Formulations comprising variants of Actria polypeptide

Abstract

In certain aspects, the present disclosure provides lyophilized pharmaceutical formulations comprising a recombinant fusion protein comprising an extracellular domain (ECD) of human activin receptor type-IIA (ActRIIa) proteins or derivatives thereof linked to a constant domain of an immunoglobulin, such as human IgG1 Fc domain and one or more pharmaceutical additives and / or excipients.

Description

1 / 109 Formulations comprising variants of Actria polypeptide CROSS-REFERENCE TO RELATED REQUESTS

[001] This application claims the benefit of U.S. Provisional Application No. 63 / 451,198, filed March 9, 2023, the contents of which are incorporated herein by reference in their entirety. REFERENCE TO THE LIST OF SEQUENCES SENT ELECTRONICALLY

[002] This application contains a Sequence Listing that was submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML file, created on January 29, 2024, is named 1848179-0002-169-WO1_SL.XML and is 37,058 bytes in size. FIELD

[003] The present invention describes lyophilized pharmaceutical formulations comprising a recombinant fusion protein comprising an extracellular domain (ECD) of human activin receptor type IIA protein (ActRIIa) or derivatives thereof linked to a constant domain of an immunoglobulin, such as the Fc domain of human IgG1, and one or more pharmaceutical additives and / or excipients as defined in the present invention. BACKGROUND

[004] Pulmonary hypertension (PH) is a disease characterized by high pressure in the pulmonary vasculature, including pulmonary arteries, pulmonary veins, and pulmonary capillaries. In general, PH is defined as a mean pulmonary arterial pressure (PA) ≥25 mm Hg at rest or ≥30 mm Hg with exercise (Hill et al., Respiratory Care 54(7):958-68 (2009)). The main symptom of PH is difficulty breathing or shortness of breath, and other symptoms include fatigue, dizziness, fainting, peripheral edema (swelling in the feet, legs, or ankles), Petition 870250077590, dated 01 / 09 / 2025, pages 480 / 595 2 / 109 Bluish lips and skin, chest pain, angina pectoris, dizziness during exercise, non-productive cough, rapid pulse, and palpitations. Pulmonary hypertension (PH) can be a serious disease that causes heart failure, one of the most common causes of death in people with pulmonary hypertension. Postoperative pulmonary hypertension can complicate many types of surgeries or procedures and represent a challenge associated with high mortality.

[005] PH can be grouped based on different manifestations of the disease that share similarities in pathophysiological mechanisms, clinical presentation, and therapeutic approaches (Simonneau et al., JACC 54(1):S44-54 (2009)). The clinical classification of PH was first proposed in 1973, and a recently updated clinical classification was endorsed by the World Health Organization (WHO) in 2008. According to the updated clinical classification of PH, there are five main groups of PH: (1) pulmonary arterial hypertension (PAH), characterized by a wedge pressure of BP ≥15 mm Hg; (2) PH due to left heart disease (also known as pulmonary venous hypertension or congestive heart failure); (3) PH characterized by a wedge pressure of BP >15 mm Hg; (4) PH due to pulmonary disease and / or hypoxia; chronic thromboembolic PH; and (5) PH with unclear or multifactorial etiologies (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 (HAPI), a sporadic disease in which there is no family history of PAH or identified risk factor; hereditary PAH; drug- and toxin-induced PAH; PAH associated with connective tissue diseases, HIV infection, portal hypertension, congenital heart disease, schistosomiasis, and chronic hemolytic anemia; and persistent PH of newborns (Simonneau et al., JACC 54(1):S44-54 (2009)). Diagnosis of various types of PH requires a series of tests. Petition 870250077590, dated 01 / 09 / 2025, pp. 481 / 595 3 / 109

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

[007] Thus, an objective of the present disclosure is to provide pharmaceutical formulations comprising an ActRIIa fusion protein and corresponding methods for treating, preventing or reducing the rate of progression and / or severity of PH, particularly treating, preventing or reducing the rate of progression and / or severity of one or more complications associated with PH. SUMMARY

[008] In the present invention, pharmaceutical formulations comprising a recombinant fusion protein comprising a Petition 870250077590, dated 01 / 09 / 2025, pp. 482 / 595 4 / 109 extracellular domain (ECD) of the human activin receptor type IIA (ActRIIa) or a variant thereof linked to a constant domain of an immunoglobulin, such as an Fc domain of human IgG1, and one or more pharmaceutical additives and / or excipients, in which the formulation is lyophilized.

[009] In some embodiments, the ActRIIa protein comprises an amino acid sequence that is at least 70% (for example, at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to an amino acid sequence that begins at any of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 of SEQ ID NO: 9 and ends at any of amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134 or 135 of SEQ ID NO: 9. In some embodiments, the ActRIIa protein comprises an amino acid sequence that is at least 70% (for example, at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 10.In some embodiments, the ActRIIa protein comprises an amino acid sequence that is at least 70% (for example, at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the ActRIIa protein is a fusion protein comprising an extracellular ActRIIa domain and one or more heterologous ActRIIa protein domains. In some embodiments, the ActRIIa protein is a fusion protein comprising an Fc domain of an immunoglobulin. In some embodiments, the Fc domain of the immunoglobulin is an Fc domain of an IgG1 immunoglobulin. In some embodiments, the ActRIIa fusion protein additionally comprises a binding domain positioned between the ActRIIa protein domain and one or more other domains. Petition 870250077590, dated 01 / 09 / 2025, pp. 483 / 595 5 / 109 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 comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 32. In some embodiments, the ActRIIa fusion protein comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the ActRIIa fusion protein consists of the amino acid sequence of SEQ ID NO: 32. In some embodiments, the ActRIIa fusion protein consists of the amino acid sequence of SEQ ID NO: 41.In some embodiments, the ActRIIa fusion protein consists of the amino acid sequence of SEQ ID NO: 32 or 41. In some embodiments, the ActRIIa fusion protein consists of a variant of the amino acid sequence of SEQ ID NO: 32, in which the sequence lacks the C-terminal lysine residue of SEQ ID NO: 32. In some embodiments, the variant of the ActRIIa fusion protein lacking the C-terminal lysine residue comprises or consists of the amino acid sequence of SEQ ID NO: 41. In some embodiments, the ActRIIa fusion protein is part of a homodimeric protein complex. In some embodiments, the ActRIIa fusion protein is glycosylated. In some embodiments, the ActRIIa fusion protein has a glycosylation pattern obtained by expression in a Chinese hamster ovary cell.

[010] The pharmaceutical formulations described in this invention comprise an ActRIIa fusion protein and one or more pharmaceutical additives and / or excipients. In certain embodiments, the pharmaceutical formulations described in this invention comprise a protein of Petition 870250077590, dated 01 / 09 / 2025, pp. 484 / 595 6 / 109 fusion of human ActRIIa or a variant thereof, a buffer, a surfactant and a stabilizer.

[011] In some embodiments, one or more pharmaceutical additives and / or excipients are buffering agents. In some embodiments, the buffering agent is selected to be physiologically compatible and to maintain a pH above 2. In some embodiments, the buffering agent is selected to be physiologically compatible and capable of maintaining a reconstituted lyophilized solution described in the present invention at a pH above 2. In some embodiments, the buffering agent is selected to be physiologically compatible and to maintain the pH of the pharmaceutical formulation at a pH of 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9. 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0.In some embodiments, the buffering agent is selected to be physiologically compatible and to maintain a pH of the pharmaceutical formulation between 5-7 (i.e., 5.0-7.0). In some embodiments, the buffering agent is selected to be physiologically compatible and to maintain a pH of the pharmaceutical formulation at a pH of 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0. In some embodiments, the buffering agent is selected to be physiologically compatible and to maintain a pH of the pharmaceutical formulation between 5.5 and 6.5 (i.e., from 5.5 to 6.5). In some embodiments, the buffering agent is selected to be physiologically compatible and to maintain a pH of the pharmaceutical formulation of 5.8.

[012] In some embodiments, the buffering agent comprises organic acids, succinate, phosphate, acetate, citrate, citric acid, Tris, HEPES, amino acids or mixtures of amino acids. In certain embodiments, the buffer is selected from the group consisting of sodium citrate, succinate and Petition 870250077590, dated 01 / 09 / 2025, pages 485 / 595 7 / 109 histidine. In some embodiments, the buffering agent comprises citrate. In certain embodiments, where the buffer is citrate, the citrate buffer comprises trisodium citrate dihydrate and citric acid monohydrate. In some embodiments, the buffering agent comprises trisodium citrate dihydrate. In some embodiments, the buffering agent comprises citric acid monohydrate. In some embodiments, the buffering agent comprises 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.

[013] In some embodiments, the buffering agent is present in an amount between 4 mM and 50 mM. In some embodiments, the buffering agent 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 comprises at least 10 mM of buffering agent.

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

[015] In some embodiments, the buffering agent is 10 mM of a Petition 870250077590, dated 01 / 09 / 2025, pages 486 / 595 8 / 109 citrate buffer that maintains the pH of the pharmaceutical formulation at a pH of about 5.5 to about 6.5. In some embodiments, the buffering agent is 10 mM of a succinate buffer that maintains the pH of the pharmaceutical formulation at a pH of about 5.5 to about 6.5. In some embodiments, the buffering agent is 10 mM of a histidine buffer that maintains the pH of the pharmaceutical formulation at a pH of about 5.5 to about 6.0. In some embodiments, the buffering agent is 10 mM of a citrate buffer that maintains the pH of the pharmaceutical formulation at about 5.8. In some embodiments, the buffering agent is 10 mM of a succinate buffer that maintains the pH of the pharmaceutical formulation at about 5.8.

[016] In some embodiments, one or more pharmaceutical additives and / or excipients is a surfactant. 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 salt of N-lauroylsarcosine, lithium dodecyl sulfate, sodium salt of 1-octanesulfonic acid, sodium cholate hydrate, sodium deoxycholate and sodium salt of glycodeoxycholic acid, benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride monohydrate, hexadecyltrimethylammonium bromide, CHAPS, CHAPSO, SB3-10, SB3-12, digitonin, Triton X-100, Triton X-114, TWEEN20, TWEEN-80, lauromacrogol 400, polyoxyl stearate 40, hydrogenated castor oil polyoxyethylene 10, 40, 50 and 60, glycerol monostearate, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80 and soy lecithin.In some formulations, the surfactant is polysorbate 80 or polysorbate 20. In some formulations, the surfactant is polysorbate 80.

[017] In some embodiments, the surfactant is present in an amount of 0.05-0.3 mg / mL. In some embodiments, the pharmaceutical formulation comprises at least 0.001, 0.002, 0.003, 0.004, 0.005, 0.01, Petition 870250077590, dated 01 / 09 / 2025, pp. 487 / 595 9 / 109 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% by weight / volume of surfactant. In some embodiments, the pharmaceutical formulation comprises 0.01 to 0.05% by weight / volume of surfactant. In some embodiments, the pharmaceutical formulation comprises at least 0.02% by weight / volume of surfactant. In some embodiments, the pharmaceutical formulation comprises 0.02% by weight / volume of surfactant. In some embodiments, the pharmaceutical formulation comprises 0.02% of polysorbate 80 or polysorbate 20.

[018] In some embodiments, one or more 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, polyhydroxylated compounds, polysaccharides, dextran, starch, hydroxyethyl starch, cyclodextrins, N-methylpyrrolidene, cellulose, and hyaluronic acid. In some embodiments, the stabilizer is sucrose.

[019] In some embodiments, the stabilizer is present in the pharmaceutical formulation at a concentration between 2-16% weight / volume. In some embodiments, the pharmaceutical formulation comprises between about 8% and about 10% stabilizer by weight / volume. In some embodiments, the pharmaceutical formulation comprises at least 0.005% weight / volume, 0.01% weight / volume, 0.02% weight / volume, 0.03% weight / volume, 0.05% weight / volume, 0.06% weight / volume, 0.07% weight / volume, 0.08% weight / volume, 0.09% weight / volume, 0.1% weight / volume, 0.5% weight / volume, 0.7% weight / volume, 0.8% weight / volume, 0.9% weight / volume, 1.0% weight / volume, 1.2% weight / volume, 1.5% weight / volume, 1.7% weight / volume, 2% weight / volume, 3% weight / volume, 4% weight / volume, 5% weight / volume, 6% weight / volume, Stabilizer of 7% Petition 870250077590, dated 01 / 09 / 2025, pages 488 / 595 10 / 109 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% by weight / volume.

[020] In certain embodiments, the pharmaceutical formulations described in the present invention do not contain a salt.

[021] The present invention also describes lyophilized pharmaceutical formulations made by lyophilizing the liquid formulations described in the present invention. The present invention also describes pharmaceutical formulations in which the formulation is reconstituted from a lyophilized formulation.

[022] In some embodiments, the lyophilized pharmaceutical formulation is reconstituted with Sterile Water for Injection. In some embodiments, the lyophilized pharmaceutical formulation is reconstituted with Sterile Water for Injection to a final protein concentration of 10-100 mg / mL. In some embodiments, the lyophilized pharmaceutical formulation is reconstituted with Sterile Water for Injection to a final protein concentration of 45-55 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 fusion protein ActRIIa is present in an amount of approximately 75 mg / mL or less.In certain forms, the human fusion protein ActRIIa is present in one. Petition 870250077590, dated 01 / 09 / 2025, pages 489 / 595 11 / 109 quantity of approximately 50 mg / mL.

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

[024] In some embodiments, the lyophilized pharmaceutical formulation is reconstituted with Sterile Water for Injection. In some embodiments, the lyophilized pharmaceutical formulation is reconstituted with Sterile Water for Injection to a final protein concentration of 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 formulation is reconstituted with Sterile Water for Injection to a final protein concentration of approximately 50 mg / mL.

[025] In certain embodiments, the lyophilized formulation is reconstituted with approximately 0.5 mL to 1.8 mL of Sterile Water for Injection. In some embodiments, the lyophilized pharmaceutical formulation is reconstituted with approximately 0.5 mL, 0.56 mL, 0.58 mL, 0.6 mL, 0.62 mL, 0.64 mL, 0.66 mL, 0.68 mL, 0.70 mL, 0.72 mL, 0.74 mL, 0.76 mL, 0.78 mL, 0.8 mL, 0.82 mL, 0.84 mL, 0.86 mL, 0.88 mL, 0.9 mL, 0.92 mL, 0.94 mL, 0.96 mL, 0.98 mL, 1 mL, 1.1 mL, 1.15 mL, 1.2 mL, 1.25 mL, 1.3 mL, 1.35 mL, 1.4 mL, 1.45 mL, 1.5 mL, 1.55 mL, 1.6 mL, 1.65 mL, 1.7 mL, 1.75 mL, or 1.8 mL of Sterile Water for Injection. In some embodiments, the lyophilized pharmaceutical formulation comprises an ActRIIa fusion protein supplied in an amount of 45 mg / vial, and the formulation is reconstituted with 0.9 mL, 0.95 mL, 1 mL, or 1.1 mL of Sterile Water for Injection. In some embodiments, the lyophilized pharmaceutical formulation comprises an ActRIIa fusion protein supplied in an amount Petition 870250077590, dated 01 / 09 / 2025, pages 490 / 595 12 / 109 of 45 mg / vial, and the formulation is reconstituted with 1.1 mL of Sterile Water for Injection. In some embodiments, the lyophilized pharmaceutical formulation comprises an ActRIIa fusion protein which is supplied in an amount of 60 mg / vial, and wherein the formulation is reconstituted with 0.90 mL, 0.95 mL, 1.00 mL, 1.05 mL, 1.10 mL, 1.15 mL, 1.20 mL, 1.25 mL, 1.30 mL, 1.35 mL, 1.40 mL, 1.45 mL or 1.50 mL of Sterile Water for Injection. In some embodiments, the lyophilized pharmaceutical formulation comprises an ActRIIa fusion protein which is supplied in an amount of 60 mg / vial, and wherein the formulation is reconstituted with 1.30 mL of Sterile Water for Injection.

[026] In certain embodiments, the lyophilized formulation is supplied in a quantity of 45 mg / vial, and in which the formulation is reconstituted with between 0.65 mL and 0.75 mL of Sterile Water for Injection. In certain embodiments, the lyophilized formulation is supplied in a quantity of 60 mg / vial, and in which the formulation is reconstituted with between 0.65 mL and 1.75 mL of Sterile Water for Injection.

[027] In certain embodiments, the lyophilized pharmaceutical formulation comprises a human ActRIIa fusion protein of SEQ ID NO: 32 or a variant of SEQ ID NO: 32 lacking the C-terminal lysine (SEQ ID NO: 41), a buffer, a surfactant, and a stabilizer, wherein the buffering agent is selected to be physiologically compatible and maintain a pH of 5.8 when reconstituted with Sterile Water for Injection. In certain embodiments, the buffering agent comprises citrate. In certain cases, 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 buffering agent is selected to be physiologically compatible and maintain a pH of 5.8 when reconstituted. Petition 870250077590, dated 01 / 09 / 2025, pp. 491 / 595 13 / 109 with Sterile Water for Injection.

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

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

[030] In certain embodiments, the lyophilized pharmaceutical formulation comprises a human ActRIIa fusion protein of SEQ ID NO: Petition 870250077590, dated 01 / 09 / 2025, pp. 492 / 595 14 / 109 or a variant of SEQ ID NO:32 lacking the C-terminal lysine residue (SEQ ID NO: 41), 0.64 mg of citric acid monohydrate, 3.37 mg of trisodium citrate dihydrate, 0.29 mg of polysorbate 80, and 116.0 mg of sucrose. In some embodiments, the human ActRIIa fusion protein consists of the amino acid sequence of SEQ ID NO:41. In some embodiments, the lyophilized formulation comprises a human ActRIIa fusion protein comprising the amino acid sequence of SEQ ID NO: 32 and a human ActRIIa fusion protein comprising the amino acid sequence of SEQ ID NO:41. In some embodiments, the lyophilized pharmaceutical formulation comprises sotatercept.

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

[032] In certain embodiments, the pharmaceutical formulations described in this invention contain a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 lacking the C-terminal lysine residue (SEQ ID NO: 41). In certain embodiments, the pharmaceutical formulations described in this invention contain a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 Petition 870250077590, dated 01 / 09 / 2025, pp. 493 / 595 15 / 109 devoid of C-terminal lysine residue (SEQ ID NO: 41) where the mixture contains 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57% 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42% 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27% 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12% 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% by weight of SEQ ID NO:32.

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

[034] In certain embodiments, the pharmaceutical formulations described in the present invention contain a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 devoid of the C-terminal lysine residue (SEQ ID NO: 41) wherein the mixture contains 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58% 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43% 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28% Petition 870250077590, dated 01 / 09 / 2025, pp. 494 / 595 16 / 109 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% by weight of SEQ ID NO:41.

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

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

[037] The present invention also describes methods for treating pulmonary arterial hypertension (PAH), comprising administering to a patient in need thereof a pharmaceutical formulation described in the present invention.

[038] The present invention also describes the uses of a pharmaceutical formulation for the treatment of pulmonary arterial hypertension. Petition 870250077590, dated 01 / 09 / 2025, pages 495 / 595 17 / 109 (PAH), comprising administering to a patient in need thereof the pharmaceutical formulation described in the present invention.

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

[040] In some embodiments, once reconstituted with sterile water, the vial comprises 50 mg / mL of the ActRIIa fusion protein comprising the amino acid sequence of SEQ ID NO: 32, 10 mM citrate, 0.2 mg / mL polysorbate 80 and 8% by weight sucrose. In some embodiments, the protein Petition 870250077590, dated 01 / 09 / 2025, pp. 496 / 595 Human ActRIIa fusion 18 / 109 comprises or consists of the amino acid sequence of SEQ ID NO: 41, 10 mM citrate, 0.2 mg / mL polysorbate 80 and 8% by weight sucrose. In some embodiments, once reconstituted with sterile water, the vial comprises 50 mg / mL sotatercept, 10 mM citrate, 0.2 mg / mL polysorbate 80 and 8% by weight sucrose.

[041] In some embodiments, the reconstituted pharmaceutical formulation comprises 50 mg / mL of an ActRIIa fusion protein comprising the amino acid sequence of SEQ ID NO: 32, 10 mM citrate, 0.02 mg / mL polysorbate 80 and 80 mg / mL sucrose at a pH of 5.8. In some embodiments, the human ActRIIa protein comprises the amino acid sequence of SEQ ID NO: 41, 10 mM citrate, 0.02 mg / mL polysorbate 80 and 80 mg / mL sucrose at a pH of 5.8. In some embodiments, the reconstituted pharmaceutical formulation comprises 50 mg / mL of sotatercept, 10 mM citrate, 0.02 mg / mL polysorbate 80 and 80 mg / mL sucrose at a pH of 5.8. BRIEF DESCRIPTION OF THE DRAWINGS

[042] Figure 1 shows components of a kit comprising a lyophilized pharmaceutical formulation comprising an ActRIIa fusion protein and an injection device. A vial (1) contains a lyophilized pharmaceutical formulation comprising an ActRIIa fusion protein, a reconstituted sterile injectable solution, or a sterile injectable solution. A pre-filled syringe (2) containing a reconstitution solution is used to reconstitute a lyophilized pharmaceutical formulation comprising an ActRIIa fusion protein from (1) into a sterile injectable solution. A vial adapter (3) connects the vial (1) to the pre-filled syringe (2) via attachment to the vial at one end and attachment to the pre-filled syringe at the opposite end. A syringe (4) and a needle (5) are provided for administration of sterile injectable solution. Moistened wipes (6) are provided for sterilization. Petition 870250077590, dated 01 / 09 / 2025, pages 497 / 595 19 / 109 of the individual components of the kit.

[043] Figure 2 shows a multiple sequence alignment of several vertebrate ActRIIa proteins and human ActRIIa (SEQ ID NOs: 62-68).

[044] Figures 3A and 3B show the purification of ActRIIa-hFc expressed in CHO cells. The protein is purified as a single, well-defined peak, as visualized by the scaling column (top panel) and Coomassie-stained SDSPAGE (bottom panel) (left band: molecular weight standards; right band: ActRIIa-hFc).

[045] Figures 4A and 4B show the binding of ActRIIa-hFc to activin (top panel) and GDF-11 (bottom panel b), as measured by BiacoreTM assay.

[046] Figure 5 shows the effect of pH and buffer on the percentage of monomeric species by PH-SEC after 6M at 25 °C.

[047] Figure 6 shows the effect of pH and buffer on Biacore™ binding activity after 6M stabilization at 25 °C.

[048] Figure 7 shows the effect of buffer and pH on % HMW, monomer and % LMW of the species by PH-SEC after 3 months of stability.

[049] Figure 8 shows the effect of pH and buffer on % of minor peak and % of major peak by CE-SDS NR after 1 month of stability.

[050] Figure 9 shows the effect of pH and buffer on % of acidic species, % of basic species and % of total major species per desialylated icIEF after 3 months of stability.

[051] Figure 10 shows the effect of the concentration in % of monomeric species by PH-SEC.

[052] Figure 11 shows the effect of buffer and concentration in % of monomeric species by PH-SEC on stability.

