Formulations comprising ACTRIIA polypeptide variants

Abstract

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

Description

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

[0002] Reference to electronically submitted sequence lists This application contains a sequence list, which is submitted electronically in XML format and incorporated herein by reference in its entirety. The XML file, created on January 29, 2024, is named 1848179-0002-169-WO1_SL.XML and is 37,058 bytes in size. Technical Field

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

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

[0005] Physiological disorders (PH) can be classified based on their similarities in pathophysiological mechanisms, clinical manifestations, and treatment methods (Simonneau et al., JACC 54(1):S44-54 (2009)). The clinical classification of PH was first proposed in 1973, and the most recent updated clinical classification was approved by the World Health Organization (WHO) in 2008. According to the updated clinical classification of PH, PH is mainly divided into five categories: (1) pulmonary hypertension (PAH) characterized by PA wedge pressure ≤15 mm Hg; (2) PH caused by left heart disease (also known as pulmonary venous hypertension or congestive heart failure); (3) PH characterized by PA wedge pressure >15 mm Hg; (4) PH caused by lung disease and / or hypoxia; chronic thromboembolic PH; and (5) PH of unknown etiology or multifactorial etiology (Simonneau et al., JACC 54(1):S44-54 (2009); Hill et al., Respiratory Care 54(7):958-68 (2009)). PAH is further classified into idiopathic PAH (IPAH), a sporadic disease without a family history of PAH and no clear risk factors; hereditary PAH; PAH induced by drugs and toxins; PAH associated with connective tissue diseases, HIV infection, portal hypertension, congenital heart disease, schistosomiasis, and chronic hemolytic anemia; and neonatal persistent PH (Simonneau et al., JACC 54(1):S44-54 (2009)). Diagnosis of each type of PH requires a series of tests.

[0006] Generally, PH treatment depends on the cause or classification of PH. In cases where PH is caused by a known medication or medical condition, it is termed secondary PH, and its treatment typically targets the underlying disease. Treatment for pulmonary venous hypertension usually involves optimizing left ventricular function through the administration of diuretics, beta-blockers, and ACE inhibitors, or repairing or replacing the mitral or aortic valve. PAH therapy includes 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 PH, and they do not directly address the underlying vascular remodeling and vascular myositis observed in many PH patients.

[0007] Therefore, the objective of this disclosure is to provide pharmaceutical formulations comprising the ActRIIa fusion protein and corresponding methods for treating, preventing, or reducing the rate of progression and / or severity of PH, particularly for treating, preventing, or reducing the rate of progression and / or severity of one or more PH-related complications. Summary of the Invention

[0008] This document provides pharmaceutical formulations comprising a recombinant fusion protein and one or more pharmaceutical additives and / or excipients, wherein the recombinant fusion protein comprises an extracellular domain (ECD) of human activin receptor type IIA (ActRIIa) or a variant thereof linked to a constant domain of an immunoglobulin, such as the human IgG1 Fc domain, wherein the formulation is lyophilized.

[0009] In some embodiments, the ActRIIa protein comprises an amino acid sequence that begins at any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of SEQ ID NO: 9 and ends at any one of amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135 of SEQ ID NO: 9, having at least 70% of the amino acid sequence. (e.g., at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) amino acid sequence identity with the amino acid sequence of SEQ ID NO: 10. In some embodiments, the ActRIIa protein comprises an amino acid sequence having at least 70% (e.g., at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) amino acid sequence identity with the amino acid sequence of SEQ ID NO: 10. In some embodiments, the ActRIIa protein comprises an amino acid sequence having at least 70% (e.g., at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with the amino acid sequence of SEQ ID NO: 11. In some embodiments, the ActRIIa protein is a fusion protein comprising an ActRIIa extracellular domain and one or more protein domains that are heterologous to ActRIIa. In some embodiments, the ActRIIa protein is a fusion protein comprising the Fc domain of an immunoglobulin. In some embodiments, the Fc domain of the immunoglobulin is the Fc domain of IgG1 immunoglobulin. In some embodiments, the ActRIIa fusion protein further comprises a linker domain located between the ActRIIa protein domain and the one or more heterologous domains (e.g., the Fc immunoglobulin domain). In some embodiments, the connector structure domain is selected from: 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 having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the 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 shown in SEQ ID NO: 32, wherein the sequence lacks the C-terminal lysine residue of SEQ ID NO: 32. In some embodiments, the ActRIIa fusion protein variant lacking a C-terminal lysine residue comprises or is composed of the amino acid sequence of SEQ ID NO:41. In some embodiments, the ActRIIa fusion protein is part of a homodimeric protein complex. In some embodiments, the ActRIIa fusion protein is glycosylated. In some embodiments, the ActRIIa fusion protein has a glycosylation pattern obtainable by expression in Chinese hamster ovary cells.

[0010] The pharmaceutical formulations described herein comprise the ActRIIa fusion protein and one or more pharmaceutical additives and / or excipients. In some embodiments, the pharmaceutical formulations described herein comprise the human ActRIIa fusion protein or a variant thereof, buffers, surfactants, and stabilizers.

[0011] In some embodiments, one or more of the pharmaceutical additives and / or excipients are buffers. In some embodiments, the buffer is selected to be physiologically compatible and maintain a pH above 2. In some embodiments, the buffer is selected to be physiologically compatible and capable of maintaining the reconstituted lyophilized solution described herein at a pH above 2. In some embodiments, the buffer is selected to be physiologically compatible and maintain the pH of the pharmaceutical formulation at pH 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0. In some embodiments, the buffer is selected to be physiologically compatible and maintain the pH of the pharmaceutical preparation between 5 and 7 (i.e., 5.0-7.0). In some embodiments, the buffer is selected to be physiologically compatible and maintain the pH of the pharmaceutical preparation at pH 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0. In some embodiments, the buffer is selected to be physiologically compatible and maintain the pH of the pharmaceutical preparation between 5.5 and 6.5 (i.e., 5.5-6.5). In some embodiments, the buffer is selected to be physiologically compatible and maintain the pH of the pharmaceutical preparation at 5.8.

[0012] In some embodiments, the buffer comprises an organic acid, succinate, phosphate, acetate, citrate, citric acid, Tris, HEPES, an amino acid, or a mixture of amino acids. In some embodiments, the buffer is selected from sodium citrate, succinate, and histidine. In some embodiments, the buffer comprises citrate. In some embodiments where the buffer is a citrate, the citrate buffer comprises trisodium citrate dihydrate and citrate monohydrate. In some embodiments, the buffer comprises trisodium citrate dihydrate. In some embodiments, the buffer comprises citrate monohydrate. In some embodiments, the buffer comprises trisodium citrate dihydrate and citrate monohydrate. In some embodiments where the protein has a negative charge due to the presence of glycans, the buffer is not histidine.

[0013] In some embodiments, the buffer is present in an amount of 4 mM to 50 mM. In some embodiments, the buffer is present 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 the buffer.

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

[0015] In some embodiments, the buffer is a 10 mM citrate buffer that maintains the pH of the pharmaceutical preparation at about 5.5 to about 6.5. In some embodiments, the buffer is a 10 mM succinate buffer that maintains the pH of the pharmaceutical preparation at about 5.5 to about 6.5. In some embodiments, the buffer is a 10 mM histidine buffer that maintains the pH of the pharmaceutical preparation at about 5.5 to about 6.0. In some embodiments, the buffer is a 10 mM citrate buffer that maintains the pH of the pharmaceutical preparation at about 5.8. In some embodiments, the buffer is a 10 mM succinate buffer that maintains the pH of the pharmaceutical preparation at about 5.8.

[0016] In some embodiments, one or more of the pharmaceutical additives and / or excipients are surfactants. In some embodiments, the surfactant is selected from: sodium lauryl sulfate, sodium dioctyl sulfosuccinate and sodium dioctyl sulfonate, chenodeoxycholic acid, sodium N-lauroyl sarcosinate, lithium dodecyl sulfate, sodium 1-octane sulfonate, sodium cholate hydrate, sodium deoxycholate and sodium glycinate deoxycholate, benzalkonium chloride, benzyl chloride, hexadecylpyridine chloride monohydrate, hexadecyltrimethylammonium bromide, CHAPS, CHAPSO, SB3-10, SB3-12, digitalis saponins, Triton X-100, Triton X-114, TWEEN-20, TWEEN-80, lauromacrogol 400. The surfactant comprises: polyoxyethylene 40 stearate, polyoxyethylene hydrogenated castor oil 10, 40, 50 and 60, glyceryl monostearate, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80 and soybean lecithin. In some embodiments, the surfactant is polysorbate 80 or polysorbate 20. In some embodiments, the surfactant is polysorbate 80.

[0017] In some embodiments, the surfactant is present in an amount of 0.05-0.3 mg / mL. In some embodiments, the pharmaceutical formulation comprises at least 0.001, 0.002, 0.003, 0.004, 0.005, 0.01, 0.02, 0.03, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0% by weight / volume of surfactant. In some embodiments, the pharmaceutical formulation comprises 0.01-0.05% 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.

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

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

[0020] In some implementations, the pharmaceutical formulations described herein do not contain salt.

[0021] This article also describes lyophilized pharmaceutical formulations prepared by lyophilizing the liquid formulations described herein. This article also describes pharmaceutical formulations wherein the formulations are reconstituted from the lyophilized formulations.

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

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

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

[0025] In some 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 preparation 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 ActRIIa fusion protein provided in a 45 mg / vial amount, 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 ActRIIa fusion protein provided in a 45 mg / vial amount, and the formulation is reconstituted with 1.1 mL of sterile water for injection. In some embodiments, the lyophilized pharmaceutical formulation comprises ActRIIa fusion protein provided in a 60 mg / vial amount, and the formulation is reconstituted with 0.90 mL, 0.95 mL, 1.00 mL, 1.05 mL, 1.10 mL, 1.15 mL, 1.20 mL, 1.25 mL, 1.30 mL, 1.35 mL, 1.40 mL, 1.45 mL, or 1.50 mL of sterile water for injection. In some embodiments, the lyophilized pharmaceutical formulation comprises ActRIIa fusion protein provided in an amount of 60 mg / vial, and wherein the formulation is reconstituted with 1.30 mL of sterile water for injection.

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

[0027] In some 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 a C-terminal lysine (SEQ ID NO: 41), a buffer, a surfactant, and a stabilizer, wherein the buffer is selected to be physiologically compatible and maintains pH 5.8 when reconstituted with sterile water for injection. In some embodiments, the buffer comprises citrate. In some embodiments, the stabilizer is sucrose. In some embodiments, the surfactant is polysorbate 20 or polysorbate 80. In some embodiments, the surfactant is polysorbate 80. In some embodiments, the lyophilized pharmaceutical formulation comprises sotacicept, a buffer, a surfactant, and a stabilizer, wherein the buffer is selected to be physiologically compatible and maintains pH 5.8 when reconstituted with sterile water for injection.

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

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

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

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

[0032] In some embodiments, the pharmaceutical formulation described herein comprises a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 lacking a C-terminal lysine residue (SEQ ID NO: 41). In some embodiments, the pharmaceutical formulation described herein comprises a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 lacking a C-terminal lysine residue (SEQ ID NO: 41), wherein the mixture 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.

[0033] In some embodiments, the pharmaceutical formulations described herein comprise a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 lacking a C-terminal lysine residue (SEQ ID NO: 41), wherein the mixture comprises approximately 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, and 53%. SEQ ID NO:32 of 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.

[0034] In some embodiments, the pharmaceutical formulations described herein comprise a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 lacking a C-terminal lysine residue (SEQ ID NO: 41), wherein the mixture comprises 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%, and 53%. SEQ ID NO:41 of 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.

[0035] In some embodiments, the pharmaceutical formulations described herein comprise a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 lacking a C-terminal lysine residue (SEQ ID NO: 41), wherein the mixture comprises approximately 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, and 53%. The pharmaceutical preparations described herein contain SEQ ID NO: 41 at 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. In some embodiments, the pharmaceutical preparations described herein contain 100% by weight of SEQ ID NO: 32. In some embodiments, the pharmaceutical preparations described herein contain 100% by weight of SEQ ID NO: 41.

[0036] In some embodiments, the pharmaceutical preparation is administered parenterally (e.g., via intravenous infusion). In some embodiments, the pharmaceutical preparation is administered subcutaneously. In some embodiments, the preparation is contained in a glass vial or injection device.

[0037] This article also describes methods for treating pulmonary arterial hypertension (PAH), including administering the pharmaceutical preparations described herein to patients in need.

[0038] This article also describes the use of pharmaceutical formulations for the treatment of pulmonary arterial hypertension (PAH), including administering the pharmaceutical formulations described herein to patients in need.

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

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

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

[0042] Figure 1 This kit contains components including a lyophilized pharmaceutical preparation and an injection device, wherein the lyophilized pharmaceutical preparation contains the ActRIIa fusion protein. A vial (1) contains the lyophilized pharmaceutical preparation containing the ActRIIa fusion protein, a reconstituted sterile injection solution, or a sterile injection solution. A pre-filled syringe (2) containing the reconstituted solution is used to reconstitute the lyophilized pharmaceutical preparation containing the ActRIIa fusion protein from (1) into the sterile injection solution. A vial adapter (3) couples the vial (1) to the pre-filled syringe (2) by attaching the vial to one end and the pre-filled syringe to the opposite end. A syringe (4) and a needle (5) are provided for administering the sterile injection solution. A cotton swab (6) is provided for sterilizing the individual kit components.

[0043] Figure 2 This shows multiple sequence alignments of ActRIIa proteins from various vertebrates and human ActRIIa (SEQ ID NO: 62-68).

[0044] Figure 3A and 3B Purification of ActRIIa-hFc expressed in CHO cells. The protein was purified as a single, clear peak, as seen through a sieving column (top panel) and Coomassie-stained SDS-PAGE (bottom panel) (left lane: molecular weight standard; right lane: ActRIIa-hFc).

[0045] Figure 4A and 4B The binding of ActRIIa-hFc to activin (top) and GDF-11 (bottom) is shown, as via Biacore. TM Measured by a determination method.

[0046] Figure 5This shows the effect of pH and buffer on the percentage of monomers determined by HP-SEC after 6 months at 25°C.

[0047] Figure 6 After demonstrating stability at 25°C for 6 months, pH and buffer effects were assessed via Biacore. TM The effect of the measured binding activity.

[0048] Figure 7 The effects of buffer and pH on % HMW, monomer and % LMW of the product were shown after 3 months of stability.

[0049] Figure 8 The effects of pH and buffer on the % minor and % major peaks as determined by CE-SDS NR after 1 month of stability were shown.

