEF, particulates by HIAC, moisture content by Karl-Fischer pH, and reconstitution time, and are summarized in Tables 28-38.

[0235] [Table 32]

[0236] [Table 33]

[0237] Table 34

[0238] Table 35

[0239] Table 36

[0240] Table 37

[0241] Table 38

[0242] Table 39

[0243] Table 40

[0244] Table 41

[0245] Table 42

[0246] There were no clear trends in reconstitution time, pH, or water content of the prototypes screened in this study at any of the test conditions. There were no significant differences in the stability profiles of the formulation prototypes with respect to HMW, monomer, or LMW content at storage temperatures of 5°C and 25°C, which is consistent with the fragmentation patterns seen with CE-SDS non-reducing. As shown in Figure 12, Hi-Lo, a formulation with a high protein concentration (55 mg / mL) and low sucrose concentration (7%), had slightly more HMW species (~0.5%) on average than the other formulations, with only minor differences between the formulations at 45°C storage.

[0247] There were no significant differences in the stability profiles of each formulation with respect to the content of acidic, major, or basic species at each storage temperature. There was the expected variability in the various charge species, with acidic species varying between 20-30%, total major species between 55-70%, and basic species between 10-20%.

[0248] With a nominal fill volume of 1.45 mL for 60 mg / vial, particulate matter / mL monitored by HIAC was greater than or equal to 10 μm and greater than or equal to 25 μm in size, with no discernible trends in any of the formulation prototypes, and was within USP limits under all conditions. <787> In conclusion, this study demonstrated the robustness of the formulation composition within the range of potential compositional variability (i.e., ±5 mg / mL of the target protein concentration of 50 mg / mL and ±1% of the target sucrose concentration of 8% (wt / vol)).

[0249] array [Table 43] TIFF2026501040000045.tif245169TIFF2026501040000046.tif250168TIFF2026501040000047.tif246169TIFF2026501040000048.tif25169

[0250] Preferred methods and materials are described herein, although methods and materials similar or equivalent to those described herein can 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 a human ActRIIa fusion protein, a buffer, a surfactant, and a stabilizer, wherein the ActRIIa fusion protein comprises the amino acid sequence of SEQ ID NO:32 or SEQ ID NO:

41.

2. 2. The pharmaceutical formulation of claim 1, wherein the buffering agent is selected from the group consisting of sodium citrate, succinate, and histidine.

3. 3. The pharmaceutical formulation of claim 1, wherein the buffering agent is present in an amount of from 4 mM to 50 mM.

4. 3. The pharmaceutical formulation of claim 1, wherein the buffering agent is present in an amount of about 10 mM.

5. 3. The pharmaceutical formulation of claim 1, wherein the buffer is a citrate buffer at a pH of about 5.5 to about 6.

5.

6. 3. The pharmaceutical formulation of claim 1, wherein the buffer is a succinate buffer at a pH of about 5.5 to about 6.

5.

7. 3. The pharmaceutical formulation of claim 1, wherein the buffer is a histidine buffer at a pH of about 5.5 to about 6.

0.

8. 3. The pharmaceutical formulation of claim 1, wherein the buffer is a citrate buffer at pH 5.

8.

9. 3. The pharmaceutical formulation of claim 1, wherein the buffer is a succinate buffer at pH 5.

8.

10. 9. The pharmaceutical formulation of claim 5 or 8, wherein the citrate buffer comprises citric acid monohydrate and trisodium citrate dihydrate.

11. The pharmaceutical formulation according to any one of claims 1 to 10, wherein the surfactant is polysorbate 80 or polysorbate 20.

12. 12. The pharmaceutical formulation of claim 11, wherein the surfactant is present in an amount of about 0.01 to about 0.05 (wt / vol).

13. 12. The pharmaceutical formulation of claim 11, wherein the surfactant is present in an amount of about 0.02% (weight / volume).

14. The pharmaceutical formulation according to any one of claims 1 to 13, wherein the stabilizer is sucrose.

15. 15. The pharmaceutical formulation of claim 14, wherein sucrose is present in an amount of about 8% (wt / vol) to about 10% (wt / vol).

16. 15. The pharmaceutical formulation of claim 14, wherein sucrose is present in an amount of about 8% (weight / volume).

17. 17. The pharmaceutical formulation of any one of claims 1 to 16, wherein the human ActRIIa fusion protein is present in an amount of about 75 mg / mL or less.

18. 17. The pharmaceutical formulation of any one of claims 1 to 16, wherein the human ActRIIa fusion protein is present in an amount of about 50 mg / mL.

19. The pharmaceutical formulation of any one of claims 1 to 18, wherein the pharmaceutical formulation is salt-free.

20. 20. The pharmaceutical formulation of any one of claims 1 to 19, wherein the ActRIIa fusion protein is sotatercept.

