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 benefits of U.S. Provisional Application No. 63 / 451,198, filed on 9 March 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] References to electronically submitted sequence listings This application includes a sequence listing submitted electronically in XML format, which is incorporated herein by reference in its entirety. The XML file was created on January 29, 2024, and its filename is 1848179-0002-169-WO1_SL.XML, with a size of 37,058.

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

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

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

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

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

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

[0009] summary Provided herein are a recombinant fusion protein comprising a human activin receptor type IIA (ActRIIa) extracellular domain (ECD) or a variant thereof linked to a constant domain of an immunoglobulin such as a human IgG1 Fc domain, and one or more pharmaceutical excipients and / or additives, 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 is also one of amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 12 The ActRIIa protein includes an amino acid sequence that is at least 70% (e.g., at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence ending at any one of 8, 129, 130, 131, 132, 133, 134, or 135. In some embodiments, the ActRIIa protein includes an amino acid sequence that is at least 70% (e.g., at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the ActRIIa protein contains an amino acid sequence that is at least 70% (e.g., at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the ActRIIa protein is a fusion protein comprising the ActRIIa extracellular domain and one or more protein domains heterologous to ActRIIa. In some embodiments, the ActRIIa protein is a fusion protein comprising the Fc domain of an immunoglobulin. In some embodiments, the Fc domain of the immunoglobulin is the Fc domain of IgG1 immunoglobulin. In some embodiments, the ActRIIa fusion protein further includes a linker domain positioned between the ActRIIa protein domain and one or more heterologous domains (e.g., 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 contains an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 32. In some embodiments, the ActRIIa fusion protein contains the amino acid sequence of SEQ ID NO: 32. In some embodiments, the ActRIIa fusion protein consists of the amino acid sequence of SEQ ID NO: 32. In some embodiments, the ActRIIa fusion protein 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 described in SEQ ID NO: 32, where the sequence lacks the C-terminal lysine residue of SEQ ID NO: 32. In some embodiments, the ActRIIa fusion protein variant lacking the C-terminal lysine residue contains or consists of the amino acid sequence of SEQ ID NO: 41. In some embodiments, the ActRIIa fusion protein is part of a homodimeric protein complex. In some embodiments, the ActRIIa fusion protein is glycosylated. In some embodiments, the ActRIIa fusion protein has a glycosylation pattern obtained by expression in Chinese hamster ovary cells.

[0011] The pharmaceutical formulations described herein comprise an ActRIIa fusion protein and one or more pharmaceutical excipients and / or additives. 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 are buffers. In some embodiments, the buffer is selected to be physiologically compatible and to maintain a pH of 2 or higher. In some embodiments, the buffer is selected to be physiologically compatible and to be able to maintain the reconstituted lyophilized solution described herein at a pH of 2 or higher. In some embodiments, the buffer 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 buffer is selected to be physiologically compatible and to maintain the pH of the pharmaceutical formulation at 5 to 7 (i.e., 5.0 to 7.0). In some embodiments, the buffer is selected to be physiologically compatible and to maintain the pH of the pharmaceutical formulation at 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0. In some embodiments, the buffer is selected to be physiologically compatible and to maintain the pH of the pharmaceutical formulation at 5.5 to 6.5 (i.e., 5.5 to 6.5). In some embodiments, the buffer 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, amino acids, 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, if 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 buffer is present in an amount of 4 mM to 50 mM. In some embodiments, the buffer is present in 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 contains at least 10 mM of the buffer.

[0014] In some embodiments, the buffer is a citrate buffer that maintains the pH of the pharmaceutical formulation at approximately pH 5.5 to approximately pH 6.5. In some embodiments, the buffer is a succinate buffer that maintains the pH of the pharmaceutical formulation at approximately pH 5.5 to approximately pH 6.5. In some embodiments, the buffer is a histidine buffer that maintains the pH of the pharmaceutical formulation at approximately pH 5.5 to approximately pH 6.0. In some embodiments, the buffer is a citrate buffer that maintains the pH of the pharmaceutical formulation at approximately pH 5.8. In some embodiments, the buffer is a succinate buffer that maintains the pH of the pharmaceutical formulation at approximately 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 a pH of about 5.8. In some embodiments, the buffer is a 10 mM succinate buffer that maintains the pH of the pharmaceutical formulation at a pH of about 5.8.

[0016] In some embodiments, one or more of the pharmaceutical additives and / or excipients are surfactants. In some embodiments, the surfactant is selected from the group consisting of: sodium lauryl sulfate, sodium dioctyl sulfosuccinate and sodium dioctyl sulfonate, chenodeoxycholic acid, sodium N-lauroyl sarcosinate, lithium dodecyl sulfate, sodium 1-octanesulfonate, sodium cholate hydrate, sodium deoxycholate and sodium glycodeoxycholate, benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride monohydrate, hexadecyltrimethylammonium bromide, CHAPS, CHAPSO, SB3-10, SB3-12, digitonin, Triton X-100, Triton X-114, TWEEN®-20, TWEEN®-80, lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 40, 50 and 60, glyceryl 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 to 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 a surfactant. In some embodiments, the pharmaceutical formulation comprises 0.01 to 0.05% (weight / volume) of a surfactant. In some embodiments, the pharmaceutical formulation comprises at least 0.02% (weight / volume) of a surfactant. In some embodiments, the pharmaceutical formulation comprises 0.02% (weight / volume) of a surfactant. In some embodiments, the pharmaceutical formulation comprises 0.02% of polysorbate 80 or polysorbate 20.

[0018] In some embodiments, one or more of the pharmaceutical additives and / or excipients are stabilizers. 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 2–16% (weight / volume). In some embodiments, the pharmaceutical formulation contains about 8%–10% (weight / volume) of the stabilizer. In some embodiments, the pharmaceutical formulation contains at least 0.005% (weight / volume), 0.01% (weight / volume), 0.02% (weight / volume), 0.03% (weight / volume), 0.05% (weight / volume), 0.06% (weight / volume), 0.07% (weight / volume), 0.08% (weight / volume), 0.09% (weight / volume), 0.1% (weight / volume), 0.5% (weight / volume), 0.7% (weight / volume), 0.8% (weight / volume), 0.9% (weight / volume), 1.0% (weight / volume), 1.2% (weight / volume), and 1.5%. The product contains a stabilizer in the following concentrations: (weight / volume), 1.7% (weight / volume), 2% (weight / volume), 3% (weight / volume), 4% (weight / volume), 5% (weight / volume), 6% (weight / volume), 7% (weight / volume), 8% (weight / volume), 9% (weight / volume), 10% (weight / volume), 11% (weight / volume), 12% (weight / volume), 13% (weight / volume), 14% (weight / volume), 15% (weight / volume), 16% (weight / volume), 17% (weight / volume), 18% (weight / volume), 19% (weight / volume), or 20% (weight / volume). In some embodiments, the stabilizer is present at a concentration of at least 8% (weight / volume).

[0020] In certain embodiments, the pharmaceutical formulations described herein do not contain salt.

[0021] Furthermore, this specification also describes lyophilized pharmaceutical preparations produced by freeze-drying the liquid preparations described herein. This specification also describes pharmaceutical preparations, which are reconstituted from the lyophilized preparations.

[0022] In some embodiments, the lyophilized pharmaceutical preparation is reconstituted using sterile water for injection. In some embodiments, the lyophilized pharmaceutical preparation is reconstituted using sterile water for injection to a final protein concentration of 10 to 100 mg / mL. In some embodiments, the lyophilized pharmaceutical preparation is reconstituted using 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 approximately 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 approximately 75 mg / mL or less. In certain embodiments, the human ActRIIa fusion protein is present in an amount of approximately 50 mg / mL.

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

[0024] In some embodiments, the lyophilized pharmaceutical preparation is reconstituted using sterile water for injection. In some embodiments, the lyophilized pharmaceutical preparation is reconstituted using sterile water for injection to a final protein concentration of approximately 45 mg / mL, 46 mg / mL, 47 mg / mL, 48 mg / mL, 49 mg / mL, 50 mg / mL, 51 mg / mL, 52 mg / mL, 53 mg / mL, 54 mg / mL, or 55 mg / mL. In some embodiments, the lyophilized pharmaceutical preparation is reconstituted using sterile water for injection to a final protein concentration of approximately 50 mg / mL.

[0025] In certain embodiments, the lyophilized formulation is reconstituted using approximately 0.5 mL to 1.8 mL of sterile water for injection. In some embodiments, the lyophilized pharmaceutical preparation is reconstituted using 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, 0.9 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 ActRIIa fusion protein provided in an amount of 45 mg / vial, and the formulation is reconstituted using 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 ActRIIa fusion protein provided in an amount of 45 mg / vial, and the formulation is reconstituted using 1.1 mL of sterile water for injection. In some embodiments, the lyophilized pharmaceutical formulation comprises ActRIIa fusion protein provided in an amount of 60 mg / vial, and the formulation is reconstituted using 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 contains 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, where 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, where 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 the 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 when reconstituted with sterile water for injection. In certain embodiments, the buffer comprises a 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 when reconstituted with sterile water for injection.

[0028] In certain embodiments, the lyophilized pharmaceutical formulation comprises a human ActRIIa fusion protein containing the amino acid sequence of SEQ ID NO: 32 or 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 containing the amino acid sequence of SEQ ID NO: 32 and a human ActRIIa fusion protein containing 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 containing 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., containing the amino acid sequence of SEQ ID NO: 41), 0.48 mg of citrate 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 human ActRIIa fusion protein containing the amino acid sequence of SEQ ID NO: 32 and human ActRIIa fusion protein containing the amino acid sequence of SEQ ID NO: 41. In some embodiments, the lyophilized pharmaceutical formulation contains sotatercept.

[0030] In certain embodiments, the lyophilized pharmaceutical formulation comprises 72.5 mg of human ActRIIa fusion protein containing SEQ ID NO: 32 or a variant of SEQ ID NO: 32 lacking a C-terminal lysine residue (SEQ ID NO: 41), 0.64 mg of citrate 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 human ActRIIa fusion protein containing the amino acid sequence of SEQ ID NO: 32 and human ActRIIa fusion protein containing the amino acid sequence of SEQ ID NO: 41. In some embodiments, the lyophilized pharmaceutical formulation contains sotatercept.

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

[0032] In certain embodiments, the pharmaceutical formulation described herein comprises a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 lacking a C-terminal lysine residue (SEQ ID NO: 41). In certain embodiments, the pharmaceutical formulation described herein comprises a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 lacking a C-terminal lysine residue (SEQ ID NO: 41), wherein the mixture is in the following proportions by weight: 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 6 Includes SEQ ID NO: 32 with values ​​of 1%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

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

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

[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 (SEQ ID NO: 41) lacking a C-terminal lysine residue, wherein the mixture comprises, by weight, approximately 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, Includes SEQ ID NO: 41 with 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In certain embodiments, the pharmaceutical formulation described herein includes 100% by weight of SEQ ID NO: 32. In certain embodiments, the pharmaceutical formulation described herein includes 100% by weight of SEQ ID NO: 41.

[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] Furthermore, this specification also describes a method for treating pulmonary arterial hypertension (PAH), which includes administering a pharmaceutical preparation described herein to a patient in need of treatment.

[0038] Furthermore, this specification also describes the use of pharmaceutical formulations for the treatment of pulmonary arterial hypertension (PAH), wherein such use includes administering the pharmaceutical formulations described herein to a patient in need of such treatment.