[0050] Figure 9 The effects of pH and buffer on the percentage of acidic compounds, percentage of basic compounds, and percentage of total major compounds as determined by desialylated icIEF after 3 months of stability were shown.

[0051] Figure 10 This shows the effect of concentration on the percentage of monomers determined by HP-SEC.

[0052] Figure 11 This shows the effects of stability, buffer, and concentration on the percentage of monomers as determined by HP-SEC.

[0053] Figure 12 This shows the effects of stability, protein, and sucrose concentrations on purity as determined by HP-SEC. Detailed Implementation

[0054] definition The terms used in this specification generally have their ordinary meaning in the art, within the context of this disclosure and in the specific context in which they are used. Certain terms are discussed below or elsewhere in the specification to provide practitioners with additional guidance on the formulations and methods described in this disclosure and on how they are prepared and used. The scope or meaning of any use of a term will be apparent from the specific context in which it is used.

[0055] "About" and "approximately" should generally refer to the acceptable degree of error for the quantity of the measurement, given the nature or precision of the measurement. Typically, an exemplary degree of error is within 20 percent (%) of a given value or range of values, preferably within 10 percent, and more preferably within 5 percent.

[0056] Or, and particularly in biological systems, the terms “about” and “approximately” may mean a value within an order of magnitude of a given value, preferably within five times, and more preferably within two times. Unless otherwise specified, the numerical quantities provided herein are approximate, meaning that the terms “about” or “approximately” can be inferred when not explicitly stated.

[0057] The terms "an" and "a" include plural objects unless the context in which the term is used explicitly indicates otherwise. The terms "an" (or "a kind"), "an / kind or multiple / kinds," and "at least one / kind" are used interchangeably herein. Furthermore, "and / or," when used herein, should be construed as explicitly disclosing each of the two or more specified features or components, with or without the other. Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of: 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).

[0058] The numerical ranges disclosed herein include the numbers that define the range. For example, pH between 5.0 and 7.0 includes pH 5.0 and pH 7.0, as well as values ​​between 5.0 and 7.0.

[0059] The proteins disclosed herein may contain amino acid sequences that are not naturally occurring. Such variants necessarily have less than 100% sequence identity or similarity to the starting molecule. In some embodiments, the variant has an amino acid sequence that has at least 75% amino acid sequence identity or similarity to the starting (e.g., naturally occurring or wild-type) protein, more preferably at least 80%, more preferably at least 100%, more preferably at least 85%, more preferably at least 100%, more preferably at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), and most preferably at least 95% over the entire length of the variant molecule.

[0060] pharmaceutical preparations This document provides pharmaceutical formulations comprising a recombinant fusion protein comprising an extracellular domain (ECD) of human activin receptor type IIA (ActRIIa) protein or a derivative thereof linked to a constant domain of an immunoglobulin, such as a human IgG1 Fc domain, wherein the pharmaceutical formulation is lyophilized. In some aspects, this disclosure relates to pharmaceutical formulations comprising an extracellular domain (ECD) of human ActRIIa protein or a derivative thereof linked to a constant domain of an immunoglobulin, such as a human IgG1 Fc domain, wherein the pharmaceutical formulation is reconstituted from a lyophilized formulation into a sterile solution for injection.

[0061] In some aspects, this disclosure relates to lyophilized pharmaceutical formulations comprising an extracellular domain (ECD) of human ActRIIa protein or a derivative thereof linked to a constant domain of an immunoglobulin, such as the Fc domain of human IgG1, for reconstitution into a sterile solution for injection. During lyophilization, the liquid formulation is transformed from an aqueous phase to an amorphous solid phase, which is believed to protect the protein from chemical and / or conformational instability. Lyophilization is performed using techniques commonly used in the art, and the lyophilized formulations are optimized for stability, shelf life, and reduction in high molecular weight (HMW) groups and aggregate levels. (Tang et al., Pharm Res. 21:191-200, (2004) and Chang et al., Pharm Res. 13:243-9 (1996). The pharmaceutical formulations help stabilize the protein against stress during manufacturing, transport, and storage. Excipients and additives used in the lyophilized formulations are essential components of the formulation and therefore need to be safe and well-tolerated by patients. For protein drugs, the selection of excipients and additives is particularly important because they can affect drug efficacy and immunogenicity. Excipients and additives can also be used to reduce the viscosity of high-concentration protein formulations to enable their delivery and improve patient convenience. The formulation excipients and additives disclosed herein provide stability against these stresses. Commonly used excipients are known in the art and can be found in Powell et al., Compendium of Excipients fir Parenteral Formulations (1998), PDA J. Pharm. Sci. Technology, 52:238-311.

[0062] In another aspect, this document provides pharmaceutical formulations comprising the ActRIIa fusion protein. In some embodiments, the formulation is lyophilized. In further embodiments, the formulation is reconstituted from a lyophilized formulation. In some embodiments, this disclosure provides pharmaceutical formulations comprising the ActRIIa protein, wherein the pharmaceutical formulation is in lyophilized form in a vial. In some embodiments, the pharmaceutical formulation comprises 15 mg to 100 mg of the ActRIIa fusion protein. In some embodiments, the pharmaceutical formulation comprises 20 mg to 80 mg of the ActRIIa fusion protein. In some embodiments, the pharmaceutical formulation comprises 40 mg to 70 mg of the ActRIIa fusion protein. In some embodiments, the pharmaceutical formulation comprises about 15 mg, about 17.5 mg, about 20 mg, about 22.5 mg, about 25 mg, about 27.5 mg, about 30 mg, about 32.5 mg, about 35 mg, about 37.5 mg, about 40 mg, about 42.5 mg, about 45 mg, about 47.5 mg, about 50 mg, about 52.5 mg, about 55 mg, about 57.5 mg, about 60 mg, about 62.5 mg, about 65 mg, about 67.5 mg, about 70 mg, about 72.5 mg, about 75 mg, about 77.5 mg, about 80 mg, about 82.5 mg, about 85 mg, about 90 mg, about 92.5 mg, about 95 mg, about 97.5 mg, or about 100 mg of the ActRIIa fusion protein.

[0063] In some embodiments, the lyophilized pharmaceutical formulation containing the ActRIIa fusion protein is provided in vials as a lyophilized powder or cake. In a particular embodiment, the lyophilized pharmaceutical formulation comprises 45 mg / vial or 60 mg / vial of the ActRIIa fusion protein. In a more specific embodiment, each of the 45 mg / vial or 60 mg / vial is reconstituted with sterile water for injection, each containing a final concentration of 45-55 mg / mL of the reconstituted ActRIIa fusion protein. In a more specific embodiment, each of the 45 mg / vial or 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).

[0064] In some embodiments, the lyophilized pharmaceutical formulation comprises 45 mg / vial or 60 mg / vial of ActRIIa fusion protein and is reconstituted with sterile water for injection. In more specific embodiments, the lyophilized pharmaceutical formulations provided in the 45 mg / vial or 60 mg / vial are 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 reconstituted ActRIIa fusion protein. In more specific embodiments, the lyophilized pharmaceutical formulations provided in the 45 mg / vial or 60 mg / vial are 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 ActRIIa fusion protein. In some embodiments, the lyophilized pharmaceutical formulations provided in the 45 mg / vial or 60 mg / vial are reconstituted with sterile water for injection to a final concentration of approximately 50 mg / mL of the ActRIIa fusion protein. In some embodiments, the lyophilized pharmaceutical formulations provided in the 45 mg / vial or 60 mg / vial are reconstituted with sterile water for injection to a final concentration of 50 mg / mL of the ActRIIa fusion protein.

[0065] In some embodiments, the lyophilized pharmaceutical formulations provided in the 45 mg / vial or 60 mg / vial are reconstituted with 0.5 to 2 mL of sterile water for injection. In some embodiments, the lyophilized pharmaceutical formulation, each in the form of 45 mg / vial or 60 mg / vial, is dispensed in 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. Reconstitute with sterile water for injection (mL). In some embodiments, the lyophilized pharmaceutical formulations provided in the 45 mg / vial or the 60 mg / vial are dispensed in solutions of 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, etc. mL, 1.54 mL, 1.55 mL, 1.56 mL, 1.57 mL, 1.58 mL, 1.59 mL, 1.6mL, 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 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 (0.75 mL). In some embodiments, the lyophilized pharmaceutical formulation provided in the 45 mg / vial is reconstituted with 0.9 mL, 0.95 mL, 1 mL, or 1.1 mL of sterile water for injection. In some embodiments, the lyophilized pharmaceutical formulation provided in the 45 mg / vial is reconstituted with 1.1 mL of sterile water for injection. In some embodiments, the lyophilized pharmaceutical formulation provided in the 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 of sterile water for injection (0.75 mL). In some embodiments, the lyophilized pharmaceutical formulation provided in the 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 60 mg / vial is reconstituted with 1.3 mL of sterile water for injection.

[0066] In some embodiments, the lyophilized pharmaceutical formulations provided herein comprise an ActRIIa fusion protein and one or more pharmaceutical additives and / or excipients. In some embodiments, the one or more pharmaceutical additives and / or excipients comprise buffers, stabilizers, and surfactants. Buffers may be selected to maintain the pH of the formulation during treatment and upon reconstitution. Stabilizers may include freeze-drying and lyophilizing protectants, such as polyols, sugars, and polysaccharides, and may be selected to protect the formulation from freeze / thaw cycle stress and stabilize the formulation in the lyophilized state. Surfactants may be selected based on their ability to function as emulsifiers, wetting agents, solubilizers, and / or dispersants.

[0067] The formulations described herein contain buffers, surfactants, and sugars, which are described in more detail below.

[0068] Those skilled in the art will recognize that the concentrations of the excipients described herein are dependent within a particular formulation. For example, the concentration of a filler may decrease in one respect, for example, in the presence of a high protein concentration. Excipients and other additives are added to provide or improve manufacturability and / or the quality of the final product, such as the stability and delivery of pharmaceutical products (e.g., proteins). The formulations provided herein contain suitable excipients that improve stability and safety.

[0069] buffer Typically, the stability of pharmacologically active protein formulations is highest within a narrow pH range. This pH range of optimal stability needs to be identified early in the pre-formulation study phase. Several methods, such as accelerated stability studies and calorimetric screening studies, can be used in such attempts (Remmele RL Jr., et al., Biochemistry, 38(16): 5241-7(1999)). Once the formulation is finalized, the protein must be manufactured and maintained throughout its entire shelf life. Therefore, buffers are almost always used to control the pH of the formulation.

[0070] Several factors must be considered when selecting a buffer. First and foremost, the type of buffer and its concentration must be defined based on its pKa and the desired formulation pH. Equally important is ensuring that the buffer is compatible with the protein and other formulation excipients and does not catalyze any degradation reactions. A third important aspect to consider is the stinging and irritation that the buffer may induce upon administration. For drugs administered via subcutaneous (SC) or intramuscular (IM) routes, where the drug solution remains at the site for a relatively longer period, the potential for stinging and irritation is greater than for administration via IV routes, where the formulation is rapidly diluted into the bloodstream upon administration. For formulations administered via direct IV infusion, the total amount of buffer (and any other formulation components) needs to be monitored.

[0071] Buffers used in lyophilized formulations require additional consideration. For example, certain buffers, such as sodium phosphate, tend to crystallize from the amorphous phase of the protein during freezing, causing pH changes. In some embodiments, exemplary buffers for buffering pharmaceutical formulations as described herein include, but are not limited to, organic acids, succinates, phosphates, acetates, citrates, Tris, HEPES, and amino acids or mixtures of amino acids, including but not limited to aspartic acid, arginine, and glycine. In some embodiments, the buffer comprises trisodium citrate dihydrate. In some embodiments, the buffer comprises citrate monohydrate. In some embodiments, the buffer comprises citrate. In one embodiment, the buffer comprises trisodium citrate dihydrate and citrate monohydrate. In another embodiment, the buffer is trisodium citrate dihydrate and citrate monohydrate. In some embodiments where the protein is negatively charged due to the presence of glycans, the buffer is not histidine. In some embodiments, the buffer comprises citrate, succinate, or histidine.

[0072] In some embodiments, the amount of buffer in the lyophilized formulation is from 0.3 mg to 5 mg. In some 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 some embodiments, the amount of buffer in the lyophilized formulation is about 0.5 mg, about 1.0 mg, about 1.5 mg, about 2.0 mg, about 2.5 mg, about 3.0 mg, about 3.5 mg, about 4.0 mg, about 4.5 mg, or about 5.0 mg.

[0073] In some embodiments, the buffer comprises citrate monohydrate and trisodium citrate dihydrate, wherein the amount of citrate monohydrate is from 0.1 mg to 1.0 mg. In some embodiments, the amount of citrate 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 some embodiments, the amount of citrate 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 some embodiments, the amount of citrate monohydrate is 0.48 mg. In some embodiments, the amount of citrate monohydrate is 0.48 mg. In some embodiments, the amount of citrate monohydrate is 0.48 mg. In some implementations, the amount of citric acid monohydrate is 0.64 mg.

[0074] In some embodiments, the buffer comprises citrate monohydrate and trisodium citrate dihydrate, wherein the amount of trisodium citrate dihydrate is from 1.0 mg to 5.0 mg. In some 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 some 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 some embodiments, the amount of trisodium citrate dihydrate is 2.56 mg. In some embodiments, the amount of trisodium citrate dihydrate is 3.37 mg.

[0075] In one embodiment, the buffer present in the formulation is selected to be physiologically compatible and maintain the desired pH of the pharmaceutical formulation when reconstituted with sterile water for injection. In another embodiment, the pH of the solution is greater than 2. In yet another embodiment, the pH of the solution is between pH 2.0 and pH 12.0. For example, in various embodiments, the pH of the reconstituted solution is 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0. In some embodiments, when reconstituted in solution, the pH of the solution is between pH 5 and pH 7. In some embodiments, the pH of the reconstituted solution is 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0. In one embodiment, the pH of the reconstituted solution is... < 6.5. In one embodiment, the pH of the reconstituted solution is 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.

[0076] 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.

[0077] 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.

[0078] pH buffering compounds can be present in any amount suitable for maintaining the pH of the formulation at a predetermined level. Crystallization and pH changes can be avoided when using appropriately low levels of buffers. In one embodiment, the concentration of the buffer is from 0.1 mM to 500 mM (1 M). For example, the buffer is contemplated to be at least 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 500 mM. In some embodiments, the buffer is 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 500 mM. In one embodiment, the concentration of the buffer is from 0.1 mM to 20 mM. In some embodiments, the buffer is 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mM. In some embodiments, the buffer is present in an amount of 10 mM.