[053] Figure 12 shows the effect of protein and sucrose concentration on purity by PH-SEC on stability. Petition 870250077590, dated 01 / 09 / 2025, pages 498 / 595 20 / 109 DETAILED DESCRIPTION Definitions

[054] The terms used in this specification generally have their common meanings in the art, within the context of this disclosure and in the specific context where each term is used. Certain terms are discussed below or elsewhere in the specification to provide additional guidance to the practitioner in describing disclosure formulations and methods and how to make and use them. The scope or meaning of any use of a term will be apparent from the specific context in which the term is used.

[055] “About” and “approximately” generally mean an acceptable degree of error for the measured quantity, given the nature or precision of the measurements. Typically, exemplary error degrees are within 20 percent (%), preferably within 10 percent, and most preferably within 5 percent of a given value or range of values.

[056] Alternatively, and particularly in biological systems, the terms “about” and “approximately” may mean values ​​that are within an order of magnitude, preferably within 5 times and more preferably within 2 times of a given value. The numerical quantities given in this invention are approximate unless otherwise indicated, meaning that the term “about” or “approximately” may be inferred when not expressly stated.

[057] The terms "a" and "an" include plural referents unless the context in which the term is used clearly indicates otherwise. The terms "a" (or "an"), as well as the terms "one or more" and "at least one" may be used interchangeably in the present invention. Furthermore, "and / or," where used in the present invention, shall be considered as specific disclosure of each of the two or more specified attributes or components, with or without Petition 870250077590, dated 01 / 09 / 2025, pp. 499 / 595 21 / 109 the other. Thus, the term “and / or”, as used in a phrase such as “A and / or B” in the present invention, is intended to include “A and B”, “A or B”, “A” (alone) and “B” (alone). Similarly, the term and / or, as used in a phrase such as A, B and / or C, is intended to encompass each of the following aspects: 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).

[058] The numerical ranges disclosed in the present invention include the numbers that define the ranges. For example, pH between 5.0 and 7.0 includes pH of 5.0 and pH 7.0 and values ​​between 5.0 and 7.0.

[059] A protein disclosed in the present invention may comprise an amino acid sequence that does not occur naturally. Such variants necessarily have less than 100% sequence identity or similarity to the initial molecule. In certain embodiments, the variant will have an amino acid sequence of about 75% to less than 100% identity or similarity to the amino acid sequence of the initial protein (e.g., naturally occurring or wild type), more preferably from about 80% to less than 100%, more preferably from about 85% to less than 100%, more preferably from about 90% to less than 100% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) and most preferably from about 95% to less than 100%, for example, along the length of the variant molecule. Pharmaceutical Formulation

[060] The present invention provides pharmaceutical formulations comprising a recombinant fusion protein comprising an extracellular domain (ECD) of human activin receptor type IIA (ActRIIa) proteins or derivatives thereof linked to a constant domain of an immunoglobulin, such as the Fc domain of human IgG1, wherein the formulations Petition 870250077590, dated 01 / 09 / 2025, pages 500 / 595 22 / 109 pharmaceuticals are lyophilized. In certain respects, the disclosure refers to a pharmaceutical formulation comprising an extracellular domain (ECD) of a human ActRIIa protein or a derivative thereof linked to a constant domain of an immunoglobulin, such as the Fc domain of human IgG1, wherein the pharmaceutical formulation is reconstituted from a lyophilized formulation in a sterile solution for injection.

[061] In certain aspects, the discovery refers to a lyophilized pharmaceutical formulation comprising an extracellular domain (ECD) of the human ActRIIa protein or a derivative thereof linked to a constant domain of an immunoglobulin, such as an Fc domain of human IgG1, for reconstitution in a sterile solution for injection. During lyophilization, a liquid formulation is converted from an aqueous phase to an amorphous solid phase, which protects the protein from chemical and / or conformational instability. Lyophilization is performed using techniques common in the field, and lyophilized formulations are optimized for stability, shelf life, and reduced 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). Pharmaceutical formulations have aided in stabilizing the protein against the stresses of manufacturing, transport, and storage.The excipients and additives used in lyophilized formulations are integral components of a formulation and therefore need to be safe and well-tolerated by patients. For protein drugs, the choice of excipients and additives is particularly important because they can affect both the efficacy and immunogenicity of the drug. Excipients and additives are also useful for reducing the viscosity of high-concentration protein formulations in order to facilitate their administration and increase patient convenience. The formulation excipients and additives disclosed in the present invention provide stability against... Petition 870250077590, dated 01 / 09 / 2025, pages 501 / 595 23 / 109 these tensions. Common excipients are known in the art and can be found in Powell et al., Compendium of Excipients for Parenteral Formulations (1998), PDA J. Pharm. Sci. Technology, 52:238-311.

[062] In another aspect, pharmaceutical formulations comprising an ActRIIa fusion protein are provided in the present invention. In some embodiments, the formulation is lyophilized. In other 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 between 15 mg and 100 mg of ActRIIa fusion protein. In certain embodiments, the pharmaceutical formulation comprises between 20 mg and 80 mg of ActRIIa fusion protein. In certain embodiments, the pharmaceutical formulation comprises between 40 mg and 70 mg of ActRIIa fusion protein.In certain embodiments, the pharmaceutical formulation comprises 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. mg, approximately 92.5 mg, approximately 95 mg, approximately 97.5 mg, or approximately 100 mg of the ActRIIa fusion protein.

[063] In certain embodiments, the lyophilized pharmaceutical formulation comprising an ActRIIa fusion protein is supplied as a lyophilized powder or cake in a vial. In specific embodiments, the lyophilized pharmaceutical formulation comprises an ActRIIa fusion protein in an amount of 45 Petition 870250077590, dated 01 / 09 / 2025, pp. 502 / 595 24 / 109 mg / vial or 60 mg / vial. In more specific embodiments, each of the aforementioned 45 mg / vial or aforementioned 60 mg / vial is reconstituted with Sterile Water for Injection, each containing a final concentration of 45 to 55 mg / mL of the reconstituted ActRIIa fusion protein. In a more specific embodiment, each of the aforementioned 45 mg / vial or aforementioned 60 mg / vial is reconstituted with Sterile Water for Injection, each containing a final concentration of 50 mg / mL of the reconstituted ActRIIa fusion protein (active pharmaceutical ingredient).

[064] In certain embodiments, the lyophilized pharmaceutical formulation comprises an ActRIIa fusion protein in an amount of 45 mg / vial or 60 mg / vial and is reconstituted with Sterile Water for Injection. In a more specific embodiment, the lyophilized pharmaceutical formulation provided in each of the aforementioned 45 mg / vial or aforementioned 60 mg / vial is reconstituted with Sterile Water for Injection to a final 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 of the reconstituted ActRIIa fusion protein. In a more specific embodiment, the lyophilized pharmaceutical formulation provided in each of the aforementioned 45 mg / vial or aforementioned 60 mg / vial is reconstituted with Sterile Water for Injection to a final concentration of 45 mg / mL, 46 mg / mL, 47 mg / mL, 48 mg / mL, 49 mg / mL, 50 mg / mL, 51 mg / mL, 52 mg / mL, 53 mg / mL, 54 mg / mL or 55 mg / mL of the reconstituted ActRIIa fusion protein.In some embodiments, the lyophilized pharmaceutical formulation provided in each of the aforementioned 45 mg / vial or aforementioned 60 mg / vial doses is reconstituted with Sterile Water for Injection to a final concentration of approximately 50 mg / mL of the reconstituted ActRIIa fusion protein. In some embodiments, the lyophilized pharmaceutical formulation provided in each of the aforementioned 45 mg / vial or aforementioned 60 mg / vial doses is reconstituted. Petition 870250077590, dated 01 / 09 / 2025, pp. 503 / 595 25 / 109 with Sterile Water for Injection to a final concentration of 50 mg / mL of reconstituted ActRIIa fusion protein.

[065] In some embodiments, the lyophilized pharmaceutical formulation provided in each of the aforementioned 45 mg / vial or aforementioned 60 mg / vial is reconstituted with 0.5 to 2 mL of Sterile Water for Injection. In some embodiments, the lyophilized pharmaceutical formulation in each of the aforementioned 45 mg / vial or aforementioned 60 mg / vial is reconstituted with approximately 0.5 mL, 0.56 mL, 0.58 mL, 0.6 mL, 0.62 mL, 0.64 mL, 0.66 mL, 0.68 mL, 0.70 mL, 0.72 mL, 0.74 mL, 0.76 mL, 0.78 mL, 0.8 mL, 0.82 mL, 0.84 mL, 0.86 mL, 0.88 mL, 0.9 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 provided in each of the aforementioned 45 mg / vial or aforementioned 60 mg / vial is reconstituted with 0.5 mL, 0.55 mL, 0.56 mL, 0.57 mL, 0.58 mL, 0.59 mL, 0.6 mL, 0.65 mL, 0.66 mL, 0.67 mL, 0.68 mL, 0.69 mL, 0.70 mL, 0.71 mL, 0.72 mL, 0.73 mL, 0.74 mL, 0.75 mL, 0.76 mL, 0.77 mL, 0.78 mL, 0.79 mL, 0.8 mL, 0.85 mL, 0.9 mL, 0.95 mL, 1 mL, 1.1 mL, 1.15 mL, 1.2 mL. 1.25 mL, 1.3 mL, 1.35 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 the aforementioned 45 mg / vial is reconstituted with 0.8 mL, 0.85 mL, 0.9 mL, 0.95 mL, 1 mL, 1.1 mL, 1.15 mL, 1.2 mL, 1.25 mL, 1.3 mL, 1.35 mL or 1.4 mL of Sterile Water for Injection. In some embodiments, the lyophilized pharmaceutical formulation provided in the aforementioned 45 mg / vial is reconstituted with 0.9 mL, 0.95 mL, 1 mL or 1.1 mL of Sterile Water. Petition 870250077590, dated 01 / 09 / 2025, pp. 504 / 595 26 / 109 for Injection. In some embodiments, the lyophilized pharmaceutical formulation provided in the aforementioned 45 mg / vial is reconstituted with 1.1 mL of Sterile Water for Injection. In some embodiments, the lyophilized pharmaceutical formulation provided in the aforementioned 60 mg / vial is reconstituted with 0.8 mL, 0.85 mL, 0.9 mL, 0.95 mL, 1 mL, 1.1 mL, 1.15 mL, 1.2 mL, 1.25 mL, 1.3 mL, 1.35 mL or 1.4 mL 0.75 mL of Sterile Water for Injection. In some embodiments, the lyophilized pharmaceutical formulation provided in the aforementioned 60 mg / vial is reconstituted with 1 mL, 1.1 mL, 1.2 mL or 1.3 mL of Sterile Water for Injection. In some embodiments, the lyophilized pharmaceutical formulation provided in the aforementioned 60 mg / vial is reconstituted with 1.3 mL of Sterile Water for Injection.

[066] In some embodiments, the lyophilized pharmaceutical formulations provided in the present invention comprise an ActRIIa fusion protein and one or more pharmaceutical additives and / or excipients. In certain embodiments, one or more pharmaceutical additives and / or excipients comprise a buffer, a stabilizer, and a surfactant. Buffering agents may be selected to maintain the pH of the formulation during processing and reconstitution. Stabilizers may include cryo- and lyoprotectants, such as polyols, sugars, and polysaccharides, and may be selected to protect the formulation from freeze / thaw cycle stresses and stabilize the formulation in the lyophilized state. Surfactants may be selected based on their ability to serve as an emulsifier, wetting agent, solubilizer, and / or dispersant.

[067] The formulations provided in the present invention comprise buffering agents, surfactants and sugars, which are described in greater detail below.

[068] A person skilled in the art would recognize that the concentrations of the excipients described in the present invention share a Petition 870250077590, dated 01 / 09 / 2025, pages 505 / 595 27 / 109 interdependence within a specific formulation. By way of example, the concentration of a bulking agent is, in one aspect, reduced where, for example, there is a high concentration of protein. Excipients and other additives are added to impart or improve the manufacturability and / or final quality of the product, such as the stability and administration of a drug (e.g., protein). The formulations provided in the present invention comprise suitable excipients that enhance stability and safety. Buffering Agents

[069] Typically, the stability of a pharmacologically active protein formulation is observed to be maximal within a narrow pH range. This ideal stability pH range needs to be identified early during pre-formulation studies. Several approaches, such as accelerated stability studies and calorimetric screening studies, are useful in this endeavor (Remmele RL Jr., et al., Biochemistry, 38(16): 5241-7 (1999)). After the formulation is finalized, the protein must be manufactured and maintained throughout its shelf life. Therefore, buffering agents are almost always employed to control the pH in the formulation.

[070] Several factors must be considered when choosing a buffering agent. First, the buffer species and its concentration must be defined based on its pKa and the desired pH of the formulation. Equally important is ensuring that the buffer is compatible with the protein and other excipients in the formulation and does not catalyze any degradation reactions. A third important aspect to consider is the burning and irritation sensation that the buffer may induce during administration. The potential for burning and irritation is greater for drugs administered subcutaneously (SC) or intramuscularly (IM), where the drug solution remains at the site for Petition 870250077590, dated 01 / 09 / 2025, pages 506 / 595 28 / 109 a relatively longer period of time than when administered intravenously, where the formulation is rapidly diluted in the blood after administration. For formulations administered by direct intravenous infusion, the total amount of buffer (and any other component of the formulation) needs to be monitored.

[071] Buffers for lyophilized formulations require further consideration. For example, specific buffers, such as sodium phosphate, have a propensity to crystallize outside the amorphous protein phase during freezing, resulting in pH shifts. In certain embodiments, exemplary buffering agents used to buffer pharmaceutical formulations as set forth in the present invention include, but are not limited to, organic acids, succinate, phosphate, acetate, citrate, Tris, HEPES, and amino acids or mixtures of amino acids, including, but not limited to, aspartate, arginine, and glycine. In some embodiments, the buffering agent comprises trisodium citrate dihydrate. In some embodiments, the buffering agent comprises citric acid monohydrate. In some embodiments, the buffering agent comprises citrate. In one embodiment, the buffering agent comprises trisodium citrate dihydrate and citric acid monohydrate.In another embodiment, the buffering agents are trisodium citrate dihydrate and citric acid monohydrate. In certain embodiments, where the protein has a negative charge due to the presence of glycans, the buffer is not histidine. In some embodiments, the buffer comprises citrate, succinate, or histidine.

[072] In certain embodiments, the amount of buffer in the lyophilized formulation is between 0.3 mg and 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 Petition 870250077590, dated 01 / 09 / 2025, pages 507 / 595 29 / 109 1.0 mg, about 1.5 mg, about 2.0 mg, about 2.5 mg, about 3.0 mg, about 3.5 mg, about 4.0 mg, about 4.5 mg or about 5.0 mg.

[073] In certain embodiments, the buffer comprises citric acid monohydrate and trisodium citrate dihydrate, wherein the amount of citric acid monohydrate is between 0.1 mg and 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 certain embodiments, the amount of citric acid monohydrate is 0.48 mg. In certain embodiments, the amount of citric acid monohydrate is 0.64 mg.

[074] In certain embodiments, the buffer comprises citric acid monohydrate and trisodium citrate dihydrate, with the amount of trisodium citrate dihydrate being between 1.0 mg and 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 formulations, the amount of trisodium citrate dihydrate is 3.37 mg.

[075] In one embodiment, the buffering agent present in the formulation is selected to be physiologically compatible and to maintain a desired pH of the pharmaceutical formulation when reconstituted with Sterile Water for Injection. Petition 870250077590, dated 01 / 09 / 2025, pages 508 / 595 30 / 109 In another embodiment, the pH of the solution is above 2. In another embodiment, the pH of the solution is between pH 2.0 and pH 12.0. For example, in several 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, 9.0. In some embodiments, the pH of the solution is between pH 5 and pH 7 when reconstituted in solution. 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 from about 5.5 to about 6.5. In one embodiment, the pH of the reconstituted solution is from 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 from about 6.0 to about 6.5. In one embodiment, the pH of the reconstituted solution is 5.8.

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

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

[078] The pH buffering compound may be present in any amount suitable to maintain the pH of the formulation at a predetermined level. When appropriately low levels of buffer are used, Petition 870250077590, dated 01 / 09 / 2025, pp. 509 / 595 31 / 109 Crystallization and pH changes can be avoided. In one embodiment, the concentration of the buffering agent is between 0.1 mM and 500 mM (1 M). For example, it is contemplated that the buffering agent be at least 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 500 mM. In certain embodiments, the buffering agent is 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 500 mM. In one embodiment, the concentration of the buffering agent is between 0.1 mM and 20 mM. In certain embodiments, the buffering agent is 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mM. In certain embodiments, the buffering agent is present in an amount of 10 mM.

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

[080] In some embodiments, the buffering agent comprises trisodium citrate dihydrate. In some embodiments, the buffering agent comprises citric acid monohydrate. In some embodiments, the buffering agent comprises citrate. In some embodiments, the agent Petition 870250077590, dated 01 / 09 / 2025, pages 510 / 595 32 / 109 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 buffering agent is present in the pharmaceutical formulation at a concentration of at least 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200 or 500 mM. In some embodiments, the pharmaceutical formulation comprises at least 10 mM of buffering agent. Stabilizers

[081] In certain embodiments, the pharmaceutical formulations provided in the present invention comprise stabilizers. These stabilizers can be classified based on the mechanisms by which they stabilize proteins against various chemical and physical stresses. Some stabilizers are used to alleviate the effects of a specific stress or to regulate a particular susceptibility of a specific protein. Other stabilizers have more general effects on the physical and covalent stabilities of proteins. Considering the teachings and guidance provided in the present invention, those skilled in the art will know what amount or range of stabilizer can be included in any specific formulation to obtain a formulation of the discovery that is likely to promote the retention and stability of the ActRIIa protein.

[082] In some embodiments, a stabilizer (or a combination of stabilizers) is added to the formulation to prevent or reduce storage-induced aggregation and chemical degradation. A cloudy or turbid solution after reconstitution usually indicates that the protein has precipitated or at least aggregated. Stabilizers are able to prevent aggregation or chemical degradation (e.g., autolysis, deamidation, oxidation, etc.). Some stabilizers are also able to act as anticoagulants when... Petition 870250077590, dated 01 / 09 / 2025, pages 511 / 595 33 / 109 administration of the formulation to a patient. In certain embodiments, the pharmaceutical formulations provided in the present invention include stabilizers, including, but not limited to, sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, glucosamine, fructose, mannitol, sorbitol, polyhydroxylated compounds, including polysaccharides such as dextran, starch, hydroxyethyl starch, cyclodextrins, N-methylpyrrolidene, cellulose and hyaluronic acid (Carpenter et al., Develop. Biol. Standard 74:225, (1991)). In one embodiment of the disclosure, sucrose is used as a stabilizer.

[083] In certain embodiments, the lyophilized formulations described in the present invention contain a stabilizer in an amount between 50 mg and 150 mg. In certain embodiments, the lyophilized formulations described in the present invention contain a stabilizer in an amount of 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg or 150 mg. In certain embodiments, the lyophilized formulations described in the present invention contain a stabilizer in an amount of about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg or about 150 mg.

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

[085] In certain embodiments, the reconstituted formulation comprises a stabilizer at a concentration of about 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 700, 900 or 1000 mM. Similarly, in certain development methods, the stabilizer is incorporated at a concentration of approximately 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 Petition 870250077590, dated 01 / 09 / 2025, pp. 512 / 595 34 / 109 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% weight / volume.

[086] In some embodiments, the formulation comprises about 8% to about 10% by weight / volume of sucrose. In some embodiments, the formulation comprises 8-10% by weight / volume of sucrose. In some embodiments, the formulation comprises 8%, 9% or 10% by weight / volume of sucrose. In some embodiments, the formulation comprises 8% by weight / volume of sucrose. Surfactants

[087] In certain embodiments, the pharmaceutical formulations provided in the present invention may additionally include surfactants. Surfactants are commonly used in protein formulations to prevent surface-induced degradation. Surfactants are amphipathic molecules with the ability to overcome proteins at interfacial positions (and / or promote the proper refolding of a structurally altered protein molecule). Hydrophobic portions of the surfactant molecules occupy interfacial positions (e.g., air / liquid), while hydrophilic portions of the molecules remain oriented towards the bulk solvent. At sufficient concentrations (typically around the critical micelle concentration of the detergent), a surface layer of surfactant molecules serves to prevent protein molecules from being adsorbed at the interface. In this way, surface-induced degradation is minimized.The surfactants contemplated in the present invention include, without limitation, sorbitan polyethoxylate fatty acid esters, namely polysorbate 20 and polysorbate 80. The two differ only in the length of the aliphatic chain that confers hydrophobic character to the molecules, C-12 and C-18, respectively. Consequently, polysorbate-80 is more active on the surface and has a micellar concentration. Petition 870250077590, dated 01 / 09 / 2025, pages 513 / 595 35 / 109 lower critical value than polysorbate-20.

[088] Detergents can also affect the thermodynamic conformational stability of proteins. Non-ionic surfactants are generally useful in stabilizing proteins. Ionic surfactants (detergents) typically destabilize proteins. Here again, the effects of a given detergent excipient will be protein-specific. For example, polysorbates have been shown to reduce the stability of some proteins and increase the stability of others. The destabilization of proteins by detergents can be rationalized in terms of the hydrophobic tails of detergent molecules that can engage in specific binding with partially or fully unfolded protein states. These types of interactions can cause a shift in conformational equilibrium toward the more expanded protein states (i.e., increasing the exposure of hydrophobic portions of the protein molecule in addition to polysorbate binding).Alternatively, if the native state of the protein exhibits some hydrophobic surfaces, the binding of the detergent to the native state can stabilize this conformation. Another aspect of polysorbates is that they are inherently susceptible to oxidative degradation. Frequently, as raw materials, they contain sufficient amounts of peroxides to cause the oxidation of side chains of protein residues, especially methionine. The potential for oxidative damage resulting from the addition of stabilizers emphasizes the point that the lowest effective concentrations of excipients should be used in formulations. For surfactants, the effective concentration of a given protein will depend on the stabilization mechanism.

[089] Surfactants are also added in appropriate amounts to prevent the surface-related aggregation phenomenon during Petition 870250077590, dated 01 / 09 / 2025, pages 514 / 595 36 / 109 freezing and drying (Chang, B, J. Pharm. Sci. 85:1325, (1996)). Thus, exemplary surfactants include, without limitation, anionic, cationic, nonionic, zwitterionic and amphoteric surfactants, including surfactants derived from natural amino acids. Anionic surfactants include, but are not limited to, sodium lauryl sulfate, sodium dioctyl sulfosuccinate and sodium dioctyl sulfonate, chenodeoxycholic acid, sodium salt of N-lauroylsarcosine, lithium dodecyl sulfate, sodium salt of 1-octanesulfonic acid, sodium cholate hydrate, sodium deoxycholate and sodium salt of glycodeoxycholic acid. Cationic surfactants include, but are not limited to, benzalkonium chloride or benzethonium chloride, cetylpyridinium chloride monohydrate, and hexadecyltrimethylammonium bromide. Zwitterionic surfactants include, but are not limited to, CHAPS, CHAPSO, SB3-10, and SB3-12.Non-ionic surfactants include, but are not limited to, digitonin, Triton X-100, Triton X114, TWEEN-20 and TWEEN-80. Surfactants also include, but are not limited to, lauromacrogol 400, polyoxyl stearate 40, hydrogenated castor oil with polyoxyethylene 10, 40, 50 and 60, glyceryl monostearate, polysorbate 40, polysorbate 60, polysorbate 65 and polysorbate 80, soy lecithin and other phospholipids such as dioleyl phosphatidylcholine (DOPC), dimyristoyl phosphatidyl glycerol (DMPG), dimyristoyl phosphatidylcholine (DMPC) and (dioleyl phosphatidyl glycerol) DOPG; Sucrose fatty acid ester, methylcellulose, and carboxymethylcellulose. Therefore, formulations comprising these surfactants, individually or as a mixture in different ratios, are additionally provided. In one embodiment of the present disclosure, the surfactant is polysorbate 80 or polysorbate 20. In another embodiment of the present disclosure, the surfactant is polysorbate 80.