[0039] In some embodiments, the disclosure provides a lyophilized pharmaceutical formulation for treating pulmonary arterial hypertension in a subject of interest, wherein the lyophilized pharmaceutical formulation comprises the human ActRIIa fusion protein of SEQ ID NO: 32, citrate, polysorbate 80, and sucrose, wherein the formulation is lyophilized. In some embodiments, the vial comprises a lyophilized pharmaceutical formulation comprising the human ActRIIa fusion protein of SEQ ID NO: 32, citrate monohydrate, trisodium citrate dihydrate, polysorbate 80, and sucrose, wherein the formulation is lyophilized. In some embodiments, the vial comprises a lyophilized pharmaceutical formulation comprising 55 mg of protein (i.e., ActRIIa fusion protein), 0.48 mg of citrate 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 citrate 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 a 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 containing the amino acid sequence of SEQ ID NO: 32, 10 mM citrate, 0.2 mg / mL of polysorbate 80, and 8% by weight of sucrose. In some embodiments, the human ActRIIa fusion protein contains or consists of the amino acid sequence of 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.

[0041] In some embodiments, the reconstituted pharmaceutical formulation contains 50 mg / mL of ActRIIa fusion protein containing 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 contains 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 contains 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 drawing]

[0042] [Figure 1]Figure 1 shows the components of a kit comprising a lyophilized pharmaceutical formulation containing the ActRIIa fusion protein and an injection device. Vial (1) contains the lyophilized pharmaceutical formulation containing the ActRIIa fusion protein, a reconstituted sterile injection solution, or a sterile injection solution. A pre-filled syringe (2) containing the reconstituted solution is used to reconstitute the lyophilized pharmaceutical formulation containing the ActRIIa fusion protein from (1) into the sterile injection solution. A vial adapter (3) connects vial (1) to pre-filled syringe (2) by having one end attached to the vial and the other end attached to the pre-filled syringe. A syringe (4) and injection needle (5) are provided for administering the sterile injection solution. Swab wipes (6) are provided for sterilizing the individual components of the kit. [Figure 2] Figure 2 shows the multiple sequence alignment of ActRIIa proteins from various vertebrates and 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, distinct peak visualized by a sizing column (upper panel) and Coomassie-stained SDS-PAGE (lower panel) (left lane: molecular weight standard; right lane: ActRIIa-hFc). [Figure 3B] Figures 3A and 3B show the purification of ActRIIa-hFc expressed in CHO cells. The protein is purified as a single, distinct peak visualized by a sizing column (upper panel) and Coomassie-stained SDS-PAGE (lower panel) (left lane: molecular weight standard; right lane: ActRIIa-hFc). [Figure 4A] Figures 4A and 4B show the binding of ActRIIa-hFc to activin (upper panel) and GDF-11 (lower panel b) as measured by the Biacore™ assay. [Figure 4B] Figures 4A and 4B show the binding of ActRIIa-hFc to activin (upper panel) and GDF-11 (lower panel b) as measured by the Biacore™ assay. [Figure 5] Figure 5 shows the effect of pH and buffer on the percentage of monomer species (%) after 6M at 25°C using HP-SEC. [Figure 6] Figure 6 shows the effect of pH and buffer on the binding activity of Biacore™ after stabilization at 6M and 25°C. [Figure 7] Figure 7 shows the effects of buffering agent and pH on %HMW species, monomer species, and %LMW species after 3 months of HP-SEC, based on stability. [Figure 8] Figure 8 shows the effects of pH and buffer on the % minor and % major peaks measured by CE-SDS NR after 1 month of stability. [Figure 9] Figure 9 shows the effects of pH and buffering on % acidic species, % basic species, and % total major species after 3 months of stability following desialized icIEF. [Figure 10] Figure 10 shows the effect of HP-SEC on concentration of % monomer species. [Figure 11] Figure 11 shows the effect of buffer and concentration on % monomer species based on stability using HP-SEC. [Figure 12] Figure 12 shows the effect of protein and sucrose concentrations on purity, based on stability, as measured by HP-SEC. [Modes for carrying out the invention]

[0043] Detailed explanation definition The terms used herein generally have their common meanings in the art within the context of this disclosure and in the specific context in which each term is used. Certain terms are discussed below or elsewhere in this specification to provide additional guidance to those skilled in the art when describing the formulations and methods of this disclosure, as well as their manufacturing methods and uses. The scope or meaning of any term in its use will become apparent from the specific context in which it is used.

[0044] "About" and "approximately" generally refer to the acceptable degree of error in a measured quantity, taking into account the nature or precision of the measurement. Typically, an exemplary degree of error is 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” may mean a value within a range of decimal places of a given value, preferably within five times, and more preferably within two times. Unless otherwise specified, the numerical quantities shown herein are approximate values; that is, the terms “about” and “approximately” mean that they can be estimated unless explicitly stated otherwise.

[0046] The terms “a” and “an” encompass multiple references unless the context in which the terms are used clearly indicates a different context. The terms “a” (or “an”) and “one or more” and “at least one” are interchangeable within this specification. Furthermore, “and / or” is considered a specific disclosure for each of two or more designated features or components, with or without the other, wherever it is used within this specification. Accordingly, within this specification, the term “and / or” as used in phrases such as “A and / or B” is intended to encompass “A and B,” “A or B,” “A (alone),” and “B (alone).” Similarly, the term “and / or” as used in phrases such as “A, B and / or C” is intended to encompass each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0047] Numerical ranges disclosed herein encompass the numerical values ​​that define those ranges. For example, a pH of 5.0 to 7.0 includes values ​​of pH 5.0, pH 7.0, and values ​​between 5.0 and 7.0.

[0048] The proteins disclosed herein may include amino acid sequences that do not exist in nature. Such variants will inevitably have less than 100% sequence identity or similarity to the starting molecule. In certain embodiments, the variant has an amino acid sequence that has, for example, about 75% to less than 100% amino acid sequence identity or similarity, more preferably about 80% to less than 100%, even more preferably about 85% to less than 100%, even more preferably about 90% to less than 100% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), and most preferably about 95% to less than 100% amino acid sequence identity or similarity over the length of the variant molecule.

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

[0050] In certain embodiments, the disclosure relates to lyophilized pharmaceutical formulations comprising the extracellular domain (ECD) or derivative thereof of a human ActRIIa protein ligated to a constant domain of an immunoglobulin, such as the human IgG1 Fc domain, for reconstitution in a sterile solution for injection. During lyophilization, the liquid formulation is converted from an aqueous phase to an amorphous solid phase, which is thought to protect the protein from chemical and / or steric instability. Lyophilization is carried out using techniques common in the art, and the lyophilized formulation is optimized with respect to stability, shelf life, and to reduce the levels 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 has helped stabilize the protein against manufacturing, transport, and storage stresses. Excipients and additives used in lyophilized formulations are essential components of the formulation and therefore need to be safe and well-tolerated by patients. In the case of protein pharmaceuticals, the selection 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 to enable the delivery of high-concentration protein formulations and to improve patient convenience. The excipients and additives for formulations 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 fir Parenteral Formulations (1998), PDA J. Pharm. Sci. Technology, 52:238-311".

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

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

[0053] In certain embodiments, the lyophilized pharmaceutical formulation contains an amount of ActRIIa fusion protein in the form of 45 mg / vial or 60 mg / vial and is reconstituted with sterile water for injection. In even more specific embodiments, the lyophilized pharmaceutical formulation provided in the respective amounts of 45 mg / vial or 60 mg / vial is reconstituted with sterile water for injection to a final concentration of reconstituted ActRIIa fusion protein of approximately 45 mg / mL, 46 mg / mL, 47 mg / mL, 48 mg / mL, 49 mg / mL, 50 mg / mL, 51 mg / mL, 52 mg / mL, 53 mg / mL, 54 mg / mL, or 55 mg / mL. In more specific embodiments, the lyophilized pharmaceutical formulations provided in 45 mg / vial or 60 mg / vial are reconstituted with sterile water for injection to a final concentration of reconstituted ActRIIa fusion protein of 45 mg / mL, 46 mg / mL, 47 mg / mL, 48 mg / mL, 49 mg / mL, 50 mg / mL, 51 mg / mL, 52 mg / mL, 53 mg / mL, 54 mg / mL, or 55 mg / mL. In some embodiments, the lyophilized pharmaceutical formulations provided in 45 mg / vial or 60 mg / vial are reconstituted with sterile water for injection to a final concentration of reconstituted ActRIIa fusion protein of approximately 50 mg / mL. In some embodiments, the lyophilized pharmaceutical formulations provided in 45 mg / vial or 60 mg / vial are reconstituted with sterile water for injection to a final concentration of reconstituted ActRIIa fusion protein of 50 mg / mL.

[0054] In some embodiments, the lyophilized pharmaceutical formulations provided in 45 mg / vial or 60 mg / vial are reconstituted using 0.5 to 2 mL of sterile water for injection. In some embodiments, the lyophilized pharmaceutical formulations in 45 mg / vial or 60 mg / vial are reconstituted using 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 It is reconstituted using 1 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 formulations provided in 45 mg / vial or 60 mg / vial are available in 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, and 1.25 mL. It is reconstituted using 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 in 45 mg / vial is reconstituted using 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 of sterile water for injection. In some embodiments, the lyophilized pharmaceutical formulation provided in 45 mg / vial is reconstituted using 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 in 45 mg / vial is reconstituted using 1.1 mL of sterile water for injection. In some embodiments, the lyophilized pharmaceutical preparation provided in 60 mg / vial is reconstituted using 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 of sterile water for injection. In some embodiments, the lyophilized pharmaceutical preparation provided in 60 mg / vial is reconstituted using 1 mL, 1.1 mL, 1.2 mL, or 1.3 mL of sterile water for injection. In some embodiments, the lyophilized pharmaceutical preparation provided in 60 mg / vial is reconstituted using 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 comprise buffers, stabilizers, and surfactants. Buffers may be selected to maintain the pH of the formulation during processing and reconstitution. Stabilizers may include cryoprotectants and lioprotectants such as polyols, sugars, and polysaccharides. Stabilizers may be selected to protect the formulation from freeze / thaw cycle stress and to stabilize the formulation in a lyophilized state. Surfactants may be selected based on their ability to function as emulsifiers, wetting agents, solubilizers, and / or dispersants.

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

[0057] Those skilled in the art will recognize that the concentrations of excipients described herein share interdependence within a particular formulation. For example, the concentration of a volume extender is, in one embodiment, lower, for instance, when the protein concentration is high. Excipients and other additives are added to impart or improve manufacturability and / or the quality of the final product, such as the stability and delivery of a drug product (e.g., a protein). The formulations provided herein include appropriate excipients that enhance stability and safety.

[0058] cushioning agent Typically, the stability of pharmacologically active protein formulations is observed to be maximized within a narrow pH range. This pH range, which exhibits optimal stability, needs to be identified early in pre-formulation studies. Several approaches are useful in this endeavor, including accelerated stability studies and calorific screening studies (Remmele RL Jr., et al., Biochemistry, 38(16): 5241-7 (1999)). Once the formulation is finalized, the protein must be manufactured and maintained throughout its storage period. Therefore, buffers are almost always used to control the pH of the formulation.

[0059] When selecting a buffer, several factors must be considered. First and foremost, the type and concentration of the buffer must be determined based on the buffer's pKa and the desired pH of the formulation. Equally important is ensuring that the buffer is reliably compatible with the protein and other formulation excipients, and that it does not catalyze any degradation reactions. A third important aspect to consider is the tingling and irritation that the buffer may induce upon administration. The likelihood of tingling and irritation is greater for drugs administered via subcutaneous (SC) or intramuscular (IM) routes, where the drug solution remains at the administration site for a relatively longer period, than for drugs administered via IV routes, where the formulation is rapidly diluted into the bloodstream upon administration. For formulations administered by direct IV infusion, it is necessary to monitor the total amount of buffer (and any other formulation components).

[0060] Buffers for lyophilized formulations require further consideration. For example, certain buffers, such as sodium phosphate, tend to crystallize from the amorphous phase of proteins 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 (this includes, but is 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 buffering agent includes citrate, succinate, or histidine.