[0079] In some embodiments, the buffer is a 10 mM citrate buffer that maintains the pH of the pharmaceutical preparation at about 5.5 to about 6.5. In some embodiments, the buffer is a 10 mM succinate buffer that maintains the pH of the pharmaceutical preparation at about 5.5 to about 6.5. In some embodiments, the buffer is a 10 mM histidine buffer that maintains the pH of the pharmaceutical preparation at about 5.5 to about 6.0. In some embodiments, the buffer is a 10 mM citrate buffer that maintains the pH of the pharmaceutical preparation at about 5.8. In some embodiments, the buffer is a 10 mM succinate buffer that maintains the pH of the pharmaceutical preparation at about 5.8.

[0080] In some embodiments, the buffer comprises trisodium citrate dihydrate. In some embodiments, the buffer comprises citrate monohydrate. In some embodiments, the buffer comprises citrate salt. In some embodiments, the buffer comprises both trisodium citrate dihydrate and citrate monohydrate. In some embodiments, the protein is negatively charged due to the presence of glycans, and the buffer is not histidine. In some embodiments, the buffer 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 buffer.

[0081] stabilizer In some embodiments, the pharmaceutical formulations provided herein include stabilizers. These stabilizers can be classified according to their mechanisms of stabilizing proteins in response to various chemical and physical stresses. Some stabilizers are used to mitigate the effects of specific stresses or modulate specific susceptibility of a particular protein. Other stabilizers have a more general effect on the physical and covalent stability of proteins. In view of the teachings and guidance provided herein, those skilled in the art will recognize that the amount or range of stabilizers that may be included in any particular formulation to achieve the formulation of this disclosure has the potential to promote the retention and stability of the ActRIIa protein.

[0082] In some embodiments, a stabilizer (or combination of stabilizers) is incorporated into the formulation to prevent or reduce storage-induced aggregation and chemical degradation. A turbid or unclear solution upon reconstitution typically indicates that the protein has precipitated or at least aggregated. Stabilizers are capable of preventing aggregation or chemical degradation (e.g., autolysis, deamidation, oxidation, etc.). Some stabilizers can also function as anticoagulants when the formulation is administered to a patient. In some embodiments, the pharmaceutical formulations provided herein include stabilizers, including but not limited to sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, glucosamine, fructose, mannitol, sorbitol, polyhydroxy compounds, including polysaccharides such as dextran, starch, hydroxyethyl starch, cyclodextrin, N-methylpyrrolidone, cellulose, and hyaluronic acid (Carpenter et al., Develop. Biol. Standard 74:225, (1991)). In one embodiment of this disclosure, sucrose is used as a stabilizer.

[0083] In some embodiments, the lyophilized formulations described herein contain a stabilizer in an amount of 50 mg to 150 mg. In some embodiments, the lyophilized formulations described herein contain a stabilizer in an amount of 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, or 150 mg. In some embodiments, the lyophilized formulations described herein contain a stabilizer in an amount of about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, or about 150 mg.

[0084] In some embodiments, the stabilizer is 88.0 mg of sucrose. In some embodiments, the stabilizer is 116.0 mg of sucrose.

[0085] In some 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 some embodiments of this disclosure, the stabilizer is incorporated at a concentration of about 0.005, 0.01, 0.02, 0.03, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% by weight / volume.

[0086] In some embodiments, the formulation comprises about 8% to about 10% sucrose by weight / volume. In some embodiments, the formulation comprises 8-10% sucrose by weight / volume. In some embodiments, the formulation comprises 8%, 9%, or 10% sucrose by weight / volume. In some embodiments, the formulation comprises 8% sucrose by weight / volume.

[0087] surfactants In some embodiments, the pharmaceutical formulations provided herein may additionally include surfactants. Surfactants are commonly used in protein formulations to prevent surface-induced degradation. A surfactant is an amphiphilic molecule that has the ability to override the protein at interfacial sites (and / or promote the proper refolding of protein molecules that undergo structural changes). The hydrophobic portion of the surfactant molecule occupies the interfacial site (e.g., air / liquid), while the hydrophilic portion of the molecule remains oriented toward the bulk solvent. At sufficient concentrations (typically near the critical micelle concentration of a detergent), the surface layer of the surfactant molecule serves to prevent protein molecules from adsorbing at the interface. Thus, surface-induced degradation is minimized. Surfactants contemplated herein include, but are not limited to, fatty acid esters of dehydrated sorbitol polyoxyethylene ether, namely polysorbate 20 and polysorbate 80. The only difference between the two is the length of the aliphatic chain that imparts the hydrophobic characteristic to the molecule, which are C-12 and C-18, respectively. Therefore, polysorbate 80 has higher surface activity and a lower critical micelle concentration compared to polysorbate 20.

[0088] Detergents can also affect the thermodynamic conformational stability of proteins. Nonionic surfactants are commonly used for protein stabilization. Ionic surfactants (detergents) typically destabilize proteins. Again, the effect of a given detergent excipient is protein-specific. For example, polysorbates have been shown to decrease the stability of some proteins and increase the stability of others. Detergent instability of proteins can be reasonably explained by the hydrophobic tails of detergent molecules, which can participate in specific binding to partially or completely unfolded protein states. These types of interactions can lead to a conformational shift towards a more extended protein state (i.e., increased exposure of the hydrophobic portions of the protein molecule to facilitate polysorbate binding). Alternatively, if the native state of the protein exhibits some hydrophobic surface, the binding of the detergent to the native state can stabilize that conformation. Another aspect of polysorbates is their inherent susceptibility to oxidative degradation. Typically, as raw materials, they contain sufficient amounts of peroxides to cause oxidation of the protein residue side chains, particularly methionine. The potential for oxidative damage due to the addition of stabilizers underscores the view that the minimum effective concentration of excipients should be used in formulations. For surfactants, the effective concentration for a given protein will depend on the stabilizing mechanism.

[0089] Surfactants are also added in appropriate amounts to prevent surface-related aggregation during freeze-drying (Chang, B, J. Pharm. Sci. 85:1325, (1996)). Therefore, exemplary surfactants include, but are not limited to, anionic, cationic, nonionic, amphoteric, and amphoteric surfactants, including surfactants derived from naturally occurring amino acids. Anionic surfactants include, but are not limited to, sodium lauryl sulfate, sodium dioctyl sulfosuccinate and sodium dioctyl sulfonate, chenodeoxycholic acid, sodium N-lauroyl sarcosinate, lithium dodecyl sulfate, sodium 1-octane sulfonate, sodium cholate hydrate, sodium deoxycholate, and sodium glycinate deoxycholate. Cationic surfactants include, but are not limited to, benzalkonium chloride or benzyl chloride, hexadecylpyridine chloride monohydrate, and hexadecyltrimethylammonium bromide. Amphoteric surfactants include, but are not limited to, CHAPS, CHAPSO, SB3-10, and SB3-12. Nonionic surfactants include, but are not limited to, digitalis saponins, Triton X-100, Triton X-114, TWEEN-20, and TWEEN-80. Surfactants also include, but are not limited to, lauryl polyoxyethylene ether 400, polyoxyethylene 40 stearate, polyoxyethylene hydrogenated castor oil 10, 40, 50, and 60, glyceryl monostearate, polysorbate 40, polysorbate 60, polysorbate 65, and polysorbate 80, soybean lecithin, and other phospholipids such as dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylglycerol (DMPG), dimyristoylphosphatidylcholine (DMPC), and (dioleoylphosphatidylglycerol) DOPG; sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. Formulations comprising these surfactants (either individually or as mixtures in different ratios) are further provided. In one embodiment of this disclosure, the surfactant is polysorbate 80 or polysorbate 20. In another embodiment of this disclosure, the surfactant is polysorbate 80.

[0090] In some embodiments, the surfactant is polysorbate 80 or polysorbate 20, and the amount of polysorbate 80 or polysorbate 20 is from 0.1 mg to 1.0 mg. In some 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 some embodiments, the amount of polysorbate 80 or polysorbate 20 is about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, or about 1.0 mg. In some embodiments, the amount of polysorbate 80 is 0.22 mg. In some embodiments, the amount of polysorbate 80 is 0.29 mg. In some embodiments, the amount of polysorbate 20 is 0.22 mg. In some embodiments, the amount of polysorbate 20 is 0.29 mg.

[0091] In the reconstituted formulation, the concentration of the surfactant is from about 0.01 to about 0.5 g / L. In various embodiments of the pharmaceutical formulations provided herein, the surfactant concentration is 0.005, 0.01, 0.02, 0.03, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 g / L. Similarly, in some embodiments of this disclosure, the surfactant is incorporated at a concentration of about 0.001, 0.002, 0.003, 0.004, 0.005, 0.01, 0.02, 0.03, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 0.8, 0.9, or 1.0% by weight / volume. In some embodiments of this disclosure, the surfactant is incorporated at a concentration of about 0.01 to about 0.05% by weight / volume. In some embodiments, the surfactant is incorporated at a concentration of about 0.02% by weight / volume.

[0092] 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% by weight / volume of surfactant. In some embodiments, the surfactant is present in the pharmaceutical formulation at a concentration of at least 0.02% by weight / volume. In some embodiments, polysorbate 80 or polysorbate 20 is incorporated at a concentration of about 0.01 to about 0.05% by weight / volume. In some embodiments, polysorbate 80 or polysorbate 20 is incorporated at a concentration of about 0.02% by weight / volume.

[0093] ActRIIa formulation In some embodiments of the pharmaceutical formulations provided herein, the vials of the ActRIIa fusion protein provided herein contain the ActRIIa fusion protein and one or more pharmaceutical additives and / or excipients. In some embodiments, the pharmaceutical formulation is lyophilized. In some embodiments, the formulation is reconstituted from a lyophilized formulation. In one embodiment of the pharmaceutical formulations provided herein, a vial of lyophilized pharmaceutical formulation contains 55 mg of ActRIIa fusion protein; 0.48 mg of citrate monohydrate, 2.56 mg of trisodium citrate dihydrate, 0.22 mg of polysorbate 80, and 88 mg of sucrose. In another embodiment, the vial is rehydrated with 1.0 mL of liquid, such as sterile water for injection. In another embodiment of the pharmaceutical formulations provided herein, the vial contains 72.5 mg of ActRIIa fusion protein; 0.64 mg of citrate monohydrate, 3.37 mg of trisodium citrate dihydrate, 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, such as sterile water for injection. In a further embodiment, the vial contains one, two, or all three citrates, such as 10 mM citrate; sucrose, such as 8% (w / v) sucrose; and / or polysorbate 80, such as pH 5.0-7.0, such as 0.02% (w / v) polysorbate 80, pH 5.8.

[0094] In some embodiments, once reconstituted with sterile water, the vial contains 50 mg / mL of the ActRIIa fusion protein of SEQ ID NO: 32 or SEQ ID NO: 41, 10 mM citrate, 0.2 mg / mL polysorbate 80, and 8% by weight sucrose. In some embodiments, once reconstituted with sterile water, the vial contains 50 mg / mL sotaspire, 10 mM citrate, 0.2 mg / mL polysorbate 80, and 8% by weight sucrose.

[0095] 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 pH 5.8. In some embodiments, the reconstituted pharmaceutical formulation comprises 50 mg / mL of sotacicept, 10 mM citrate, 0.2 mg / mL polysorbate 80, and 80 mg / mL sucrose, at pH 5.8.

[0096] In some embodiments, the dose is administered parenterally. In some embodiments, the dose is administered subcutaneously. In some embodiments, the dose is administered intradermally. In some embodiments, the dose is administered intramuscularly. In some embodiments, the dose is administered intravenously. In some embodiments, the dose is self-administered.

[0097] medicine box This disclosure provides a kit comprising a lyophilized pharmaceutical formulation and an injection device. In some embodiments, the lyophilized pharmaceutical formulation comprises an ActRIIa protein (e.g., a protein having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 9 or SEQ ID NO: 32) or a fragment thereof, a functional variant, or a modified form. In some embodiments, the protein binds to one or more ligands selected from activin A, activin B, and GDF11. In some such embodiments, the protein further binds to one or more ligands selected from BMP10, GDF8, and BMP6. In some embodiments, the protein binds to activin and / or GDF11.

[0098] In some embodiments, the lyophilized pharmaceutical formulation comprises a protein having an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 9 or SEQ ID NO: 32, and is substantially composed of or constitutes thereof. In some such embodiments, the protein comprises an amino acid sequence having at least 90%, 95%, or 99% identity with SEQ ID NO: 9 or SEQ ID NO: 32, wherein the protein binds activin and / or GDF11. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 32. In other embodiments, the protein is composed of the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 32. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 41. In other embodiments, the protein is composed of the amino acid sequence of SEQ ID NO: 41.

[0099] In some embodiments, the lyophilized pharmaceutical formulation comprises a protein having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 32. In some embodiments, the protein consists essentially of the amino acid sequence of SEQ ID NO: 32. In other embodiments, the protein consists of the amino acid sequence of SEQ ID NO: 32. In other embodiments, the protein consists of a variant of SEQ ID NO: 32 lacking a C-terminal lysine residue (i.e., SEQ ID NO: 41).

[0100] In some of the foregoing embodiments, the lyophilized pharmaceutical formulation comprises a fusion protein, which further comprises an Fc domain of an immunoglobulin. In some such embodiments, the Fc domain of the immunoglobulin is the Fc domain of IgG1 immunoglobulin. In other embodiments, the fusion protein further comprises a linker domain located between the protein domain and the Fc domain of the immunoglobulin. In some embodiments, the linker domain is a polyglycine linker.

[0101] In some embodiments, the protein is part of a homodimeric protein complex.

[0102] In some implementations, the protein is glycosylated.

[0103] This disclosure further provides a kit comprising a sterile lyophilized pharmaceutical preparation and an injection device, said sterile lyophilized pharmaceutical preparation comprising a protein as disclosed herein. In some embodiments of the kit disclosed herein, the sterile lyophilized pharmaceutical preparation comprising the protein is pre-filled in one or more containers, such as one or more vials. Figure 1 )middle.

[0104] In some embodiments, the pH range of the sterile lyophilized pharmaceutical formulation containing the ActRIIa fusion protein is 5-7. In some embodiments, the sterile lyophilized pharmaceutical formulation containing the ActRIIa fusion protein further comprises a buffer. In some embodiments, the buffer is added in an amount of at least 10 mM. In some embodiments, the buffer is added in an amount ranging from about 10 mM to about 200 mM. In some embodiments, the buffer comprises citrate.