[090] In certain embodiments, the surfactant is polysorbate 80 or polysorbate 20, and the amount of polysorbate 80 or polysorbate 20 is between Petition 870250077590, dated 01 / 09 / 2025, pages 515 / 595 37 / 109 0.1 mg and 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 approximately 0.1 mg, approximately 0.2 mg, approximately 0.3 mg, approximately 0.4 mg, approximately 0.5 mg, approximately 0.6 mg, approximately 0.7 mg, approximately 0.8 mg, approximately 0.9 mg, or approximately 1.0 mg. In certain embodiments, the amount of polysorbate of 0.22 mg. In certain formulations, the amount of polysorbate of 0.29 mg. In certain formulations, the amount of polysorbate of 0.22 mg. In certain formulations, the amount of polysorbate 20 is 0.29 mg.

[091] In reconstituted formulations, the surfactant is at a concentration of about 0.01 to about 0.5 g / L. In various embodiments of the pharmaceutical formulations provided in the present invention, the surfactant concentration is 0.005, 0.01, 0.02, 0.03, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 g / L. Similarly, in certain development modalities, the surfactant is incorporated at a concentration of approximately 0.001 g / L. 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 development, 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.

[092] In some embodiments, the surfactant is polysorbate 20 or polysorbate 80. In some embodiments, the surfactant is polysorbate 80. In some embodiments, the pharmaceutical formulation comprises 0.05-0.3 mg / mL of surfactant. In some embodiments, the pharmaceutical formulation comprises at least 0.001, 0.002, 0.003, 0.004, 0.005, 0.01, 0.02, 0.03, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0% in Petition 870250077590, dated 01 / 09 / 2025, pp. 516 / 595 38 / 109 weight / volume of surfactant. In some embodiments, the surfactant is present in the pharmaceutical formulation at a concentration of at least 0.02% weight / volume. In certain embodiments, polysorbate 80 or polysorbate 20 is incorporated at a concentration of about 0.01 to about 0.05% weight / volume. In certain embodiments, polysorbate 80 or polysorbate 20 is incorporated at a concentration of about 0.02% weight / volume. ActRIIa formulations

[093] In some embodiments of the pharmaceutical formulations provided in the present invention, the ActRIIa fusion protein vials provided in the present invention comprise an ActRIIa fusion protein and one or more pharmaceutical additives and / or excipients. In some embodiments, the pharmaceutical formulations are lyophilized. In some embodiments, the formulations are reconstituted from a lyophilized formulation. In one embodiment of the pharmaceutical formulations provided in the present invention, a vial of a lyophilized pharmaceutical formulation comprises 55 mg of ActRIIa fusion protein; 0.48 mg of citric acid monohydrate, 2.56 mg of trisodium citrate dehydrate, 0.22 mg of polysorbate 80 and 88 mg of sucrose. In another embodiment, the vial is rehydrated with 1.0 mL of liquid, for example, Sterile Water for Injection.In another embodiment of the pharmaceutical formulations provided in the present invention, a vial comprises 72.5 mg of ActRIIa fusion protein; 0.64 mg of citric acid monohydrate, 3.37 mg of trisodium citrate dehydrate, 0.29 mg of polysorbate 80 and 116 mg of sucrose, wherein the formulation is lyophilized. In another embodiment, the vial is rehydrated with 1.3 mL of liquid, for example, Sterile Water for Injection. In other embodiments, the vials comprise one, two or all three of citrate, for example, 10 mM citrate; sucrose, for example, 8% (weight / volume) sucrose; and / or polysorbate 80, for example, at a pH of 5.0 to 7.0, for example. Petition 870250077590, dated 01 / 09 / 2025, pages 517 / 595 39 / 109 0.02% (weight / volume) of polysorbate 80 at pH 5.8.

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

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

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

[097] The present disclosure provides a kit comprising a lyophilized pharmaceutical formulation and an injection device. In certain embodiments, the lyophilized pharmaceutical formulation comprises an ActRIIa protein (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 fragments, functional variants or modified forms thereof. In certain embodiments, the protein binds to Petition 870250077590, dated 01 / 09 / 2025, pp. 518 / 595 40 / 109 one or more ligands selected from the group consisting of activin A, activin B and GDF11. In certain embodiments, the protein additionally 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.

[098] In some embodiments, the lyophilized pharmaceutical formulation comprises a protein comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 9 or to SEQ ID NO: 32. 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 to 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.

[099] In some embodiments, the lyophilized pharmaceutical formulation comprises a protein comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 32. In certain embodiments, the protein consists essentially of the amino acid sequence of SEQ ID NO: 32. In other embodiments, the protein consists of the amino acid sequence of SEQ ID NO: 32. In other embodiments, the protein consists of a variant of SEQ ID NO:32 lacking the C-terminal lysine residue (i.e., SEQ ID NO:41). Petition 870250077590, dated 01 / 09 / 2025, pp. 519 / 595 41 / 109

[100] In certain embodiments of the above, the lyophilized pharmaceutical formulation comprises a fusion protein further comprising an Fc domain of an immunoglobulin. In certain such embodiments, the Fc domain of the immunoglobulin is an Fc domain of an 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.

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

[102] In certain forms, the protein is glycosylated.

[103] The present disclosure further provides a kit comprising a sterile lyophilized pharmaceutical formulation comprising a protein as disclosed in the present invention and an injection device. In some embodiments of the kits disclosed in the present invention, the sterile lyophilized pharmaceutical formulation comprising a protein is pre-filled in one or more containers, such as one or more vials Figure 1.

[104] In certain embodiments, the pH range for the sterile lyophilized pharmaceutical formulation comprising an ActRIIa fusion protein is 5 to 7. In some embodiments, the sterile lyophilized pharmaceutical formulation comprising an ActRIIa fusion protein additionally comprises a buffering agent. In some embodiments, the buffering agent is added in an amount of at least 10 mM. In some embodiments, the buffering agent is added in an amount in the range of about 10 mM to about 200 mM. In some embodiments, the buffering agent comprises citrate.

[105] In some forms, the pharmaceutical formulation Petition 870250077590, dated 01 / 09 / 2025, pp. 520 / 595 42 / 109 additionally 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.

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

[107] In certain embodiments of the kits disclosed in the present invention, the injection device comprises a syringe, as shown in Figure 1. In some of these embodiments, the syringe is pre-filled with a reconstitution solution. In some embodiments, the reconstitution solution comprises a pharmaceutically acceptable carrier and / or excipient. In some embodiments, the pharmaceutically acceptable carrier comprises Petition 870250077590, dated 01 / 09 / 2025, pages 521 / 595 43 / 109 an aqueous solution, such as water, physiologically buffered saline solution or other solvent or vehicle, such as glycol, glycerol, an oil or an injectable organic ester. In some embodiments, the pharmaceutically acceptable excipient comprises a pharmaceutically acceptable excipient selected from calcium phosphate, calcium carbonate, calcium sulfate, a halite, a metal oxide, a sugar, a sugar alcohol, starch, glycol, povidone, a mineral hydrocarbon, an acrylic polymer, a fatty alcohol, mineral stearate, glycerin and / or a lipid. In certain embodiments, the reconstitution solution comprises sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or pharmaceutically acceptable emulsions. In certain of these embodiments, the reconstitution solution comprises an antioxidant, a buffer, a bacteriostat and / or a solute that makes the formulation isotonic with the blood of the intended recipient.In other forms, the reconstitution solution comprises a suspending or thickening agent.

[108] In certain embodiments of the kits disclosed in the present invention, the kit further comprises a bottle adapter, as shown in Figure 1. In some embodiments, the bottle (1) pre-filled with a lyophilized pharmaceutical formulation of the invention is attached to one end of the bottle adapter (3). In some embodiments, the pre-filled syringe (2) with a reconstitution solution as disclosed in the present invention is attached to one end of the bottle adapter (3). In some embodiments, the pre-filled syringe (2) with a reconstitution solution as disclosed in the present invention and the bottle (1) pre-filled with the lyophilized pharmaceutical formulation are attached to opposite ends of the bottle adapter (3). In some embodiments, the reconstitution solution is transferred from the pre-filled syringe to the bottle. In some embodiments, the transfer Petition 870250077590, dated 01 / 09 / 2025, pages 522 / 595 44 / 109 of the reconstitution solution for the pre-filled vial with the lyophilized pharmaceutical formulation reconstitutes the lyophilized formulation into a sterile injectable solution. In some embodiments, the lyophilized formulation is reconstituted into a sterile injectable solution. In some embodiments, the lyophilized formulation is reconstituted into a sterile injectable solution prior to use.

[109] In other embodiments of the kits disclosed in the present invention, the kit further comprises a pump apparatus. In certain embodiments, the pump apparatus comprises an electromechanical pumping assembly. In certain embodiments, the pump apparatus comprises a reservoir for containing a sterile injectable solution. In certain embodiments, the reservoir contains 1 mL of sterile injectable solution. In certain embodiments, the pump apparatus comprises one or more vials or cartridges containing a sterile injectable solution. In certain embodiments, the vials or cartridges are pre-filled with sterile injectable solution. In certain embodiments, the vials or cartridges comprise sterile injectable solution reconstituted from a lyophilized formulation. In certain embodiments, the reservoir is attached to the vial or cartridge. In certain embodiments, the vial or cartridge contains 1-20 mL of sterile injectable solution.In certain embodiments, the electromechanical pumping assembly comprises a pump chamber. In certain embodiments, the electromechanical pumping assembly is coupled to the reservoir. In certain embodiments, the sterile injectable solution is received from the reservoir into the pump chamber. In some embodiments, the electromechanical pumping assembly comprises a plunger that is arranged so that the sterile injectable solution in the pump chamber is in direct contact with the plunger. In certain embodiments, a sterile injectable solution is received from the reservoir into the pump chamber during a first phase of... Petition 870250077590, dated 01 / 09 / 2025, pp. 523 / 595 45 / 109 pumping and is delivered from the pump chamber to an individual during a second pumping phase. In certain embodiments, the electromechanical pumping assembly comprises a control circuitry. In certain embodiments, the control circuitry actuates the plunger to (a) aspirate the sterile injectable solution into the pump chamber during the first pumping phase and (b) deliver the sterile injectable solution from the pump chamber in a plurality of discrete plunger movements during the second pumping phase, thus delivering the therapeutic substance to the individual in a plurality of controlled and discrete dosages throughout the second pumping phase. In certain embodiments, an alternating cycle of the first and second pumping phases is repeated until a desired dose is administered. In certain embodiments, the pump apparatus is coupled to a wearable patch.In certain applications, the pump device is a wearable pump device.

[110] The present 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 in the present invention.

[111] In certain embodiments of the kits disclosed in the present invention, the kit further comprises an injectable device for use in the parenteral administration of the sterile injectable solution. In some embodiments, the sterile injectable solution is administered via subcutaneous injection. In some embodiments, the sterile injectable solution is administered via intradermal injection. In some embodiments, the sterile injectable solution is administered via intramuscular injection. In some embodiments, the sterile injectable solution is administered via intravenous injection. In some embodiments, the sterile injectable solution is self-administered. In some embodiments, the solution Petition 870250077590, dated 01 / 09 / 2025, pp. 524 / 595 46 / 109 sterile injectable comprises a therapeutically effective dose. In some embodiments, the therapeutically effective dose comprises a weight-based dose. ActRIIa polypeptides

[112] In certain embodiments, the present disclosure refers to ActRIIa fusion proteins. As used in the present invention, the term ActRIIa refers to a family of activin receptor type IIA (ActRIIa) proteins of any kind and variants derived from such ActRIIa fusion proteins by mutagenesis or other modification. Reference to ActRIIa in the present invention should be understood as a reference to any of the currently identified forms. Members of the ActRIIa family are generally transmembrane proteins, composed of an extracellular ligand-binding domain comprising a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine / threonine kinase activity.

[113] The term ActRIIa fusion protein includes proteins comprising any natural protein of a member of the ActRIIa family, as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain useful activity. Examples of such variant ActRIIa fusion proteins are provided throughout the present disclosure, as well as in International Patent Application Publications No. WO 2006 / 012627 and WO 2007 / 062188, which are incorporated herein by reference in their entirety. The amino acid numbering for all ActRIIa-related proteins described in this invention is based on the sequence numbering of the human ActRIIa precursor protein provided below (SEQ ID NO: 9), unless specifically designated otherwise.

[114] The canonical sequence of the human ActRIIa precursor protein is Petition 870250077590, dated 01 / 09 / 2025, pp. 525 / 595 47 / 109 as follows: MGAAAKLAFA VFLISCSSGA ILGRSETQEC LFFNANWEKD RTNQTGVEPC YGDKDKRRHC FATWKNISGS IEIVKQGCWL DDINCYDRTD CVEKKDSPEV 101 YFCCCEGNMC NEKFSYFPEM EVTQPTSNPV TPKPPYYNIL LYSLVPLMLI 151 AGIVICAFWV YRHHKMAYPP VLVPTQDPGP PPPSPLLGLK PLQLLEVKAR 201 GRFGCVWKAQ LLNEYVAVKI FPIQDKQSWQ NEYEVYSLPG MKHENILQFI 251 GAEKRGTSVD VDLWLITAFH EKGSLSDFLK ANVVSWNELC HIAETMARGL 301 AYLHEDIPGL KDGHKPAISH RDIKSKNVLL KNNLTACIAD FGALKFEAG 351 KSAGDTHGQV GTRRYMAPEV LEGAINFQRD AFLRIDMYAM GLVLWELASR 401 CTAADGPVDE YMLPFEEIG QHPSLEDMQE VVVHKKKRPV LRDYWQKHAG 451 MAMLCETIEE CWDHDAEARL SAGCVGERIT QMQRLTNIIT TEDIVTVVTM 501 VTNVDFPPKE SSL (SEQ ID NO: 9)

[115] The signal peptide is indicated by a single underline; the extracellular domain is indicated in bold; and potential endogenous N-linked glycosylation sites are indicated by a double underline.

[116] The sequence of the processed (mature) human extracellular ActRIIa protein is as follows: ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQ GCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPP (SEQ ID NO: 10)

[117] The C-terminal tail of the extracellular domain is indicated by a single underline. The sequence with the deleted tail (a Δ15 sequence) is as follows: ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQ GCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM (SEQ ID NO: 11) Petition 870250077590, dated 01 / 09 / 2025, pp. 526 / 595 48 / 109

[118] The nucleic acid sequence encoding the precursor protein of human ActRIIa is shown below (SEQ ID NO: 12), as follows nucleotides 159-1700 of Genbank Reference Sequence NM_001616.4. The signal sequence is underlined. ATGGGAGCTG CTGCAAAGTT GGCGTTTGCC GTCTTTCTTA TCTCCTGTTC TTCAGGTGCT ATACTTGGTA GATCAGAAAC TCAGGAGTGT CTTTTCTTTA 101 ATGCTAATTG GGAAAAAGAC AGAACCAATC AAACTGGTGT TGAACCGTGT 151 TATGGTGACA AAGATAAACG GCGGCATTGT TTTGCTACCT GGAAGAATAT 201 TTCTGGTTCC ATTGAAATAG TGAAACAAGG TTGTTGGCTG GATGATATCA 251 ACTGCTATGA CAGGACTGAT TGTGTAGAAA AAAAAGACAG CCCTGAAGTA 301 TATTTTTGTT GCTGTGAGGG CAATATGTGT AATGAAAAGT TTTCTTATTT 351 TCCGGAGATG GAAGTCACAC AGCCCACTTC AAATCCAGTT ACACCTAAGC 401 CACCCTATTA CAACATCCTG CTCTATTCCT TGGTGCCACT TATGTTAATT 451 GCGGGGATTG TCATTTGTGC ATTTTGGGTG TACAGGCATC ACAAGATGGC 501 CTACCCTCCT GTACTTGTTC CAACTCAAGA CCCAGGACCA CCCCCACCTT 551 CTCCATTACT AGGTTTGAAA GAAAGCAAGG 601 GGAAGATTTG GTTGTGTCTG AATATGTGGC 651 TGTCAAAATA TTTCCAATAC AATGAATACG CCACTGCAGT TATTAGAAGT GAAAGCCCAG TTGCTTAACG AGGACAAACA GTCATGGCAA Petition 870250077590, dated 01 / 09 / 2025, pages 527 / 595 49 / 109 701 AAGTCTACAG TTTGCCTGGA ATGAAGCATG AGAACATATT ACAGTTCATT 751 GGTGCAGAAA AACGAGGCAC CAGTGTTGAT GTGGATCTTT GGCTGATCAC 801 AGCATTTCAT GAAAAGGGTT CACTATCAGA CTTTCTTAAG GCTAATGTGG 851 ​​TCTCTTGGAA TGAACTGTGT CATATTGCAG AAACCATGGC TAGAGGATTG 901 GCATATTTAC ATGAGGATAT ACCTGGCCTA AAAGATGGCC ACAAACCTGC 951 CATATCTCAC AGGGACATCA AAAGTAAAAA TGTGCTGTTG AAAAACAACC 1001 TGACAGCTTG CATTGCTGAC TTTGGGTTGG CCTTAAAATT TGAGGCTGGC 1051 AAGTCTGCAG GCGATACCCA TGGACAGGTT GGTACCCGGA GGTACATGGC 1101 TCCAGAGGTA TTAGAGGGTG CTATAAACTT CCAAAGGGAT GCATTTTTGA 1151 GGATAGATAT GTATGCCATG GGATTAGTCC TATGGGAACT GGCTTCTCGC 1201 TGTACTGCTG CAGATGGACC TGTAGATGAA TACATGTTGC CATTTGAGGA 1251 GGAAATTGGC CAGCATCCAT CTCTTGAAGA CATGCAGGAA GTTGTTGTGC 1301 ATAAAAAAAA GAGGCCTGTT TTAAGAGATT ATTGGCAGAA ACATGCTGGA 1351 ATGGCAATGC TCTGTGAAAC CATTGAAGAA TGTTGGGATC Petition 870250077590, of 01 / 09 / 2025, p. 528 / 595 50 / 109 ACGACGCAGA 1401 AGCCAGGTTA CAUTIONGCAGA 1451 PUBLICATION GGTCACAATG TCAGCTGGAT GTGTAGGTGA AAGAATTACC TATTTACC ACAGAGGACA TTGTAACAGT 1501 GTGACAAATG TTGACTTTCC TCCCAAAGAA TCTAGTCTA (SEQ ID NO: 12)

[119] The nucleic acid sequence encoding the processed soluble (extracellular) human ActRIIa protein is as follows: ATACTTGGTA GATCAGAAAC TCAGGAGTGT CTTTTCTTTA ATGCTAATTG GGAAAAAGAC TATGGTGACA 101 AAGATAAACG TTCTGGTTCC 151 ATTGAAATAG ACTGCTATGA 201 CAGGACTGAT TATTTTTGTT 251 GCTGTGAGGG TCCGGAGATG AGAACCAATC AAACTGGTGT TGAACCGTGT GCGGCATTGT TTTGCTACCT GGAAGAATAT TGAAACAAGG TTGTTGGCTG GATGATATCA TGTGTAGAAA AAAAAGACAG CCCTGAAGTA CAATATGTGT AATGAAAAGT TTTCTTATTT 301 gaagtcacac agcccacttc aaatccagtt acacctaagc caccc(SEQ ID NO: 13)

[120] ActRIIa is well conserved among vertebrates, with large portions of the extracellular domain completely conserved. For example, Figure 2 depicts a multisequence alignment of a human ActRIIa extracellular domain compared to several ActRIIa orthologs. Many of the ligands that bind to ActRIIa are also highly conserved. Thus, from these alignments, it is possible to predict the positions of key amino acids. Petition 870250077590, dated 01 / 09 / 2025, pp. 529 / 595 51 / 109 within the ligand-binding domain that are important for normal ActRIIa ligand-binding activities, as well as predicting amino acid positions that are likely to be tolerant to substitution without significantly altering normal ActRIIa ligand-binding activities. Therefore, a useful active human ActRIIa variant protein, according to currently disclosed methods, may include one or more amino acids at corresponding sequence positions of another vertebrate ActRIIa, or may include a residue similar to human or other vertebrate sequences.

[121] Without intending to be limiting, the following examples illustrate this approach to defining an active ActRIIa variant. As illustrated in Figure 2, F13 in the human extracellular domain is Y in ActRIIa from Ovis aries (SEQ ID NO: 62), Gallus gallus (SEQ ID NO: 65), Bos taurus (SEQ ID NO: 66), Tyto alba (SEQ ID NO: 67), and Myotis davidii (SEQ ID NO: 68), indicating that aromatic residues are tolerated at this position, including F, W, and Y. Q24 in the human extracellular domain is R in ActRIIa from Bos taurus, indicating that charged residues will be tolerated at this position, including D, R, K, H, and E. S95 in the human extracellular domain is F in ActRIIa from Gallus gallus and Tyto alba, indicating that this site may be tolerant to a wide variety of changes, including polar residues such as E, D, K, R, H, S, T, P, G, Y, and probably hydrophobic residues such as L, I, or F.E52 in the human extracellular domain is D in ActRIIa from Ovis aries, indicating that acidic residues are tolerated at this position, including D and E. P29 in the human extracellular domain is relatively poorly conserved, appearing as S in ActRIIa from Ovis aries and L in ActRIIa from Myotis davidii; therefore, essentially any amino acid should be tolerated at this position.

[122] In addition, as discussed above, the ActRIIa proteins were Petition 870250077590, dated 01 / 09 / 2025, pages 530 / 595 52 / 109 characterized in the art in terms of structural / functional characteristics, particularly with regard to ligand binding (Attisano et al. (1992) Cell 68(1):97-108; Greenwald et al. (1999) Nature Structural Biology 6(1): 18-22; Allendorph et al. (2006) PNAS 103(20): 7643-7648; Thompson et al. (2003) The EMBO Journal 22(7): 1555-1566; as well as US Patents Nos: 7,709,605, 7,612,041 and 7,842,663). e.g., ligand binding activity).