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

[0062] In certain embodiments, the buffer comprises citric acid monohydrate and trisodium citrate dihydrate, where 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 buffer comprises citric acid monohydrate and trisodium citrate dihydrate, where 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 when reconstituted with sterile water for injection. In another embodiment, the pH of the solution is greater than 2. In yet 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, and 9.0. In some embodiments, when reconstituted in solution, the pH of the solution is pH 5 to 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 contains citrate, and the pH of the reconstituted solution is about 5.5 to about 6.5. In some embodiments, the buffer contains citrate, and the pH of the reconstituted solution is about 5.3 to about 6.3. In some embodiments, the buffer contains citrate, and the pH of the reconstituted solution is about 5.8.

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

[0067] The pH buffer compound can be present in any amount suitable for maintaining the pH of the formulation at a predetermined level. Crystallization and pH shift can be avoided if an appropriate low level of buffer is used. In one embodiment, the concentration of the buffer is 0.1 mM to 500 mM (1 M). For example, the buffer is intended to be at least 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 500 mM. In certain embodiments, the buffer is 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 500 mM. In one embodiment, the concentration of the buffer is 0.1 mM to 20 mM. In certain embodiments, the amount of the buffer is 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mM. In certain embodiments, the buffer 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 approximately pH 5.5 to approximately pH 6.5. In some embodiments, the buffer is a 10 mM succinate buffer that maintains the pH of the pharmaceutical formulation at approximately pH 5.5 to approximately pH 6.5. In some embodiments, the buffer is a 10 mM histidine buffer that maintains the pH of the pharmaceutical formulation at approximately pH 5.5 to approximately pH 6.0. In some embodiments, the buffer is a 10 mM citrate buffer that maintains the pH of the pharmaceutical formulation at approximately pH 5.8. In some embodiments, the buffer is a 10 mM succinate buffer that maintains the pH of the pharmaceutical formulation at approximately pH 5.8.

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

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

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

[0072] In certain embodiments, the lyophilized formulations described herein contain 50 mg to 150 mg of stabilizer. In certain embodiments, the lyophilized formulations described herein contain 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, or 150 mg of stabilizer. In certain embodiments, the lyophilized formulations described herein contain 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 of stabilizer.

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

[0074] In certain embodiments, the reconstituted formulation contains a stabilizer at a concentration of approximately 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% (by weight / volume).

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

[0076] surfactants In certain embodiments, the pharmaceutical formulations provided herein may further include surfactants. Surfactants are commonly used in protein formulations to prevent surface-induced degradation. Surfactants are amphiphilic molecules that have the ability to compete with proteins for interfacial location (and / or to facilitate the proper refolding of structurally altered protein molecules). The hydrophobic portion of the surfactant molecule occupies the interfacial location (e.g., air / liquid), while the hydrophilic portion remains oriented toward the bulk solvent. At sufficient concentrations (typically near the critical micelle concentration of the surfactant), the surface layer of the surfactant molecule plays a role in preventing protein molecules from adsorbing to the interface. This minimizes surface-induced degradation. Surfactants considered herein include, but are not limited to, fatty acid esters of sorbitan polyethoxylate, i.e., polysorbate 20 and polysorbate 80. These two types differ only in the length of the aliphatic chains that confer hydrophobicity to the molecules, C-12 and C-18, respectively. Therefore, polysorbate 80 has higher surface activity and a lower critical micelle concentration than polysorbate 20.

[0077] Surfactants also affect the thermodynamic structural stability of proteins. Nonionic surfactants are generally useful in stabilizing proteins. Ionic surfactants (detergents) typically destabilize proteins. Here again, the effect of a given surfactant excipient is specific to the protein. For example, polysorbates have been shown to decrease the stability of some proteins and increase the stability of others. The destabilization of proteins by surfactants can be reasonably explained in terms of the hydrophobic tails of surfactant molecules that may be involved in specific binding to proteins in a partially or completely unfolded state. This type of interaction may cause a conformational shift to a more expanded protein state (i.e., increasing the exposure of hydrophobic portions of the protein molecule that complement the binding with the polysorbate). Alternatively, if the native state of a protein exhibits some hydrophobic surfaces, a surfactant that binds to that native state may stabilize its conformation. Another aspect of polysorbates is that they are inherently susceptible to oxidative degradation. In many cases, the raw materials contain enough peroxides to cause oxidation of the side chains of protein residues (especially methionine). The potential for oxidative damage resulting from the addition of stabilizers highlights the need to use the lowest effective concentration of excipients in formulations. In the case of surfactants, the effective concentration for a given protein depends on the stabilization mechanism.

[0078] Surfactants are also added in appropriate amounts to prevent surface-related aggregation during freezing and drying (Chang, B, J. Pharm. Sci. 85:1325, (1996)). Therefore, exemplary surfactants include, but are not limited to, anionic, cationic, nonionic, zwitterionic, and amphoteric surfactants, which include surfactants derived from naturally occurring amino acids. Anionic surfactants include, but are not limited to, sodium lauryl sulfate, sodium dioctyl sulfosuccinate and sodium dioctyl sulfonate, chenodeoxycholic acid, sodium N-lauroyl sarcosinate, lithium dodecyl sulfate, sodium 1-octanesulfonate, sodium cholate hydrate, sodium deoxycholate, and sodium glycodeoxycholate. Cationic surfactants include, but are not limited to, benzalkonium chloride or benzethonium chloride, cetylpyridinium chloride monohydrate, and hexadecyltrimethylammonium bromide. Examples of zwitterionic surfactants include, but are not limited to, CHAPS, CHAPSO, SB3-10, and SB3-12. Examples of nonionic surfactants include, but are not limited to, digitonin, Triton X-100, Triton X-114, TWEEN(registered trademark)-20, and TWEEN(registered trademark)-80. The surfactants include, but are not limited to, the following: lauromacrogol 400, polyoxyl stearate 40, polyoxyethylene hydrogenated castor oil 10, 40, 50 and 60, glycerol monostearate, polysorbate 40, polysorbate 60, polysorbate 65 and polysorbate 80, soy lecithin and other phospholipids, such as dioleylphosphatidylcholine (DOPC), dimyristoylphosphatidylglycerol (DMPG), dimyristoylphosphatidylcholine (DMPC) and (dioleylphosphatidylglycerol)DOPG; sucrose fatty acid esters, methylcellulose and carboxymethylcellulose. Accordingly, formulations containing these surfactants individually, or formulations containing these surfactants as mixtures 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 formulations, 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 concentration of the surfactant 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 contains 0.05 to 0.3 mg / mL of surfactant. In some embodiments, the pharmaceutical formulation contains 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 approximately 0.01 to approximately 0.05% (weight / volume). In certain embodiments, polysorbate 80 or polysorbate 20 is incorporated at a concentration of approximately 0.02% (weight / volume).

[0082] ActRIIa preparation In some embodiments of the pharmaceutical formulations provided herein, the vial of the ActRIIa fusion protein provided herein comprises the 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 the lyophilized formulation. In one embodiment of the pharmaceutical formulations provided herein, the vial of the lyophilized pharmaceutical formulation comprises 55 mg of ActRIIa fusion protein; 0.48 mg of citrate monohydrate; 2.56 mg of trisodium citrate dihydrate (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, the vial contains 72.5 mg of ActRIIa fusion protein; 0.64 mg of citrate monohydrate; 3.37 mg of trisodium citrate dihydrate (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% (by weight / volume) sucrose; and / or polysorbate 80 (e.g., pH 5.0 to 7.0), e.g., 0.02% (by weight / volume) polysorbate 80 (pH 5.8).

[0083] In some embodiments, once reconstituted with sterile water, the vial contains 50 mg / mL of SEQ ID NO: 32 or SEQ ID NO: 41 ActRIIa fusion protein, 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 contains 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 contains 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 This disclosure provides a kit comprising a lyophilized pharmaceutical formulation and an injection device. In certain embodiments, the lyophilized pharmaceutical formulation comprises an ActRIIa protein (for example, a protein that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9 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 an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9 or SEQ ID NO: 32, a protein consisting essentially of that amino acid sequence, or a protein consisting of that amino acid sequence. In certain such embodiments, the protein comprises an amino acid sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 9 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 having 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 a C-terminal lysine residue (i.e., SEQ ID NO: 41).

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

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

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

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

[0092] In certain embodiments, the pH range of the sterile lyophilized pharmaceutical formulation containing the ActRIIa fusion protein is 5 to 7. In certain embodiments, the sterile lyophilized pharmaceutical formulation containing the ActRIIa fusion protein further includes a buffer. In some embodiments, the buffer is added in an amount of at least 10 mM. In some embodiments, the buffer is added in an amount ranging from about 10 mM to about 200 mM. In some embodiments, the buffer contains a citrate anion.

[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 lioprotectant. In some embodiments, the lioprotectant comprises a sugar such as a disaccharide (e.g., sucrose). In some embodiments, the lioprotectant comprises sucrose, trehalose, mannitol, polyvinylpyrrolidone (PVP), dextrose, and / or glycine. In some embodiments, the lioprotectant comprises sucrose. In some embodiments, the lyophilized pharmaceutical formulation comprises lioprotectant and protein in a weight ratio of at least 1:1 protein to lioprotectant. In some embodiments, the lyophilized pharmaceutical formulation comprises lioprotectant and protein in a weight ratio of 1:1 to 1:10 protein to lioprotectant. In some embodiments, the lyophilized pharmaceutical formulation comprises lioprotectant and protein in a weight ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10 protein to lioprotectant. In some embodiments, the lyophilized pharmaceutical formulation comprises a lioprotectant and a protein in a weight ratio of 1:6 protein to lioprotectant. In the aforementioned specific embodiments, the lyophilized pharmaceutical formulation comprises an amount of lioprotectant sufficient to stabilize the protein.

[0095] In certain embodiments of the kits disclosed herein, the injection device includes a syringe, as shown in Figure 1. In certain such embodiments, the syringe is pre-filled with a reconstituted solution. In some embodiments, the reconstituted solution includes a pharmaceutically acceptable carrier and / or excipient. In some embodiments, the pharmaceutically acceptable carrier includes an aqueous solution, e.g., water, physiologically buffered saline, or another 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, starch, glycol, povidone, mineral hydrocarbons, acrylic polymers, fatty alcohols, mineral stearates, glycerin, and / or lipids. In certain embodiments, the reconstituted solution includes a pharmaceutically acceptable sterile isotonic aqueous solution or non-aqueous solution, dispersion, suspension, or emulsion. In certain such embodiments, the reconstituted solution contains antioxidants, buffers, bacteriostatic agents, and / or solutes that make the formulation isotonic with the blood of the recipient to which it is intended. In other embodiments, the reconstituted solution contains suspending agents or thickeners.

[0096] In certain embodiments of the kits disclosed herein, the kit further includes a vial adapter, as shown in Figure 1. In some embodiments, a vial (1) pre-filled with the lyophilized pharmaceutical formulation of the present invention is attached to one end of the vial adapter (3). In some embodiments, a syringe (2) pre-filled with the reconstituted solution disclosed herein is attached to one end of the vial adapter (3). In some embodiments, the syringe (2) pre-filled with the reconstituted solution disclosed herein and the vial (1) pre-filled with the lyophilized pharmaceutical formulation are attached to the opposite end of the vial adapter (3). In some embodiments, the reconstituted solution is transferred from the pre-filled syringe to the vial. In some embodiments, the lyophilized formulation is reconstituted into a sterile injection by transferring the reconstituted solution to the vial pre-filled with the lyophilized pharmaceutical formulation. In some embodiments, the lyophilized formulation is reconstituted into a sterile injection. In some embodiments, the lyophilized formulation is reconstituted into a sterile injection before use.