[0105] In some embodiments, the pharmaceutical formulation further comprises a surfactant. In some embodiments, the surfactant comprises polysorbate. In some embodiments, the surfactant comprises polysorbate 20 or polysorbate 80. In some embodiments, the surfactant comprises polysorbate 80.

[0106] In some embodiments, the lyophilized pharmaceutical formulation further comprises a cryoprotectant. In some embodiments, the cryoprotectant comprises a sugar, such as a disaccharide (e.g., sucrose). In some embodiments, the cryoprotectant comprises sucrose, trehalose, mannitol, polyvinylpyrrolidone (PVP), glucose, and / or glycine. In some embodiments, the cryoprotectant comprises sucrose. In some embodiments, the lyophilized pharmaceutical formulation comprises a cryoprotectant and protein in a weight ratio of at least 1:1 protein:cryoprotectant. In some embodiments, the lyophilized pharmaceutical formulation comprises a cryoprotectant and protein in a weight ratio of 1:1 to 1:10 protein:cryoprotectant. In some embodiments, the lyophilized pharmaceutical formulation comprises a cryoprotectant and protein in a weight ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10 protein:cryoprotectant. In some embodiments, the lyophilized pharmaceutical formulation comprises a cryoprotectant and protein in a weight ratio of 1:6 protein:cryoprotectant. In some of the foregoing embodiments, the lyophilized pharmaceutical formulation contains a cryoprotectant in an amount sufficient to stabilize the protein.

[0107] In some embodiments of the medicine box disclosed herein, the injection device includes a syringe, such as Figure 1As shown in the diagram. In some such embodiments, the syringe is pre-filled with a reconstitution solution. In some embodiments, the reconstitution solution contains a pharmaceutically acceptable carrier and / or excipient. In some embodiments, the pharmaceutically acceptable carrier comprises an aqueous solution, such as water, physiologically buffered saline, or other solvents or media, such as ethylene glycol, glycerol, oil, or injectable organic esters. In some embodiments, the pharmaceutically acceptable excipient comprises a pharmaceutically acceptable excipient selected from calcium phosphate, calcium carbonate, calcium sulfate, rock salt, metal oxides, sugars, sugar alcohols, starch, ethylene glycol, povidone, mineral hydrocarbons, acrylic polymers, fatty alcohols, mineral stearates, glycerol, and / or lipids. In some embodiments, the reconstitution solution comprises a pharmaceutically acceptable sterile isotonic or non-aqueous solution, dispersion, suspension, or emulsion. In some such embodiments, the reconstitution solution comprises an antioxidant, buffer, antibacterial agent, and / or a solute that imparts isotonicity to the blood of the intended recipient. In other embodiments, the reconstitution solution comprises a suspending agent or thickener.

[0108] In some embodiments of the pillbox disclosed herein, the pillbox further includes a bottle adapter, such as... Figure 1 As shown in the diagram. In some embodiments, a vial (1) prefilled with the lyophilized pharmaceutical formulation of the present invention is connected to one end of a vial adapter (3). In some embodiments, a syringe (2) prefilled with the reconstitution solution disclosed herein is connected to one end of the vial adapter (3). In some embodiments, the syringe (2) prefilled with the reconstitution solution disclosed herein and the vial (1) prefilled with the lyophilized pharmaceutical formulation are connected to the opposite end of the vial adapter (3). In some embodiments, the reconstitution solution is transferred from the prefilled syringe to the vial. In some embodiments, the reconstitution solution is transferred to the vial prefilled with the lyophilized pharmaceutical formulation to reconstitute the lyophilized formulation into a sterile injection solution. In some embodiments, the lyophilized formulation is reconstituted into a sterile injection solution. In some embodiments, the lyophilized formulation is reconstituted into a sterile injection solution before use.

[0109] In other embodiments of the pillbox disclosed herein, the pillbox further includes a pump device. In some embodiments, the pump device includes an electromechanical pumping assembly. In some embodiments, the pump device includes a reservoir for containing a sterile injectable solution. In some embodiments, the reservoir contains 1 mL of sterile injectable solution. In some embodiments, the pump device includes one or more bottles or cartridges containing sterile injectable solution. In some embodiments, the bottles or cartridges are pre-filled with sterile injectable solution. In some embodiments, the bottles or cartridges contain sterile injectable solution reconstituted from a lyophilized formulation. In some embodiments, the reservoir is connected to the bottle or cartridge. In some embodiments, the bottle or cartridge contains 1-20 mL of sterile injectable solution. In some embodiments, the electromechanical pumping assembly includes a pump chamber. In some embodiments, the electromechanical pumping assembly is connected to the reservoir. In some embodiments, sterile injectable solution is received from the reservoir into the pump chamber. In some embodiments, the electromechanical pumping assembly includes a piston arranged such that the sterile injectable solution in the pump chamber is in direct contact with the piston. In some embodiments, a sterile injectable solution is received from a reservoir into a pump chamber during a first pumping phase and delivered from the pump chamber to the subject during a second pumping phase. In some embodiments, the electromechanical pumping assembly includes control circuitry. In some embodiments, the control circuitry drives a piston to (a) draw sterile injectable solution into the pump chamber during the first pumping phase and (b) deliver sterile injectable solution from the pump chamber with multiple discontinuous piston movements during the second pumping phase, thereby delivering the therapeutic substance to the subject in multiple controlled and discrete doses throughout the second pumping phase. In some embodiments, the alternating cycle of the first and second pumping phases is repeated until the desired dose is administered. In some embodiments, the pump device is attached to a wearable patch. In some embodiments, the pump device is a wearable pump device.

[0110] This disclosure provides a kit for reconstituted lyophilized pharmaceutical formulations into a sterile injectable solution. In some embodiments, the resulting sterile injectable solution can be used in the methods disclosed herein.

[0111] In some embodiments of the kit disclosed herein, the kit further includes an injection device for 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 sterile injectable solution contains a therapeutically effective dose. In some embodiments, the therapeutically effective dose contains a weight-based dose.

[0112] ActRIIa peptide In some embodiments, this disclosure relates to ActRIIa fusion proteins. As used herein, the term "ActRIIa" refers to the activin receptor type IIA (ActRIIa) protein family from any species, as well as variants derived from such ActRIIa fusion proteins through mutagenesis or other modifications. References to ActRIIa herein should be understood as references to any currently identified form. Members of the ActRIIa family are typically transmembrane proteins consisting of a ligand-binding extracellular domain containing a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain having predicted serine / threonine kinase activity.

[0113] The term "ActRIIa fusion protein" includes any naturally occurring protein comprising a member of the ActRIIa family, as well as any variants (including mutants, fragments, fusions, and peptide-like forms) that retain useful activity. Examples of such variant ActRIIa fusion proteins are provided throughout this disclosure and in International Patent Application Publications 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 herein is based on the sequence number of the human ActRIIa precursor protein (SEQ ID NO: 9) provided below, unless explicitly stated otherwise.

[0114] The standard human ActRIIa precursor protein sequence is as follows: Signal peptides via single underscore Indicated; extracellular domains are indicated in bold; and potential endogenous N-linked glycosylation sites are indicated via express.

[0115] The processed (mature) extracellular human ActRIIa protein sequence is as follows: The C-terminal "tail" of the extracellular domain passes through single underscore The sequence with the missing "tail" (Δ15 sequence) is shown below: ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM (SEQ ID NO: 11) The nucleic acid sequence encoding the human ActRIIa precursor protein is shown below (SEQ ID NO: 12), corresponding to nucleotides 159-1700 of the Genbank reference sequence NM_001616.4. The signal sequence is... underline express.

[0116] The nucleic acid sequence encoding the processed soluble (extracellular) human ActRIIa protein is as follows: ActRIIa is highly conserved in vertebrates, with large segments of its extracellular domains being completely conserved. For example, Figure 2 Multiple sequence alignments of the human ActRIIa extracellular domain with various ActRIIa orthologs are described. Many ActRIIa-binding ligands are also highly conserved. Therefore, based on these alignments, it is possible to predict key amino acid positions within the ligand-binding domain that are important for normal ActRIIa-ligand binding activity, as well as amino acid positions that may tolerate substitutions without significantly altering normal ActRIIa-ligand binding activity. Therefore, active human ActRIIa variant proteins usable according to the methods disclosed herein may include one or more amino acids at corresponding positions from sequences of ActRIIa from another vertebrate, or may include residues similar to residues in human or other vertebrate sequences.

[0117] This is not intended to be restrictive; the following examples illustrate this method of defining active ActRIIa variants. For example... Figure 2 As explained in the text, F13 in the extracellular domain of human cells is present in sheep (…). Ovis aries (SEQ ID NO: 62), Red Junglefowl ( Gallus gallus (SEQ ID NO: 65), scalpers ( Bos Taurus ) (SEQ ID NO: 66), Barn Owl ( Tyto alba (SEQ ID NO: 67) and David's mouse-eared bat ( Myotis davidiiThe presence of Y in ActRIIa (SEQ ID NO: 68) indicates that aromatic residues are tolerant at this position, including F, W, and Y. Q24 in the human extracellular domain is R in the cattle ActRIIa, indicating that charged residues are tolerant at this position, including D, R, K, H, and E. S95 in the human extracellular domain is F in the junglefowl and barn owl ActRIIa, indicating that this site is tolerant to a wide variety of variations, including polar residues such as E, D, K, R, H, S, T, P, G, and Y, as well as possible hydrophobic residues such as L, I, or F. E52 in the human extracellular domain is D in the sheep ActRIIa, indicating that acidic residues are tolerant at this position, including D and E. P29 in the human extracellular domain exhibits relatively poor conservation, appearing as S in the sheep ActRIIa and as L in the David's mouse eared bat ActRIIa, thus essentially any amino acid will be tolerant at this position.

[0118] Furthermore, as discussed above, ActRIIa proteins have been characterized in this field based on their structural / functional features, particularly regarding 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) PNAS103(20: 7643-7648; Thompson). et al (2003) The EMBO Journal 22(7): 1555-1566; and U.S. Patent Nos. 7,709,605, 7,612,041 and 7,842,663). In addition to the teachings of this paper, these references provide ample guidance on how to generate ActRIIa variants that retain one or more desired activities (e.g., ligand-binding activity).

[0119] For example, a crucial structural motif called the tridigital toxin fold is important for ligand binding via type I and type II receptors and is formed by conserved cysteine ​​residues located at different positions within the extracellular domains of each monomeric receptor (Greenwald et al. (1999) Nat Struct Biol 6:18-22; and Hinck (2012) FEBS Lett 586:1860-1870). Thus, the core ligand-binding domain of human ActRIIa, defined by the outermost of these conserved cysteine ​​residues, corresponds to positions 30-110 of SEQ ID NO: 9 (ActRIIa precursor). Therefore, the more structurally disordered amino acids flanking these cysteine-defined core sequences can be truncated by 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 by 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 ligand binding. Exemplary ActRIIa extracellular domain truncations include SEQ ID NO: 10 and 11.

[0120] Therefore, the general formula for the active portion (e.g., ligand binding) of ActRIIa is a protein comprising or consisting substantially of amino acids 30-110 of SEQ ID NO: 9. Thus, an ActRIIa protein may, for example, comprise residues starting at any of amino acids 21-30 corresponding to SEQ ID NO: 9 (e.g., starting at any of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) and corresponding to SEQ ID NO: The ActRIIa portion of 9 that ends at any one of the positions of amino acids 110-135 (e.g., ending at any one of amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134 or 135) has an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to, substantially composed of or composed of. Other examples include starting at positions selected from SEQ ID NO: 9, 21-30 (e.g., starting at any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30), 22-30 (e.g., starting at any one of amino acids 22, 23, 24, 25, 26, 27, 28, 29, or 30), 23-30 (e.g., starting at any one of amino acids 23, 24, 25, 26, 27, 28, 29, or 30), and 24-30 (e.g., starting at any one of amino acids 24, 25, 26, 27, 28, 29, or 30), and at positions selected from SEQ ID NO: 9, 111-135. (e.g., ending at any of amino acids 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135), 112-135 (e.g., ending at any of amino acids 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135), 113-135 (e.g.,Ending at any of amino acids 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135), 120-135 (e.g., ending at any of amino acids 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135), 130-135 (e.g., ending at any of amino acids 130, 131, 132, 133, 134, or 135), 111-134 (e.g., ending 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, or 134), 111-133 (e.g., ending 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, or 133), 111-132 A construct that 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, or 132) or 111-131 (e.g., ending 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, or 131). Variants within these ranges are also envisioned, particularly those comprising, substantially comprising, or composed of, an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the corresponding portion of SEQ ID NO: 9. Therefore, in some embodiments,The ActRIIa protein may comprise, consist substantially of, or be composed of, a protein having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with amino acids 30-110 of SEQ ID NO: 9. Optionally, the ActRIIa protein comprises a protein having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with amino acids 30-110 of SEQ ID NO: 9 and containing no more than 1, 2, 5, 10, or 15 conserved amino acid variations in the ligand-binding pouch.

[0121] In some embodiments, this disclosure relates to GDF / BMP antagonists (inhibitors) comprising the ActRIIa protein (including fragments, functional variants, and modified forms thereof) and their uses (e.g., enhancing immune responses in patients of need and treating cancer). Preferably, the ActRIIa protein is soluble (e.g., the extracellular domain of ActRIIa). In some embodiments, the ActRIIa protein inhibits (e.g., Smad signaling) one or more GDF / BMP ligands (e.g., GDF11, GDF8, activins (activin A, activin B, activin AB, activin C, activin E), BMP6, GDF3, BMP15, and / or BMP10). In some embodiments, the ActRIIa protein binds to one or more GDF / BMP ligands (e.g., GDF11, GDF8, activins (activin A, activin B, activin AB, activin C, activin E), BMP6, GDF3, BMP15, and / or BMP10). In some embodiments, the ActRIIa protein of this disclosure comprises residues starting at amino acids 21-30 corresponding to SEQ ID NO: 9 (e.g., starting at any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) and corresponding to SEQ ID NO: The ActRIIa portion of amino acid 9 ending at any one of amino acids 110-135 (e.g., ending at any one of amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135) has an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, substantially composed of, or composed of. In some embodiments, the ActRIIa protein comprises, consists of, or is substantially composed of an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with amino acids 30-110 of SEQ ID NO:9.In some embodiments, the ActRIIa protein comprises, consists of, or is substantially composed of an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with amino acids 21-135 of SEQ ID NO: 9.