[123] For example, a defining structural motif known as the three-finger toxin fold is important for ligand binding by type I and type II receptors and is formed by conserved cysteine ​​residues located at varying 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). Consequently, the central ligand-binding domains of human ActRIIa, as demarcated by the outermost of these conserved cysteines, correspond to positions 30-110 of SEQ ID NO: 9 (precursor of ActRIIa).Therefore, the structurally less ordered amino acids flanking these central cysteine-demarcated sequences can be truncated at approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 residues at the N-terminus and at approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 residues at the C-terminus without necessarily altering the Ligand binding. Truncations of extracellular domains of exemplary ActRIIa include SEQ ID NOs: 10 and 11.

[124] Thus, a general formula for an active (e.g., ligand-binding) moiety of ActRIIa is a protein comprising, consisting Petition 870250077590, dated 01 / 09 / 2025, pp. 531 / 595 53 / 109 essentially in, or consists of, amino acids 30-110 of SEQ ID NO: 9. Therefore, ActRIIa proteins may, for example, comprise, consist essentially of, or consist of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a portion of ActRIIa beginning at a residue corresponding to any of the amino acids 2130 (for example, beginning at any of the amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) of SEQ ID NO: 9 and ending at a corresponding position. any of the amino acids 110-135 (for example, ending in any of the amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135) of SEQ ID NO: 9. Other examples include constructs that begin at a selected position from 21 to 30 (for example, beginning in any of the amino acids 21, 22, 23, 24, 25,26, 27, 28, 29 or 30), 22-30 (for example, starting at any of the amino acids 22, 23, 24, 25, 26, 27, 28, 29 or 30), 23-30 (for example, starting at any of the amino acids 23, 24, 25, 26, 27, 28, 29 or 30), 24-30 (for example, starting at any of the amino acids 24, 25, 26, 27, 28, 29 or 30) of SEQ ID NO: 9, and terminate at a position selected from 111-135 (for example, terminating at any of the amino acids 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134 or 135), 112-135 (for example, ending in any of the amino acids 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134 or 135), 113-135 (for example, ending in any of the amino acids 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134 or 135), 120-135 (for example,ending in any of the amino acids 120, 121, 122, 123, 124, 125, 126, Petition 870250077590, dated 01 / 09 / 2025, pp. 532 / 595 54 / 109 127, 128, 129, 130, 131, 132, 133, 134 or 135), 130-135 (for example, ending in any of the amino acids 130, 131, 132, 133, 134 or 135), 111-134 (for example, ending in any of the amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133 or 134), 111-133 (for for example, ending in any of the amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132 or 133), 111-132 (for example, ending in any of the amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 or 132) or 111-131 (for example, ending in any of the amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130 or 131) from SEQ ID NO: 9.Variants within these ranges are also contemplated, particularly those comprising, essentially consisting of, or consisting of an amino acid sequence that has 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. Thus, in some embodiments, an ActRIIa protein may comprise, essentially consist of, or consist of a protein that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 30-110 of SEQ ID NO: 9. Optionally, ActRIIa proteins comprise a protein that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 30-110 of SEQ ID NO: 9, and comprising no more than 1, 2, 5, 10 or 15 amino acid-conserving changes at the ligand binding site.

[125] In certain forms, the revelation refers to antagonists of Petition 870250077590, dated 01 / 09 / 2025, pp. 533 / 595 55 / 109 GDF / BMP (inhibitors) comprising an ActRIIa protein, which includes fragments, functional variants and modified forms thereof, as well as uses thereof (e.g., enhancing an immune response in a patient in need thereof and treating cancer). Preferably, the ActRIIa proteins are soluble (e.g., an extracellular domain of ActRIIa). In some embodiments, the ActRIIa proteins inhibit (e.g., Smad signaling) one or more GDF / BMP ligands (e.g., GDF11, GDF8, activin (activin A, activin B, activin AB, activin C, activin E), BMP6, GDF3, BMP15 and / or BMP10). In some embodiments, ActRIIa proteins bind 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 development protein comprises, consists essentially of, or consists of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a portion of ActRIIa beginning at a residue corresponding to amino acids 21-30 (e.g., beginning at any of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) of SEQ ID NO: 9 and terminating at a position corresponding to any of amino acids 110-135 (e.g., terminating at any of amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134 or 135) of SEQ ID NO: 9.In some embodiments, the ActRIIa proteins comprise, consist of, or consist essentially of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 30-110 of SEQ ID NO: 9. In certain embodiments, the ActRIIa proteins comprise, consist of, or consist essentially of an amino acid sequence that... Petition 870250077590, dated 01 / 09 / 2025, pp. 534 / 595 56 / 109 is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 21-135 of SEQ ID NO: 9.

[126] In certain embodiments, the extracellular domain (ECD) of human activin receptor type IIA (ActRIIa) proteins or their derivatives are linked to a constant domain of an immunoglobulin, such as the Fc domain of human IgG1. In some embodiments, the ActRIIa protein is a fusion protein comprising an ActRIIa domain and one or more heterologous ActRIIa protein domains. In some embodiments, the ActRIIa protein is a fusion protein comprising an Fc domain of an immunoglobulin. In some embodiments, the immunoglobulin Fc domain is an Fc domain of an IgG1 immunoglobulin. In some embodiments, the ActRIIa fusion protein additionally comprises a linker domain positioned between the ActRIIa protein domain and one or more heterologous domains (e.g., an immunoglobulin Fc domain).In some embodiments, the linker domain is selected from the group consisting of: TGGG (SEQ ID NO: 23), TGGGG (SEQ ID NO: 21), SGGGG (SEQ ID NO: 22), GGGGS (SEQ ID NO: 25), GGG (SEQ ID NO: 19), GGGG (SEQ ID NO: 20), and SGGG (SEQ ID NO: 24). In some embodiments, the ActRIIa fusion protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 32. In some embodiments, the ActRIIa fusion protein comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the ActRIIa fusion protein consists of the amino acid sequence of SEQ ID NO: 32. In some embodiments, the ActRIIa fusion protein consists of the amino acid sequence of SEQ ID NO: 41. In some embodiments, the ActRIIa fusion protein consists of the amino acid sequence of SEQ ID NO:. Petition 870250077590, dated 01 / 09 / 2025, pp. 535 / 595 57 / 109 or 41.

[127] In some embodiments, the ActRIIa fusion proteins comprise, consist of, or essentially consist of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any of the SEQ ID NOs: 9, 10, 11, 32, 36, and 39.

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

[129] In some alternative embodiments, the ActRIIa fusion protein (e.g., SEQ ID NO: 32) may lack the C-terminal lysine. In some embodiments, the ActRIIa fusion protein lacking the C-terminal lysine is SEQ ID NO: 41. For example, in certain embodiments of the pharmaceutical formulations described in the present invention, the ActRIIa fusion protein may be composed of 1-100% of SEQ ID NO: 32. For example, in certain embodiments of the pharmaceutical formulations described in the present invention, the ActRIIa fusion protein is composed of 1-100% of SEQ ID NO: 41.

[130] In certain embodiments, the pharmaceutical formulations described in this invention contain a mixture of SEQ ID NO: 32 and a variant of SEQ Petition 870250077590, dated 01 / 09 / 2025, pp. 536 / 595 58 / 109 ID NO:32 devoid of the C-terminal lysine residue (SEQ ID NO: 41). In certain embodiments, the pharmaceutical formulations described in the present invention contain a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO:32 devoid of the C-terminal lysine residue (SEQ ID NO: 41) wherein the mixture contains 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57% 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42% 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27% 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12% 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% by weight of SEQ ID NO:32.

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

[132] In certain embodiments, the pharmaceutical formulations described in the present invention contain a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 devoid of the C-terminal lysine residue (SEQ ID NO: 41) wherein the mixture contains 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, Petition 870250077590, dated 01 / 09 / 2025, pp. 537 / 595 59 / 109 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58% 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43% 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28% 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13% 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% by weight of SEQ ID NO:41.

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

[134] In certain embodiments, the pharmaceutical formulations described in this invention contain 100% of SEQ ID NO: 32 by weight. In certain embodiments, the pharmaceutical formulations described in this invention contain 100% of SEQ ID NO: 41 by weight.

[135] In certain respects, the present disclosure relates to GDF trap proteins (also referred to as “GDF traps”). In some embodiments, the GDF traps of the present disclosure are variant ActRIIa proteins (e.g., ActRIIa) comprising one or more mutations (e.g., amino acid additions, deletions, substitutions, and combinations thereof) in the extracellular domain (also referred to as the ligand-binding domain) of an ActRIIa protein (e.g., an ActRIIa protein of the “type”). Petition 870250077590, dated 01 / 09 / 2025, pp. 538 / 595 60 / 109 wild-type” or unmodified) such that the variant ActRIIa protein has one or more ligand-binding activities altered compared to the corresponding wild-type ActRIIa protein. In certain embodiments, the GDF trap proteins of the present disclosure retain at least one activity similar to that of a corresponding wild-type ActRIIa protein. For example, preferred GDF traps bind to and inhibit (e.g., antagonize) the function of GDF11 and / or GDF8. In some embodiments, the GDF traps of the present disclosure additionally bind to and inhibit one or more GDF / BMP ligands.

[136] To illustrate, one or more mutations can be selected to increase the selectivity of the altered ligand-binding domain for GDF11 and / or GDF8 over one or more ActRIIa-binding ligands, such as activins, particularly activin A. Optionally, the altered ligand-binding domain has a Kd ratio for activin binding to Kd for GDF11 and / or GDF8 binding that is at least 2, 5, 10, 20, 50, 100 or even 1000 times greater than the ratio for the wild-type ligand-binding domain. Optionally, the altered ligand-binding domain has an IC50 ratio for inhibiting activin to the IC50 ratio for inhibiting GDF11 and / or GDF8 that is at least 2, 5, 10, 20, 50, 100, or up to 1000 times greater than the wild-type ligand-binding domain. Optionally, the altered ligand-binding domain inhibits GDF11 and / or GDF8 with an IC50 at least 2, 5, 10, 20, 50, 100, or up to 1000 times less than the IC50 for inhibiting activin. Methods of Use

[137] In certain respects, the revelation provides a method of treating pulmonary arterial hypertension (PAH), comprising administering a Petition 870250077590, dated 01 / 09 / 2025, pages 539 / 595 61 / 109 pharmaceutical formulation described in the present invention to a patient in need thereof.

[138] In certain respects, the disclosure provides a method of treating pulmonary arterial hypertension (PAH) in a patient in need thereof, comprising reconstituting a lyophilized pharmaceutical formulation described in the present invention to create a reconstituted formulation and administering the reconstituted formulation to the patient.

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

[140] In certain aspects, the disclosure provides a method of treating pulmonary arterial hypertension (PAH), comprising administering a pharmaceutical formulation described in the present invention to a patient in need thereof, wherein the administration of the pharmaceutical formulation results in an alteration in one or more of the following hemodynamic or functional parameters: a reduction in pulmonary vascular resistance (PVR); an increase in 6-minute walk distance (6MWD); a decrease in levels of N-terminal B-type natriuretic propeptide (NTproBNP); the prevention or reduction of progression of Functional Class of Petition 870250077590, dated 01 / 09 / 2025, pages 540 / 595 62 / 109 pulmonary hypertension, as recognized by the World Health Organization (WHO); the promotion or increase in the regression of the Functional Class of pulmonary hypertension, as recognized by the WHO; an improvement in right ventricular function; an improvement in pulmonary artery pressure; and / or an improvement in mean right atrial pressure.

[141] In certain respects, the disclosure provides a method of treating pulmonary arterial hypertension (PAH), comprising administering a pharmaceutical formulation described in the present invention to a patient in need thereof, wherein the administration of the pharmaceutical formulation results in increased exercise capacity, provides clinical improvement, improves WHO functional class (FC) and slows disease progression, including reducing the risk of death and hospitalization due to PAH.

[142] In certain aspects, 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, comprising administering to a patient in need thereof a pharmaceutical formulation described in the present invention, wherein the administration of said formulation results in a change in one or more of the following hemodynamic or functional parameters: a reduction in pulmonary vascular resistance (PVR); an increase in 6-minute walk distance (6MWD); a decrease in levels of N-terminal B-type natriuretic propeptide (NT-proBNP); the prevention or reduction of the progression of the Functional Class of pulmonary hypertension, as recognized by the World Health Organization (WHO); the promotion or enhancement of the regression of the Functional Class of pulmonary hypertension, as recognized by the WHO; an improvement in right ventricular function; an improvement in pulmonary artery pressure;and / or an improvement in mean right atrial pressure. In some modalities, one or more complications of hypertension; Petition 870250077590, dated 01 / 09 / 2025, pages 541 / 595 63 / 109 pulmonary artery lesions are selected from the group consisting of: proliferation of smooth muscle and / or endothelial cells in the pulmonary artery, angiogenesis in the pulmonary artery, dyspnea, chest pain, pulmonary vascular remodeling, right ventricular hypertrophy, and pulmonary fibrosis.

[143] In some embodiments, administration of the pharmaceutical formulations described in the present invention reduces the patient's PVR. In some embodiments, administration of the pharmaceutical formulations described in the present invention reduces the patient's PVR by at least 10% (for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or at least 50%). In some embodiments, administration of the pharmaceutical formulations described in the present invention reduces the patient's PVR by at least 20%. In some embodiments, the reduction in PVR results from a decrease in mean pulmonary artery pressure. In some embodiments, administration of the pharmaceutical formulations described in the present invention increases the patient's 6-minute walk distance.In some embodiments, administration of the pharmaceutical formulations described in the present invention increases the patient's 6-minute walk distance by at least 10 meters (for example, at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300 or more than 400 meters). In some embodiments, administration of the pharmaceutical formulations described in the present invention increases the patient's 6-minute walk distance by at least 30 meters. In some embodiments, administration of the pharmaceutical formulations described in the present invention decreases NT-proBNP levels in the patient. In some embodiments, administration of the pharmaceutical formulations described in the present invention decreases NT-proBNP levels in the patient by at least 10% (for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or at least 80%). In some embodiments, administration... Petition 870250077590, dated 01 / 09 / 2025, pages 542 / 595 64 / 109 of the pharmaceutical formulations described in the present invention decreases NT-proBNP levels in the patient by at least 30%. In some embodiments, administration of the pharmaceutical formulations described in the present invention decreases NT-proBNP levels to normal levels. In some embodiments, the normal NT-proBNP level is <100 pg / ml.

[144] In some embodiments, the administration of the pharmaceutical formulations described in the present invention prevents or reduces the progression of Functional Class of pulmonary hypertension, as recognized by the WHO. In some embodiments, the administration of the pharmaceutical formulations described in the present invention prevents or reduces the regression of Functional Class of pulmonary hypertension from Class I to Functional Class II, as recognized by the WHO. In some embodiments, the administration of the pharmaceutical formulations described in the present invention prevents or reduces the regression of Functional Class of pulmonary hypertension from Class II to Functional Class III, as recognized by the WHO.In some embodiments, the administration of the pharmaceutical formulations described in the present invention prevents or reduces the regression of pulmonary hypertension from Functional Class III to Functional Class IV, as recognized by the WHO. In some embodiments, the administration of the pharmaceutical formulations described in the present invention promotes or increases the regression of pulmonary hypertension, as recognized by the WHO. In some embodiments, the administration of the pharmaceutical formulations described in the present invention promotes or increases the regression of pulmonary hypertension from Functional Class IV to Class III, as recognized by the WHO. In some embodiments, the administration of the pharmaceutical formulations described in the present invention... Petition 870250077590, dated 01 / 09 / 2025, pages 543 / 595 65 / 109 promotes or increases the regression of pulmonary hypertension Functional Class from Class III to Class II, as recognized by the WHO. In some embodiments, the administration of the pharmaceutical formulations described in the present invention promotes or increases the regression of pulmonary hypertension Functional Class from Class II to Class I, as recognized by the WHO.

[145] In some embodiments, administration of the pharmaceutical formulations described in the present invention improves right ventricular function in the patient. In some embodiments, the improvement in right ventricular function is due to an increase in the change in the fractional area of ​​the right ventricle. 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 the ejection fraction. In some embodiments, the improvement in right ventricular function is due to an increase in the change in the fractional area of ​​the right ventricle and in the ejection fraction.

[146] In some embodiments, administration of the pharmaceutical formulations described in the present invention improves pulmonary artery pressure in the patient. In some embodiments, the improvement in pulmonary artery pressure is a reduction in mean pulmonary artery pressure (mPAP). In some embodiments, administration of the pharmaceutical formulations described in the present invention reduces mPAP in the patient by at least 10% (for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or at least 50%). In some embodiments, administration of the pharmaceutical formulations described in the present invention reduces mPAP by at least 3 mmHg (for example, at least 3, 5, 7, 10, 12, 15, 20 or 25 mmHg) in the patient. In some embodiments, administration of the pharmaceutical formulations described Petition 870250077590, dated 01 / 09 / 2025, pages 544 / 595 The present invention, 66 / 109, improves the mean right atrial pressure (mRAP) in the patient. In some embodiments, the improvement in mRAP is a reduction in mRAP. In some embodiments, administration of the pharmaceutical formulations described in the present invention reduces the patient's mRAP by at least 10% (for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%). In some embodiments, administration of the pharmaceutical formulations described in the present invention reduces the patient's mRAP by at least 1 mmHg (for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mmHg).

[147] In some modalities, the patient has a pulmonary vascular resistance (PVR) greater than or equal to 3 Wood Units. In some modalities, the patient has a walking distance of 150 to 550 meters in 6 minutes. In some modalities, the patient has elevated NT-proBNP levels compared to a healthy patient. In some modalities, the patient has an NT-proBNP level of at least 100 pg / mL (e.g., 100, 150, 200, 300, 400, 500, 1000, 3000, 5000, 10,000, 15,000, or 20,000 pg / mL). In some modalities, the patient has elevated brain natriuretic peptide (BNP) levels compared to a healthy patient. In some modalities, the patient has a BNP level of at least 100 pg / mL (for example, 100, 150, 200, 300, 400, 500, 1000, 3000, 5000, 10,000, 15,000, or 20,000 pg / mL).In some embodiments, administration of the pharmaceutical formulations described in the present invention decreases BNP levels in the patient by at least 10% (for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or at least 80%). In some embodiments, administration of the pharmaceutical formulations described in the present invention decreases BNP levels to normal levels (i.e., <100 pg / ml). In some embodiments, the patient has a mean pulmonary arterial pressure (mPAP) selected from the group consisting of: an mPAP of at least... Petition 870250077590, dated 01 / 09 / 2025, pages 545 / 595 67 / 109 minus 20 mmHg; an mPAP of at least 25 mmHg; an mPAP of at least 30 mmHg; an mPAP of at least 35 mmHg; an mPAP of at least 40 mmHg; an mPAP of at least 45 mmHg; and an mPAP of at least 50 mmHg. In some modalities, the patient has a mean right atrial pressure (mRAP) selected from the group consisting of: an mRAP of at least 5 mmHg; an mRAP of at least 6 mmHg; an mRAP of at least 8 mmHg; an mRAP of at least 10 mmHg; an mRAP of at least 12 mmHg; an mRAP of at least 14 mmHg; and an mRAP of at least 16 mmHg.

[148] In some modalities, PAH is idiopathic pulmonary arterial hypertension (PAH). In some modalities, PAH is hereditary PAH. In some modalities, PAH is drug- or toxin-induced PAH. In some modalities, PAH is PAH associated with simple congenital systemic pulmonary shunts, at least 1 year after shunt repair. In some modalities, the patient has pulmonary hypertension functional class II or class III, according to the World Health Organization's functional classification system for pulmonary hypertension. In some modalities, the patient has pulmonary hypertension functional class I, class II, class III, or class IV, as recognized by the World Health Organization. In some modalities, the patient has pulmonary hypertension functional class I, class II, class III, or class IV, according to the World Health Organization's functional classification system for pulmonary hypertension.In some embodiments, the patient has pulmonary hypertension of functional class IV, according to the World Health Organization's functional classification system for pulmonary hypertension. In some embodiments, administration of the pharmaceutical formulations described in the present invention increases transplant-free survival in the patient. In some embodiments, Petition 870250077590, dated 01 / 09 / 2025, pages 546 / 595 68 / 109 The administration of the pharmaceutical formulations described in the present invention increases transplant-free survival in the patient by at least 10% (for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or at least 50%). In some embodiments, the administration of the pharmaceutical formulations described in the present invention decreases right ventricular hypertrophy in the patient. In some embodiments, the administration of the pharmaceutical formulations described in the present invention decreases right ventricular hypertrophy in the patient by at least 10% (for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or at least 50%). In some embodiments, the administration of the pharmaceutical formulations described in the present invention decreases smooth muscle hypertrophy in the patient.In some embodiments, administration of the pharmaceutical formulations described in the present invention decreases smooth muscle hypertrophy in the patient by at least 10% (for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or at least 50%). In some embodiments, administration of the pharmaceutical formulations described in the present invention decreases pulmonary arteriole muscularity in the patient. In some embodiments, administration of the pharmaceutical formulations described in the present invention decreases pulmonary arteriole muscularity in the patient by at least 10% (for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or at least 50%).

[149] In some embodiments, administration of the pharmaceutical formulations described in the present invention improves the patient's exercise capacity. In some embodiments, administration of the pharmaceutical formulations described in the present invention decreases the patient's Borg dyspnea index (BDI). In some embodiments, administration of the pharmaceutical formulations described in the present invention reduces the patient's BDI by at least 0.5 index points (for example, 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). Petition 870250077590, dated 01 / 09 / 2025, pages 547 / 595 69 / 109 In some embodiments, the patient presents with decreased renal function. In some embodiments, the administration of the pharmaceutical formulations described in the present invention additionally improves renal function. In some embodiments, the administration of the pharmaceutical formulations described in the present invention delays the clinical worsening of pulmonary arterial hypertension. In some embodiments, the administration of the pharmaceutical formulations described in the present invention delays the clinical worsening of pulmonary arterial hypertension according to the World Health Organization functional classification system for pulmonary hypertension. In some embodiments, the administration of the pharmaceutical formulations described in the present invention reduces the risk of hospitalization for one or more complications associated with pulmonary arterial hypertension.In some embodiments, administration of the pharmaceutical formulations described in the present invention reduces the risk of morbidity from one or more complications associated with pulmonary arterial hypertension. In some embodiments, morbidity comprises a change in one or more of the following: increased need for lung and / or heart transplantation; need to initiate salvage therapy with a known treatment for PAH; need to increase prostacyclin by at least 10%; need for atrial septostomy; PAH-specific hospitalization for at least 24 hours; and deterioration of PAH. In some embodiments, the deterioration of PAH comprises a worsening of the WHO functional class and a decrease in 6MWD of at least 15%. In some embodiments, administration of the pharmaceutical formulations described in the present invention reduces the risk of death associated with pulmonary arterial hypertension.In some embodiments, the administration of the pharmaceutical formulations described in this invention reduces the risk of death associated with pulmonary arterial hypertension by at least 10% (per 20%). Petition 870250077590, dated 01 / 09 / 2025, pp. 548 / 595 70 / 109 example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or at least 50%). In some modalities, the patient has a hemoglobin level of >8 and <15 g / dl. In some modalities, the patient's hemoglobin levels are <18 g / dl.