[0097] In other embodiments of the kits disclosed herein, the kit further includes a pumping device. In certain embodiments, the pumping device includes an electromechanical pump assembly. In certain embodiments, the pumping device includes a reservoir for holding sterile injection solution. In certain embodiments, the reservoir holds 1 mL of sterile injection solution. In certain embodiments, the pumping device includes one or more vials or cartridges containing sterile injection solution. In certain embodiments, the vials or cartridges are pre-filled with sterile injection solution. In certain embodiments, the vials or cartridges contain sterile injection solution reconstituted from a lyophilized preparation. In certain embodiments, the reservoir is connected to the vials or cartridges. In certain embodiments, the vials or cartridges hold 1 to 20 mL of sterile injection solution. In certain embodiments, the electromechanical pump assembly includes a pump chamber. In certain embodiments, the electromechanical pump assembly is connected to the reservoir. In certain embodiments, the sterile injection solution is received from the reservoir into the pump chamber. In some embodiments, the electromechanical pump assembly includes a plunger positioned so that the sterile injection solution in the pump chamber is in direct contact with the plunger. In certain embodiments, the sterile injection solution is received from a reservoir into the pump chamber during a first pumping phase and delivered from the pump chamber to the subject during a second pumping phase. 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 injection solution into the pump chamber during a first pumping phase, and (b) deliver the sterile injection solution from the pump chamber through a plurality of discrete movements of the plunger during a second pumping phase, thereby delivering the therapeutic substance to the subject in a plurality of controlled discrete doses through the second pumping phase. In certain embodiments, a cycle of alternating first and second pumping phases 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] This disclosure provides a kit used to reconstitute a lyophilized pharmaceutical formulation into a sterile injectable solution. In certain embodiments, the resulting sterile injectable solution is useful in the methods disclosed herein.

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

[0100] ActRIIa polypeptide In certain embodiments, this disclosure relates to ActRIIa fusion proteins. Where used herein, the term “ActRIIa” refers to the activin receptor type IIA (ActRIIa) protein family derived from any species, and to variants derived from such ActRIIa fusion proteins by mutagenesis or other modifications. References to ActRIIa herein are understood to refer to any one of the currently identified forms. Members of the ActRIIa family are generally transmembrane proteins comprising a ligand-binding extracellular domain containing a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain in which serine / threonine kinase activity is expected.

[0101] The term “ActRIIa fusion protein” encompasses any naturally occurring protein of an ActRIIa family member and any variant thereof that retains useful activity (e.g., mutants, fragments, fusions, and peptide-mimicking forms). Examples of such variant ActRIIa fusion proteins are provided throughout this disclosure and in International Patent Application Publications WO2006 / 012627 and WO2007 / 062188 (which are incorporated herein by reference in their entirety). The amino acid numbering for all ActRIIa-related proteins described herein is based on the numbering of the human ActRIIa precursor protein sequence (SEQ ID NO: 9) provided below, unless otherwise specifically designated.

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

[0103] Signal peptides are single underline The extracellular domain is shown in bold; and the potential endogenous N-linked glycosylation site is double underline This is shown.

[0104] The sequence of the processed (mature) extracellular human ActRIIa protein is as follows: ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM EVTQPTSNPVTPKPP (Sequence ID: 10).

[0105] The C-terminal "tail" of the extracellular domain is single underline As shown above, the sequences from which the "tail" has been removed (Δ15 sequences) are as follows: ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM (Sequence ID: 11).

[0106] The nucleic acid sequence encoding the human ActRIIa precursor protein is shown below (SEQ ID NO: 12), followed by nucleotides 159-1700 of Genbank Reference Sequence NM_001616.4. The signal sequence is: underline It has been deducted.

[0107] [Table 2]

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

[0109] ActRIIa is well-conserved among vertebrates, and most of its extracellular domain is completely conserved. For example, Figure 2 shows multi-sequence alignments of the human ActRIIa extracellular domain compared to various ActRIIa orthologues. Many of the ligands that bind to ActRIIa are also highly conserved. Therefore, from these alignments, it is possible to predict the key amino acid positions within the ligand-binding domain that are important for normal ActRIIa-ligand-binding activity, and also to predict amino acid positions that are likely to be resistant to substitution without significantly altering normal ActRIIa-ligand-binding activity. Thus, according to the methods disclosed, useful active human ActRIIa mutant proteins may contain one or more amino acids at corresponding positions in the sequences of other vertebrate ActRIIa, or may contain residues similar to those in the human or other vertebrate sequences.

[0110] While not limiting, the following examples illustrate this approach to defining active ActRIIa variants. As shown in Figure 2, F13 in the human extracellular domain corresponds to Y in ActRIIa of sheep (Ovis aries) (SEQ ID NO: 62), red junglefowl (Gallus gallus) (SEQ ID NO: 65), cattle (Bos Taurus) (SEQ ID NO: 66), barn owl (Tyto alba) (SEQ ID NO: 67), and bat (Myotis davidii) (SEQ ID NO: 68), indicating that aromatic residues containing F, W, and Y are acceptable at this position. Q24 in the human extracellular domain corresponds to R in Bos Taurus ActRIIa, indicating that charged residues containing D, R, K, H, and E are acceptable at this position. S95 in the human extracellular domain is F in ActRIIa of the red junglefowl (Gallus gallus) and barn owl (Tyto alba), indicating that this site can tolerate a wide variety of changes, including polar residues such as E, D, K, R, H, S, T, P, G, and Y, and possibly hydrophobic residues such as L, I, or F. E52 in the human extracellular domain is D in ActRIIa of the sheep (Ovis aries), indicating that acidic residues including D and E are tolerated at this position. P29 in the human extracellular domain is relatively poorly conserved, appearing as S in ActRIIa of the sheep (Ovis aries) and as L in ActRIIa of the bat (Myotis davidii). Therefore, 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 their structural and functional properties, particularly with respect to ligand binding (Attisano et al. (1992) Cell 68(1):97-108; Greenwald et al. (1999) Nature Structural Biology 6(1): 18-22; Allendorph et al. (2006) PNAS 103(20: 7643-7648; Thompson et al. (2003); and U.S. Patents 7,709,605, 7,612,041 and 7,842,663). In addition to the teachings herein, these references provide ample guidance on how to generate ActRIIa variants that retain one or more desired activities (e.g., ligand-binding activity).

[0112] For example, the structural motif known as the three-finger toxin fold is crucial for ligand binding by type I and type II receptors and is formed by conserved cysteine ​​residues at various positions within the extracellular domain of each monomeric receptor ("Greenwald et al. (1999) Nat Struct Biol 6:18-22" and "Hinck (2012) FEBS Lett 586:1860-1870"). Therefore, the core ligand-binding domain of human ActRIIa, defined by the outermost of these conserved cysteines, corresponds to positions 30-110 of SEQ ID NO: 9 (ActRIIa precursor). Therefore, structurally disordered amino acids adjacent to the core sequences defined by these cysteines can cleave approximately residues 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 at the N-terminus and approximately residues 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 at the C-terminus, without necessarily altering ligand binding. Exemplary cleavage of the ActRIIa extracellular domain includes SEQ ID NOs: 10 and 11.

[0113] Therefore, the general formula for the active region of ActRIIa (e.g., ligand binding of ActRIIa) is either a protein containing amino acids 30-110 of SEQ ID NO: 9, or a protein consisting essentially of amino acids 30-110, or a protein consisting of amino acids 30-110. Thus, the ActRIIa protein begins with a residue corresponding to any one of amino acids 21-30 of SEQ ID NO: 9 (e.g., any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) and ends at a position corresponding to any one of amino acids 110-135 of SEQ ID NO: 9 (e.g., amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123 It may contain, or may consist of, or may consist of, a portion of ActRIIa (ending in any one of 24, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135) an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or essentially consist of, or may consist of, such amino acid sequences. Other examples include starting at a position selected from 21-30 of SEQ ID NO: 9 (for example, starting with any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30), or starting at a position selected from 22-30 (for example, starting with any one of amino acids 22, 23, 24, 25, 26, 27, 28, 29, or 30), or starting at a position selected from 23-30 (for example, starting with any one of amino acids 23, 24, 25, 26, 27, 28, 29, or 30). ), or starting at a position selected from 24-30 (for example, starting with any one of amino acids 24, 25, 26, 27, 28, 29, or 30), and ending at a position selected from 111-135 of SEQ ID NO: 9 (for example, ending with any one of amino acids 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135), or,It ends at a position selected from 112-135 (for example, ending with any one of amino acids 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134 or 135), or it ends at a position selected from 113-135 (for example, amino acids 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 1 (Ends with any one of 32, 133, 134, or 135) or ends at a position selected from 120-135 (e.g., ending with any one of amino acids 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135) or ends at a position selected from 130-135 (e.g., ending with any one of amino acids 130, 131, 132, 133, 134, or 135) or ends at a position selected from 111-134 (e.g., ending with any one of amino acids 110, 111, 1 (ending in any one of 12, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133 or 134), or (ending in any one of 111-133 (for example, ending in any one of amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132 or 133), or , or end at a position selected from 111-132 (for example, ending at any one of amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 or 132), or end at a position selected from 111-131 (for example, amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,There are constructs, etc., ending in either 130 or 131. Variants within these ranges are also intended, in particular, variants containing amino acid sequences having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the corresponding portion of SEQ ID NO: 9, variants consisting essentially of those amino acid sequences, or variants consisting of those amino acid sequences are also intended. Accordingly, in some embodiments, the ActRIIa protein may include, essentially consist of, or be composed of, proteins that are at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 30-110 of SEQ ID NO: 9. In some cases, ActRIIa proteins are at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 30-110 of SEQ ID NO: 9, and contain 1 or fewer, 2 or fewer, 5 or fewer, 10 or fewer, or 15 or fewer conservative amino acid changes in the ligand-binding pocket.

[0114] In certain embodiments, this disclosure relates to GDF / BMP antagonists (inhibitors) comprising the ActRIIa protein (which includes its fragments, functional variants and modified forms), and their uses (e.g., to enhance the immune response in patients who require it, and to treat cancer). Preferably, the ActRIIa protein is soluble (e.g., the extracellular domain of ActRIIa). In some embodiments, the ActRIIa protein inhibits (e.g., Smad signaling) one or more GDF / BMP ligands (e.g., GDF11, GDF8, activin (activin A, activin B, activin AB, activin C, activin E), BMP6, GDF3, BMP15, and / or BMP10). In some embodiments, the ActRIIa protein binds to one or more GDF / BMP ligands (e.g., GDF11, GDF8, activin (activin A, activin B, activin AB, activin C, activin E), BMP6, GDF3, BMP15, and / or BMP10). In some embodiments, the ActRIIa protein of this disclosure begins at a residue corresponding to amino acids 21-30 of SEQ ID NO: 9 (e.g., any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) and ends at a position corresponding to any one of amino acids 110-135 of SEQ ID NO: 9 (e.g., amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123) It contains, essentially consists of, or consists of amino acid sequences that are at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the portion of ActRIIa (ending in any one of 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135).In some embodiments, the ActRIIa protein contains, consists of, or essentially consists of amino acid sequences that are at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 30-110 of SEQ ID NO: 9. In certain embodiments, the ActRIIa protein contains, consists of, or essentially consists of amino acid sequences that are at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 21-135 of SEQ ID NO: 9.

[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 heterogeneous protein domains. 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 positioned between the ActRIIa protein domain and one or more heterogeneous domains (e.g., 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 contains an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 32. In some embodiments, the ActRIIa fusion protein contains the amino acid sequence of SEQ ID NO: 32. In some embodiments, the ActRIIa fusion protein consists of the amino acid sequence of SEQ ID NO: 32. In some embodiments, the ActRIIa fusion protein 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 contains, consists of, or essentially consists of amino acid sequences that are at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to any one of the amino acid sequences of SEQ ID NOs: 9, 10, 11, 32, 36, and 39.