[0122] In some embodiments, the extracellular domain (ECD) of a human activin receptor type IIA (ActRIIa) protein or a derivative thereof is linked to a constant domain of an immunoglobulin, such as the human IgG1 Fc domain. In some embodiments, the ActRIIa protein is a fusion protein comprising the ActRIIa domain and one or more heterologous protein domains. In some embodiments, the ActRIIa protein is a fusion protein comprising the Fc domain of an immunoglobulin. In some embodiments, the Fc domain of the immunoglobulin is the Fc domain of IgG1 immunoglobulin. In some embodiments, the ActRIIa fusion protein further comprises a linker domain located between the ActRIIa protein domain and one or more heterologous domains (e.g., the Fc immunoglobulin domain). In some embodiments, the linker domain is selected from: 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 having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the 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.

[0123] In some embodiments, the ActRIIa fusion protein comprises, consists of, or is substantially composed of an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identity with, the amino acid sequence of any one of SEQ ID NO: 9, 10, 11, 32, 36, and 39.

[0124] In some embodiments, the ActRIIa fusion protein comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 32. In some embodiments, the ActRIIa fusion protein comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the ActRIIa fusion protein consists of the amino acid sequence of SEQ ID NO: 32. In some embodiments, the ActRIIa fusion protein is part of a homodimeric protein complex. In some embodiments, the ActRIIa fusion protein is glycosylated. In some embodiments, the ActRIIa fusion protein has a glycosylation pattern obtainable by expression in Chinese hamster ovary cells.

[0125] In some alternative embodiments, the ActRIIa fusion protein (e.g., SEQ ID NO: 32) may lack a C-terminal lysine. In some embodiments, the ActRIIa fusion protein lacking a C-terminal lysine is SEQ ID NO: 41. For example, in some embodiments of the pharmaceutical formulations described herein, the ActRIIa fusion protein may contain 1-100% of SEQ ID NO: 32. For example, in some embodiments of the pharmaceutical formulations described herein, the ActRIIa fusion protein contains 1-100% of SEQ ID NO: 41.

[0126] In some embodiments, the pharmaceutical formulation described herein comprises a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 lacking a C-terminal lysine residue (SEQ ID NO: 41). In some embodiments, the pharmaceutical formulation described herein comprises a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 lacking a C-terminal lysine residue (SEQ ID NO: 41), wherein the mixture 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.

[0127] In some embodiments, the pharmaceutical formulations described herein comprise a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 lacking a C-terminal lysine residue (SEQ ID NO: 41), wherein the mixture comprises approximately 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, and 53%. SEQ ID NO:32 of 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.

[0128] In some embodiments, the pharmaceutical formulations described herein comprise a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 lacking a C-terminal lysine residue (SEQ ID NO: 41), wherein the mixture comprises 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%, and 53%. SEQ ID NO:41 of 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.

[0129] In some embodiments, the pharmaceutical formulations described herein comprise a mixture of SEQ ID NO: 32 and a variant of SEQ ID NO: 32 lacking a C-terminal lysine residue (SEQ ID NO: 41), wherein the mixture comprises approximately 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, and 53%. SEQ ID NO:41 of 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.

[0130] In some embodiments, the pharmaceutical formulation described herein contains 100% by weight of SEQ ID NO: 32. In some embodiments, the pharmaceutical formulation described herein contains 100% by weight of SEQ ID NO: 41.

[0131] In some aspects, this disclosure relates to GDF trapping proteins (also referred to as "GDF trappers"). In some embodiments, the GDF trappers of this disclosure are variant ActRIIa proteins (e.g., ActRIIa) containing 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., "wild-type" or unmodified ActRIIa protein), such that the variant ActRIIa protein has one or more altered ligand-binding activities compared to the corresponding wild-type ActRIIa protein. In some embodiments, the GDF trapping protein of this disclosure retains at least one activity similar to that of the corresponding wild-type ActRIIa protein. For example, preferred GDF trappers bind to GDF11 and / or GDF8 and inhibit (e.g., antagonize) their function. In some embodiments, the GDF trappers of this disclosure further bind to and inhibit one or more GDF / BMP ligands. Therefore, this disclosure provides GDF capture proteins with altered binding specificity for one or more ActRIIa ligands.

[0132] For illustration, one or more mutations can be selected to increase the selectivity of the altered ligand-binding domain for GDF11 and / or GDF8 relative to one or more ActRIIa-binding ligands, such as activin, particularly activin A. Optionally, the altered ligand-binding domain has an activin-binding K ratio of at least 2, 5, 10, 20, 50, 100, or even 1000 times that of the wild-type ligand-binding domain. d Combined with GDF11 and / or GDF8 K d The ratio. Optionally, the modified ligand-binding domain has an IC50 of at least 2, 5, 10, 20, 50, 100, or even 1000 times greater inhibitory effect on activin than the wild-type ligand-binding domain. 50 ICs that suppress GDF11 and / or GDF8 50 The ratio. Optionally, the ligand-binding domain is modified to inhibit the IC50 of activin. 50 At most 1 / 2, 1 / 5, 1 / 10, 1 / 20, 1 / 50, 1 / 100 or even 1 / 1000 of the IC 50 Inhibit GDF11 and / or GDF8.

[0133] How to use In some respects, this disclosure provides a method for treating pulmonary arterial hypertension (PAH), including administering the pharmaceutical preparation described herein to a patient in need.

[0134] In some respects, this disclosure provides a method for treating pulmonary arterial hypertension (PAH) in patients of need, comprising reconstituted a lyophilized pharmaceutical formulation described herein to produce a reconstituted formulation and administering the reconstituted formulation to the patient.

[0135] In some aspects, this disclosure provides pharmaceutical formulations for treating PAH in subjects of need, comprising reconstituted a lyophilized pharmaceutical formulation comprising a human ActRIIa fusion protein linked to a constant domain of an immunoglobulin and administering the reconstituted formulation to a subject, wherein the dosing regimen comprises: 1) administering an initial dose of 0.3 mg / kg; 2) monitoring the subject's response; and 3) administering a subsequent dose of 0.7 mg / kg; and wherein the subsequent dose is administered to the subject every three weeks. In some embodiments, the subsequent dose is modified based on the subject's response.

[0136] In some respects, this disclosure provides a method for treating pulmonary arterial hypertension (PAH), comprising administering a pharmaceutical preparation described herein to a patient in need, wherein administration of said pharmaceutical preparation results in changes in one or more of the following hemodynamic or functional parameters: a decrease in pulmonary vascular resistance (PVR); an increase in 6-minute walk distance (6MWD); a decrease in N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels; prevention or reduction of progression to the WHO-recognized functional classification of pulmonary hypertension; promotion or increase of progression to the WHO-recognized functional classification of pulmonary hypertension; improvement in right ventricular function; improvement in pulmonary artery pressure; and / or improvement in mean right atrial pressure.

[0137] In some respects, this disclosure provides a method for treating pulmonary arterial hypertension (PAH) comprising administering a pharmaceutical preparation described herein to a patient in need, wherein administration of said pharmaceutical preparation results in increased exercise capacity, provides clinical improvements, improves WHO functional classification (FC), and delays disease progression, including reducing the risk of death and hospitalization in PAH.

[0138] In some aspects, this disclosure provides a method for treating, preventing, or reducing the rate of progression and / or severity of one or more complications of pulmonary hypertension, comprising administering a pharmaceutical preparation described herein to a patient in need, wherein administration of said preparation results in changes in one or more of the following hemodynamic or functional parameters: reduced pulmonary vascular resistance (PVR); increased 6-minute walk distance (6MWD); decreased N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels; prevention or reduction of progression of pulmonary hypertension functional classification as determined by the World Health Organization (WHO); promotion or increase of progression of pulmonary hypertension functional classification as determined by the WHO; improvement of right ventricular function; improvement of pulmonary artery pressure; and / or improvement of mean right atrial pressure. In some embodiments, one or more complications of pulmonary hypertension are selected from: smooth muscle and / or endothelial cell proliferation in the pulmonary artery, pulmonary angiogenesis, dyspnea, chest pain, pulmonary vascular remodeling, right ventricular hypertrophy, and pulmonary fibrosis.

[0139] In some embodiments, administration of the pharmaceutical formulation described herein reduces a patient's PVR. In some embodiments, administration of the pharmaceutical formulation described herein reduces a patient's PVR by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%). In some embodiments, administration of the pharmaceutical formulation described herein reduces a patient's PVR by at least 20%. In some embodiments, the reduction in PVR is due to a decrease in mean pulmonary artery pressure. In some embodiments, administration of the pharmaceutical formulation described herein increases a patient's 6-minute walking distance. In some embodiments, administration of the pharmaceutical formulation described herein increases a patient's 6-minute walking distance by at least 10 meters (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, or more than 400 meters). In some embodiments, administration of the pharmaceutical formulation described herein increases a patient's 6-minute walking distance by at least 30 meters. In some embodiments, administration of the pharmaceutical formulation described herein reduces a patient's NT-proBNP level. In some embodiments, administration of the pharmaceutical formulation described herein reduces a patient's NT-proBNP level by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least 80%). In some embodiments, administration of the pharmaceutical formulation described herein reduces a patient's NT-proBNP level by at least 30%. In some embodiments, administration of the pharmaceutical formulation described herein reduces NT-proBNP levels to normal levels. In some embodiments, the normal level of NT-proBNP is <100 pg / ml.

[0140] In some embodiments, the administration of the pharmaceutical preparations described herein prevents or reduces the progression of WHO-recognized pulmonary hypertension functional classification. In some embodiments, the administration of the pharmaceutical preparations described herein prevents or reduces the progression of WHO-recognized pulmonary hypertension functional classification from functional class I to II. In some embodiments, the administration of the pharmaceutical preparations described herein prevents or reduces the progression of WHO-recognized pulmonary hypertension functional classification from functional class II to III. In some embodiments, the administration of the pharmaceutical preparations described herein prevents or reduces the progression of WHO-recognized pulmonary hypertension functional classification from functional class III to IV. In some embodiments, the administration of the pharmaceutical preparations described herein promotes or increases the progression of WHO-recognized pulmonary hypertension functional classification. In some embodiments, the administration of the pharmaceutical preparations described herein promotes or increases the progression of WHO-recognized pulmonary hypertension functional classification from class IV to III. In some embodiments, the administration of the pharmaceutical preparations described herein promotes or increases the progression of WHO-recognized pulmonary hypertension functional classification from class III to II. In some embodiments, the administration of the pharmaceutical preparations described herein promotes or increases the progression of WHO-recognized pulmonary hypertension functional classification from class II to I.

[0141] In some embodiments, administration of the pharmaceutical formulation described herein improves right ventricular function in patients. In some embodiments, the improvement in right ventricular function is due to an increase in the fractional change in right ventricular area. In some embodiments, the improvement in right ventricular function is due to a reduction 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 both an increase in the fractional change in right ventricular area and an increase in the ejection fraction.

[0142] In some embodiments, administration of the pharmaceutical formulation described herein improves a patient's pulmonary artery pressure. 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 formulation described herein reduces a patient's mPAP by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%). In some embodiments, administration of the pharmaceutical formulation described herein reduces a patient's mPAP by at least 3 mmHg (e.g., at least 3, 5, 7, 10, 12, 15, 20, or 25 mmHg). In some embodiments, administration of the pharmaceutical formulation described herein improves a patient's mean right atrial pressure (mRAP). In some embodiments, the improvement in mRAP is a reduction in mRAP. In some embodiments, administration of the pharmaceutical formulation described herein reduces a patient's mRAP by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%). In some implementations, administration of the pharmaceutical formulation described herein reduces a patient's mRAP by at least 1 mmHg (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mmHg).

[0143] In some embodiments, the patient has pulmonary vascular resistance (PVR) greater than or equal to 3 Wood units. In some embodiments, the patient has a 6-minute walk distance of 150-550 meters. In some embodiments, the patient has elevated NT-proBNP levels compared to healthy patients. In some embodiments, the patient has NT-proBNP levels of at least 100 pg / mL (e.g., 100, 150, 200, 300, 400, 500, 1000, 3000, 5000, 10,000, 15,000, or 20,000 pg / mL). In some embodiments, the patient has elevated brain natriuretic peptide (BNP) levels compared to healthy patients. In some embodiments, the patient has a BNP level of at least 100 pg / mL (e.g., 100, 150, 200, 300, 400, 500, 1000, 3000, 5000, 10,000, 15,000, or 20,000 pg / mL). In some embodiments, administration of the pharmaceutical formulation described herein reduces the patient's BNP level by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least 80%). In some embodiments, administration of the pharmaceutical formulation described herein reduces the BNP level to a normal level (i.e., <100 pg / mL). In some embodiments, the patient has a mean pulmonary artery pressure (mPAP) selected from the following: at least 20 mmHg; at least 25 mmHg; at least 30 mmHg; at least 35 mmHg; at least 40 mmHg; at least 45 mmHg; and at least 50 mmHg. In some embodiments, the patient has a mean right atrial pressure (mRAP) selected from the following: at least 5 mmHg; at least 6 mmHg; at least 8 mmHg; at least 10 mmHg; at least 12 mmHg; at least 14 mmHg; and at least 16 mmHg.

[0144] In some embodiments, PAH is idiopathic pulmonary arterial hypertension (PAH). In some embodiments, PAH is hereditary PAH. In some embodiments, PAH is drug- or toxin-induced PAH. In some embodiments, PAH is PAH associated with isolated congenital body-lung shunt at least 1 year after shunt repair. In some embodiments, the patient has functional grade II or III pulmonary hypertension according to the World Health Organization's functional classification system for pulmonary hypertension. In some embodiments, the patient has functional grade I, II, III, or IV pulmonary hypertension as defined by the World Health Organization. In some embodiments, the patient has functional grade I, II, III, or IV pulmonary hypertension according to the World Health Organization's functional classification system for pulmonary hypertension. In some embodiments, the patient has functional grade IV pulmonary hypertension according to the World Health Organization's functional classification system for pulmonary hypertension. In some embodiments, administration of the pharmaceutical formulation described herein increases the patient's transplant-free survival. In some embodiments, administration of the pharmaceutical formulation described herein increases the patient's transplant-free survival by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%). In some embodiments, administration of the pharmaceutical formulation described herein reduces right ventricular hypertrophy in patients. In some embodiments, administration of the pharmaceutical formulation described herein reduces right ventricular hypertrophy in patients by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%). In some embodiments, administration of the pharmaceutical formulation described herein reduces smooth muscle hypertrophy in patients. In some embodiments, administration of the pharmaceutical formulation described herein reduces smooth muscle hypertrophy in patients by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%). In some embodiments, administration of the pharmaceutical formulation described herein reduces pulmonary arteriole muscularization in patients. In some embodiments, administration of the pharmaceutical formulation described herein reduces pulmonary arteriole muscularization in patients by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%).