[150] In certain embodiments, the patient treated according to the methods described in the present invention is female. In certain embodiments, the patient treated according to the methods described in the present invention is male. In certain embodiments, the patient treated according to the methods described in the present invention may be of any age. In certain embodiments, the patient treated according to the methods described in the present invention is under 18 years of age. In one specific embodiment, the patient treated according to the methods described in the present invention is under 13 years of age. In another specific embodiment, the patient treated according to the methods described in the present invention is under 12, under 11, under 10, under 9, under 8, under 7, under 6, or under 5 years of age.In another specific embodiment, the patient treated according to the methods described in the present invention is 1-3 years old, 3-5 years old, 5-7 years old, 7-9 years old, 9-11 years old, 11-13 years old, 13-15 years old, 15-20 years old, 20-25 years old, 25-30 years old, or over 30 years old. In another specific embodiment, the patient treated according to the methods described in the present invention is 30-35 years old, 35-40 years old, 40-45 years old, 45-50 years old, 50-55 years old, 55-60 years old, or over 60 years old. In another specific embodiment, the patient treated according to the methods described in the present invention is between 18 and 64 years old, between 65 and 74 years old, or over 75 years old.

[151] In certain embodiments, the hemoglobin levels in a patient treated according to the dosage forms and methods in the present invention Petition 870250077590, dated 01 / 09 / 2025, pages 549 / 595 71 / 109 provided are less than 10 g / dL, 9 g / dL, 8 g / dL, or 7 g / dL. In certain embodiments, the hemoglobin levels in a patient treated according to the dosage forms and methods provided in the present invention are between 7 g / dL and 7.5 g / dL, between 7.5 g / dL and 8 g / dL, between 8 g / dL and 8.5 g / dL, between 8.5 g / dL and 9.0 g / dL, between 9.0 g / dL and 9.5 g / dL, or between 9.5 g / dL and 10.0 g / dL. EXAMPLES

[152] The revelation now being generally described will be more readily understood by reference to the following examples, which are included merely for the purpose of illustrating certain aspects of the present revelation and are not intended to limit the revelation.

[153] The invention now being generally described will be more readily understood by reference to the following examples, which are included merely for the purpose of illustrating certain embodiments of the invention, and are not intended to limit the invention. Example 1 Fusion proteins ActRIIa-Fc

[154] A soluble ActRIIa fusion protein has been constructed that has the extracellular domain of human ActRIIa fused to a human or mouse Fc domain with a minimal linker between them. The constructs are referred to as ActRIIa-hFc and ActRIIa-mFc, respectively.

[155] The following ActRIIa-hFc shown below is sotatercept purified from CHO cell lines (SEQ ID NO: 32): ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQ GCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPP TGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Petition 870250077590, dated 01 / 09 / 2025, pp. 550 / 595 72 / 109

[156] The ActRIIa-hFc and ActRIIa-mFc proteins were expressed in CHO cell lines. Three different leader sequences were considered: (i) Bee melittin (HBML): MKFLVNVALVFMVVYISYIYA (SEQ ID NO: 33) (ii) Tissue plasminogen activator (TPA): MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO: 34) (iii) Native: MGAAAKLAFAVFLISCSSGA (SEQ ID NO: 35).

[157] The selected form employs the TPA leader and has the following unprocessed amino acid sequence: MDAMKRGLCCVLLLCGAVFVSPGAAILGRSETQECLFFNANWEKDRTNQTGVEPC YGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNM CNEKFSYFPEMEVTQPTSNPVTPKPPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK (SEQ ID NO: 36)

[158] This protein is encoded by the following nucleic acid sequence: ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGT CTTCGTTTCGCCCGGCGCCGCTATACTTGGTAGATCAGAAACTCAGGAGTGTCTTTTTT TAATGCTAATTGGGAAAAAGACAGAACCAATCAAACTGGTGTTGAACCGTGTTATGG TGACAAAGATAAACGGCGGCATTGTTTTGCTACCTGGAAGAATATTTCTGGTTCCATT GAATAGTGAAACAAGGTTGTTGGCTGGATGATATCAACTGCTATGACAGGACTGATT GTGTAGAAAAAAAAGACAGCCCTGAAGTATATTTCTGTTGCTGTGAGGGCAATATGT GTAATGAAAAGTTTTCTTATTTTCCGGAGATGGAAGTCACACAGCCCACTTCAAATCC AGTTACACCTAAGCCACCCACCGGTGGTGGAACTCACACATGCCCACCGTGCCCAGC Petição 870250077590, de 01 / 09 / 2025, pág. 551 / 595 73 / 109 ACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACC CTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAA GACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAA GACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCA CCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACA AAGCCCTCCCAGTCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAG AACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCA GCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGA GCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG GCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGA ACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAG CCTCTCCCTGTCTCCGGGTAAATGAGAATTC (SEQ ID NO: 37)

[159] Both ActRIIa-hFc and ActRIIa-mFc were remarkably receptive to recombinant expression. As shown in Figures 3A and 3B, the protein was purified as a single, well-defined protein peak. N-terminal sequencing revealed a single -ILGRSETQE sequence (SEQ ID NO: 38). Purification could be achieved by a series of column chromatography steps, including, for example, three or more of the following, in any order: protein A chromatography, sepharose Q chromatography, phenylsepharose chromatography, size exclusion chromatography, and cation exchange chromatography. Purification could be completed with viral filtration and buffer exchange. The ActRIIa-hFc protein was purified to a purity of >98%, as determined by size exclusion chromatography, and >95%, as determined by SDS PAGE.

[160] ActRIIa-hFc and ActRIIa-mFc showed high affinity for ligands. GDF11 or activin A were immobilized on a Biacore™ CM5 chip using a standard amine docking procedure. The ActRIIa-hFc and proteins Petition 870250077590, dated 01 / 09 / 2025, pp. 552 / 595 74 / 109 ActRIIa-mFc were loaded into the system and binding was measured. ActRIIa-hFc bound to activin with a dissociation constant (Kd) of 5 x 10-12 and bound to GDF11 with a Kd of 9.96 x 10-9. See Figures 4A and 4B. Using a similar binding assay, it was determined that ActRIIa-hFc has high to moderate affinity for other ligands of the TGF-beta superfamily, including, for example, activin B, GDF8, BMP6, and BMP10. ActRIIa-mFc behaved similarly.

[161] ActRIIa-hFc was very stable in pharmacokinetic studies. Rats received doses of 1 mg / kg, 3 mg / kg, or 10 mg / kg of ActRIIahFc protein, and plasma protein levels were measured at 24, 48, 72, 144, and 168 hours. In a separate study, rats received doses of 1 mg / kg, 10 mg / kg, or 30 mg / kg. In rats, ActRIIa-hFc had a serum half-life of 11 to 14 days, and circulating drug levels were quite high after two weeks (11 μg / ml, 110 μg / ml, or 304 μg / ml for initial administrations of 1 mg / kg, 10 mg / kg, or 30 mg / kg, respectively). In cynomolgus monkeys, the plasma half-life was substantially longer than 14 days, and circulating drug levels were 25 μg / ml, 304 μg / ml, or 1440 μg / ml for initial administrations of 1 mg / kg, 10 mg / kg, or 30 mg / kg, respectively. Example 2: Characterization of an ActRIIa-hFc protein

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

[163] The material expressed in CHO cells has a higher affinity for Petition 870250077590, dated 01 / 09 / 2025, pages 553 / 595 75 / 109 activin B ligand than that reported for an ActRIIahFc fusion protein expressed in human cells 293 (see, del Re et al. (2004) J Biol Chem. 279(51):53126-53135). Furthermore, the use of the TPA leader sequence provided higher yield than other leader sequences and, unlike ActRIIa-Fc expressed with a native leader, provided a highly pure N-terminal sequence. The use of the native leader sequence resulted in two main ActRIIa-Fc species, each with a different N-terminal sequence.

[164] Additional ActRIIa ligand traps (ActRIIa-Fc fusion proteins modified to reduce the activin A binding ratio relative to myostatin or GDF11) are described in International Patent Application Publications No. WO 2006 / 012627 and WO 2007 / 062188, incorporated by reference in the present invention. Example 3 Preparation of a Lyophilized ActRIIa-hFc Fusion Protein Formulation

[165] Initially, the ActRIIa-hFc fusion protein SEQ ID NO: 32 (sotatercept) was initially formulated in phosphate-buffered saline as a frozen solution. Based on subsequent development studies performed, a lyophilized citrate buffer formulation of the ActRIIa-hFc fusion protein SEQ ID NO: 32 was developed containing sucrose and polysorbate 80, to enable a stable formulation with adequate shelf life for commercialization.

[166] Two formulations of the fusion protein ActRIIa-hFc SEQ ID NO: 32, 45 mg / vial and 60 mg / vial, were developed. The 45 mg / vial formulation contained 55.0 mg of SEQ ID NO: 32 or a variant of SEQ ID NO: 32 lacking the C-terminal lysine residue, 0.48 mg of citric acid monohydrate, 2.56 mg of trisodium citrate dihydrate, 0.22 mg of polysorbate 80, and 88.0 mg of sucrose. The 60 mg / ml formulation contained 72.5 mg of SEQ ID Petition 870250077590, dated 01 / 09 / 2025, pages 554 / 595 76 / 109 NO: 32 or a variant of SEQ ID NO:32 devoid of the C-terminal lysine residue, 0.64 mg of citric acid monohydrate, 3.37 mg of trisodium citrate dihydrate, 0.29 mg of polysorbate 80, and 116.0 mg of sucrose. The formulations have identical composition before lyophilization and after reconstitution with sWFI. They differ only in their filling volumes. The 45 mg / vial has a filling volume of 1.1 mL, while the 60 mg / vial has a filling volume of 1.45 mL. Both formulations are lyophilized and reconstituted before subcutaneous administration. The 45 mg / vial is reconstituted with 1.0 mL of sWFI and the 60 mg / vial with 1.3 mL of sWFI.

[167] The development of the ActRIIa-hFc fusion protein formulation SEQ ID NO: 32 was guided by the results of several screening studies described below. These studies focused on selecting the ideal pH, buffer system, protein concentration, and excipients for stabilizing the ActRIIa-hFc fusion protein SEQ ID NO: 32. The screening studies were conducted under accelerated or stressed conditions to observe differences in the stability of the ActRIIa-hFc fusion protein SEQ ID NO: 32 in the test samples. Preliminary pH screening study: Early evaluation and identification of a suitable buffer and pH range for the ActRIIa-hFc fusion protein SEQ ID NO: 32 for optimal biophysical stability and binding capacity.

[168] An initial buffer and pH screening was performed to understand the impact of pH and buffer system on the stability of the ActRIIa-hFc fusion protein SEQ ID NO: 32. In this study, the ActRIIa-hFc fusion protein SEQ ID NO: 32 was formulated to a concentration of 5 mg / mL in acetate, citrate, histidine, phosphate, succinate, and tris buffers at a concentration of 50 mM, with pH levels ranging from 4 to 8. These formulations were filled into Type I glass vials and placed under short-term stability at 5 °C, 25 °C, and 45 °C for up to 6 months and analyzed for purity by chromatography. Petition 870250077590, dated 01 / 09 / 2025, pages 555 / 595 77 / 109 high-efficiency size exclusion (PH-SEC), thermal stability using differential scanning calorimetry (DSC), and binding activity using surface plasmon resonance (Biacore). The data from these analyses are summarized in Table 1. Table 1: Preliminary pH 1 Screening Study: Effect of Buffer and pH on % of Monomeric Species by pH-SEC, Tm1 by DSC, Kd by Biacore Buffer System Tm1 (°C) Time Point (months, M); Monomer (%) Time Point (months, M); Kd (pM) Initial 5 °C 25 °C 45 °C Initial 5 °C 25 °C 45 °C 3M 6M 3M 6M 1M 2M 6M 2M 6M 1M 2M Acetate, pH 4.0 55.6 99.7 99.9 99.7 99.8 99.8 89.4 88.4 8.4 21.1 47 192 127 272 Acetate, pH 4.5 59.7 99.7 99.9 99.7 99.8 99.8 95.3 91.7 8.0 17.1 41 115 138 184 Acetate, pH 5.0 63.1 99.8 99.9 100.0 99.8 100.0 99.4 96.0 7.5 11.3 23 50.0 77.5 186 Acetate, pH 5.5 66.8 99.8 99.9 100.0 99.9 100.0 99.8 97.9 9.7 10.6 13 23.2 41.8 53.4 Citrate, pH 4.5 57.9 99.8 99.9 99.8 99.9 99.8 85.2 66.2 9.2 16.4 46 181 251 24.9 Citrate, pH 5.0 64.0 99.8 99.9 100.0 99.9 99.7 98.6 94.3 9.9 13.0 29 70.0 307 84.2 Citrate, pH 5.5 66.2 99.8 99.9 100.0 99.9 100.0 99.8 97.8 10.7 10.1 13 27.3 46.4 56.2 Citrate, pH 6.0 68.5 99.8 99.9 100.0 99.9 100.0 99.8 97.2 9.4 9.7 11 17.7 21.5 33.0 Citrate, pH 6.5 69.4 99.8 99.9 100.0 99.9 100.0 99.1 96.7 10.0 11.5 8.6 12.4 13.4 8.3 Citrate, pH 7.0 69.5 99.8 99.9 100.0 99.9 98.1 99.0 93,7 9.4 8.4 7.2 13.1 11.5 18.0 Histidine, pH 5.5 62.7 99.8 99.9 100.0 99.9 100.0 99.6 96.3 10.9 10.9 14 31.3 82.4 170 Histidine, pH 6.0 65.8 99.8 99.9 100.0 99.9 100.0 99.8 97.6 10.4 10.5 11 20.2 50.7 55.3 Histidine, pH 6.5 67.5 99.8 99.9 100.0 98.8 98.3 99.9 90.0 9.3 8.8 9.3 14.9 29.2 32.0 Histidine, pH 7.0 67.8 99.8 99.9 100.0 99.9 98.0 99.9 88.8 8.7 8.3 10 16.0 21.0 29.3 Histidine, pH 7.4 67.9 99.8 99.9 100.0 99.9 95.9 100.0 87.1 10.1 9.4 9.1 15.9 19.2 26.2 Phosphate, pH 5.8 69.1 99.8 99.9 100.0 99.9 100.0 99.8 97.7 8.0 9.8 10 19.3 34.7 41.8 Phosphate, pH 6.3 69.4 99.8 99.9 100.0 99.9 100.0 99.8 97.0 9.0 10.7 11 15.8 20.6 27.7 Phosphate, pH 6.8 69.9 99.8 99.9 99.6 99.9 96.7 98.3 94.3 10.0 8.6 6.5 11.9 15.5 16.7 Phosphate, pH 7.3 70.2 99.8 99.8 100.0 99.9 95.8 92.3 87.9 10.0 10.0 7.3 8.6 13.8 20.7 Petition 870250077590, dated 01 / 09 / 2025, pages 556 / 595 78 / 109 Buffer System Tm1 (°C) Time Point (months, M); Monomer (%) Time Point (months, M); Kd (pM) Initial 5 °C 25 °C 45 °C Initial 5 °C 25 °C 45 °C 3M 6M 3M 6M 1M 2M 6M 2M 6M 1M 2M Phosphate, pH 7.8 70.0 99.8 99.9 100.0 99.5 90.4 87.7 63.7 9.0 9.4 7.7 12.9 17.7 18.0 Succinate, pH 4.5 59.4 99.8 99.8 99.7 99.8 99.7 95.2 91.5 9.5 18.3 42 114.0 108 65.8 Succinate, pH 5.0 64.0 99.8 99.8 100.0 99.9 99.8 99.5 95.4 12.1 13.8 25 48.9 73.4 106 Succinate, pH 5.5 67.1 99.8 99.8 100.0 99.8 99.8 99.8 96.4 10.5 10.8 15 25.0 31.6 65.6 Succinate, pH 6.0 68.6 99.8 99.9 100.0 99.8 99.8 99.7 95.9 11.5 5.8 8.4 12.0 17.7 5.7 Succinate, pH 6.5 69.5 99.8 99.9 100.0 99.7 99.7 98.8 96.2 9.8 10.4 7.6 10.2 10.9 14.3 Tris, pH 7.0 68.9 99.8 99.9 100.0 99.9 97.3 99.8 97.1 9.7 8.4 6.5 11.3 12.6 25.3 Tris, pH 7.3 69.2 99.8 99.9 100.0 99.9 97.1 98.1 96.4 11.2 8.1 10 9.3 11.1 24.5 Tris, pH 7.5 69.1 99.8 99.9 99.3 99.8 95.8 99.2 95.3 10.6 10.7 8.1 10.8 15.5 19.8 Tris, pH 7.8 69.0 99.8 99.9 100 99.9 95.7 99.0 94,6 10.5 6.8 9.8 12.3 10.9 21.3 Tris, pH 8.0 69.0 99.8 99.9 100 99.8 95.7 98.9 94.0 9.6 7.4 10 12.7 10.4 22.6,

[169] Observation of changes in % of monomeric species (purity) by PH-SEC revealed that there was no significant change in purity after 6 months at 5 °C, with all formulations retaining >99.0% monomer. After 6 months at 25 °C, there was a significant decrease in % monomer for formulations with pH > 6.5, while there were no significant changes for any formulation below pH 6.5, as shown in Figure 5. After 2 months at 45 °C, there was a similar decrease in % monomer for formulations with pH > 6.5, but there was also a decrease in % monomer correlated with lower pH < 5.5.

[170] Based on these observations under accelerated (25 °C) and stressed (45 °C) conditions, the fusion protein ActRIIa-hFc SEQ ID NO: 32 in citrate and succinate buffer in the pH range of 5.5 to 6.5 had the greatest stabilizing effect. Histidine at pH 5.5 - 6.0 also showed similar stability to citrate and succinate at lower pH and less stability at higher pH.

[171] Binding affinity is normally measured by the equilibrium dissociation constant (Kd), which is used to assess and classify the strengths of bimolecular interactions. In this case, the interaction or affinity Petition 870250077590, dated 01 / 09 / 2025, pages 557 / 595 The binding affinity of the ActRIIa-hFc fusion protein SEQ ID NO: 32 to its Biacore activin target, 79 / 109, was used as a measure of product stability. The lower the Kd value, the greater the binding affinity of the ligand to its target.

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

[173] Based on these observations, the fusion protein ActRIIa-hFc SEQ ID NO: 32 can retain its binding affinity at pH >5.5 in many different buffer systems.

[174] The unfolding temperature of the ActRIIa-hFc fusion protein SEQ ID NO: 32 increased consistently with pH, ​​stabilizing near 67 °C - 70 °C around pH >5.5. All buffers with equivalent pH values ​​performed identically, except for the histidine and tris systems, which have significantly lower melting points.

[175] Overall, the results of this experiment suggested that a pH range of 5.5 to 6.5 would be the ideal range for SEQ ID NO: 32 in terms of physical stability monitored by PH-SEC and binding affinity retention by Biacore. Furthermore, citrate and succinate buffer systems outperformed histidine at the upper end of this range. Additionally, acetate and phosphate buffers were not further developed due to the potential for pH changes due to precipitation during freezing or lyophilization. Determining the ideal buffer concentration: Evaluating the effect of buffer concentration with one of the main buffer systems, citrate, on Petition 870250077590, dated 01 / 09 / 2025, pages 558 / 595 80 / 109 Conformational and colloidal stability of the ActRIIa-hFc fusion protein SEQ ID NO: 32

[176] In the initial pH screening study, a variety of buffer systems were evaluated at a concentration of 50 mM to provide a high buffering capacity. However, this concentration may not be ideal for stabilizing the ActRIIa-hFc fusion protein SEQ ID NO: 32 and is not ideal for subcutaneous injections, where the higher ionic strength of the buffer, especially citrate, is known to cause injection pain. In this study, the ActRIIa-hFc fusion protein SEQ ID NO: 32 was prepared in a variety of citrate buffer concentrations, one of the main buffer systems from the previous study. Citrate buffer (5-50 mM) at pH 5.8 with 50 mg / mL of the fusion protein ActRIIa-hFc SEQ ID NO: 32 and 8% (w / v) sucrose were evaluated for the effect of buffer strength on thermal stability using DSC and diffusion interaction parameter (kD) by DLS, which is a measure of a molecule's propensity to self-associate. The results are summarized in Table 2. Table 2: Study of Buffer Concentration Variation: Effect of Buffer Concentration at Melting Points and kD Citrate (mM) Tm1 (°C) kD (mL / g) 5 67.9 6.7 10 68.8 7.4 25 69.0 1.4 50 68.8 -4.5

[177] The study results showed that the highest dissociation constant (kD) of the fusion protein ActRIIa-hFc SEQ ID NO: 32 was at 10 mM (+6.7 mL / g) and would decrease with higher concentrations up to 50 mM, resulting in a negative kD (-4.5 mL / g). The more positive the kD value of a molecule, the greater the repulsion between the molecules, indicating better colloidal stability, while a negative kD value indicates a Petition 870250077590, dated 01 / 09 / 2025, pp. 559 / 595 81 / 109 propensity for self-association, which can lead to aggregation. Furthermore, the melting points of the ActRIIa-hFc fusion protein SEQ ID NO: 32 were largely identical and sufficiently high for all buffer concentrations. Based on these data, a buffer concentration of 10 mM was chosen for the subsequent development of the formulation. Optimal Stability pH Selection