[0117] In some embodiments, the ActRIIa fusion protein contains an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 32. In some embodiments, the ActRIIa fusion protein contains the amino acid sequence of SEQ ID NO: 32. In some embodiments, the ActRIIa fusion protein consists of the amino acid sequence of SEQ ID NO: 32. In some embodiments, the ActRIIa fusion protein is part of a homodimeric protein complex. In some embodiments, the ActRIIa fusion protein is glycosylated. In some embodiments, the ActRIIa fusion protein has a glycosylation pattern obtained by expression in Chinese hamster ovary cells.

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

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

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

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

[0123] In certain embodiments, the pharmaceutical formulation described herein includes 100% by weight of SEQ ID NO: 32. In certain embodiments, the pharmaceutical formulation described herein includes 100% by weight of SEQ ID NO: 41.

[0124] In certain embodiments, this disclosure relates to GDF trap proteins (also referred to as "GDF traps"). In some embodiments, the GDF traps of this disclosure are mutant ActRIIa proteins (e.g., ActRIIa) comprising one or more mutations (e.g., addition, deletion, substitution, and combination thereof) in the extracellular domain (also referred to as the ligand-binding domain) of the ActRIIa protein (e.g., "wild-type" or unmodified ActRIIa protein) such that the mutant ActRIIa protein has one or more altered ligand-binding activities compared to the corresponding wild-type ActRIIa protein. In certain embodiments, the GDF trap proteins of this disclosure retain at least one similar activity to the corresponding wild-type ActRIIa protein. For example, a preferred GDF trap binds to GDF11 and / or GDF8 and inhibits their function (e.g., antagonizes them). In some embodiments, the GDF traps of this disclosure further bind to one or more ligands of GDF / BMP and inhibit them. Accordingly, this disclosure provides a GDF trap protein having altered binding specificity for one or more ActRIIa ligands.

[0125] For example, one or more mutations may be selected that increase the selectivity of the modified ligand-binding domain for GDF11 and / or GDF8 compared to one or more ActRIIa-binding ligands (e.g., activin, especially activin A). In some cases, the modified ligand-binding domain may have a K ratio for GDF11 and / or GDF8 binding that is at least 2, 5, 10, 20, 50, 100, or 1000 times greater than the ratio for the wild-type ligand-binding domain. d K related to activin binding d The ratio is such that, in some cases, the modified ligand-binding domain is at least 2 times, 5 times, 10 times, 20 times, 50 times, 100 times, or even 1000 times larger than the wild-type ligand-binding domain in terms of IC for inhibiting GDF11 and / or GDF8. 50 IC to inhibit activin 50It has the ratio of . In some cases, the modified ligand-binding domain is used to inhibit IC for activin 50 ICs that are at least 2 times, 5 times, 10 times, 20 times, 50 times, and 100 times smaller than the others. 50 And, or even, an IC that is 1000 times smaller. 50 This inhibits GDF11 and / or GDF8.

[0126] How to use In certain embodiments, the Disclosure provides a method for treating pulmonary arterial hypertension (PAH), the method comprising administering a pharmaceutical formulation described herein to a patient in need of treatment for pulmonary arterial hypertension.

[0127] In certain embodiments, the Disclosure provides a method for treating pulmonary arterial hypertension (PAH) in a patient in need, the method comprising reconstituting a lyophilized pharmaceutical formulation described herein to produce a reconstituted formulation, and administering the reconstituted formulation to the patient.

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

[0129] In certain embodiments, the Disclosure provides a method for treating pulmonary arterial hypertension (PAH), wherein the method comprises administering a pharmaceutical formulation described herein to a patient in need, wherein the administration of the pharmaceutical formulation results in a change in one or more of the following hemodynamic or functional parameters: a reduction in pulmonary vascular resistance (PVR); an increase in the 6-minute walk distance (6MWD); a decrease in N-terminal pro-B natriuretic peptide (NT-proBNP) levels; prevention or reduction of progression of the World Health Organization (WHO) recognized functional classification of pulmonary hypertension; promotion or increase of regression of the WHO recognized functional classification of pulmonary hypertension; improvement of right ventricular function; improvement of pulmonary artery pressure; and / or improvement of mean right atrial pressure.

[0130] In certain embodiments, the Disclosure provides a method for treating pulmonary arterial hypertension (PAH), the method comprising administering a pharmaceutical formulation described herein to a patient in need of treatment for pulmonary arterial hypertension, wherein the administration of the pharmaceutical formulation results in increased exercise capacity, provides clinical improvement, improves WHO functional class (FC), and slows disease progression (this includes reducing the risk of death and hospitalization related to PAH).

[0131] In certain embodiments, the Disclosure provides a method for treating, preventing or reducing the rate of progression and / or severity of one or more complications of pulmonary arterial hypertension, wherein the method comprises administering a pharmaceutical formulation described herein to a patient in need, wherein the administration of the formulation results in one or more changes in the following hemodynamic or functional parameters: reduction of pulmonary vascular resistance (PVR); increase of 6-minute walk distance (6MWD); decrease of N-terminal pro-B natriuretic peptide (NT-proBNP) levels; prevention or reduction of progression of the World Health Organization (WHO) recognized functional classification of pulmonary hypertension; promotion or increase of regression of the WHO recognized functional classification of pulmonary hypertension; improvement of right ventricular function; improvement of pulmonary artery pressure; and / or improvement of mean right atrial pressure. In some embodiments, the one or more complications of pulmonary arterial hypertension are selected from the group consisting of proliferation of smooth muscle and / or endothelial cells in the pulmonary arteries, angiogenesis in the pulmonary arteries, dyspnea, chest pain, pulmonary vascular remodeling, right ventricular hypertrophy, and pulmonary fibrosis.

[0132] In some embodiments, administration of the pharmaceutical formulations described herein reduces the patient's PVR. In some embodiments, administration of the pharmaceutical formulations described herein reduces the patient's PVR by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%). In some embodiments, administration of the pharmaceutical formulations described herein reduces the patient's PVR by at least 20%. In some embodiments, the reduction in PVR is a result of a decrease in mean pulmonary artery pressure. In some embodiments, administration of the pharmaceutical formulations described herein increases the patient's 6-minute walking distance. In some embodiments, administration of the pharmaceutical formulations described herein increases the patient's 6-minute walking distance by at least 10 meters (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300 meters, or 400 meters or more). In some embodiments, administration of the pharmaceutical formulation described herein increases a patient's 6-minute walking distance by at least 30 meters. In some embodiments, administration of the pharmaceutical formulation described herein lowers a patient's NT-proBNP levels. In some embodiments, administration of the pharmaceutical formulation described herein lowers a patient's NT-proBNP levels by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least 80%). In some embodiments, administration of the pharmaceutical formulation described herein lowers a patient's NT-proBNP levels by at least 30%. In some embodiments, administration of the pharmaceutical formulation described herein lowers NT-proBNP levels to normal levels. In some embodiments, a normal level of NT-proBNP is <100 pg / mL.

[0133] In some embodiments, administration of the pharmaceutical formulations described herein prevents or reduces the progression of pulmonary hypertension functional classification as recognized by the WHO. In some embodiments, administration of the pharmaceutical formulations described herein prevents or reduces the progression of pulmonary hypertension functional classification from functional class I to class II pulmonary hypertension as recognized by the WHO. In some embodiments, administration of the pharmaceutical formulations described herein prevents or reduces the progression of pulmonary hypertension functional classification from functional class II to class III pulmonary hypertension as recognized by the WHO. In some embodiments, administration of the pharmaceutical formulations described herein prevents or reduces the progression of pulmonary hypertension functional classification from functional class III to class IV pulmonary hypertension as recognized by the WHO. In some embodiments, administration of the pharmaceutical formulations described herein promotes or increases the regression of pulmonary hypertension functional classification as recognized by the WHO. In some embodiments, administration of the pharmaceutical formulations described herein promotes or increases the regression of pulmonary hypertension functional classification from class IV to class III pulmonary hypertension as recognized by the WHO. In some embodiments, administration of the pharmaceutical formulations described herein promotes or increases the regression of the functional classification of pulmonary hypertension from Class III to Class II pulmonary hypertension as recognized by the WHO. In some embodiments, administration of the pharmaceutical formulations described herein promotes or increases the regression of the functional classification of pulmonary hypertension from Class II to Class I pulmonary hypertension as recognized by the WHO.

[0134] In some embodiments, administration of the pharmaceutical formulations described herein improves the right ventricular function of a patient. In some embodiments, the improvement in right ventricular function is due to an increase in the change in right ventricular volume. In some embodiments, the improvement in right ventricular function is due to a decrease in right ventricular hypertrophy. In some embodiments, the improvement in right ventricular function is due to an increase in ejection fraction. In some embodiments, the improvement in right ventricular function is due to an increase in both the change in right ventricular volume and the ejection fraction.

[0135] In some embodiments, administration of the pharmaceutical formulations described herein improves the patient's pulmonary artery pressure. In some embodiments, the improvement in pulmonary artery pressure is a decrease in mean pulmonary artery pressure (mPAP). In some embodiments, administration of the pharmaceutical formulations described herein reduces the patient's mPAP by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%). In some embodiments, administration of the pharmaceutical formulations described herein reduces the patient's mPAP by at least 3 mmHg (e.g., at least 3, 5, 7, 10, 12, 15, 20, or 25 mmHg). In some embodiments, administration of the pharmaceutical formulations described herein improves the patient's mean right atrial pressure (mRAP). In some embodiments, the improvement in mRAP is a decrease in mRAP. In some embodiments, administration of the pharmaceutical formulations described herein reduces a patient's mRAP by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%). In some embodiments, administration of the pharmaceutical formulations described herein reduces a patient's mRAP by at least 1 mmHg (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mmHg).

[0136] In some embodiments, the patient has a pulmonary vascular resistance (PVR) of 3 Wood units or more. In some embodiments, the patient has a 6-minute walking distance of 150 to 550 meters. In some embodiments, the patient has elevated NT-proBNP levels compared to healthy patients. In some embodiments, the patient has an NT-proBNP level of at least 100 pg / mL (e.g., 100, 150, 200, 300, 400, 500, 1000, 3000, 5000, 10000, 15000, or 20000 pg / mL). In some embodiments, the patient has elevated brain natriuretic peptide (BNP) levels compared to healthy patients. In some embodiments, the patient has a BNP level of at least 100 pg / mL (e.g., 100, 150, 200, 300, 400, 500, 1000, 3000, 5000, 10000, 15000, or 20000 pg / mL). In some embodiments, administration of the pharmaceutical formulation described herein reduces the patient's BNP level by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least 80%). In some embodiments, administration of the pharmaceutical formulation described herein reduces the BNP level to a normal level (i.e., <100 pg / mL). In some embodiments, the patient has a mean pulmonary artery pressure (mPAP) selected from the group consisting of at least 20 mmHg mPAP; at least 25 mmHg mPAP; at least 30 mmHg mPAP; at least 35 mmHg mPAP; at least 40 mmHg mPAP; at least 45 mmHg mPAP; and at least 50 mmHg mPAP. In some embodiments, the patient has a mean right atrial pressure (mRAP) selected from the group consisting of at least 5 mmHg mRAP; at least 6 mmHg mRAP; at least 8 mmHg mRAP; at least 10 mmHg mRAP; at least 12 mmHg mRAP; at least 14 mmHg mRAP; and at least 16 mmHg mRAP.