[0145] In some embodiments, administration of the pharmaceutical formulation described herein improves a patient's exercise capacity. In some embodiments, administration of the pharmaceutical formulation described herein reduces a patient's Borg Dyspnea Index (BDI). In some embodiments, administration of the pharmaceutical formulation described herein reduces a patient's BDI by at least 0.5 index points (e.g., at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 index points). In some embodiments, the patient has reduced renal function. In some embodiments, administration of the pharmaceutical formulation described herein further improves renal function. In some embodiments, administration of the pharmaceutical formulation described herein delays the clinical exacerbation of pulmonary hypertension. In some embodiments, administration of the pharmaceutical formulation described herein delays the clinical exacerbation of pulmonary hypertension according to the World Health Organization's functional classification system for pulmonary hypertension. In some embodiments, administration of the pharmaceutical formulation described herein reduces the risk of hospitalization for one or more complications associated with pulmonary hypertension. In some embodiments, administration of the pharmaceutical formulation described herein reduces the risk of onset of one or more complications associated with pulmonary hypertension. In some embodiments, the onset of illness includes one or more of the following changes: increased need for lung and / or heart transplantation; need to initiate rescue therapy with known PAH treatment; need to increase prostacyclin by at least 10%; need for atrial septalostomy; PAH-specific hospitalization for at least 24 hours; and PAH exacerbation. In some embodiments, PAH exacerbation includes a worsening of WHO functional classification and a reduction of at least 15% in 6MWD. In some embodiments, administration of the pharmaceutical formulation described herein reduces the risk of death associated with pulmonary hypertension. In some embodiments, administration of the pharmaceutical formulation described herein reduces the risk of death associated with pulmonary hypertension by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%). In some embodiments, the patient has a hemoglobin level >8 and <15 g / dL. In some embodiments, the patient's hemoglobin level is <18 g / dL.

[0146] In some embodiments, the patient treated according to the method described herein is female. In some embodiments, the patient treated according to the method described herein is male. In some embodiments, the patient treated according to the method described herein may be of any age. In some embodiments, the patient treated according to the method described herein is less than 18 years old. In a particular embodiment, the patient treated according to the method described herein is less than 13 years old. In another particular embodiment, the patient treated according to the method described herein is less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, or less than 5 years old. In yet another particular embodiment, the patient treated according to the method described herein 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 older than 30 years old. In another specific embodiment, patients treated according to the methods described herein are 30-35 years old, 35-40 years old, 40-45 years old, 45-50 years old, 50-55 years old, 55-60 years old, or older than 60 years old. In another specific embodiment, patients treated according to the methods described herein are 18-64 years old, 65-74 years old, or older than 75 years old.

[0147] In some embodiments, patients treated according to the dosage forms and methods provided herein have hemoglobin levels of less than 10 g / dL, 9 g / dL, 8 g / dL, or 7 g / dL. In some embodiments, patients treated according to the dosage forms and methods provided herein have hemoglobin levels of 7 g / dL to 7.5 g / dL, 7.5 g / dL to 8 g / dL, 8 g / dL to 8.5 g / dL, 8.5 g / dL to 9.0 g / dL, 9.0 g / dL to 9.5 g / dL, or 9.5 g / dL to 10.0 g / dL. Example

[0148] Although this disclosure is now described in general terms, it will be more readily understood by referring to the following embodiments, which are included for the purpose of illustrating certain implementations of this disclosure and are not intended to limit the disclosure.

[0149] Although the invention has now been generally described, it will be more readily understood by referring to the following embodiments, which are included for the purpose of illustrating certain embodiments of the invention and are not intended to limit the invention.

[0150] Example 1: ActRIIa-Fc fusion protein A soluble ActRIIa fusion protein was constructed in which the extracellular domain of human ActRIIa was fused with the human or mouse Fc domain via a minimal linker. The constructs were designated ActRIIa-hFc and ActRIIa-mFc, respectively.

[0151] The ActRIIa-hFc shown below is sotacept (SEQ ID NO: 32) purified from the CHO cell line: ActRIIa-hFc and ActRIIa-mFc proteins are expressed in CHO cell lines. Consider three different leader sequences: (i) Bee melittin (HBML): MKFLVNVALVFMVVYISYIYA (SEQ ID NO: 33) (ii) Tissue plasminogen activator (TPA): MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO: 34) (iii) Natural: MGAAAKLAFAVFLISCSSGA (SEQ ID NO: 35).

[0152] The chosen form uses a TPA precursor and has the following unprocessed amino acid sequence: This protein is encoded by the following nucleic acid sequence: Both ActRIIa-hFc and ActRIIa-mFc are highly suitable for recombination expression. Figure 3A and 3B The protein is shown to be purified as a single, clear peak. N-terminal sequencing revealed a single sequence of –ILGRSETQE (SEQ ID NO: 38). Purification can be achieved via a series of column chromatography steps, including, for example, three or more steps in any order: protein A chromatography, Q agarose chromatography, phenyl agarose chromatography, size exclusion chromatography, and cation exchange chromatography. Purification can be accomplished via viral filtration and buffer exchange. ActRIIa-hFc protein is purified to >98% purity, as determined by size exclusion chromatography, and >95% purity, as determined by SDS-PAGE.

[0153] ActRIIa-hFc and ActRIIa-mFc exhibited high affinity for their ligands. GDF11 or activin A was immobilized on a Biacore™ CM5 chip using a standard amine coupling procedure. ActRIIa-hFc and ActRIIa-mFc proteins were loaded onto the system and binding was measured. ActRIIa-hFc was loaded at 5 x 10⁻⁶ mmol / L. -12 dissociation constant (K) D ) binds to activin, and at 9.96 x 10 -9 K D Combined with GDF11. See also Figure 4A and 4B Using a similar binding assay, ActRIIa-hFc was found to have high to moderate affinity for other TGF-β superfamily ligands, including, for example, activin B, GDF8, BMP6, and BMP10. ActRIIa-mFc exhibited similar behavior.

[0154] ActRIIa-hFc was highly stable in pharmacokinetic studies. Rats were administered ActRIIa-hFc protein at doses of 1 mg / kg, 3 mg / kg, or 10 mg / kg, and plasma levels of the protein were measured at 24, 48, 72, 144, and 168 hours. In another study, rats were administered 1 mg / kg, 10 mg / kg, or 30 mg / kg. In rats, ActRIIa-hFc had a serum half-life of 11–14 days, and circulating levels of the drug were very high after two weeks (11 μg / ml, 110 μg / ml, or 304 μg / ml for initial administration of 1 mg / kg, 10 mg / kg, or 30 mg / kg, respectively). In cynomolgus monkeys, the plasma half-life was significantly greater than 14 days, and circulating levels of the drug were 25 μg / ml, 304 μg / ml, or 1440 μg / ml for initial administration of 1 mg / kg, 10 mg / kg, or 30 mg / kg, respectively.

[0155] Example 2: Characterization of ActRIIa-hFc protein The ActRIIa-hFc fusion protein was expressed from the pAID4 vector (SV40 ori / enhancer, CMV promoter) using the tissue plasminogen leader sequence of SEQ ID NO: 34 in stably transfected CHO-DUKX B11 cells. The purified protein, as described above in Example 1, has the sequence of SEQ ID NO: 32. The Fc portion is the human IgG1 Fc sequence as shown in SEQ ID NO: 32. Protein analysis revealed that the ActRIIa-hFc fusion protein forms as a homodimer with disulfide bonds.

[0156] Compared to the ActRIIa-hFc fusion protein reported for expression in human 293 cells, the material expressed in CHO cells exhibited a higher affinity for the activin B ligand (see del Re et al. (2004) J Biol Chem. 279(51):53126-53135). Furthermore, the use of the TPA leader sequence provided greater yield than other leader sequences and yielded a higher purity N-terminal sequence, unlike ActRIIa-Fc expressed via the natural leader. The use of the natural leader sequence resulted in two main classes of ActRIIa-Fc, each with a different N-terminal sequence.

[0157] Other ActRIIa ligand traps (ActRIIa-Fc fusion proteins modified to reduce the activin A binding ratio relative to myosin or GDF11) are described in International Patent Application Publications WO 2006 / 012627 and WO 2007 / 062188, which are incorporated herein by reference.

[0158] Example 3: Preparation of freeze-dried ActRIIa-hFc fusion protein formulation Initially, the ActRIIa-hFc fusion protein SEQ ID NO: 32 (sotasicept) was first prepared as a frozen solution in phosphate-buffered saline. Based on subsequent development studies, a lyophilized citrate buffer formulation of the ActRIIa-hFc fusion protein SEQ ID NO: 32 containing sucrose and polysorbate 80 was developed to form a stable formulation with a commercially viable shelf life.

[0159] Two formulations of the ActRIIa-hFc fusion protein SEQ ID NO: 32 were developed, in 45 mg / vial and 60 mg / vial. The 45 mg / vial formulation contains 55.0 mg of SEQ ID NO: 32 or a variant of SEQ ID NO: 32 lacking a C-terminal lysine residue, 0.48 mg of citrate monohydrate, 2.56 mg of trisodium citrate dihydrate, 0.22 mg of polysorbate 80, and 88.0 mg of sucrose. The 60 mg / ml formulation contains 72.5 mg of SEQ ID NO: 32 or a variant of SEQ ID NO: 32 lacking a C-terminal lysine residue, 0.64 mg of citrate monohydrate, 3.37 mg of trisodium citrate dihydrate, 0.29 mg of polysorbate 80, and 116.0 mg of sucrose. The formulations are identical compositions before lyophilization and after reconstitution with sWFI. They differ only in their packing volume. The 45 mg / vial has a 1.1 mL filling volume, while the 60 mg / vial has a 1.45 mL filling volume. 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 is reconstituted with 1.3 mL of sWFI.

[0160] Formulation development of the ActRIIa-hFc fusion protein SEQ ID NO: 32 was guided by the results of various screening studies described below. These studies focused on the selection of optimal pH, buffer systems, protein concentrations, and excipients to stabilize the ActRIIa-hFc fusion protein SEQ ID NO: 32. Screening studies were conducted under accelerated or stress conditions to observe differences in the stability of the ActRIIa-hFc fusion protein SEQ ID NO: 32 in test samples.

[0161] Preliminary pH screening study: ActRIIa-hFc fusion protein for optimal biophysical stability and binding capacity. Early evaluation and identification of suitable buffers and pH ranges for SEQ ID NO: 32 Initial buffer and pH screening was performed to understand the effects of pH and buffer systems 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 50 mM acetate, citrate, histidine, phosphate, succinate, and tris buffers, with pH levels ranging from 4 to 8. These formulations were filled into Type I glass vials and stored at 5°C, 25°C, and 45°C for up to 6 months for short-term stability. Purity was analyzed by high performance size exclusion chromatography (HP-SEC), thermal stability was analyzed by differential scanning calorimetry (DSC), and binding activity was analyzed by surface plasmon resonance (Biacore). Data from these analyses are summarized in Table 1.

[0162] Table 1: Preliminary pH Screening Study 1: Effects of Buffer and pH on % of monomers as determined by HP-SEC, Tm1 as determined by DSC, and Kd as determined by Biacore. Observations of changes in % monomer purity as determined by HP-SEC revealed no significant change in purity after 6 months at 5°C, with all formulations retaining >99.0% monomers. After 6 months at 25°C, the % monomer content of formulations with pH ≥ 6.5 decreased significantly, while no significant change was observed for any formulation with pH below 6.5. Figure 5 As shown, after 2 months at 45°C, the percentage of monomers in formulations with pH ≥ 6.5 decreased similarly, but the percentage of monomers associated with lower pH < 5.5 also decreased.

[0163] Based on these observations under accelerated (25°C) and stress (45°C) conditions, the ActRIIa-hFc fusion protein SEQ ID NO: 32 exhibited maximum stabilizing effect in citrate and succinate buffers within a pH range of 5.5–6.5. Histidine buffers at pH 5.5–6.0 also showed similar stability to citrate and succinate buffers at lower pH levels, but exhibited poorer stability at higher pH levels.

[0164] Binding affinity is typically measured by the equilibrium dissociation constant (Kd), which is used to evaluate and rank the strength of the interaction between two molecules. In this case, the interaction or binding affinity between the ActRIIa-hFc fusion protein SEQ ID NO: 32 and its target activin, as determined by Biacore, is used as a measure of product stability. The smaller the Kd value, the greater the binding affinity of the ligand to its target.

[0165] The equilibrium dissociation constant (Kd) of SEQ ID NO: 32 did not change significantly after 6 months at 5°C. After 6 months at 25°C, binding was significantly reduced for formulations with pH < 5.5, as indicated by... Figure 6 The sharp increase in Kd value indicates this. Formulations with pH < 6.0 showed decreased binding after 2 months at 45°C.

[0166] Based on these observations, the ActRIIa-hFc fusion protein SEQ ID NO: 32 retains its binding affinity in many different buffer systems at pH ≥ 5.5.

[0167] The unfolding temperature of the ActRIIa-hFc fusion protein SEQ ID NO: 32 increases with pH, ​​reaching a plateau of approximately 67°C-70°C near pH ≥ 5.5. All buffers exhibited similar behavior at equivalent pH values, except for the histidine and tris systems, which have significantly lower melting points.

[0168] Overall, the results of this experiment suggest that a pH range of 5.5–6.5 is optimal for SEQ ID NO: 32 in terms of physical stability as monitored by HP-SEC and retention of binding affinity as determined by Biacore. Furthermore, at the upper limit of this range, citrate and succinate buffer systems outperformed histidine. Additionally, acetate and phosphate buffers were not further pursued in development due to the potential for pH changes caused by precipitation during freeze-drying or lyophilization.

[0169] Determining the optimal buffer concentration: Evaluating the effect of buffer concentration on the buffer's effectiveness using citrate, one of the main buffer systems. Effects of ActRIIa-hFc fusion protein SEQ ID NO: 32 on conformation and colloidal stability In the initial pH screening study, various buffer systems were evaluated at a concentration of 50 mM to provide high buffering capacity. However, this concentration may not be optimal for stabilizing the ActRIIa-hFc fusion protein SEQ ID NO: 32 and is not ideal for subcutaneous injection, where higher buffer ionic strength, particularly from citrate, is known to cause injection pain. In this study, ActRIIa-hFc fusion protein SEQ ID NO: 32 was prepared at a range of citrate buffer concentrations, one of the leading buffer systems from previous studies. The effect of buffer strength on thermal stability determined by DSC and diffusion interaction parameters (kD) (a measure of molecular self-association tendency) determined by DLS was evaluated for citrate buffers (5–50 mM) at pH 5.8, as well as 50 mg / mL of ActRIIa-hFc fusion protein SEQ ID NO: 32 and 8% (w / v) sucrose. The results are summarized in Table 2.