[178] A short-term stability study was conducted where the fusion protein ActRIIa-hFc SEQ ID NO: 32 was formulated to a concentration of 5 mg / mL in succinate, citrate, and histidine at a concentration of 10 mM with pH levels ranging from 5 to 7. These formulations were filled into Type I glass vials and subjected to short-term stability at 5 °C, 25 °C, and 40 °C for up to 3 months. The formulations were analyzed by biophysical methods for purity by high-performance size exclusion chromatography (PH-SEC), thermal stability using differential scanning calorimetry (DSC), fragmentation by unreduced CE-SDS, chemical degradation by desialylated iCIEF, and pH. The data from these analyses are summarized in Table 3, Table 4, and Table 5, and the following observations were made: Table 3: pH Selection for Optimal Stability Study: Effect of buffer and pH on % HMW, Monomer, and % LMW Species by pH-SEC, Tm1 by DSC DSC Buffer System Time Point (months, M); HMW (%) Time Point (months, M); Monomer (%) Time Point (months, M); LMW (%) Tm1 Initial 5 °C 25 °C 40 °C Initial 5 °C 25 °C 40 °C Initial 5 °C 25 °C 40 °C 3M 3M 3M 3M 3M 3M 3M 3M 3M Succinate pH 5.0 61.2 0.7 0.5 0.5 N / A 98.7 98.9 98.4 N / A 0.7 0.6 1.0 N / A Succinate pH 5.5 65.3 0.5 0.5 0.5 0.7 98.9 99.0 98.7 96.1 0.7 0.6 0.9 3.1 Succinate pH 6.0 68.1 0.5 0.4 0.4 0.5 98.9 99.0 98.7 96.3 0.6 0.5 0.9 3.3 Petition 870250077590, dated 01 / 09 / 2025, pages 560 / 595 82 / 109 DSC Buffer System Time Point (months, M); HMW (%) Time Point (months, M); Monomer (%) Time Point (months, M); LMW (%) Tm1 Initial 5 °C 25 °C 40 °C Initial 5 °C 25 °C 40 °C Initial 5 °C 25 °C 40 °C 3M 3M 3M 3M 3M 3M 3M 3M 3M Succinate pH 6.5 69.7 0.5 0.5 0.4 0.4 98.9 99.0 98.6 95.5 0.6 0.6 1.0 4.1 Citrate pH 5.0 61.7 0.5 0.5 0.5 1.5 98.9 99.0 98.5 95.0 0.6 0.5 1.0 3.6 Citrate pH 5.5 65.4 0.5 0.5 0.4 0.7 98.9 99.1 98.7 96.2 0.6 0.5 0.9 3.2 Citrate pH 6.0 68.6 0.5 0.4 0.4 0.5 98.9 99.1 98.7 96.1 0.6 0.5 0.9 3.4 Citrate pH 6.5 70.0 0.5 0.5 0.4 0.4 99.0 99.0 98.6 95.1 0.6 0.5 1.0 4.5 Citrate pH 7.0 70.4 0.5 0.5 0.4 0.4 98.9 99.0 98.4 93.8 0.6 0.5 1.2 5.8 Histidine pH 5.5 60.9 0.5 0.5 0.4 1.1 98.9 99.1 98.7 95.6 0.6 0.5 0.9 3.3 Histidine pH 6.0 63.7 0.5 0.4 0.3 0.5 99.0 99.1 98.8 96.3 0.6 0.5 0.9 3.1 Histidine pH 6.5 66.3 0.5 0.4 0.3 0.3 99.0 99.1 98.7 96.4 0.6 0.5 1.0 3.3 Histidine pH 7.0 66.9 0.5 0.4 0.3 0.3 99.0 99.0 98.7 96.1 0.6 0.6 1.0 3.6 Table 4: Selection of pH for Optimal Stability Study: Effect of Buffer and pH on % of minor peak species and % of major peak species by CE-SDS NR pH Buffer System Point in Time (months, M); Lower Peak (%) Point in Time (months, M); Peak (%) Initial Initial 5 °C 25 °C 40 °C Initial 5 °C 25 °C 40 °C 1M 1M 1M 1M 1M 1M Succinate pH 5.0 5.0 1.9 2.2 3.4 6.3 98.0 97.7 96.6 93.8 Succinate pH 5.5 5.5 1.9 2.2 2.3 4.5 98.0 97.8 97.7 95.4 Succinate pH 6.0 5.9 2.0 2.8 2.4 3.2 98.0 97.1 97.6 96.8 Succinate pH 6.5 6.5 2.2 2.3 2.4 3.4 97.8 97.7 97.6 96.7 Citrate pH 5.0 4.9 3.5 2.2 3.4 6.9 96.5 97.9 96.6 93.0 Petition 870250077590, dated 01 / 09 / 2025, pages 561 / 595 83 / 109 pH Buffer System Point in Time (months, M); Lower Peak (%) Point in Time (months, M); Peak (%) Initial Initial 5 °C 25 °C 40 °C Initial 5 °C 25 °C 40 °C 1M 1M 1M 1M 1M 1M Citrate pH 5.5 5.4 3.4 2.4 3.0 3.5 96.5 97.6 97.0 96.4 Citrate pH 6.0 5.7 2.1 1.8 2.4 4.1 97.9 98.2 97.6 95.9 Citrate pH 6.5 6.3 1.9 1.8 2.1 2.9 98.1 98.1 97.9 97.0 Citrate pH 7.0 6.7 1.7 2.1 2.1 3.6 98.3 97.9 97.9 96.3 Histidine pH 5.5 5.7 1.9 2.1 2.8 4.5 98.1 97.8 97.2 95.4 Histidine pH 6.0 6.1 1.9 2.0 1.9 3.3 98.1 98.0 98.1 96.7 Histidine pH 6.5 6.6 1.8 2.0 2.1 3.2 98.3 98.0 97.8 96.8 Histidine pH 7.0 6.9 2.0 2.3 2.3 3.4 98.0 97.6 97.8 96.5 Table 5: Selection of pH for the Optimal Stability Study: Effect of Buffer and pH on % of Acidic Species, % of Basic Species, and % of Species Main Totals by icIEF Desialylated Buffer System Time Point (months, M); Acid Species (%) Time Point (months, M); Total Main Species (%) Time Point (months, M); Basic Species (%) Initial 5 °C 25 °C 40 °C Initial 5 °C 25 °C 40 °C Initial 5 °C 25 °C 40 °C 3M 3M 3M 3M 3M 3M 3M 3M Succinate pH 5.0 19.6 18.5 12.3 DNT 64.1 65.3 50.9 DNT 16.4 16.2 36.8 DNT Succinate pH 5.5 17.2 17.8 15.1 13.9 65.8 67.3 60.8 45.1 17.0 15.0 24.1 41.0 Succinate pH 6.0 16.8 16.2 16.4 19.1 65.5 69.7 65.3 53.6 17.7 14.1 18.3 27.3 Succinate pH 6.5 17.3 17.3 17.9 DNT 65.1 67.7 66.5 DNT 17.6 15.1 15.6 DNT Citrate pH 5.0 18.1 15.7 11.6 16.3 64.1 65.5 51.2 28.6 17.7 18.8 37.3 55.1 Citrate pH 5.5 17.7 16.6 14.9 24.0 65.2 68.4 59.8 37.5 17.1 15.0 25.3 38.4 Citrate pH 6.0 17.5 16.3 15.7 22.2 64.9 69.1 65.9 52.1 17.6 14.7 18.4 25.7 Citrate pH 6.5 17.6 17.4 18.0 26.8 65.1 69.0 66.3 55.0 17.2 13.6 15.7 18.2 Petition 870250077590, dated 01 / 09 / 2025, pages 562 / 595 84 / 109 Buffer System Time Point (months, M); Acid Species (%) Time Point (months, M); Total Main Species (%) Time Point (months, M); Basic Species (%) Initial 5 °C 25 °C 40 °C Initial 5 °C 25 °C 40 °C Initial 5 °C 25 °C 40 °C 3M 3M 3M 3M 3M 3M 3M 3M Citrate pH 7.0 18.2 18.7 18.1 36.0 64.9 69.1 68.0 49.6 16.9 12.3 13.8 14.4 Histidine pH 5.5 19.1 18.9 16.0 13.7 64.2 68.0 59.4 31.4 16.7 13.1 24.6 54.9 Histidine pH 6.0 17.6 17.8 16.0 13.1 65.6 69.5 64.8 47.7 16.8 12.8 19.2 39.2 Histidine pH 6.5 17.0 17.8 17.5 19.6 66.0 69.1 66.9 53.2 17.0 13.1 15.6 27.1 Histidine pH 7.0 17.8 20.0 19.9 26.2 65.3 67.6 65.7 53.8 17.0 12.4 14.4 20.0

[179] The results of this study shown in Figure 7 showed that citrate and succinate have comparable performance between pH 5.3 and 6.3 (measured pH) for better thermal stability compared to PH-SEC based on 25 °C stability results. Higher amounts of fragmentation (LMW%) observed by PH-SEC at 40 °C in citrate and succinate buffer systems were attributed to the nature of the stress and the integration challenges of the method. The observations made with PH-SEC were also consistent with the unreduced CE-SDS data shown in Figure 8. Characterization of the loading profile by desialylated iCIEF shown in Figure 9 also confirmed that the optimal stability pH range is between 5.3 and 6.3. The histidine buffer formulation requires a slightly wider pH range, between 6.0 and 6.5, to have comparable performance to citrate and succinate; However, this buffer system also exhibited a lower Tm1, indicative of lower biophysical stability.

[180] The results of this study demonstrated that the fusion protein ActRIIa-hFc SEQ ID NO: 32 is biophysically and chemically stable in the range of 5.3 to 6.3 (measured pH) in citrate and succinate buffers. Furthermore, this range offers the potential to decrease the rate of possible deamidation by selecting a pH that is less basic in nature, as typically observed with Petition 870250077590, dated 01 / 09 / 2025, pages 563 / 595 85 / 109 biological products. Thus, a target pH of 5.8 was selected with citrate as a buffer system for further product development. Physical Stability of the ActRIIa-hFc Fusion Protein from SEQ ID NO: 32

[181] A study was conducted to evaluate the physical stability of the ActRIIa-hFc fusion protein from SEQ ID NO: 32 under physical stress of agitation in the absence of any excipients in one of two buffer systems: lead, 10 mM citrate, as the worst-case scenario. The ActRIIa-hFc fusion protein from SEQ ID NO: 32 was diluted to concentrations of 5 and 50 mg / mL in 10 mM citrate, pH 5.8. The two formulations were agitated together with un-agitated control samples, where 4 mL of sample were filled into 10 mL Type I glass vials with an analog shaker for up to 4 days and analyzed for monomer loss by PH-SEC and summarized in Table 6. Table 6 Physical Stability by Agitation and Impact of the Monomer (%) Protein Concentration (mg / mL) Condition Time Point; Monomer (%) 0 1 h 4 h 8 h 4 days 5 agitation 99.3 99.4 99.4 99.5 99.6 5 control 99.4 99.4 99.4 99.5 99.3 50 agitation 99.5 99.4 99.4 99.4 99.3 50 control 99.4 99.4 99.2 99.4 99.0

[182] No significant change in monomer content was observed between control and stressed samples for up to 4 days of agitation, regardless of protein concentration. The results of this study suggest that, even in the absence of a bulk sugar and a surfactant, the ActRIIa-hFc fusion protein from SEQ ID NO: 32 is biophysically stable against shear and interfacial stress. ActRIIa-hFc fusion protein SEQ ID NO: 32 Concentration Variation Study

[183] ​​A study was conducted to evaluate the short-term biophysical stability of the ActRIIa-hFc fusion protein from SEQ ID NO: 32 at various concentrations. The ActRIIa-hFc fusion protein from SEQ ID NO: 32 was prepared Petition 870250077590, dated 01 / 09 / 2025, pp. 564 / 595 86 / 109 of 20 mg / mL to 200 mg / mL in 10 mM citrate buffer, pH 5.8, without any other excipients, and filled into Type I glass vials and placed in short-term stability at 5 °C and 25 °C for up to 7 days. The purity of the formulations was measured by PH-SEC analysis throughout the study and summarized in Table 7. Table 7. pH-SEC data for concentration variation study. Protein Concentration (mg / mL) Point in Time (days, D); Monomer (%) 5 °C 25 °C Initial 1D 3D 7D Initial 1D 3D 7D 200 99.6 99.6 99.4 99.4 99.5 99.3 98.8 98.7 150 99.6 99.6 99.5 99.5 99.6 99.5 99.2 99.1 100 99.6 99.6 99.6 99.6 99.6 99.6 99.4 99.3 75 99.7 99.6 99.6 99.6 99.6 99.6 99.5 99.5 50 99.6 99.7 99.6 99.6 99.6 99.6 99.6 99.5 20 99.6 99.7 99.2 99.7 99.7 99.7 99.6 99.6

[184] There was no significant change in the purity of any of the formulations when stored at 5 °C for up to 7 days. However, it was observed that concentrations above 75 mg / mL showed greater monomer loss during short-term storage at 25 °C, and this trend continued with increasing concentration up to 200 mg / mL, as shown in Figure 10. The results of this study demonstrated that protein concentrations of 75 mg / mL and below had a negligible impact on 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 chosen to be <75 mg / mL based on the shaking and concentration variation study. Evaluation of the Long-Term Stability of Lead Buffers for Aiding in the Compounding of Pharmaceutical Substances

[185] A long-term stability study was conducted to compare the performance of two buffer systems at two different concentrations (based on the initial understanding of the need for dosing). Petition 870250077590, dated 01 / 09 / 2025, pages 565 / 595 87 / 109 clinical). The ActRIIa-hFc fusion protein from SEQ ID NO: 32 was prepared at 75 and 50 mg / mL in 10 mM succinate and 10 mM citrate at a pH of 5.8 and filled into Type I glass vials and stored at -80 °C, -20 °C, 5 °C and 25 °C for up to 30 months under each storage condition.

[186] Stability data from PH-SEC analysis are provided in Table 8 and the following observations are made: Table 8 HP-SEC data for long-term stability of lead buffers Protein Concentration (mg / mL) Buffer System Time Point (months, M); Monomer (%) Initial -80 °C 5 °C 25 °C 6M 12M 24M 6M 12M 24M 6M 12M 75 10 mM Citrate, pH 5.8 97.9 98.2 98.1 98.2 96.0 95.7 94.6 93.3 91.2 50 10 mM Citrate, pH 5.8 98.0 98.1 98.4 98.2 96.6 96.7 95.8 94.8 93.4 75 10 mM Succinate, pH 5.8 97.5 98.3 97.9 98.6 95.5 94.4 93.2 91.8 90.1 50 10 mM of succinate, pH of 5.8 97.5 97.8 98.0 97.8 96.4 95.6 94.4 94.0 92.2

[187] There was no significant change in purity by PH-SEC after 24 months at -80 °C with all formulations retaining >97.8% monomer. After 24 months at 5 °C, the ActRIIa-hFc fusion protein from SEQ ID NO: 32 decreased in monomer to 94-95% for citrate formulations and 93-94% for succinate formulations. These results demonstrate that the ActRIIa-hFc fusion protein from SEQ ID NO: 32 is biophysically stable as a solution, even in the absence of any surfactant or stabilizing sugar, since none of the formulations showed a loss of % monomer below 94%, even after 2 years at 2-8 °C. After 12 months at 25 °C, the ActRIIa-hFc fusion protein from SEQ ID NO: 32 decreased in monomer to 91-93% for citrate formulations and 90-92% for succinate formulations. In the storage data at 5 °C and 25 °C, the 75 mg / mL drug substance degraded slightly more than when at 50 mg / mL for each buffer system, as expected, and Petition 870250077590, dated 01 / 09 / 2025, pages 566 / 595 88 / 109 these trends are shown in Figure 11.

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

[189] The ActRIIa-hFc fusion protein from SEQ ID NO: 32 was prepared in a range of sodium chloride concentrations from 0 to 150 mM in 10 mM citrate pH from 5.8 to 50 mg / mL of the ActRIIa-hFc fusion protein from SEQ ID NO: 32 with 8% (w / v) sucrose to evaluate the effect of buffer intensity on its biophysical properties. These formulations were evaluated for their thermal stability using DSC and their diffusion interaction parameter (kD) by DLS. The results are summarized in Table 9. Table 9: Study of salt concentration variation: Effect of Chloride Sodium Melting Point and Diffusion Interaction Parameter (kD) Sodium chloride (mM) DSC DLS Tm1 (°C) kD (mL / g) 0 69.8 16.2 30 70.1 9.6 50 70.0 5.8 70 70.0 3.5 100 69.8 5.3 150 69.4 4.5

[190] The study results showed that the highest kD of the ActRIIa-hFc fusion protein from SEQ ID NO: 32 did not contain sodium chloride (16.2 mL / g) and would decrease with higher concentrations down to about 5 mL / g, stabilizing at concentrations above 50 mM. The more positive the kD value of a Petition 870250077590, dated 01 / 09 / 2025, pp. 567 / 595 89 / 109 molecule, the greater the repulsion between molecules, which indicates better colloidal stability, suggesting that the ActRIIa-hFc fusion protein from SEQ ID NO: 32 has the best colloidal stability without any sodium chloride. Furthermore, the melting points of the ActRIIa-hFc fusion protein from SEQ ID NO: 32 were largely identical and sufficiently high for all sodium chloride concentrations. Based on these data, sodium chloride was not considered suitable for further development work on stabilizing the ActRIIa-hFc fusion protein from SEQ ID NO: 32. Excipient Screening Study

[191] A screening study was developed to evaluate a variety of excipients with the aim of increasing the biophysical stability of the ActRIIa-hFc fusion protein from SEQ ID NO: 32 in solution. In this excipient screening, the ActRIIa-hFc fusion protein from SEQ ID NO: 32 was formulated to a concentration of 2 mg / mL with a variety of excipients: a salt (sodium chloride), sugars (sucrose, mannitol), and amino acids (arginine, histidine). These solutions, as summarized in Table 10, were placed in stability at 5 °C, 25 °C, and 45 °C for up to 12 months, 9 months, and 3 months, respectively, and subsequently tested for their purity (% monomer content) by PHSEC. Table 10: Excipient Screening Study: Effect of Excipients as a percentage of Monomeric Species by pH-Sec Excipient Concentration of excipient Initial 12 months 5 °C 9 months 25 °C 3 months 45 °C Arginine 0.1% (weight / volume) 99.7 99.8 99.5 97.8 0.5% (weight / volume) 99.7 99.8 99.6 97.7 2.0% (weight / volume) 99.7 99.8 99.0 97.7 Histidine 0.1% (weight / volume) 99.8 99.8 99.2 96.8 Petition 870250077590, dated 01 / 09 / 2025, pages 568 / 595 90 / 109 Excipient Concentration of excipient Initial 12 months 5 °C 9 months 25 °C 3 months 45 °C 0.5% (weight / volume) 99.7 99.8 98.2 94.7 2.0% (weight / volume) 99.7 99.9 96.7 90.5 Mannitol 0.1% (weight / volume) 99.7 99.8 99.5 98.8 0.5% (weight / volume) 99.7 99.8 99.2 97.9 2.0% (weight / volume) 99.7 99.8 99.3 98.0 Sucrose 0.1% (weight / volume) 99.8 99.8 99.5 97.8 0.5% (weight / volume) 99.8 99.8 99.3 97.9 2.0% (weight / volume) 99.8 99.8 99.5 98.0

[192] All formulations showed biophysical stability at storage temperatures of 5 °C and 25 °C, with >99% of monomer retained after 12 months at 5 °C and 9 months at 25 °C. The excipients evaluated demonstrated comparable biophysical stability under stress conditions (45 °C) after 3 months, retaining 97-99% of monomer, except for histidine. Increasing histidine concentration was correlated with a decrease in biophysical stability, with up to 2% (weight / volume) of histidine resulting in 6% less monomer than the 0.1% (weight / volume) histidine formulation.

[193] In conclusion, the ActRIIa-hFc fusion protein from SEQ ID NO: 32 was identified as biophysically stable in all prototypes tested. Surfactant Screening Study

[194] Surfactants are widely used to stabilize proteins against shear and interfacial stresses encountered during manufacturing, transport, and handling. Therefore, a series of experiments was conducted to evaluate polysorbate 80 (PS80) as a surfactant to stabilize SEQ ID NO: 32 under various stress conditions. Study 1: Screening of Surfactants by Thermal Stability Petition 870250077590, dated 01 / 09 / 2025, pp. 569 / 595 91 / 109

[195] A PS80 concentration-ranging stability study was conducted to evaluate the effect of PS80 on the long-term stability of the ActRIIa-hFc fusion protein from SEQ ID NO: 32. In this study, the ActRIIa-hFc fusion protein from SEQ ID NO: 32 was formulated to a concentration of 50 mg / mL with varying levels of PS80 ranging from 0.001% to 0.05% in 10 mM citrate at a pH of 5.8 with 8% (w / v) sucrose. These formulations were filled into Type I glass vials and stabilized at 5°C and 25°C for up to 24 months and at 45°C for up to 3 months. The purity of the formulations was measured by PH-SEC throughout the study to monitor their physical stability. PH-SEC stability data are provided in Table 11. Table 11: PS80 concentration range: Effect of concentration of PS80 as a percentage of monomeric species under different storage conditions. PS80 (%) Time Point (months, M); Monomer %) Initial 5 °C 25 °C 45 °C 6M 12M 24M 12M 24M 1M 3M 0.001 99.5 99.3 99.3 99.2 98.1 96.6 97.7 93.5 0.020 99.5 99.3 99.3 99.2 98.2 96.5 97.8 93.5 0.050 99.5 99.4 99.3 99.2 98.1 96.6 97.8 93.6

[196] No significant differences were observed in the biophysical stability profiles between the different levels of PS80, where all formulations showed >99% monomer after 24 months at 5 °C, >96% monomer after 24 months at 25 °C and >93% monomer after 3 months at 45 °C. The results of this study demonstrated that PS80 concentrations between 0.001% and 0.05% were comparable in terms of biophysical stability under thermal stress. Study 2: Screening of Surfactants by Agitation and Freeze-Thaw