[0137] In some embodiments, PAH is idiopathic pulmonary arterial hypertension (PAH). In some embodiments, the PAH is hereditary PAH. In some embodiments, the PAH is drug-induced or toxin-induced PAH. In some embodiments, the PAH is PAH associated with a simple congenital systemic-pulmonary shunt at least one year after shunt repair. In some embodiments, the patient has functional class II or III pulmonary hypertension according to the World Health Organization's functional classification system for pulmonary hypertension. In some embodiments, the patient has functional class I, class II, class III or class IV pulmonary hypertension as recognized by the World Health Organization. In some embodiments, the patient has functional class I, class II, class III or class IV pulmonary hypertension according to the World Health Organization's functional classification system for pulmonary hypertension. In some embodiments, the patient has functional class IV pulmonary hypertension according to the World Health Organization's functional classification system for pulmonary hypertension. In some embodiments, administration of the pharmaceutical formulations described herein increases the transplant-free survival in the patient. In some embodiments, administration of the pharmaceutical formulations described herein increases the transplant-free survival rate in patients by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%). In some embodiments, administration of the pharmaceutical formulations described herein reduces right ventricular hypertrophy in patients. In some embodiments, administration of the pharmaceutical formulations described herein reduces right ventricular hypertrophy in patients by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%). In some embodiments, administration of the pharmaceutical formulations described herein reduces smooth muscle hypertrophy in patients. In some embodiments, administration of the pharmaceutical formulations described herein reduces smooth muscle hypertrophy in patients by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%). In some embodiments, administration of the pharmaceutical formulations described herein reduces pulmonary arteriole muscularity in patients.In some embodiments, administration of the pharmaceutical formulations described herein reduces pulmonary arteriole muscle hypertrophy in a patient by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%).

[0138] In some embodiments, administration of the pharmaceutical formulations described herein improves the patient's exercise capacity. In some embodiments, administration of the pharmaceutical formulations described herein reduces the patient's Borg dyspnea index (BDI). In some embodiments, administration of the pharmaceutical formulations described herein reduces the patient's BDI by at least 0.5 index points (e.g., at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 index points). In some embodiments, the patient has impaired renal function. In some embodiments, administration of the pharmaceutical formulations described herein further improves renal function. In some embodiments, administration of the pharmaceutical formulations described herein delays the clinical progression of pulmonary arterial hypertension. In some embodiments, administration of the pharmaceutical formulations described herein delays the clinical progression of pulmonary arterial hypertension according to the World Health Organization's functional classification system for pulmonary hypertension. In some embodiments, administration of the pharmaceutical formulations described herein reduces the risk of hospitalization for one or more complications associated with pulmonary arterial hypertension. In some embodiments, administration of the pharmaceutical formulations described herein reduces the risk of morbidity for one or more complications associated with pulmonary arterial hypertension. In some embodiments, the morbidity includes changes in one or more of the following: increased need for lung transplantation and / or heart transplantation; need to initiate salvage therapy with known treatments for PAH; need to increase prostacyclin by at least 10%; need for atrial septal resection; PAH-specific hospitalization for at least 24 hours; and worsening of PAH. In some embodiments, worsening of PAH includes worsening of WHO functional class and a decrease of at least 15% in 6MWD. In some embodiments, administration of the pharmaceutical formulations described herein reduces the risk of death associated with pulmonary arterial hypertension. In some embodiments, administration of the pharmaceutical formulations described herein reduces the risk of death associated with pulmonary arterial hypertension by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%).In some embodiments, the patient has hemoglobin levels of >8 g / dL and <15 g / dL. In some embodiments, the patient's hemoglobin level is <18 g / dL.

[0139] In certain embodiments, the patient treated according to the method described herein is female. In certain embodiments, the patient treated according to the method described herein is male. In certain embodiments, the patient treated according to the method described herein may be of any age. In certain embodiments, the patient treated according to the method described herein is under 18 years of age. In certain embodiments, the patient treated according to the method described herein is under 13 years of age. In another particular embodiment, the patient treated according to the method described herein is under 12 years of age, under 11 years of age, under 10 years of age, under 9 years of age, under 8 years of age, under 7 years of age, under 6 years of age, or under 5 years of age. In another particular embodiment, the patient treated according to the method described herein is between 1 and 3 years of age, 3 and 5 years of age, 5 and 7 years of age, 7 and 9 years of age, 9 and 11 years of age, 11 and 13 years of age, 13 and 15 years of age, 15 and 20 years of age, 20 and 25 years of age, 25 and 30 years of age, or 30 years of age or older. In another specific embodiment, the patients treated according to the method described herein are 30–35 years old, 35–40 years old, 40–45 years old, 45–50 years old, 50–55 years old, 55–60 years old, or 60 years old or older. In another specific embodiment, the patients treated according to the method described herein are 18–64 years old, 65–74 years old, or 75 years old or older.

[0140] In certain embodiments, the hemoglobin level of a patient treated according to the dosage forms and methods provided herein is less than 10 g / dL, less than 9 g / dL, less than 8 g / dL, or less than 7 g / dL. In certain embodiments, the hemoglobin level in a patient treated according to the dosage forms and methods provided herein is 7 g / dL to 7.5 g / dL, 7.5 g / dL to 8 g / dL, 8 g / dL to 8.5 g / dL, 8.5 g / dL to 9.0 g / dL, 9.0 g / dL to 9.5 g / dL, or 9.5 g / dL to 10.0 g / dL. [Examples]

[0141] Examples This disclosure is generally described, but will be more readily understood by referring to the following examples. These examples are included solely for the purpose of illustrating specific embodiments of this disclosure and are not intended to limit this disclosure.

[0142] Although the present invention is described in general terms, it will be more readily understood by referring to the following examples. These examples are included solely for the purpose of illustrating specific embodiments of the present 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 a human or mouse Fc domain and adding a minimal linker between them. These constructs are referred to as ActRIIa-hFc and ActRIIa-mFc, respectively.

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

[0145] ActRIIa-hFc and ActRIIa-mFc proteins were expressed in CHO cell lines. Three different leader sequences were investigated: (i) Honeybee melittin (HBML): MKFLVNVALVFMVVYISYIYA (Sequence ID: 33) (ii) Tissue plasminogen activator (TPA): MDAMKRGLCCVLLLCGAVFVSP (Sequence ID: 34) (iii) Native: MGAAAKLAFAVFLISCSSGA (Sequence ID: 35).

[0146] The selected form employs a TPA reader and has the following raw amino acid sequence: MDAMKRGLCCVLLLCGAVFVSPGAAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID: 36).

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

[0148] Both ActRIIa-hFc and ActRIIa-mFc were extremely suitable for recombinant expression. As shown in FIGS. 3A and 3B, the protein was purified as a single distinct peak of the protein. N-terminal sequencing revealed a single sequence of -ILGRSETQE (SEQ ID NO: 38). The 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 with virus filtration and buffer exchange. The ActRIIa-hFc protein was purified until it reached a purity of >98% as measured by size exclusion chromatography and >95% as measured by SDS PAGE.

[0149] ActRIIa-hFc and ActRIIa-mFc showed high affinity for the ligand. GDF11 or activin A was immobilized on a Biacore TM CM5 chip using a standard amine coupling method. ActRIIa-hFc protein and ActRIIa-mFc protein were loaded into the system and binding was measured. ActRIIa-hFc bound to activin with a dissociation constant (K -12 ) of 5×10 D and bound to GDF11 with a K -9 of 9.96×10 D . See FIGS. 4A and 4B. Using a similar binding assay, it was confirmed that ActRIIa-hFc has a high to moderate affinity for other TGF-β superfamily ligands including, for example, activin B, GDF8, BMP6, and BMP10. ActRIIa-mFc also showed 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 the circulating level of the drug after two weeks was quite high (11 μg / mL, 110 μg / mL, or 304 μg / mL for the initial dose of 1 mg / kg, 10 mg / kg, or 30 mg / kg, respectively). In cynomolgus monkeys, the plasma half-life was substantially more than 14 days, and the circulating levels of the drug were 25 μg / mL, 304 μg / mL, and 1440 μg / mL, respectively, after initial doses of 1 mg / kg, 10 mg / kg, and 30 mg / kg.

[0151] Example 2: Characterization of ActRIIa-hFc protein The ActRIIa-hFc fusion protein was expressed in CHO-DUKX B11 cells stably transfected from a pAID4 vector (SV40 original / enhancer, CMV promoter) using the tissue plasminogen reader sequence of SEQ ID NO: 34. The purified protein in Example 1, as described above, had the sequence of SEQ ID NO: 32. Its Fc portion is a human IgG1 Fc sequence, as shown in SEQ ID NO: 32. Protein analysis revealed that the ActRIIa-hFc fusion protein is formed as a homodimer with a disulfide bond.

[0152] The CHO cell expression exhibited higher affinity for activin B ligand than that reported for ActRIIa-hFc fusion proteins expressed in human 293 cells (see "del Re et al. (2004) J Biol Chem. 279(51):53126-53135"). Furthermore, the use of the TPA reader sequence resulted in higher yields than other reader sequences and provided a purer N-terminal sequence, unlike ActRIIa-Fc expressed with the native reader. The use of the native reader sequence resulted in two major species of ActRIIa-Fc, each with a different N-terminal sequence.

[0153] Further ActRIIa ligand traps (ActRIIa-Fc fusion proteins modified to reduce the ratio of activin A binding to myostatin or GDF11) are described in International Patent Application Publications WO2006 / 012627 and WO2007 / 062188 (these are incorporated herein by reference).

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

[0155] Two formulations of the ActRIIa-hFc fusion protein SEQ ID NO: 32, 45 mg / vial and 60 mg / vial, were developed. 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 citrate 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 citrate monohydrate, 3.37 mg of trisodium citrate dihydrate, 0.29 mg of polysorbate 80, and 116.0 mg of sucrose. These formulations had the same composition before lyophilization and after reconstitution using sWFI. The only difference between the two formulations is their filling volume. The 45 mg / vial contains 1.1 mL of filling, while the 60 mg / vial contains 1.45 mL. Both formulations are lyophilized and reconstituted before subcutaneous administration. The 45 mg / vial is reconstituted using 1.0 mL of sWFI, and the 60 mg / vial is reconstituted using 1.3 mL of sWFI.

[0156] The formulation development of ActRIIa-hFc fusion protein SEQ ID NO: 32 was guided by the results of various screening studies described below. These studies focused on selecting the optimal pH, buffer system, protein concentration, and excipients for the stabilization of 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 ActRIIa-hFc fusion protein SEQ ID NO: 32 in test samples.

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

[0158] [Table 4] TIFF0007899455000005.tif115170

[0159] Observation of changes in monomer species % (purity) using HP-SEC revealed no significant change in purity after 6 months at 5°C, indicating that all formulations retained >99.0% monomer. As shown in Figure 5, after 6 months at 25°C, the monomer % decreased significantly in formulations with pH ≥ 6.5, but there was no significant change in formulations with pH < 6.5. After 2 months at 45°C, the monomer % similarly decreased in formulations with pH ≥ 6.5, and similarly, the decrease in monomer % correlated with lower pH < 5.5.

[0160] Based on these observations under accelerated conditions (25°C) and stress conditions (45°C), ActRIIa-hFc fusion protein SEQ ID NO: 32 showed the greatest stabilizing effect in citrate buffers 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 its stability decreased at high pH.

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

[0162] There was no significant change in the equilibrium dissociation constant (Kd) of Sequence ID No. 32 after 6 months at 5°C. In formulations with pH < 5.5, there was a significant decrease in binding after 6 months at 25°C, as shown by the sharp increase in Kd value in Figure 6. In formulations with pH < 6.0, binding decreased after 2 months at 45°C.

[0163] Based on these observations, ActRIIa-hFc fusion protein SEQ ID NO:32 can maintain 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 consistently increased with pH, ​​leveling off at around 67°C–70°C at pH ≥ 5.5. All buffers with equivalent pH values ​​functioned similarly, with the exception of histidine and Tris-based buffers, which have significantly lower melting points.