[0170] Table 2: Study on buffer concentration range: Effect of buffer concentration on melting point and kD Citrate (mM) Tm1 (°C) <![CDATA[k D (mL / g)]]> 5 67.9 6.7 10 68.8 7.4 25 69.0 1.4 50 68.8 -4.5 The results showed that the highest dissociation constant (kD) of the ActRIIa-hFc fusion protein SEQ ID NO: 32 was at 10 mM (+6.7 mL / g), and decreased with increasing concentration until 50 mM, which resulted in a negative kD (-4.5 mL / g). A more positive molecular kD value indicates stronger intermolecular repulsion, suggesting better colloidal stability, while a negative kD value indicates a tendency for self-association that can lead to aggregation. Furthermore, the melting point of the ActRIIa-hFc fusion protein SEQ ID NO: 32 was largely the same and sufficiently high across all buffer concentrations. Based on this data, a 10 mM buffer concentration was selected for subsequent formulation development.

[0171] Choose the pH that provides the best stability. Short-term stability studies were conducted in which the ActRIIa-hFc fusion protein SEQ ID NO: 32 was formulated to a concentration of 5 mg / mL in 10 mM succinate, citrate, and histidine solutions at pH levels ranging from 5 to 7. These formulations were filled into Type I glass vials and stored at 5°C, 25°C, and 40°C for up to 3 months for short-term stability testing. The formulations were analyzed using biophysical methods for purity determined by high performance size exclusion chromatography (HP-SEC), thermal stability determined by differential scanning calorimetry (DSC), fragmentation determined by non-reducing CE-SDS, chemical degradation determined by desialylated iCIEF, and pH. Data from these analyses are summarized in Tables 3, 4, and 5, and the following observations were obtained: Table 3: pH for optimal stability studies: Effects of buffer and pH on % HMW, monomer and % LMW as determined by HP-SEC, and Tm1 as determined by DSC. Table 4: pH for optimal stability study: Effects of buffer and pH on % minor and % major peak types as determined by CE-SDS NR Table 5: Selection of pH for optimal stability studies: Effects of buffer and pH on % acidic compounds, % basic compounds, and % total major compounds as determined by desialylated icIEF. like Figure 7The results of this study show that, based on the stability results at 25 °C, citrate and succinate exhibit comparable performance in terms of optimal thermal stability for HP-SEC between pH 5.3 and 6.3 (measured pH). The higher amount of fragmentation (LMW%) observed in the citrate and succinate buffer systems via HP-SEC at 40 °C is attributed to the nature of the stress and the challenges of method integration. The observations obtained via HP-SEC are also consistent with… Figure 8 The displayed non-restored CE-SDS data is consistent. Figure 9 The charge distribution characterization shown by the desialylated iCIEF assay also confirms that the optimal stability is achieved in the pH range of 5.3–6.3. Formulations in histidine buffers require a slightly higher pH range between 6.0 and 6.5 to achieve performance comparable to citrate and succinate; however, this buffer system also exhibits a lower Tm1, indicating lower biophysical stability.

[0172] The results of this study confirm that the ActRIIa-hFc fusion protein SEQ ID NO: 32 is biophysically and chemically stable in citrate and succinate buffers within the range of 5.3–6.3 (measured pH). Furthermore, by selecting a pH that is inherently less alkaline, this range offers the potential to reduce the possible deamidation rate, as is commonly observed with biopharmaceuticals. Therefore, using citrate as the buffer system, a target pH of 5.8 was selected for further product development.

[0173] Physical stability of the ActRIIa-hFc fusion protein of SEQ ID NO: 32 The physical stability of the ActRIIa-hFc fusion protein of SEQ ID NO: 32 was evaluated under physical stress from stirring in the absence of any excipients in 10 mM citrate, one of the main buffer systems, as the worst-case scenario. The ActRIIa-hFc fusion protein of SEQ ID NO: 32 was diluted to concentrations of 5 and 50 mg / mL in 10 mM citrate at pH 5.8. Both formulations were stirred, and a non-stirred “control” sample was set up side-by-side, in which 4 mL of sample was filled into 10 mL Type I glass vials using a simulated shaker for up to 4 days. Monomer loss was analyzed by HP-SEC and is summarized in Table 6.

[0174] Regardless of protein concentration, no significant changes in monomer content were observed between control and stress samples after agitation for up to 4 days. The results of this study indicate that the ActRIIa-hFc fusion protein of SEQ ID NO: 32 is biophysically stable against shear and interfacial stress, even in the absence of filler sugars and surfactants.

[0175] Concentration range study of ActRIIa-hFc fusion protein of SEQ ID NO: 32 Studies were conducted to evaluate the short-term biophysical stability of the ActRIIa-hFc fusion protein of SEQ ID NO: 32 at various concentrations. Concentrations of the ActRIIa-hFc fusion protein of SEQ ID NO: 32, ranging from 20 mg / mL to 200 mg / mL, were prepared in 10 mM citrate buffer at pH 5.8 without any other excipients and filled into Type I glass vials. Short-term stability was assessed at 5°C and 25°C for up to 7 days. The purity of the preparations was measured by HP-SEC analysis during the studies and is summarized in Table 7.

[0176] When stored at 5°C for up to 7 days, purity did not change significantly for any formulation. However, higher monomer loss was observed at concentrations above 75 mg / mL during short-term storage at 25°C, and this trend continued with increasing concentration up to 200 mg / mL. Figure 10 The results of this study confirm that protein concentrations equal to or below 75 mg / mL have no significant effect on the biophysical stability of the ActRIIa-hFc fusion protein of SEQ ID NO: 32. Therefore, based on agitation and concentration range studies, the target concentration of the ActRIIa-hFc fusion protein of SEQ ID NO: 32 was selected as ≤75 mg / mL.

[0177] Evaluate the long-term stability of the primary buffer to support the pharmaceutical composition. Long-term stability studies were conducted to compare the performance of the two buffer systems at two different concentrations (based on an early understanding of clinical dosing needs). SEQ ID NO: 32 ActRIIa-hFc fusion protein at 75 and 50 mg / mL was prepared in 10 mM succinate and 10 mM citrate at pH 5.8 and filled into Type I glass vials and stored at -80°C, -20°C, 5°C, and 25°C under each storage condition for up to 30 months.

[0178] Table 8 provides the stability data obtained through HP-SEC analysis, and the following observations are made: After 24 months at -80°C, the purity as determined by HP-SEC showed no significant change, with all formulations retaining ≥97.8% monomer. After 24 months at 5°C, the monomer content of the ActRIIa-hFc fusion protein of SEQ ID NO: 32 decreased to 94-95% for the citrate formulation and to 93-94% for the succinate formulation. These results confirm that the ActRIIa-hFc fusion protein of SEQ ID NO: 32 is biophysically stable as a solution, even in the absence of any surfactant or stabilizing sugar, as no formulation exhibited a monomer loss of less than 94%, even after 2 years at 2-8°C. After 12 months at 25°C, the monomer content of the ActRIIa-hFc fusion protein of SEQ ID NO: 32 decreased to 91-93% for the citrate formulation and to 90-92% for the succinate formulation. In the storage data at both 5°C and 25°C, as expected, for each buffer system, the degradation of the 75 mg / mL drug was slightly greater than 50 mg / mL, and these trends showed... Figure 11 middle.

[0179] In summary, the data from this study confirm that the ActRIIa-hFc fusion protein of SEQ ID NO:32 exhibits slightly better long-term stability in citrate buffer than in succinate buffer when stored at 5°C and 25°C, but the stability is the same at -80°C, which is the recommended storage condition. Therefore, 10 mM citrate at pH 5.8 was selected as the target formulation for SEQ ID NO:32 DS under frozen conditions.

[0180] Salt concentration range study ActRIIa-hFc fusion protein of SEQ ID NO: 32 was prepared in 10 mM citrate at pH 5.8 with 50 mg / mL of the SEQ ID NO: 32 ActRIIa-hFc fusion protein and 8% (w / v) sucrose at sodium chloride concentrations ranging from 0 to 150 mM to evaluate the effect of buffer strength on its biophysical properties. These formulations were evaluated for thermal stability as determined by DSC and diffusion interaction parameters (kD) as determined by DLS. The results are summarized in Table 9.

[0181] Table 9: Salt Concentration Range Study: Effect of Sodium Chloride on Melting Point and Diffusion Interaction Parameter (kD) The results showed that the highest kD of the ActRIIa-hFc fusion protein of SEQ ID NO: 32 was in the absence of sodium chloride (16.2 mL / g), and decreased with increasing concentration, plateauing at approximately 5 mL / g above 50 mM. A more positive molecular kD value indicates stronger intermolecular repulsion, suggesting better colloidal stability, indicating that the ActRIIa-hFc fusion protein of SEQ ID NO: 32 has the best colloidal stability in the absence of sodium chloride. Furthermore, the melting point of the ActRIIa-hFc fusion protein of SEQ ID NO: 32 remained largely the same and sufficiently high for all sodium chloride concentrations. Based on this data, sodium chloride is considered unsuitable for further development work regarding the stability of the ActRIIa-hFc fusion protein of SEQ ID NO: 32.

[0182] Excipient Screening Study To enhance the biophysical stability of the ActRIIa-hFc fusion protein of SEQ ID NO: 32 in solution, a screening study was designed to evaluate a range of excipients. In this excipient screening, the ActRIIa-hFc fusion protein of SEQ ID NO: 32 was formulated to a concentration of 2 mg / mL using various excipients, including salts (sodium chloride), sugars (sucrose, mannitol), and amino acids (arginine, histidine). The solutions outlined in Table 10 were stored at 5°C, 25°C, and 45°C for up to 12 months, 9 months, and 3 months, respectively, and their purity (% monomer content) was subsequently determined by HP-SEC.

[0183] Table 10: Excipient Screening Study: Effect of Excipients on % Monomer Classes Determined by HP-SEC All formulations were biophysically stable at storage temperatures of 5°C and 25°C, with >99% of monomers retained after 12 months at 5°C and 9 months at 25°C. Except for histidine, the evaluated excipients demonstrated comparable biophysical stability under stress conditions (45°C) after 3 months, retaining 97-99% of monomers. Increasing histidine concentration was associated with decreased biophysical stability, with up to 2% (w / v) histidine resulting in 6% less monomers compared to formulations containing 0.1% (w / v) histidine.

[0184] In summary, among all the prototypes tested, the ActRIIa-hFc fusion protein of SEQ ID NO: 32 was identified as biophysically stable.

[0185] Surfactant screening study Surfactants are widely used to stabilize proteins from shear and interfacial stresses during manufacturing, transportation, and handling. Therefore, a series of experiments were conducted to evaluate the stabilizing effect of polysorbate 80 (PS80) as a surfactant on SEQ ID NO: 32 under various stress conditions.

[0186] Study 1: Screening of surfactants based on thermal stability A PS80 concentration range stability study was conducted to evaluate the effect of PS80 on the long-term stability of the ActRIIa-hFc fusion protein of SEQ ID NO: 32. In this study, the ActRIIa-hFc fusion protein of SEQ ID NO: 32 was formulated to a concentration of 50 mg / mL with different levels of PS80 ranging from 0.001% to 0.05% in 10 mM citrate at pH 5.8 and 8% (w / v) sucrose. These formulations were filled into Type I glass vials and their stability was assessed 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 HP-SEC during the study to monitor their physical stability. The stability data determined by HP-SEC are provided in Table 11.

[0187] Table 11: PS80 Concentration Range: Effect of PS80 Concentration on % Monomer Types under Different Storage Conditions No significant differences in biophysical stability characteristics were observed among different levels of PS80, with all formulations exhibiting >99% monomer retention after 24 months at 5°C, >96% monomer retention after 24 months at 25°C, and >93% monomer retention after 3 months at 45°C. These results confirm that PS80 concentrations between 0.001% and 0.05% are comparable in terms of biophysical stability under heat stress.

[0188] Study 2: Surfactant screening through stirring and freeze-thaw cycles Further PS80 concentration range studies were conducted to further evaluate the effect of PS80 on the stability of the ActRIIa-hFc fusion protein of SEQ ID NO: 32 under stirring and freeze-thaw stress. In this study, the ActRIIa-hFc fusion protein of SEQ ID NO: 32 was formulated to a concentration of 50 mg / mL with different levels of PS80 ranging from 0% to 0.05% in 10 mM citrate at pH 5.8 and 8% (w / v) sucrose. These formulations were filled into Type I glass vials and stirred at 300 RPM for up to 7 days. Additionally, the formulations were subjected to freeze-thaw stress for up to 8 cycles from -80°C to 25°C. During the study, the purity of the formulations was measured by UP-SEC, the particulate matter by HIAC and MFI, and the charge variants by desialylated iCIEF to monitor the biophysical and chemical stability of SEQ ID NO: 32. Stability data are provided in Tables 12 to 19.

[0189] Table 12: Screening of surfactants by stirring: Effect of PS80 concentration on HMW, monomer, LMW and major peaks determined by UP-SEC and CE-SDS NR Table 13: Screening of surfactants by stirring: Effect of PS80 concentration on particulate matter determined by MFI Table 14: Screening of surfactants by stirring: Effect of PS80 concentration on particulate matter measured by HIAC Table 15: Screening of Surfactants by Stirring: Effect of PS80 Concentration on Acidic, Basic, and Total Major Components Measured by Desialylated iCIEF Table 16: Surfactant Screening by Freeze-Thaw Cycle: Effect of PS80 Concentration on HMW, Monomer, LMW, and Major Peak Measured by Non-Reducing CE-SDS for SEQ ID NO: 32 after Freeze-Thaw Stress Table 17: Surfactant screening by freeze-thaw: Effect of PS80 concentration on charge variants of SEQ ID NO: 32 as determined by desialylated iCIEF after freeze-thaw stress Table 18: Surfactant screening by freeze-thaw cycle: Effect of PS80 concentration on particulate matter of SEQ ID NO: 32 as determined by HIAC after freeze-thaw stress Table 19: Surfactant Screening by Freeze-Thaw Cycle: Effect of PS80 Concentration on Particulate Matter of SEQ ID NO: 32 as Determined by MFI after Freeze-Thaw Stress When compared with control samples, there was no significant difference in the purity of any tested formulation for HMW, monomer, or LMW content as determined by UP-SEC. When compared with control, no discernible trend was observed between all tested formulation prototypes as determined by CE-SDS or all charge variants as determined by desialylated iCIEF. A slight reduction in 2 μm and 5 μm particles was observed starting from freeze-thaw stress at at least 0.01% PS80, compared to 0% PS80.