[197] An additional study of varying PS80 concentrations was conducted to further evaluate the effect of PS80 on stabilizing the Petition 870250077590, dated 01 / 09 / 2025, pages 570 / 595 92 / 109 ActRIIa-hFc fusion protein from SEQ ID NO: 32 against agitation and freeze-thaw stress. In this study, the ActRIIa-hFc fusion protein from SEQ ID NO: 32 was formulated to a concentration of 50 mg / mL with varying PS80 levels ranging from 0% to 0.05% in 10 mM citrate at a pH of 5.8 with 8% (weight / volume) sucrose. These formulations were filled into Type I glass vials and agitated at 300 RPM for up to 7 days. Separately, the formulations were also subjected to up to 8 freeze-thaw cycles, from -80 °C to 25 °C. The purity of the formulations was measured by UP-SEC, particulates by HIAC and MFI, and filler variants by desialylated iCIEF throughout the study to monitor the biophysical and chemical stability of SEQ ID NO: 32. Stability data are provided in Tables 12 through 19. Table 12: Surfactant Screening by Agitation: Effect of PS80 Concentration on HMW, Monomer, LMW by UP-SEC and Main Peak by CE-SDS NR PS80 UP-SEC CE-SDS NR Time Point (days, D); HMW (%) Time Point Time Point Time Point (days, D); Peak Value (%) Monomer (%' LMW (% (%) Control 3D 7D Control 3D 7D Control 3D 7D Control 3D 7D 0.000 0.8 0.8 0.8 97.3 97.3 97.3 1.9 1.9 1.9 98.1 98.0 98.2 0.005 0.8 0.8 0.8 97.4 97.2 97.3 1.9 2.0 1.9 98.0 98.2 98.4 0.010 0.8 0.8 0.8 97.3 97.3 97.3 1.9 1.9 1.9 98.1 98.2 97.9 0.020 0.8 0.8 0.8 97.3 97.3 97.3 1.9 1.9 1.9 98.0 98.0 98.0 0.030 0.8 0.8 0.8 97.3 97.3 97.3 1.9 2.0 2.0 97.8 98.2 98.0 0.050 0.8 0.8 0.8 97.2 97.3 97.2 2.0 1.9 2.0 97.9 98.1 97.9 Table 13: Surfactant Screening by Agitation: Effect of PS80 Concentration on Particulate Matter by MFI PS80 (%) Time Point (days, D); > 2 μm Particulates per mL Time Point (days, D); > 5 μm Particulates per mL Time Point (days, D); > 10 μm Particulates per mL Time Point (days, D); > 25 μm Particulates per mL Control 3D 7D Control 3D 7D Control 3D 7D Control 3D 7D 0.000 173 157 175 40 28 44 14 12 12 0 0 0 0.005 35 35 51 5 7 5 0 5 5 0 0 0 0.010 115 53 88 10 5 12 0 3 3 0 0 3 Petition 870250077590, dated 01 / 09 / 2025, pages 571 / 595 93 / 109 PS80 (%) Time Point (days, D); > 2 μm Particulates per mL Time Point (days, D); > 5 μm Particulates per mL Time Point (days, D); > 10 μm Particulates per mL Time Point (days, D); > 25 μm Particulates per mL Control 3D 7D Control 3D 7D Control 3D 7D Control 3D 7D 0.020 39 72 129 7 12 23 3 3 7 0 0 3 0.030 115 65 69 19 23 14 3 12 3 0 0 0 0.050 125 161 129 23 37 37 10 12 7 0 3 0 Table 14: Surfactant Screening by Agitation: Effect of PS80 Concentration on Particulate Matter by HIAC PS80 (%) Time Point (days, D); > 2 μm Particulates per mL Time Point (days, D); > 5 μm Particulates per mL Time Point (days, D); > 10 μm Particulates per mL Time Point (days, D); > 25 μm Particulates per mL Control 3D 7D Control 3D 7D Control 3D 7D Control 3D 7D 0.000 77 50 39 15 14 7 5 4 1 1 1 0 0.020 16 34 35 7 8 6 2 2 2 1 0 0 Table 15: Surfactant Screening by Agitation: Effect of PS80 Concentration on Acid Species, Basic Species, and Total Major Species by Desialylated iCIEF PS80 (%) Time Point (days, D); Acid Species (%) Time Point (days, D); Total Main Species (%) Time Point (days, D); Basic (%) 3D Control 7D Control 3D Control 7D Control 3D Control 7D 0.000 22.7 20.8 21.1 67.9 69.3 69.1 9.4 10.0 9.8 0.005 20.7 20.8 22.1 68.3 69.1 68.4 11.1 10.2 9.6 0.010 21.8 22.6 22.7 68.5 67.7 68.5 9.8 9.8 8.9 0.020 20.7 21.5 22.6 69.3 69.1 67.8 10.1 9.5 9.7 0.030 20.9 22.7 23.4 69.3 68.2 68.9 9.9 9.1 7.8 0.050 20.9 21.9 21.0 69.4 68.6 69.3 9.7 9.6 9.9 Table 16: Surfactant Screening by Freezing and Thawing: Effect of PS80 concentration on HMW, Monomer, LMW by UP-SEC data, Peak Higher by CE-SDS not reduced from SEQ ID NO: 32 When under Stress by Freezing and Thawing PS80 (%) UP-SEC CE-SDS NR Freeze-Thaw Cycles; HMW (%) Freeze-Thaw Cycles; Monomer (%) Freeze-Thaw Cycles; LMW (%) Freeze-Thaw Cycles; Highest Peak (%) 0 3 5 0 3 5 0 3 5 0 3 5 0.000 0.8 0.8 0.8 97.3 97.4 97.4 1.9 1.8 1.9 98.0 98.1 98.1 Petition 870250077590, dated 01 / 09 / 2025, pp. 572 / 595 94 / 109 PS80 (%) UP-SEC CE-SDS NR Freeze-Thaw Cycles; HMW (%) Freeze-Thaw Cycles; Monomer (%) Freeze-Thaw Cycles; LMW (%) Freeze-Thaw Cycles; Peak Value (%) 0 3 5 0 3 5 0 3 5 0 3 5 0.010 0.8 0.8 0.8 97.4 97.4 97.4 1.9 1.8 1.8 98.0 98.1 98.1 0.020 0.8 0.8 0.8 97.4 97.3 97.3 1.9 1.9 1.9 98.0 98.2 98.1 0.030 0.8 0.8 0.8 97.3 97.3 97.3 2.0 1.9 1.9 98.1 98.2 98.0 Table 17: Surfactant Screening by Freezing and Thawing: Effect of PS80 Concentration on Charge Variants by iCIEF Desialylated SEQ ID NO: 32 When under Freeze-Thaw Stress PS80 (%) Freeze and Defrost Cycles; Acidic Species (%) Freeze-Thaw Cycles; Total Main Species (%) Freeze-Thaw Cycles; Basic Species (%) 0 3 5 0 3 5 0 3 5 0.000 21.4 22.0 20.4 68.7 68.4 69.2 9.9 9.7 10.5 0.010 21.9 23.0 20.7 69.2 67.5 69.6 9.0 9.5 9.8 0.020 22.4 20.8 20.1 68.2 69.3 70.5 9.5 9.9 9.6 0.030 22.2 20.5 20.0 68.6 69.5 70.3 9.4 10.1 9.8 Table 18: Surfactant Screening by Freezing and Thawing: Effect of PS80 Concentration on Particulate Matter by HIAC of SEQ ID NO: When under Freezing and Thawing Stress PS80 (%) Freeze-Thaw Cycles; > 2 μm Particulates per mL Freeze-Thaw Cycles; > 5 μm Particulates per mL Freeze-Thaw Cycles; > 10 μm Particulates per mL Freeze-Thaw Cycles; > 25 μm Particulates per mL 0 3 5 0 3 5 0 3 5 0 3 5 0.000 60 81 88 15 19 14 4 6 2 1 1 1 0.020 27 65 82 6 9 12 1 2 6 0 1 1 Table 19: Surfactant Screening by Freezing and Thawing: Effect of PS80 Concentration on Particulate Matter by MFI of SEQ ID NO: When under Freezing and Thawing Stress PS80 (%) Freeze-Thaw Cycles; > 2 μm Particulates per mL Freeze-Thaw Cycles; > 5 μm Particulates per mL Freeze-Thaw Cycles; > 10 μm Particulates per mL Freeze-Thaw Cycles; > 25 μm Particulates per mL 0 3 5 0 3 5 0 3 5 0 3 5 0.000 134 230 343 37 46 104 5 7 28 0 3 5 Petition 870250077590, dated 01 / 09 / 2025, pp. 573 / 595 95 / 109 PS80 (%) Freeze-Thaw Cycles; > 2 μm Particulates per mL Freeze-Thaw Cycles; > 5 μπ Particulates per mL Freeze-Thaw Cycles; > 10 μπ Particulates per mL Freeze-Thaw Cycles; > 25 μπ Particulates per mL 0 3 5 0 3 5 0 3 5 0 3 5 0.010 33 74 113 5 14 7 0 0 0 0 0 0 0.020 56 147 115 14 26 30 7 0 5 5 0 3 0.030 44 145 129 21 35 44 5 12 12 0 3 0

[198] There was no significant difference in the purity of any of the formulations tested for HMW, monomer, or LMW content by UP-SEC when compared to the control sample. There was no perceptible trend among the formulation prototypes tested by CE-SDS or filler variants by desialylated iCIEF when compared to the control. A slight reduction in 2 μm and 5 μm particulates was observed starting with at least 0.01% PS80 due to freeze-thaw stress compared to 0% PS80.

[199] The results of this study demonstrated that no significant difference was observed between samples containing 0.01 - 0.05% PS-80 after exposure to agitation or freeze / thaw stress. These studies did not discriminate between the various concentrations of PS80; however, 0.02% (weight / volume) of PS80 was selected to ensure product quality and minimize the risk of stability, maintaining safety in support of the emerging supply chain. Study of Variation in Sucrose Concentration

[200] To ensure that an injection solution of the ActRIIa-hFc fusion protein from SEQ ID NO: 32 has adequate tonicity for parenteral administration (260 - 380 mOsm / kg), a range of sucrose concentrations described in Table 20 was prepared in 10 mM citrate at a pH of 5.8 and the osmolality was measured. Based on these preparations, 8-10% (weight / volume) was determined to be appropriate for optimization studies. Petition 870250077590, dated 01 / 09 / 2025, pages 574 / 595 96 / 109 formulation described below. Table 20: Osmolality with varying sucrose content in the platform composition Sucrose (%) (weight / volume) Osmolality (mOsm / kg) 2 103 4 169 6 239 8 320 10 388 12 489 14 560

[201] Formulations comprising 50 mg / mL of the fusion protein ActRIIa-hFc from SEQ ID NO: 32, 8% to 10% (w / v) sucrose in 10 mM citrate at a pH of 5.8 with 0.02% (w / v) PS80 were prepared and evaluated for purity by PH-SEC to monitor their physical stability under different conditions. For the thermal stability arm of the study, the formulations were stabilized at 5 °C and 25 °C for up to 24 months and at 45 °C for up to 3 months. Stability data from PH-SEC analysis are provided in Table 21. Table 21: Study of Sucrose Concentration Variation: Effect of Sucrose Concentration in % Monomer by PH-SEC on Stability Sucrose (%) Time Point (months, M); Monomer (%) Initial 5 °C 25 °C 45 °C 12M 24M 6M 12M 1M 3M 8 99.4 99.3 99.1 99.1 98.1 98.2 92.3 9 99.4 99.3 99.0 98.7 98.1 98.3 92.4 10 99.4 99.3 99.1 98.6 98.1 98.4 92.5

[202] For the shaking arm of the study, formulations were filled into 3 mL Type I glass vials and shaken at 250 RPM on a plate shaker for up to 7 days. Shaking data by PH-SEC analysis are provided in Table 22. Table 22: Study of Sucrose Concentration Variation: Effect of Sucrose Concentration in % Monomer by PH-SEC on Agitation Petition 870250077590, dated 01 / 09 / 2025, pages 575 / 595 97 / 109 Sucrose (%) Time Point (days, D); Monomer (%) Initial 1D 3D 7D 8 98.6 98.7 98.6 98.8 9 98.5 98.7 98.7 98.7 10 98.6 98.6 98.7 98.8

[203] For the photostability arm of the study, another set of vials was filled with 1 mL of each formulation and divided into two sets for incubation in a photostability chamber: one exposed to visible light at 25 kLux for 48 hours (1.2 mL lux h) and the other exposed to 20 W / m2 of UV light for 10 hours (200 Wh / m2). For each formulation, two control vials were also prepared: one protected from light by aluminum foil in the photostability chamber and the other kept at 5 °C in a refrigerator. The photostability data by PH-SEC analysis are provided in Table 23. Table 23: Study of Variation in Sucrose Concentration: Effect of Sucrose Concentration in % Monomer by PH-SEC in Photostability Sucrose (%) Monomer (%) Visible Light (1.2 mLuxh) UV Light (200 Wh / m2) Control at 5 °C Control Dark Exposed to Light Difference Control Dark Exposed to Light Difference 8 99.4 83.8 -15.6 99.5 90.1 -9.4 99.4 9 99.5 82.7 -16.8 99.4 90.3 -9.1 99.4 10 99.4 83.3 -16.1 99.3 90.5 -8.8 99.4

[204] There were no significant differences in stability profiles between the different sucrose levels, with each formulation showing 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. There was no perceptible trend in the purity of any of the formulations after 7 days of agitation, with all formulations retaining >98% monomer. In the photostability arm of the study, each formulation decreased in % monomer by approximately 16 to 17% after exposure to visible light and approximately 9 to 10% after exposure to UV light, highlighting a possible degradation pathway.

[205] In conclusion, sucrose concentrations between 8% and 10% Petition 870250077590, dated 01 / 09 / 2025, pages 576 / 595 98 / 109 (weight / volume) did not have a significant impact on the long-term stability profile of the ActRIIa-hFc fusion protein from 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 from SEQ ID NO: 32 is prone to light instability, a complex mechanism to delineate. Thus, a lyophilized formulation was developed that reduces the potential for chemical instability due to light. A sucrose concentration of 8% (weight / volume) was considered sufficient for the development of the lyophilized formulation. Protein Concentration Variation Study

[206] A protein concentration variation stability study was conducted to evaluate the effect of increasing protein concentration on the long-term stability profile. In this study, the ActRIIahFc fusion protein from SEQ ID NO: 32 was formulated at concentrations ranging from 50 to 100 mg / mL in 10 mM citrate, 8% (w / v) sucrose, and 0.02% (w / v) PS80. These formulations were packaged in Type I glass vials and kept stable for 24 months at 5 °C and 3 months at 45 °C. The purity of the formulations was measured by PH-SEC analysis throughout the study to monitor their physical stability and is summarized in Table 24. Table 24: Study of Protein Concentration Variation from SEC Data Protein Concentration (mg / mL) Time Point (months, M); Monomer (%) Initial 5 °C 45 °C 6M 12M 24M 1M 2M 3M 50 99.6 99.4 99.3 99.0 97.0 94.5 91.4 75 99.5 99.3 99.1 99.1 96.3 93.4 91.1 100 99.5 99.1 99.0 98.6 96.0 91.5 87.7

[207] There was no significant difference in stability profiles at 5 °C between the different protein concentrations, where all formulations showed 99% monomer after 24 months at 5 °C. The 100 mg / mL concentration, however, showed slightly greater degradation than the lower concentrations, which showed 88% monomer after 3 months. Petition 870250077590, dated 01 / 09 / 2025, pages 577 / 595 99 / 109 under stress conditions (45 °C), while the lowest concentrations retained 91% of monomer.

[208] The results of this study demonstrated that SEQ ID NO: 32 can be concentrated up to 75 mg / mL without any significant impact on long-term stability and these initial physical stability data suggested that a concentration as high as 100 mg / mL may also have sufficient long-term stability based on the 5°C data. Formulation Composition

[209] Based on the formulation development work summarized above and the potential for charge instability during long-term storage under recommended storage conditions as a ready-to-use solution, a lyophilized formulation was developed with the optimized composition. The pre- and post-lyophilization target composition of the ActRIIa-hFc fusion protein from SEQ ID NO: 32 is described below. Table 25: Proposed Formulation Formulation Component: Lyophilized Solution; Protein Concentration: 50 mg / mL; Buffer System and pH: 10 mM Sodium Citrate, pH 5.8; Stabilizer / Bulking Agent: 8% (weight / volume) Sucrose; Surfactant: 0.02% (weight / volume) Polysorbate 80

[210] The composition was supplied in 2 dosages for vials and, when reconstituted with the defined amount of Sterile Water for Injection (SWFI), each composition contains 50 mg / mL of ActRIIa binder trap (active pharmaceutical ingredient) and the following excipients: 10 mM citrate, 8% (weight / volume) sucrose and 0.02% (weight / volume) polysorbate 80 at a pH of 5.8.

[211] The components present in the composition of lyophilized ActRIIa-hFc fusion protein are as follows: Table 26 Petition 870250077590, dated 01 / 09 / 2025, pages 578 / 595 100 / 109 Freeze-dried cake (mg / vial) Percentage by weight Component Function 45 mg / vial 60 mg / vial Fusion protein ActRIIa-hFc Active ingredient 55 72.5 37.6% Citric acid monohydrate Buffer 0.48 0.64 0.32% Trisodium citrate dihydrate Buffer 2.56 3.37 1.75% Polysorbate 80 Surfactant 0.22 0.29 0.15% Sucrose Tonicity adjustment 88 116 60.2% Total 146.26 mg 192.8 mg

[212] For administration, the lyophilisate in the 45 mg vial is reconstituted with 1.0 mL of Sterile Water for Injection (sWFI), and the lyophilisate in the 60 mg vial is reconstituted with 1.3 mL of sWFI. Both result in an ActRIIa ligand trap solution of at least 50 mg / mL. pH Robustness of the Lyophilized Formulation

[213] Studies were conducted to evaluate the robustness of critical components in the formulation. This initial study was conducted to evaluate the effect of pH on the stability of lyophilized formulations. Formulations ranging in pH from 5.3 to 6.3 were prepared in a 10 mM citrate buffer formulation at a pH of 5.8 with 8% (w / v) sucrose and 0.02% (w / v) polysorbate 80 at 50 mg / mL protein. These formulations (2 mL) were filled into 5 mL Type I glass vials and lyophilized using an initial phase lyophilization cycle and stabilized at 25 °C, 45 °C, and 60 °C. The purity of the formulations was measured by PH-SEC throughout the study to monitor their physical stability and summarized in Table 27. Table 27: SEC Data for pH Robustness of Product Formulation pH Point in Time (months, M); Monomer (%) Pre-Lio Initial 25 °C 45 °C 3 6 9 1 3 6 5.3 99.6 99.6 99.4 99.3 99.3 98.9 98.8 98.1 5.6 99.6 99.6 99.5 99.3 99.2 98.5 99.0 98.1 5.8 99.6 99.6 99.5 99.3 99.3 98.9 99.0 98.2 Petition 870250077590, dated 01 / 09 / 2025, pages 579 / 595 101 / 109 pH Point in Time (months, M); Monomer (%) Pre-Lio Initial 25 °C 45 °C 3 6 9 1 3 6 6.0 99.6 99.6 99.6 99.5 99.3 99.2 99.0 98.4 6.3 99.6 99.5 99.5 99.2 99.3 99.1 99.0 98.3

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

[215] The results of this study did not demonstrate a significant effect on the biophysical stability of the ActRIIa-hFc fusion protein of SEQ ID NO: 32 within a pH range of 5.3 - 6.3 of the formulation compositions under lyophilized conditions. Furthermore, previous development studies showed that variation in PS80 had no impact on biophysical stability in solution form. Therefore, pH and PS80 were eliminated as parameters in formulation robustness studies under lyophilized conditions. Formulation Robustness Study

[216] The formulation robustness study was conducted to determine the robustness of the formulation by varying the protein and sucrose concentration relative to their target levels. These two components were selected for evaluation as they demonstrated to have the most significant effect on determining the stability of the ActRIIa-hFc fusion protein of SEQ ID NO: 32 as a lyophilized product. In addition, this laboratory-scale study took advantage of the primary packaging and lyophilization cycle parameters for manufacturing.

[217] Formulations varying in protein concentration from 45 (Lo) to 55 mg / mL (Hi) and sucrose concentration from 7 (Lo) to 9% (Hi) (w / volume) were prepared in 10 mM citrate buffer with 0.02% (w / volume) polysorbate 80, as described in Table 28. 60 mg / Vial, containing 1.45 mL of each formulation, were filled into 2R Type I glass vials and lyophilized using the commercial directed lyophilization cycle. Table 28: Summary of Formulations for Robustness Study of Petition 870250077590, dated 01 / 09 / 2025, pages 580 / 595 102 / 109 Formulations Protein Concentration Level (mg / mL) Sucrose (%) Low-Low 45 7 Low-High 45 9 High-Low 55 7 High-High 55 9 Target 50 8

[218] All formulation prototypes, such as lyophilized cakes, were placed under stability conditions of 5 °C, 25 °C and 40 °C for up to 18 months, 6 months and 3 months, respectively. The prototypes were analyzed for purity by PH-SEC, fragmentation by CE-SDS NR, charge variants by desialylated iCIEF, particulates by HIAC, moisture content by pH Karl-Fischer and reconstitution time and summarized in Tables 29 to 39. Table 29: Formulation Robustness: Reconstitution Time Level Point in Time (months, M): Reconstitution Time (minutes:seconds) Initial 5 °C 25 °C 40 °C 6M 18M 1M 3M 6M 1M 3M LowLow 2:09 1:44 2:07 1:46 1:58 1:54 1:29 2:01 LowHigh 2:25 1:48 2:14 2:03 2:21 1:53 1:41 2:00 HighLow 2:23 1:54 2:09 2:13 2:28 2:09 1:58 2:29 HighHigh 2:40 1:46 2:23 2:05 2:32 2:09 2:03 2:37 Target 1:53 1:38 2:16 1:44 1:53 1:43 2:06 2:12 Table 30: Formulation Robustness: HMW by PH-SEC Point in Time Level (months, M); HMW (%) Initial 5 °C 25 °C 40 °C 3M 6M 12M 18M 1M 3M 6M 1M 3M LowLow 0.5 0.6 0.6 0.6 0.7 0.6 0.7 0.8 0.9 1.2 LowHigh 0.5 0.5 0.6 0.5 0.7 0.6 0.6 0.7 0.7 0.9 HighLow 0.6 0.6 0.7 0.7 0.8 0.7 0.8 0.9 1.1 1.6 HighHigh 0.5 0.6 0.6 0.5 0.7 0.6 0.6 0.7 0.8 1.1 Target 0.5 0.5 0.6 0.5 0.7 0.6 0.6 0.7 0.8 1.0 Petition 870250077590, dated 01 / 09 / 2025, pages 581 / 595 103 / 109 Table 31: Formulation Robustness: Monomer by PH-SEC Point in Time Level (months, M); Monomer (%) Initial 5 °C 25 °C 40 °C 3M 6M 12M 18M 1M 3M 6M 1M 3M LowLow 97.9 97.5 97.6 97.5 97.9 97.1 97.0 97.4 97.0 96.4 LowHigh 98.2 97.3 97.7 98.1 97.9 97.2 97.2 97.5 97.2 96.7 HighLow 98.1 97.3 97.5 96.7 97.7 97.1 97.0 97.3 96.7 95.9 HighHigh 98.1 97.3 97.6 96.7 97.7 97.3 97.1 97.5 97.1 96.3 Target 98.1 97.3 97.6 96.8 97.8 97.5 97.1 97.5 97.2 96.6 Table 32: Formulation Robustness: LMW by PH-SEC Point in Time Level (months, M); LMW (%) Initial 5 °C 25 °C 40 °C 3M 6M 12M 18M 1M 3M 6M 1M 3M LowLow 1.6 2.0 1.8 2.0 1.4 2.3 2.4 1.8 2.1 2.4 LowHigh 1.3 2.2 1.8 1.3 1.5 2.2 2.2 1.8 2.0 2.4 HighLow 1.4 2.1 1.8 2.6 1.4 2.2 2.2 1.8 2.2 2.5 HighHigh 1.4 2.2 1.8 2.8 1.6 2.1 2.3 1.8 2.0 2.6 Target 1.4 2.2 1.9 2.7 1.5 2.0 2.3 1.8 2.0 2.4 Table 33: Formulation Robustness: Lower Peak and Upper Peak by CE-SDS NR Point in Time Level (months, M); Lowest Peak (%) Point in Time (months, M); Peak (%) Initial 5 °C 25 °C 40 °C Initial 5 °C 25 °C 40 °C 18M 3M 1M 3M 18M 3M 1M 3M LowLow 1.5 1.4 1.0 1.9 1.6 98.5 98.6 99.0 98.1 98.4 LowHigh 2.0 1.3 1.8 2.1 1.2 98.0 98.8 98.2 97.9 98.8 HighLow 1.9 1.3 1.9 1.6 2.3 98.1 98.8 98.1 98.4 97.7 High-High 2.1 1.2 1.5 2.1 2.3 97.9 98.8 98.5 97.9 97.7 Target 2.1 1.2 1.9 1.9 2.6 97.9 98.7 98.1 98.1 97.4 Table 34: Formulation Robustness: Moisture per KF Petition 870250077590, dated 01 / 09 / 2025, pages 582 / 595 104 / 109 Level Point in Time (months, M); Humidity (%) w / w Initial 5 °C 25 °C 40 °C 3M 6M 12M 18M 3M 6M 3M LowLow 0.9 0.6 0.8 0.9 0.8 0.8 0.9 0.9 LowHigh 0.9 0.8 0.9 0.9 0.8 0.9 0.8 1.0 HighLow 0.7 0.5 0.7 0.9 0.7 0.7 0.8 0.9 HighHigh 0.5 0.7 0.8 0.9 0.9 0.8 0.9 0.9 Target 0.8 0.8 0.8 0.9 0.8 0.8 0.9 0.9 Table 35: Formulation Robustness: Acid Species by iCIEF Desialylated Point in Time Level (months, M); Acid Species (%) Initial 5 °C 25 °C 40 °C 3M 6M 12M 18M 1M 3M 6M 1M 3M Low-Low 22.5 20.0 29.1 25.2 21.2 22.6 22.5 23.5 21.3 19.7 Low-High 21.9 22.8 26.8 24.2 20.6 22.0 22.2 24.7 22.1 19.6 High-Low 23.6 21.8 26.9 26.3 21.5 25.4 23.9 28.2 26.2 19.9 High-High 24.5 22.7 27.1 27.1 21.6 22.4 36.7 27.7 24.0 20.7 Target 26.6 22.3 27.0 23.2 20.6 21.8 20.1 27.4 24.2 20.4 Table 36: Formulation Robustness: Total Major Species per iCIEF Desialilado Point in Time Level (months, M); Total of Main Species (%) Initial 5 °C 25 °C 40 °C 3M 6M 12M 18M 1M 3M 6M 1M 3M Low-Low 63.2 65.1 57.1 60.4 69.4 62.6 62.2 66.3 62.7 63.5 Low-High 63.9 62.3 59.0 61.5 69.9 62.9 62.7 60.6 62.1 63.3 High-Low 61.7 63.0 58.3 56.8 68.7 59.2 61.2 57.4 57.8 61.8 High-High 61.6 62.2 59.0 59.2 69.6 63.0 51.2 57.6 60.5 62.1 Target 59.1 62.9 58.8 61.8 69.6 63.3 64.5 57.8 60.2 62.2 Table 37: Formulation Robustness: Basic Species by iCIEF De-diallylated Point in Time Level (months, M); Basic Species (%) Initial 5 °C 25 °C 40 °C 3M 6 M 12 M 18 M 1M 3M 6M 1M 3M Low-Low 14.3 15.0 13.8 14.5 9.4 14.8 15.3 10.2 16.0 17.9 Low-High 14.3 14.9 14.2 14.3 9.5 15.2 15.1 14.8 15.8 17.1 High-Low 14.7 15.3 14.7 16.9 9.7 15.4 15.0 14.4 16.0 18.3 High-High 13.9 15.1 14.0 13.7 8.8 14.6 12.0 14.7 15.5 17.2 Target 14.3 14.9 14.3 15.0 9.8 14.8 15.4 14.7 15.6 17.5 Table 38: Formulation Robustness: Particulate Matter Analysis >2 μm and >5 μm by HIAC Petition 870250077590, dated 01 / 09 / 2025, pages 583 / 595 105 / 109 Point-in-Time Level (months, M): > 2 μm Particulate Matter per mL Point-in-Time Level (months, M): > 5 μm Particulate Matter per mL Initial 5 °C 25 °C 40 °C Initial 5 °C 25 °C 40 °C 3M 6M 12M 1M 3M 6M 1M 3M 3M 6M 12M 1M 3M 6M 1M 3M LowLow 2,260 3,365 360 5,305 2,373 855 428 980 773 415 430 100 1,162 1,738 160 138 155 240 LowHigh 1,013 773 1,573 3,675 2,000 925 1,610 1,533 665 143 75 660 888 1,335 153 438 710 123 HighLow 1,205 2,073 513 3,095 1,448 913 915 1,818 1,408 350 775 38 883 610 233 230 420 425 HighHigh 1,058 828 283 3,968 1,010 1,938 638 588 2,245 205 260 40 1,142 370 768 155 150 365 Target 620 1,648 360 3,417 1,158 1,340 2,263 1,778 1,045 175 363 48 817 445 248 823 953 193 Table 39: Formulation Robustness: >10 μm and >25 μm Particulates Point-in-Time Level (months, M); > 10 μm Particulate matter per mL Point-in-Time Level (months, M); > 25 μm Particulates per mL Initial 5 °C 25 °C 40 °C Initial 5 °C 25 °C 40 °C 3M 6M 12M 1M 3M 6M 1M 3M 3M 6M 12M 1M 3M 6M 1M 3M LowLow 143 58 15 445 863 63 80 45 55 30 13 0 32 20 3 8 18 3 LowHigh 28 10 80 345 533 20 90 113 13 3 0 3 32 10 0 3 5 3 HighLow 65 245 3 353 168 43 50 105 98 3 0 0 25 0 0 3 5 5 HighHigh 30 53 3 433 105 393 28 30 55 0 3 0 30 0 218 0 3 0 Target 58 80 5 190 88 58 43 393 40 3 5 3 12 5 10 5 8 0