[0165] Overall, the results of this experiment suggest that the pH range of 5.5–6.5 is optimal for SEQ ID NO: 32 in terms of maintaining physical stability monitored by HP-SEC and binding affinity with Biacore. Furthermore, the citrate and succinate buffers showed superior performance to histidine at the higher end of this range. Acetate and phosphate buffers were not further pursued in development due to the possibility of pH changes due to precipitation during freezing or lyophilization.

[0166] Confirmation of optimal buffering concentration: Evaluation of the effect of buffering concentration using citrate, one of the lead buffering systems, on the conformational and colloidal stability of the ActRIIa-hFc fusion protein SEQ ID NO: 32. In early pH screening studies, various buffer systems were evaluated at a concentration of 50 mM to provide high buffering performance. However, this concentration may not be optimal for stabilizing 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, ActRIIa-hFc fusion protein SEQ ID NO: 32 was prepared in a range of citrate buffer concentrations, one of the lead buffer systems from previous studies. The effect of buffer strength on thermal stability was evaluated using DSC for 50 mg / mL ActRIIa-hFc fusion protein SEQ ID NO: 32 and a pH 5.8 citrate buffer (5–50 mM) containing 8% (weight / volume) sucrose, and the diffusion interaction parameter (kD), a measure of the molecular self-association tendency, was evaluated using DLS. The results are summarized in Table 2.

[0167] [Table 5]

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

[0169] Selection of pH for optimal stability Short-term stability studies were conducted, and ActRIIa-hFc fusion protein SEQ ID NO: 32 was formulated at a concentration of 5 mg / mL in 10 mM succinate, citrate, and histidine (pH level within the range of 5-7). These formulations were packed into Type I glass vials and subjected to short-term stability testing 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 obtained from these analyses are summarized in Tables 3, 4, and 5, and the following results were obtained.

[0170] [Table 6]

[0171] [Table 7]

[0172] [Table 8]

[0173] As shown in Figure 7, the results of this study showed that, based on HP-SEC stability results at 25°C, citrate and succinate performed comparably between pH 5.3 and 6.3 (measured pH) for optimal thermal stability. The citrate and succinate buffer systems showed a higher amount of fragmentation (LMW%) observed by HP-SEC at 40°C, which was attributed to the nature of the stress and challenges in method integration. The observations in HP-SEC were consistent with the unreduced CE-SDS data shown in Figure 8. Characterization of the charge profile by desialized iCIEF, shown in Figure 9, also confirmed that the optimal stability pH range was 5.3–6.3. Formulations in histidine buffers require a slightly higher pH range of 6.0–6.5 to achieve performance comparable to citrate and succinate; however, this buffer system also shows a lower Tm1, indicating lower biophysical stability.

[0174] The results of this study demonstrated that ActRIIa-hFc fusion protein SEQ ID NO:32 is biophysical and chemically stable in both citrate and succinate buffers within the range of 5.3–6.3 (measured pH). Furthermore, this range offered the possibility of reducing the rate of possible deamidation by selecting a pH lower in reality, as is commonly observed with biological products. Therefore, for further product development, a target pH of 5.8 was selected using citrate as the buffering system.

[0175] Physical stability of the ActRIIa-hFc fusion protein of Sequence ID: 32 To evaluate the physical stability of the ActRIIa-hFc fusion protein of SEQ ID NO: 32, the worst-case scenario was performed in 10 mM citrate, one of the lead buffer systems, in the absence of excipients and under physical stress by 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 an unagitated "control" sample, with 4 mL of the sample placed in a 10 mL Type I glass vial equipped with an analog stirrer for up to 4 days, and monomer loss was analyzed by HP-SEC, summarized in Table 6.

[0176] [Table 9]

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

[0178] Concentration range study of the ActRIIa-hFc fusion protein of Sequence 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 in 10 mM citrate buffer (pH 5.8) at concentrations ranging from 20 mg / mL to 200 mg / mL without other excipients, packed into Type I glass vials, and subjected to short-term stability testing at 5°C and 25°C for up to 7 days. During the study period, the purity of the formulations was measured by HP-SEC analysis, and the results are summarized in Table 7.

[0179] [Table 10]

[0180] All of the formulations showed no significant change in purity after 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 at concentrations up to 200 mg / mL, as shown in Figure 10. The results of this study indicated that protein concentrations below 75 mg / mL do 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 as ≤75 mg / mL based on stirring and concentration range studies.

[0181] Evaluation of the long-term stability of lead buffers supporting the composition of drug substances. Long-term stability studies were conducted to compare the performance of two buffer systems at two different concentrations (based on an early understanding of the need for clinical administration). The ActRIIa-hFc fusion protein of SEQ ID NO: 32 was prepared 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 for up to 30 months at -80°C, -20°C, 5°C, and 25°C, each under its own storage conditions.

[0182] The stability data obtained 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 as measured by HP-SEC, and all formulations retained over 97.8% of the monomer. After 24 months at 5°C, the monomer of the ActRIIa-hFc fusion protein of SEQ ID NO: 32 decreased to 94-95% in the citrate formulation and to 93-94% in the succinate formulation. These results indicate that the ActRIIa-hFc fusion protein of SEQ ID NO: 32 is biophysically stable in solution, even in the absence of surfactants or stabilizing sugars, as no formulation showed monomer loss (%) below 94% even after 2 years at 2-8°C. After 12 months at 25°C, the monomer of the ActRIIa-hFc fusion protein of SEQ ID NO: 32 decreased to 91-93% in the citrate formulation and to 90-92% in the succinate formulation. In both the 5°C and 25°C storage data, the 75 mg / mL drug substance decomposed slightly more in each buffer system than the 50 mg / mL drug substance, as expected, and these trends are shown in Figure 11.

[0185] In conclusion, the data from this study show that the ActRIIa-hFc fusion protein of SEQ ID NO: 32 in citrate buffer exhibits slightly better long-term stability than succinate buffer 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 freezing conditions.

[0186] Research on the salt concentration range The ActRIIa-hFc fusion protein of SEQ ID NO: 32 was prepared by adding 8% (weight / volume) sucrose to 10 mM citrate (pH 5.8) at a concentration of 50 mg / mL of ActRIIa-hFc fusion protein of SEQ ID NO: 32, and sodium chloride concentrations ranging from 0 to 150 mM. The effect of buffer strength on the biophysical properties of these preparations was evaluated. The thermal stability of these preparations was evaluated using DSC, and the diffusion interaction parameter (kD) was evaluated using DLS. The results are summarized in Table 9.

[0187] [Table 12]

[0188] The results of the study showed that the kD of the ActRIIa-hFc fusion protein of SEQ ID NO: 32 was highest in the absence of sodium chloride (16.2 mL / g), decreased to approximately 5 mL / g as the concentration increased, and leveled off at concentrations above 50 mM. The more positive the kD value of a molecule, the stronger the intermolecular repulsion, which indicates 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 the same and sufficiently high at all concentrations of sodium chloride. Based on this data, sodium chloride was considered unsuitable for further development work in stabilizing the ActRIIa-hFc fusion protein of SEQ ID NO: 32.

[0189] Excipient screening research A screening study was planned 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 screening, the ActRIIa-hFc fusion protein of SEQ ID NO: 32 was formulated at a concentration of 2 mg / mL with various excipients [salts (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 %) using HP-SEC.

[0190] [Table 13]

[0191] All formulations were biophysical stable at storage temperatures of 5°C and 25°C, retaining >99% of monomers after 12 months at 5°C and 9 months at 25°C. The evaluated excipients, with the exception of histidine, showed comparable biophysical stability even after 3 months under stress conditions (45°C), retaining 97–99% of monomers. Increasing the histidine concentration reduced biophysical stability; at up to 2% (weight / volume) histidine, monomer content decreased by 6% compared to a 0.1% (weight / volume) histidine formulation.

[0192] In conclusion, the ActRIIa-hFc fusion protein of Sequence 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, transportation, 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 of surfactants based on thermal stability To evaluate the effect of PS80 on the long-term stability of the ActRIIa-hFc fusion protein of SEQ ID NO: 32, a PS80 concentration range stability 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 using various levels of PS80 ranging from 0.001% to 0.05% in 10 mM citrate at pH 5.8 containing 8% (weight / volume) sucrose. These formulations were filled into Type I glass vials and stabilized for up to 24 months at 5°C and 25°C, and up to 3 months at 45°C. To monitor the physical stability of these formulations, their purity was measured using HP-SEC during the study period. The HP-SEC stability data are shown in Table 11.

[0195] [Table 14]

[0196] No significant differences were observed in the biophysical stability profiles among the various levels of PS80. All formulations retained over 99% (>99%) of monomer after 24 months at 5°C, >96% after 24 months at 25°C, and >93% after 3 months at 45°C. This study demonstrated that PS80 concentrations between 0.001% and 0.05% are equivalent in terms of biophysical stability under thermal stress.

[0197] Study 2: Screening of surfactants by stirring and freeze-thaw cycles To further evaluate the effect of PS80 on the stabilization of the ActRIIa-hFc fusion protein of SEQ ID NO: 32 against stirring and freeze-thaw stress, an additional PS80 concentration range study was conducted. In this study, the ActRIIa-hFc fusion protein of SEQ ID NO: 32 was formulated to a concentration of 50 mg / mL using various levels of PS80 ranging from 0% to 0.05% in 10 mM citrate at pH 5.8 containing 8% (weight / volume) sucrose. These formulations were filled into Type I glass vials and stirred at 300 RPM for up to 7 days. Separately, the formulations were subjected to freeze-thaw cycle stress of up to 8 cycles from -80°C to 25°C. Throughout the study period, the biophysical and chemical stability of SEQ ID NO: 32 was monitored by measuring the purity of the formulations by UP-SEC, measuring the fine particles by HIAC and MFI, and measuring the charge variants by desialized iCIEF. Stability data is shown in Tables 12 to 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] Regarding HMW content, monomer content, and LMW content measured by UP-SEC, there were no significant differences in the purity of any of the test formulations compared to the control sample. Compared to the control, no discernible trends were observed between the tested formulation prototypes using CE-SDS or the charge variants using desialylated iCIEF. Starting with at least 0.01% PS80, a slight decrease in 2 μm and 5 μm particulate matter due to freeze-thaw stress was observed compared to 0% PS80.

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

[0208] Sucrose concentration range study To ensure that the injection solution of the ActRIIa-hFc fusion protein of Sequence ID No. 32 has an appropriate osmotic pressure (260–380 mOsm / kg) for parenteral administration, the sucrose concentration range outlined in Table 20 was prepared in 10 mM citrate at pH 5.8, and the osmotic pressure was measured. Based on these preparations, it was determined that 8–10% (weight / volume) is appropriate for the formulation optimization studies described below.

[0209] [Table 23]

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

[0211] [Table 24]

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

[0213] [Table 25]

[0214] For the photostability of this study, 1 mL of each formulation was filled into a separate set of vials and divided into two sets for incubation in a photostability chamber: one set was exposed to 25 kLux visible light for 48 hours (1.2 mLuxhr), and the other set was exposed to 20 W / m². 2 Exposure to ultraviolet light for 10 hours (200Wh / m³) 2 ) were exposed. In addition, two control vials were prepared for each formulation: one protected from light with aluminum foil in a photostability chamber, and the other maintained at 5°C in a refrigerator. Photostability data obtained by 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 trend in purity was observed for any of the formulations, and all formulations retained >98% monomer. Regarding the photostability of this study, each formulation showed a decrease in monomer (%) of approximately 16-17% after visible light irradiation and approximately 9-10% after ultraviolet irradiation, revealing potential degradation pathways.