[0190] The results of this study confirm that no significant differences were observed among samples containing 0.01–0.05% PS-80 after exposure to stirring or freeze / thaw stress. These studies did not differentiate between various PS80 concentrations; however, 0.02% (w / v) of PS80 was chosen to ensure product quality and minimize stability risks while supporting emerging supply chains and maintaining safety.

[0191] Study on sucrose concentration range To ensure that the injectable solution of the ActRIIa-hFc fusion protein of SEQ ID NO: 32 has a suitable tonicity (260–380 mOsm / kg) for parenteral administration, sucrose concentrations within the range outlined in Table 20 were prepared in 10 mM citrate at pH 5.8, and osmolality was measured. Based on these preparations, 8–10% (w / v) was determined to be suitable for the formulation optimization studies described below.

[0192] Table 20: Osmotic Molar Concentration of Different Sucroses in the Platform Composition Sucrose (%) (weight / volume) Osmotic molar concentration (mOsm / kg) 2 103 4 169 6 239 8 320 10 388 12 489 14 560 Formulations containing 50 mg / mL of the ActRIIa-hFc fusion protein (SEQ ID NO: 32) and 8% to 10% (w / v) sucrose in 0.02% (w / v) PS80 at 10 mM citrate pH 5.8 were prepared, and their purity was evaluated by HP-SEC to monitor their physical stability under different conditions. Regarding the thermal stability studied, the formulations were stable at 5°C and 25°C for up to 24 months and at 45°C for up to 3 months. Stability data analyzed by HP-SEC are provided in Table 21.

[0193] Table 21: Sucrose Concentration Range Study: Effect of Sucrose Concentration on Stability of % Monomers as Determined by HP-SEC For the stirring aspect of the study, the formulation was filled into 3 mL Type I glass vials and shaken on a plate shaker at 250 RPM for 7 days. The stirring data analyzed by HP-SEC are provided in Table 22.

[0194] Table 22: Study on Sucrose Concentration Range: Effect of Sucrose Concentration on % Monomer Content as Determined by HP-SEC with respect to stirring For the photostability analysis, another set of vials was filled with 1 mL of each formulation and divided into two groups for incubation in a photostability chamber: one group was exposed to visible light at 25 kLux for 48 hours (1.2 mLuxhr), and the other group was exposed to UV light at 20 W / m² for 10 hours (200 Wh / m²). Two control vials were also prepared for each formulation: one protected from light by aluminum foil in the photostability chamber, and the other maintained at 5°C in a refrigerator. Photostability data analyzed by HP-SEC are provided in Table 23.

[0195] Table 23: Study on Sucrose Concentration Range: Effect of Sucrose Concentration on the % Monomer's Photostability as Determined by HP-SEC No significant differences in stability characteristics were observed among different sucrose levels, with each formulation retaining 99% monomer content after 24 months at 5°C, 98% after 24 months at 25°C, and 92% after 3 months at 45°C. After 7 days of stirring, no discernible trend was observed in the purity of any formulation, with all formulations retaining >98% monomer content. Regarding photostable properties, the percentage of monomer content decreased by approximately 16-17% after visible light exposure and by approximately 9-10% after UV light exposure, highlighting potential degradation pathways.

[0196] In summary, when protected from light or under physical stress due to aggregation, a sucrose concentration of 8%–10% (w / v) had no significant effect on the long-term stability characteristics of the ActRIIa-hFc fusion protein of SEQ ID NO: 32. The ActRIIa-hFc fusion protein of SEQ ID NO: 32 was also observed to be susceptible to photoinstability, a complex mechanism that is difficult to elucidate. Therefore, a lyophilized formulation was developed to reduce the potential for photo-induced chemical instability. An 8% (w / v) sucrose concentration is considered sufficient for the development of lyophilized formulations.

[0197] Protein concentration range study A protein concentration range stability study was conducted to evaluate the effect of increasing the protein concentration on long-term stability characteristics. In this study, the ActRIIa-hFc fusion protein of 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 filled into Type I glass vials, and stability was assessed at 5°C for 24 months and at 45°C for 3 months. The purity of the formulations was measured by HP-SEC analysis during the study to monitor their physical stability, and is summarized in Table 24.

[0198] Table 24: SEC Data on Protein Concentration Ranges There were no significant differences in stability characteristics at 5°C among different protein concentrations, with all formulations retaining 99% monomer after 24 months at 5°C. However, the 100 mg / mL concentration showed slightly more degradation than the lower concentrations, retaining 88% monomer after 3 months under stress conditions (45°C), while the lower concentrations retained 91% monomer.

[0199] The results of this study confirm that SEQ ID NO: 32 can be concentrated up to 75 mg / mL without any significant impact on long-term stability, and the early physical stability data suggest that concentrations up to 100 mg / mL can also have sufficient long-term stability based on data at 5°C.

[0200] Pharmaceutical composition Based on the formulation development work outlined above and the potential charge instability during long-term storage as a ready-to-use solution under recommended storage conditions, a lyophilized formulation with optimized composition was developed. The target composition of the ActRIIa-hFc fusion protein of SEQ ID NO: 32 before and after lyophilization is summarized below.

[0201] Table 25: Proposed Formulations Formulation components Solution for lyophilization protein concentration 50 mg / mL Buffer systems and pH 10 mM sodium citrate, pH 5.8 stabilizers / fillers 8% (by weight / volume) sucrose surfactants 0.02% (weight / volume) Polysorbate 80 The compositions are further provided in two-bottle strengths and, when reconstituted with a defined amount of sterile water for injection (SWFI), each composition contains 50 mg / mL of ActRIIa ligand capture agent (active pharmaceutical ingredient) and the following excipients: 10 mM citrate, 8% (w / v) sucrose and 0.02% (w / v) polysorbate 80, pH 5.8.

[0202] The components present in the lyophilized ActRIIa-hFc fusion protein composition are as follows: For administration, reconstitute the lyophilized material in the 45 mg vial with 1.0 mL of sterile water for injection (sWFI), and reconstitute the lyophilized material in the 60 mg vial with 1.3 mL of sWFI. Both result in an ActRIIa ligand capture solution of at least 50 mg / mL.

[0203] pH robustness of lyophilized formulations Studies were conducted to evaluate the robustness of key components in the formulations. This initial study was performed to evaluate the effect of pH on the stability of the lyophilized formulations. Formulations with a pH range of 5.3–6.3 were prepared in a formulation containing 50 mg / mL protein, 10 mM citrate buffer at pH 5.8, and 8% (w / v) sucrose and 0.02% (w / v) polysorbate 80. These formulations (2 mL) were filled into 5 mL Type I glass vials and lyophilized using early-stage lyophilization cycles, and their stability was assessed at 25°C, 45°C, and 60°C. The purity of the formulations was measured by HP-SEC during the study to monitor their physical stability, and is summarized in Table 26.

[0204] Table 26: SEC data on pH robustness for product formulation pH had no significant effect on the stability characteristics of SEQ ID NO: 32 at temperatures of 25°C or 45°C.

[0205] The results of this study confirm that lyophilization conditions had no 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 composition. Furthermore, early development studies showed that changes in PS 80 had no effect on the biophysical stability in solution form. Therefore, pH and PS 80 were removed as parameters in the formulation robustness studies under lyophilization conditions.

[0206] Formulation robustness studies Formulation robustness studies were conducted by altering the concentrations of protein and sucrose away from target levels to determine the robustness of the formulation. These two components were chosen for evaluation because they showed the most significant impact in determining the stability of the ActRIIa-hFc fusion protein of SEQ ID NO: 32 as a lyophilized product. Furthermore, this laboratory-scale study utilized primary packaging and lyophilization cycling parameters used in the manufacturing process.

[0207] Formulations with protein concentrations ranging from 45 (Lo) to 55 mg / mL (Hi) and sucrose concentrations ranging from 7 (Lo) to 9% (Hi) (by weight / volume) were prepared in 10 mM citrate buffer and 0.02% (w / v) polysorbate 80, as outlined in Table 27. 60 mg vials containing 1.45 mL of each formulation were filled into 2R I glass vials and lyophilized using the target commercial lyophilization cycle.

[0208] Table 27: Overview of formulations used in formulation robustness studies level Protein concentration (mg / mL) sucrose(%) Lo-Lo 45 7 Lo-Hi 45 9 Hi-Lo 55 7 Hi-Hi 55 9 Target 50 8 All formulation prototypes were stored as lyophilized cakes at 5°C, 25°C, and 40°C for up to 18 months, 6 months, and 3 months, respectively. Prototype purity was analyzed by HP-SEC, fragmentation by CE-SDS NR, charge variants by desialylated iCIEF, particulate matter by HIAC, water content by Karl-Fischer assay, pH, and reconstitution time, and are summarized in Tables 28 to 38.

[0209] Table 28: Formulation robustness: Reconstitution time Table 29: Formulation robustness: HMW as determined by HP-SEC Table 30: Formulation robustness: Monomers as determined by HP-SEC Table 31: Formulation robustness: LMW as determined by HP-SEC Table 32: Formulation robustness: Secondary and major peaks as determined by CE-SDS NR Table 33: Formulation robustness: Moisture content as determined by KF Table 34: Formulation robustness: Acids as determined by desialylated iCIEF Table 35: Formulation robustness: Total major compounds as determined by desialylated iCIEF Table 36: Formulation robustness: Basic substances as determined by desialylated iCIEF Table 37: Formulation robustness: Analysis of ≥2 μm and ≥5 μm particulate matter by HIAC Table 38: Formulation robustness: ≥10 μm and ≥25 μm particles Under any testing conditions, no discernible trend was observed in the reconstitution time, pH, or water content of the prototypes screened in this study. For storage temperatures of 5°C and 25°C, there were no significant differences in the stability characteristics of the formulation prototypes for HMW, monomer, or LMW content, and this was consistent with the fragmentation patterns observed by non-reducing CE-SDS. Slight differences were observed between the high protein concentration (55 mg / mL) and low sucrose concentration (7%) formulations of Hi-Lo stored at 45°C, with the latter exhibiting, on average, slightly higher HMW (~0.5%) levels than the others. Figure 12 As shown.

[0210] At various storage temperatures, there were no significant differences in the stability characteristics of the formulations regarding the content of acidic, major, or basic substances. Some expected variations were observed among the various charged substances, with acidic substances varying between 20-30%, total major substances between 55-70%, and basic substances between 10-20%.

[0211] At a nominal fill volume of 1.45 mL for 60 mg / vial, no discernible trend was observed in particulate matter / mL as monitored by HIAC for any formulation prototype at sizes ≥10 and ≥25 µm, and under all conditions, the particulate matter / mL was within the range specified by USP. <787> Within the prescribed acceptance criteria. In summary, this study confirms the robustness of the formulation composition within potential compositional variations, namely within ± 5 mg / mL of the target protein concentration of 50 mg / mL and within ± 1% of the target sucrose concentration of 8% (w / v).

[0212] sequence Table 1: Sequences This document describes preferred methods and materials, but similar or equivalent methods and materials may also be used to practice or test the methods and formulations disclosed herein. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety.

Claims

1. A pharmaceutical formulation comprising a human ActRIIa fusion protein, a buffer, a surfactant, and a stabilizer, wherein the ActRIIa fusion protein comprises the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO:

41.

2. The pharmaceutical formulation according to claim 1, wherein the buffer is selected from sodium citrate, succinate, and histidine.

3. The pharmaceutical formulation according to claim 1 or 2, wherein the buffer is present in an amount of 4 mM to 50 mM.

4. The pharmaceutical formulation according to claim 1 or 2, wherein the buffer is present in an amount of about 10 mM.

5. The pharmaceutical formulation according to claim 1 or 2, wherein the buffer is a citrate buffer with a pH of about 5.5 to about 6.

5.

6. The pharmaceutical formulation according to claim 1 or 2, wherein the buffer is a succinate buffer with a pH of about 5.5 to about 6.

5.

7. The pharmaceutical formulation according to claim 1 or 2, wherein the buffer is a histidine buffer with a pH of about 5.5 to about pH 6.

0.

8. The pharmaceutical formulation according to claim 1 or 2, wherein the buffer is a citrate buffer with a pH of 5.

8.

9. The pharmaceutical formulation according to claim 1 or 2, wherein the buffer is a succinate buffer with a pH of 5.

8.

10. The pharmaceutical formulation according to claim 5 or 8, wherein the citrate buffer comprises citrate monohydrate and trisodium citrate dihydrate.

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

12. The pharmaceutical formulation of claim 11, wherein the surfactant is present in an amount of about 0.01 to about 0.05 by weight / volume.

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

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

15. The pharmaceutical formulation of claim 14, wherein sucrose is present in an amount of about 8% to about 10% by weight / volume.

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

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

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

19. The pharmaceutical preparation according to any one of claims 1-18, wherein the pharmaceutical preparation does not contain salt.

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

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

32.

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

41.

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

41.

24. A lyophilized pharmaceutical preparation, which is prepared by lyophilizing the preparation according to any one of claims 1-23.

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

26. A reconstituted pharmaceutical formulation prepared by reconstituted lyophilized pharmaceutical formulation according to claim 24.

27. The reconstituted pharmaceutical preparation according to claim 26, wherein the lyophilized pharmaceutical preparation is reconstituted with sterile water for injection.

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

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

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

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

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

33. A lyophilized pharmaceutical formulation comprising a human ActRIIa fusion protein, a buffer, a surfactant, and a stabilizer, wherein the buffer is selected to be physiologically compatible and maintains pH 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. The lyophilized pharmaceutical formulation of claim 33, wherein the buffer comprises citrate.

35. The lyophilized pharmaceutical preparation according to claim 33 or 34, wherein the stabilizer is sucrose.

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

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

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

41.

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

41.

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

41.

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

41.

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

43. The pharmaceutical preparation according to any one of claims 1-41, wherein the preparation is contained in a glass vial or injection device.

44. A method of treating pulmonary arterial hypertension (PAH) in a patient in need, comprising administering to the patient a pharmaceutical preparation according to any one of claims 1-43.

45. A method for treating pulmonary arterial hypertension (PAH) in a patient in need, comprising reconstituted a lyophilized pharmaceutical preparation according to any one of claims 24, 25 or 33-40 to produce a reconstituted preparation, and administering the reconstituted preparation to the patient.

46. ​​Use of the pharmaceutical preparation according to any one of claims 1-43 for the treatment of pulmonary arterial hypertension (PAH) in patients in need.

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