21. 20. The pharmaceutical formulation of any one of claims 1 to 19, wherein the ActRIIa fusion protein comprises the amino acid sequence of SEQ ID NO:

32.

22. 20. The pharmaceutical preparation of any one of claims 1 to 19, wherein the ActRIIa fusion protein comprises the amino acid sequence of SEQ ID NO:

41.

23. 20. The pharmaceutical formulation of any one of claims 1 to 19, wherein the formulation comprises a first ActRIIa fusion protein comprising the amino acid sequence of SEQ ID NO: 32 and a second ActRIIa fusion protein comprising the amino acid sequence of SEQ ID NO:

41.

24. A freeze-dried pharmaceutical formulation produced by freeze-drying the formulation according to any one of claims 1 to 23.

25. 25. The lyophilized pharmaceutical formulation of claim 24, wherein the human ActRIIa fusion protein is provided in an amount of 45 mg / vial or 60 mg / vial.

26. 25. A reconstituted pharmaceutical formulation produced by reconstituting the lyophilized pharmaceutical formulation of claim 24.

27. 27. The reconstituted pharmaceutical formulation of claim 26, wherein the lyophilized pharmaceutical formulation is reconstituted with sterile water for injection.

28. 27. The reconstituted pharmaceutical formulation of claim 26, wherein the lyophilized formulation is reconstituted with sterile water for injection to a final protein concentration of about 45 mg / mL to 55 mg / mL.

29. 30. The reconstituted pharmaceutical formulation of claim 28, wherein the lyophilized formulation is reconstituted with sterile water for injection to a final protein concentration of about 50 mg / mL.

30. 27. The reconstituted pharmaceutical formulation of claim 26, wherein the lyophilized formulation is reconstituted with about 0.5 mL to 1.8 mL of sterile water for injection.

31. 27. The reconstituted pharmaceutical formulation of claim 26, wherein the lyophilized formulation is provided in an amount of 45 mg / vial, and the formulation is reconstituted with 0.65 mL to 0.75 mL of sterile water for injection.

32. 27. The reconstituted pharmaceutical formulation of claim 26, wherein the lyophilized formulation is provided in an amount of 60 mg / vial, and the formulation is reconstituted with 0.65 mL to 1.75 mL of sterile water for injection.

33. A lyophilized pharmaceutical formulation comprising a human ActRIIa fusion protein, a buffer, a surfactant and a stabilizer, wherein the buffer is selected to be physiologically compatible and to maintain a pH of 5.8 when reconstituted with sterile water for injection, and wherein the human ActRIIa fusion protein comprises the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO:

41.

34. 34. The lyophilized pharmaceutical formulation of claim 33, wherein the buffer comprises citrate.

35. 35. The lyophilized pharmaceutical formulation of claim 33 or 34, wherein the stabilizer is sucrose.

36. The freeze-dried pharmaceutical formulation according to any one of claims 33 to 35, wherein the surfactant is polysorbate 20 or polysorbate 80.

37. 37. The lyophilized pharmaceutical formulation of claim 36, wherein the surfactant is polysorbate 80.

38. A lyophilized pharmaceutical formulation comprising a human ActRIIa fusion protein, citrate, polysorbate 80 and sucrose, wherein the ActRIIa fusion protein comprises the amino acid sequence of SEQ ID NO:32 or SEQ ID NO:

41.

39. A lyophilized pharmaceutical formulation comprising 55.0 mg of human ActRIIa fusion protein, 0.48 mg of citric acid monohydrate, 2.56 mg of trisodium citrate dihydrate, 0.22 mg of polysorbate 80 and 88.0 mg of sucrose, wherein the human ActRIIa fusion protein comprises the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO:

41.

40. A lyophilized pharmaceutical formulation comprising 72.5 mg of human ActRIIa fusion protein, 0.64 mg of citric acid monohydrate, 3.37 mg of trisodium citrate dihydrate, 0.29 mg of polysorbate 80 and 116.0 mg of sucrose, wherein the human ActRIIa fusion protein comprises the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO:

41.

41. A reconstituted pharmaceutical formulation comprising 50 mg / mL human ActRIIa fusion protein, 10 mM citrate, 0.2 mg / mL polysorbate 80 and 80 mg / mL sucrose at pH 5.8, wherein the human ActRIIa fusion protein comprises the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO:

41.

42. The pharmaceutical formulation of any one of claims 1 to 41, wherein the pharmaceutical formulation is administered by subcutaneous injection.

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

44. 44. A method of treating pulmonary arterial hypertension (PAH) in a human patient in need thereof, comprising administering to said patient a pharmaceutical formulation according to any one of claims 1 to 43.

45. 41. A method of treating pulmonary arterial hypertension (PAH) in a human patient in need thereof, comprising reconstituting the lyophilized pharmaceutical formulation of any one of claims 24, 25, or 33-40 to form a reconstituted formulation, and administering the reconstituted formulation to the patient.

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

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