[0217] In conclusion, a sucrose concentration of 8% - 10% (weight / volume) 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, which is a complex mechanism to accurately summarize. Therefore, a lyophilized formulation was developed to reduce the possibility of chemical instability caused by light. An 8% (weight / volume) concentration of sucrose was considered sufficient for the development of the lyophilized formulation.

[0218] Protein concentration range study To evaluate 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 at concentrations varying from 50 mg / mL to 100 mg / mL in 10 mM citrate, 8% (weight / volume) sucrose, and 0. .02% (weight / volume) PS80. These formulations were filled into type I glass vials and subjected to stability 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 period and is summarized in Table 24.

[0219]

Table 27

[0220] There was no significant difference in the 5°C stability profile between different protein concentrations, and all formulations retained 99% monomers after 24 months at 5°C. However, the 100 mg / mL concentration showed slightly more degradation (monomer content 88%) than the lower concentrations after 3 months under stress conditions (45°C), while the lower concentrations retained 91%.

[0221] The results of this study showed that Sequence ID No. 32 can be concentrated up to 75 mg / mL without significantly affecting its long-term stability. Furthermore, based on this initial physical stability data and data from 5°C, it was suggested that it may have sufficient long-term stability even at a high concentration of 100 mg / mL.

[0222] Composition of the formulation Based on the formulation development work summarized above and the possibility of charge instability during long-term storage under the recommended storage conditions for a 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 supplied in two vial strengths, and when reconstituted with a specified volume 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, and 0.02% (weight / volume) polysorbate 80 (pH 5.8).

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

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

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

[0228] [Table 30]

[0229] At either 25°C or 45°C, pH did not have a significant effect on the stability profile of Sequence ID No. 32.

[0230] The results of this study showed that under lyophilization conditions, the biophysical stability of the ActRIIa-hFc fusion protein of Sequence ID No. 32 was not significantly affected within the pH range of 5.3 to 6.3 of the formulation composition. Furthermore, previous development studies had shown that variations in PS80 did not affect the biophysical stability in solution form. Therefore, pH and PS80 were excluded as parameters in studies of the robustness of the formulation under lyophilization conditions.

[0231] Research on the robustness of pharmaceutical formulations The robustness study of the formulation was carried out to confirm the robustness of the formulation by varying the concentrations of protein and sucrose from their target levels. These two components were selected 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. Furthermore, in this laboratory-scale study, the parameters of the primary packaging for production and the lyophilization cycle were utilized.

[0232] As outlined in Table 27, formulations with protein concentrations ranging from 45 mg / mL (Lo) to 55 mg / mL (Hi) and sucrose concentrations ranging from 7% (weight / volume) (Lo) to 9% (weight / volume) (Hi) were prepared in 10 mM citrate buffer containing 0.02% (weight / volume) polysorbate 80. Vials containing 1.45 mL of each formulation at 60 mg / vial were 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. They were analyzed for purity by HP-SEC, fragmentation by CE-SDS NR, charge variants by desialylated iCIEF, particulates by HIAC, water content by Karl-Fischer pH, and reconstitution time, and the results are summarized in Tables 28 to 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] No clear trends were observed in the reconstitution time, pH, or water content of the prototypes screened in this study under any of the test conditions. There were no significant differences in the stability profiles of the formulation prototypes regarding the content of HMW, monomer, or LMW at storage temperatures of 5°C and 25°C, which is consistent with the fragmentation pattern observed in the CE-SDS non-reduced formulation. As shown in Figure 12, the Hi-Lo formulation, with a high protein concentration (55 mg / mL) and a low sucrose concentration (7%), contained slightly more HMW species (~0.5%) on average than the other formulations, and there were slight differences among the formulations when stored at 45°C.

[0247] There were no significant differences in the stability profiles of each formulation regarding the content of acidic, major, or basic species at each storage temperature. The various charge species showed the expected variability, with acidic species ranging from 20-30%, all major species from 55-70%, and basic species from 10-20%.

[0248] Since the nominal filling volume of a 60 mg / vial is 1.45 mL, the particulate matter / mL monitored by HIAC was ≥10 μm and ≥25 μm in size, with no discernible trend in any formulation prototype, and under all conditions, USP <787> It was within the specified acceptable limits. 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 scroll concentration of 8% (weight / volume)).

[0249] array [Table 43] TIFF0007899455000045.tif245169TIFF0007899455000046.tif250168TIFF0007899455000047.tif246169TIFF0007899455000048.tif25169

[0250] Preferred methods and materials are described herein, but similar or equivalent methods and materials may also be used in the implementation or testing of the currently disclosed methods and formulations. All publications, patent applications, patents and other references mentioned herein are incorporated in their entirety by reference.

Claims

1. A pharmaceutical formulation comprising a combination of a human ActRIIa fusion protein containing the amino acid sequence of SEQ ID NO: 32 and a human ActRIIa fusion protein containing the amino acid sequence of SEQ ID NO: 41, in a concentration of 50-75 mg / mL, a citrate buffer of 4 mM-50 mM, 0.01-0.05% (weight / volume) of polysorbate 80, and 8%-10% (weight / volume) of sucrose, wherein the pharmaceutical formulation has a pH of 5.5-6.

5.

2. The pharmaceutical formulation according to claim 1, comprising a combination of a human ActRIIa fusion protein containing the amino acid sequence of SEQ ID NO: 32 and a human ActRIIa fusion protein containing the amino acid sequence of SEQ ID NO: 41 at a concentration of 50 mg / mL, a 10 mM citrate buffer, 0.02% (weight / volume) of polysorbate 80, and 8% (weight / volume) of sucrose.

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

8.

4. A lyophilized pharmaceutical preparation comprising 15-100 mg of a combination of a human ActRIIa fusion protein containing the amino acid sequence of SEQ ID NO: 32 and a human ActRIIa fusion protein containing the amino acid sequence of SEQ ID NO: 41, 0.1-1.0 mg of citrate monohydrate, 1.0-5.0 mg of trisodium citrate dihydrate, 0.1-1.0 mg of polysorbate 80, and 50-150 mg of sucrose.

5. The lyophilized pharmaceutical preparation according to claim 4, comprising 40 mg to 75 mg of a combination of a human ActRIIa fusion protein containing the amino acid sequence of SEQ ID NO: 32 and a human ActRIIa fusion protein containing the amino acid sequence of SEQ ID NO:

41.

6. The lyophilized pharmaceutical preparation according to claim 5, comprising 45 mg or 60 mg of a combination of a human ActRIIa fusion protein containing the amino acid sequence of SEQ ID NO: 32 and a human ActRIIa fusion protein containing the amino acid sequence of SEQ ID NO:

41.

7. A lyophilized pharmaceutical preparation according to claim 4, comprising 55 mg of a combination of a human ActRIIa fusion protein containing the amino acid sequence of SEQ ID NO: 32 and a human ActRIIa fusion protein containing the amino acid sequence of SEQ ID NO: 41, 0.48 mg of citrate monohydrate, 2.56 mg of trisodium citrate dihydrate, 0.22 mg of polysorbate 80, and 88.0 mg of sucrose.

8. A lyophilized pharmaceutical preparation according to claim 4, comprising 72.5 mg of a combination of a human ActRIIa fusion protein containing the amino acid sequence of SEQ ID NO: 32 and a human ActRIIa fusion protein containing the amino acid sequence of SEQ ID NO: 41, 0.64 mg of citrate monohydrate, 3.37 mg of trisodium citrate dihydrate, 0.29 mg of polysorbate 80, and 116.0 mg of sucrose.

9. The lyophilized pharmaceutical preparation according to any one of claims 4 to 8, wherein the lyophilized pharmaceutical preparation is contained in a vial.

10. A reconstituted pharmaceutical preparation comprising a lyophilized pharmaceutical preparation and water, wherein the lyophilized pharmaceutical preparation comprises 15 to 100 mg of a combination of 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, 0.1 to 1.0 mg of citrate monohydrate, 1.0 to 5.0 mg of trisodium citrate dihydrate, 0.1 to 1.0 mg of polysorbate 80 and 50 to 150 mg of sucrose.

11. The reconstituted pharmaceutical preparation according to claim 10, wherein the lyophilized pharmaceutical preparation comprises 40 mg to 75 mg of a combination of a human ActRIIa fusion protein having the amino acid sequence of SEQ ID NO: 32 and a human ActRIIa fusion protein having the amino acid sequence of SEQ ID NO:

41.

12. The reconstituted pharmaceutical preparation according to claim 11, wherein the lyophilized pharmaceutical preparation comprises 45 mg or 60 mg of a combination of a human ActRIIa fusion protein having the amino acid sequence of SEQ ID NO: 32 and a human ActRIIa fusion protein having the amino acid sequence of SEQ ID NO:

41.

13. The reconstituted pharmaceutical formulation according to claim 10, wherein the lyophilized pharmaceutical formulation comprises 55 mg of a combination of a human ActRIIa fusion protein having the amino acid sequence of SEQ ID NO: 32 and a human ActRIIa fusion protein having the amino acid sequence of SEQ ID NO: 41, 0.48 mg of citrate monohydrate, 2.56 mg of trisodium citrate dihydrate, 0.22 mg of polysorbate 80 and 88.0 mg of sucrose.

14. The reconstituted pharmaceutical formulation according to claim 10, wherein the lyophilized pharmaceutical formulation comprises 72.5 mg of a combination of a human ActRIIa fusion protein having the amino acid sequence of SEQ ID NO: 32 and a human ActRIIa fusion protein having the amino acid sequence of SEQ ID NO: 41, 0.64 mg of citrate monohydrate, 3.37 mg of trisodium citrate dihydrate, 0.29 mg of polysorbate 80 and 116.0 mg of sucrose.

15. The reconstituted pharmaceutical preparation according to claim 10, wherein the water is sterile water for injection.

16. The reconstituted pharmaceutical preparation according to claim 15, wherein the volume of the sterile water for injection is 0.8 mL to 1.5 mL.

17. The reconstituted pharmaceutical preparation according to claim 16, wherein a combination of a human ActRIIa-Fc fusion protein containing the amino acid sequence of SEQ ID NO: 32 and a human ActRIIa-Fc fusion protein containing the amino acid sequence of SEQ ID NO: 41 is provided in an amount of 45 mg, and the volume of the sterile water for injection is 0.9 mL.

18. The reconstituted pharmaceutical preparation according to claim 16, wherein a combination of a human ActRIIa-Fc fusion protein containing the amino acid sequence of SEQ ID NO: 32 and a human ActRIIa-Fc fusion protein containing the amino acid sequence of SEQ ID NO: 41 is provided in an amount of 60 mg, and the volume of the sterile water for injection is 1.2 mL.

19. A reconstituted pharmaceutical preparation comprising a lyophilized pharmaceutical preparation and water, wherein the reconstituted pharmaceutical preparation comprises 50 mg / mL of a combination of a human ActRIIa fusion protein having the amino acid sequence of SEQ ID NO: 32 and a human ActRIIa fusion protein having the amino acid sequence of SEQ ID NO: 41, 10 mM citrate, 0.2 mg / mL of polysorbate 80 and 80 mg / mL of sucrose, and has a pH of 5.

8.

20. The reconstituted pharmaceutical formulation according to claim 19, wherein the reconstituted pharmaceutical formulation is suitable for subcutaneous administration.

21. The reconstituted pharmaceutical preparation according to claim 19, wherein the reconstituted pharmaceutical preparation is contained in a vial or syringe.

22. Use of a pharmaceutical formulation according to any one of claims 1 to 3 or a reconstituted pharmaceutical formulation according to any one of claims 10 to 21 in the preparation of a pharmaceutical for the treatment of pulmonary arterial hypertension (PAH).

23. Use of a lyophilized pharmaceutical preparation according to any one of claims 4 to 8 in the preparation of a pharmaceutical for the treatment of pulmonary arterial hypertension (PAH).

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