[219] There was no perceptible trend in the reconstitution time, pH, or moisture content of the prototypes selected in this study under any of the conditions tested. There was no significant difference in the stability profiles of the prototype formulations for HMW, monomer, or LMW content for storage temperatures of 5 °C and 25 °C, and this is aligned with the fragmentation pattern observed by unreduced CE-SDS. There was a slight differentiation between the formulations at 45 °C storage, with the high protein concentration (55 mg / mL) and low sucrose concentration (7%) formulation, High-Low, having slightly more HMW species (~0.5%) than the others on average, as shown in Figure 12.

[220] There was no significant difference in the stability profiles of each formulation for acidic species, major species, or basic species content. Petition 870250077590, dated 01 / 09 / 2025, pages 584 / 595 106 / 109 at each of the storage temperatures. There was some expected variation in the various load species, where acidic species ranged between 20-30%, total major species between 55-70%, and basic species between 10-20%.

[221] There were no discernible trends in particulate matter / mL monitored by HIAC for any of the formulation prototypes in sizes >10 and >25 μm and were within the acceptance criteria specified by USP. <787> under all conditions, since the nominal filling volume for 60 mg / vial is 1.45 mL. In conclusion, this study demonstrates the robust nature of the formulation composition within the potential compositional variability, i.e., ± 5 mg / mL of the target protein concentration of 50 mg / mL and within ± 1% of the target sucrose concentration of 8% (weight / volume). Sequences Table 40: Sequences SEQ ID NO: 9 MGAAAKLAFA VFLISCSSGA ILGRSETQEC LFFNANWEKD RTNQTGVEPC YGDKDKRRHC FATWKNISGS IEIVKQGCWL DDINCYDRTD CVEKKDSPEV YFCCCEGNMC NEKFSYFPEM EVTQPTSNPV TPKPPYYNIL LYSLVPLMLI AGIVICAFWV YRHHKMAYPP VLVPTQDPGP PPPSPLLGLK PLQLLEVKAR GRFGCVWKAQ LLNEYVAVKI FPIQDKQSWQ NEYEVYSLPG MKHENILQFI GAEKRGTSVD VDLWLITAFH EKGSLSDFLK ANVVSWNELC HIAETMARGL AYLHEDIPGL KDGHKPAISH RDIKSKNVLL KNNLTACIAD FGLALKFEAG KSAGDTHGQV GTRRYMAPEV LEGAINFQRD AFLRIDMYAM GLVLWELASR CTAADGPVDE YMLPFEEEIG QHPSLEDMQE VVVHKKKRPV LRDYWQKHAG MAMLCETIEE CWDHDAEARL SAGCVGERIT QMQRLTNIIT TEDIVTVVTM VTNVDFPPKE SSL SEQ ID NO: 10 ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVK QGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNP VTPKPP SEQ ID NO: 11 ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVK QGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM SEQ ID NO: 12 ATGGGAGCTG CTGCAAAGTT GGCGTTTGCC GTCIIICIIA TCTCCTGTTC TTCAGGTGCT ATACTTGGTA GATCAGAAAC TCAGGAGTGT CIIIICIIIA ATGCTAATTG GGAAAAAGAC AGAACCAATC AAACTGGTGTTGAACCGTGT TATGGTGACA AAGATAAACG GCGGCATTGT TTTGCTACCT GGAAGAATAT TTCTGGTTCC ATTGAAATAG TGAAACAAGG TTGTTGGCTG GATGATATCA Petition 870250077590, of 01 / 09 / 2025, p. 585 / 595 107 / 109 ACTGCTATGA CAGGACTGAT TGTGTAGAAA AAAAAGACAG CCCTGAAGTA TAIIIIIIGTT GCTGTGAGGG CAATATGTGT AATGAAAAGT TTTCTTATTT TCCGGAGATG GAAGTCACAC AGCCCACTTC AAATCCAGTT ACACCTAAGC CACCCTATTA CACCCTTATCTTTCTTTCTCATTC TATGTTAATT GCGGGGATTG TCATTTGTGC AI IIIGGGTG TACAGGCATC ACAAGATGGC CTACCCTCCT GTACTTGTTC CAACTCAAGA CCCAGGACCA CCCCCACCTT CTCCATTACT AGGTTTGAAA CCACTGCAGT TATTAGAAGT GAAAGCAAGG GGAAGATTTGTTG TTGCTTAACG AATATGTGGC TGTCAAAATA TTTCCAATAC AGGACAAACA GTCATGGCAA AATGAATACG AAGTCTACAG TTTGCCTGGA ATGAAGCATG AGAACATATT ACAGTTCATT GGTGCAGAAA AACGAGGCAC CAGTGTTGAT GTGGATCII I GGACACTGATCGATTGTTGTT CACTATCAGA CIIICIIAAG GCTAATGTGG TCTCTTGGAA TGAACTGTGT CATATTGCAG AAACCATGGC TAGAGGATTG GCATATTTAC ATGAGGATAT ACCTGGCCTA AAAGATGGCC ACAAACCTGC CATATCTCAC AGGGACATCA AAAGTAAACCTAACCATTAGGACCATTAG TGAACTAATCATTA CATTGCTGAC TTTGGGTTGG CCTTAAAATT TGAGGCTGGC AAGTCTGCAG GCGATACCCA TGGACAGGTT GGTACCCGGA GGTACATGGC TCCAGAGGTA TTAGAGGGTG CTATAAACTT CCAAAGGGAT GCAI IIIIGAGGATAGATAT GTATGCCATG GGATTAGTCC TATGGGAACT GGCTTCTCGC TGTACTGCTG CAGATGGACC TGTAGATGAA TACATGTTGC CATTTGAGGA GGAAATTGGC CAGCATCCAT CTCTTGAAGA CATGCAGGAA GTTGcagaTTaGTtagttagaggacca acatgctgga ATGGCAATGC TCTGTGAAAC CATTGAAGAA TGTTGGGATC ACGACGCAGA AGCCAGGTTA TCAGCTGGAT GTGTAGGTGA AAGAATTACC CAGATGCAGA GACTAACAAA TATTATTACC ACAGAGGACA TTGTGAACAGT TCCGACAACAACCATG GTTCGACAACAAATG TCTAGTCTA SEQ ID NO:13 ATACTTGGTA GATCAGAAAC TCAGGAGTGT CIIIICIIIA ATGCTAATTG GGAAAAAGAC AGAACCAATC AAACTGGTGT TGAACCGTGT TATGGTGACA AAGATAAACG GCGGCATTGT TTTGAGCTAGTT T GATGACTAGAATAT TTGTTGGCTG GATGATTCA ACTGCTATGA CAGGACTGAT TGTGTAGAAA AAAAAGACAG CCCTGAAGTA TAIIIIIGTT GCTGTGAGGG CAATATGTGT AATGAAAGT TTTCGTATTT TCCGGAGATG GAAGTCACAC AGCCCACTAC2 ACAACTCCAG ILGRSETQECLFFNWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVK QGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPTGGGTHTCPCPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLPG SEQ ID NO: 41 ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKRRHCFATWKNISGSIEIVK QGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNP VTPKPPTGGGGTHTCPPCPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPG Petition 870250077590, de 01 / 09 / 2025, pág. 586 / 595 108 / 109 SEQ ID NO: 19 GGG SEQ ID NO: 20 GGGG SEQ ID NO: 21 TGGGG SEQ ID NO: 22 SGGGG SEQ ID NO: 23 TGGG SEQ ID NO: 24 SGGG SEQ ID NO: 25 GGGGS SEQ ID NO: 33 MKFLVNVALVFMVVYISYIYA SEQ ID NO: 34 MDAMKRGLCCVLLLCGAVFVSP SEQ ID NO: 35 MGAAAKLAFAVFLISCSSGA SEQ ID NO: 36 MDAMKRGLCCVLLLCGAVFVSPGAAILGRSETQECLFFNANWEKDRTNQTGVEP CYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCC CEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPTGGGTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 37 ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAG TCTTCGTTTCGCCCGGCG CCGCTATACTTGGTAGATCAGAAACTCAGGAGTGT Cllllll lAATGCTAATTGGGAAAAAGACAGAACCAATCAAACTGGTGTTGAA CCGTGTTATGGTGACAAAGATAAACGGCGGCATTGllllGCTACCTGGAAGAA TAlllClGGTTCCATTGAATAGTGAAACAAGGTTGTTGGCTGGATGATATCAA CTGCTATGACAGGACTGATTGTGTAGAAAAAAAAGACAGCCCTGAAGTATATTTCTGTTGCTGTGAGGGCAATATGTGTAATGAAAAGllllCllAllllCCGGAG ATGGAAGTCACACAGCCCACTTCAAATCCAGTTACACCTAAGCCACCCACCGG TGGTGGAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGA CCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG ACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGG TCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAA GCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACC GTCCTGCACCAGGACTGG CTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCA ACAAAGCCCTCCCAGTCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAG CCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCA AGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATC GCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACG CCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTG GACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATG AGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA TGAGAATTC SEQ ID NO: 38 ILGRSETQE SEQ ID NO: 62 ILGRSETQECIFYNANWERDRTNRTGVESCYGDKDKRRHCFATWKNISGSIEIVKQ GCWLDDINCYDRTDCIEKKDSPEVYFCCCEGNMCNERFSYFPEMEVTQPTSNPV TPKPP SEQ ID NO: 65ILGRSETQECIYYNANWEKDKTNRSGIEPCYGDKDKRRHCFATWKNISGSIEIVKQ GCWLDDINCYDRNDCIEKKDSPEVFFCCCEGNMCNERFFYFPEMEVTQPTSNPV TPKPP Petição 870250077590, de 01 / 09 / 2025, pág. 587 / 595 109 / 109 SEQ ID NO: 66 ILGRSETQECIFYNANWERDRTNRTGVESCYGDKDKRRHCFATWKNISGSIEIVKQ GCWLDDINCYDRTDCIEKKDSPEVYFCCCEGNMCNERFSYFPEMEVTQPTSNPV TPKPP SEQ ID NO: 67 ILGRSETQECIYYNANWEKDKTNRSGIEPCYGDKDKRRHCFATWKNISGSIEIVKQ GCWLDDINCYDRNDCIEKKDSPEVFFCCCEGNMCNERFFYFPEMEVTQPTSNPV TPKP SEQ ID NO: 68 ILGRSETQECIFYNANWERDKTNRTGVELCYGDKDKRRHCFATWKNISGSIEIVKQ GCWLDDINCYDRTDCIEKKDSPEVYFCCCEGNMCNERFSYFPEMEVTQPTSNPV TPKP

[222] Preferred methods and materials are described in the present invention, although methods and materials similar or equivalent to those described in the present invention may be used in practice or in testing the methods and formulations presently disclosed. All publications, patent applications, patents and other references mentioned in this invention are incorporated by reference in their entirety. Petition 870250077590, dated 01 / 09 / 2025, pp. 588 / 595

Claims

1 / 6 CLAIMS 1. Pharmaceutical formulation, characterized in that it comprises a human ActRIIa fusion protein, a buffer, a surfactant and a stabilizer, wherein the ActRIIa fusion protein comprises the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO:

41.

2. Pharmaceutical formulation according to claim 1, characterized in that the buffer is selected from the group consisting of sodium citrate, succinate and histidine.

3. Pharmaceutical formulation, according to claim 1 or 2, characterized in that the buffer is present in an amount between 4 mM and 50 mM.

4. Pharmaceutical formulation, according to claim 1 or 2, characterized in that the buffer is present in an amount of about 10 mM.

5. Pharmaceutical formulation, according to claim 1 or 2, characterized in that the buffer is a citrate buffer at a pH of about 5.5 to about 6.

5.

6. Pharmaceutical formulation, according to claim 1 or 2, characterized in that the buffer is a succinate buffer at a pH of about 5.5 to about 6.

5.

7. Pharmaceutical formulation, according to claim 1 or 2, characterized in that the buffer is a histidine buffer at a pH of about 5.5 to about 6.

0.

8. Pharmaceutical formulation, according to claim 1 or 2, characterized in that the buffer is a citrate buffer at a pH of 5.

8.

9. Pharmaceutical formulation, according to claim 1 or 2, characterized in that the buffer is a succinate buffer at a pH of 5.

8. Petition 870250077590, dated 01 / 09 / 2025, pp. 589 / 595 2 / 6 10. Pharmaceutical formulation, according to claim 5 or 8, characterized in that the citrate buffer comprises citric acid monohydrate and trisodium citrate dihydrate.

11. Pharmaceutical formulation, according to any one of claims 1 to 10, characterized in that the surfactant is polysorbate 80 or polysorbate 20.

12. Pharmaceutical formulation, according to claim 11, characterized in that the surfactant is present in an amount of about 0.01 to about 0.05 weight / volume.

13. Pharmaceutical formulation, according to claim 11, characterized in that the surfactant is present in an amount of approximately 0.02% weight / volume.

14. Pharmaceutical formulation, according to any one of claims 1 to 13, characterized in that the stabilizer is sucrose.

15. Pharmaceutical formulation, according to claim 14, characterized in that sucrose is present in an amount of about 8% to about 10% weight / volume.

16. Pharmaceutical formulation, according to claim 14, characterized in that sucrose is present in an amount of approximately 8% weight / volume.

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

18. Pharmaceutical formulation, according to any one of claims 1 to 16, characterized in that the human fusion protein ActRIIa is present in an amount of approximately 50 mg / mL.

19. Pharmaceutical formulation, according to any of the claims 1 to 18, characterized by the fact that it does not contain a salt.

20. Pharmaceutical formulation, according to any one of claims 1 to 19, characterized in that the fusion protein ActRIIa is sotaterceptible.

21. Pharmaceutical formulation, according to any one of claims 1 to 19, characterized in that the ActRIIa fusion protein comprises the amino acid sequence of SEQ ID NO:

32.

22. Pharmaceutical formulation, according to any one of claims 1 to 19, characterized in that the ActRIIa fusion protein comprises the amino acid sequence of SEQ ID NO:

41.

23. Pharmaceutical formulation, according to any one of claims 1 to 19, characterized in that it comprises a first ActRIIa fusion protein comprising the amino acid sequence of SEQ ID NO: 32 and a second ActRIIa fusion protein comprising the amino acid sequence of SEQ ID NO:

41.

24. Lyophilized pharmaceutical formulation, characterized in that it is made by lyophilizing the formulation defined in any one of claims 1 to 23.

25. Lyophilized pharmaceutical formulation, according to claim 24, characterized in that the human fusion protein ActRIIa is provided in an amount of 45 mg / vial or 60 mg / vial.

26. Reconstituted pharmaceutical formulation, characterized in that it is made by reconstituting the lyophilized pharmaceutical formulation defined in claim 24.

27. Reconstituted pharmaceutical formulation, according to claim 26, characterized in that the lyophilized pharmaceutical formulation is reconstituted with sterile water for injection. Petition 870250077590, dated 01 / 09 / 2025, pp. 591 / 595 4 / 6 28. Reconstituted pharmaceutical formulation according to claim 26, characterized in that the lyophilized formulation is reconstituted with sterile water for injection to a final protein concentration of approximately 45 mg / mL to 55 mg / mL.

29. Reconstituted pharmaceutical formulation according to claim 28, characterized in that the lyophilized formulation is reconstituted with sterile water for injection to a final protein concentration of approximately 50 mg / mL.

30. Reconstituted pharmaceutical formulation according to claim 26, characterized in that the lyophilized formulation is reconstituted with approximately 0.5 mL to 1.8 mL of sterile water for injection.

31. Reconstituted pharmaceutical formulation, according to claim 26, characterized in that the lyophilized formulation is supplied in an amount of 45 mg / vial, and in that the formulation is reconstituted with between 0.65 mL and 0.75 mL of sterile water for injection.

32. Reconstituted pharmaceutical formulation, according to claim 26, characterized in that the lyophilized formulation is supplied in a quantity of 60 mg / vial, and in that the formulation is reconstituted with between 0.65 mL and 1.75 mL of sterile water for injection.

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

41.

34. Lyophilized pharmaceutical formulation, according to claim 33, Petition 870250077590, dated 01 / 09 / 2025, pp. 592 / 595 5 / 6, characterized in that the buffer comprises citrate.

35. Lyophilized pharmaceutical formulation, according to claim 33 or 34, characterized in that the stabilizer is sucrose.

36. Lyophilized pharmaceutical formulation, according to any one of claims 33 to 35, characterized in that the surfactant is polysorbate 20 or polysorbate 80.

37. Lyophilized pharmaceutical formulation, according to claim 36, characterized in that the surfactant is polysorbate 80.

38. Lyophilized pharmaceutical formulation, characterized in that it comprises a human ActRIIa fusion protein, citrate, polysorbate 80 and sucrose, wherein the ActRIIa fusion protein comprises the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO:

41.

39. Lyophilized pharmaceutical formulation, characterized in that it comprises 55.0 mg of a human ActRIIa fusion protein, 0.48 mg of citric acid monohydrate, 2.56 mg of trisodium citrate dihydrate, 0.22 mg of polysorbate 80 and 88.0 mg of sucrose; wherein the human ActRIIa fusion protein comprises the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO:

41.

40. Lyophilized pharmaceutical formulation, characterized in that it comprises 72.5 mg of a human ActRIIa fusion protein, 0.64 mg of citric acid monohydrate, 3.37 mg of trisodium citrate dihydrate, 0.29 mg of polysorbate 80 and 116.0 mg of sucrose; wherein the human ActRIIa fusion protein comprises the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO:

41.

41. Reconstituted pharmaceutical formulation, characterized in that it comprises 50 mg / mL of a human ActRIIa fusion protein, 10 mM of citrate, 0.2 mg / mL of polysorbate 80 and 80 mg / mL of sucrose at a pH of Petition 870250077590, dated 01 / 09 / 2025, page 593 / 595 6 / 6 5.8, wherein the human ActRIIa fusion protein comprises the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO:

41.

42. Pharmaceutical formulation, according to any one of claims 1 to 41, characterized in that it is administered via subcutaneous injection.

43. Pharmaceutical formulation, according to any one of claims 1 to 41, characterized in that it is contained in a glass vial or injection device.

44. Use of a pharmaceutical formulation defined in any one of claims 1 to 43, characterized in that it is for the manufacture of a dosage form for treating pulmonary arterial hypertension (PAH).

45. Use of a lyophilized pharmaceutical formulation defined in any one of claims 24, 25 and 33 to 40, characterized in that it is for the manufacture of a dosage form for treating pulmonary arterial hypertension (PAH), wherein the lyophilized pharmaceutical formulation is reconstituted to create a reconstituted formulation.

46. ​​Product, process, system, kit, means or use, characterized in that it comprises one or more elements described in the descriptive report, claims, drawings, sequence listing, or summary of this application, when applicable. Petition 870250077590, dated 01 / 09 / 2025, pp. 594 / 595

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