Formulations of factor VIII chimeric proteins and uses thereof - Patents.com

Abstract

The present disclosure provides pharmaceutical compositions of chimeric proteins comprising a factor VIII (FVIII) polypeptide and a von Willebrand factor (vWF) polypeptide. Pharmaceutical kits and methods of using the disclosed pharmaceutical compositions for treating hemophilia A are also disclosed.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 256,432, filed October 15, 2021, the contents of which are incorporated herein in their entirety.

[0002] Reference to Electronically Submitted Sequence Listing The contents of the Sequence Listing, submitted electronically in XML file format (Name: SA9-483PC_SL_ST26.xml; Size: 55,697 bytes; Created: October 4, 2022), are hereby incorporated by reference in their entirety. [Background technology]

[0003] Hemophilia A is a bleeding disorder caused by a defect in the gene encoding coagulation factor VIII (FVIII), affecting 1-2 per 10,000 male births. Non-Patent Document 1. Patients suffering from hemophilia A can be treated by infusion of purified plasma FVIII or recombinantly produced FVIII. Many commercially available FVIII products are known to have a half-life of about 8-12 hours, requiring frequent intravenous administration to patients. See Non-Patent Document 2; Non-Patent Document 3. In addition, many approaches have been attempted to extend the half-life of FVIII. For example, approaches in development to extend the half-life of coagulation factors include pegylation, glycopegylation, and conjugation with albumin. See Non-Patent Document 4. Consistent results have been demonstrated in humans, for example, rFVIIIFc was reported to improve half-life by up to about 1.7-fold in patients with hemophilia A compared to ADVATE®. See Non-Patent Document 5. Thus, despite the minimal improvement, the increase in half-life indicates the presence of other half-life limiting factors, such as clearance by VWF.

[0004] Efanesoctocog alfa (also known as Efa and BIVV001) is a fusion protein designed to uncouple recombinant coagulation factor VIII from VWF in the blood circulation. This chimeric protein contains a single recombinant factor VIII protein fused to the D'D3 domain of VWF, a dimeric Fc, and two ELNN polypeptides. (Evans et al., 2002, J. Immunol. 149:1311-1323, 2002). In one early study in patients with severe hemophilia A, a single intravenous injection of efanesoctocog alfa resulted in sustained, high levels of factor VIII activity with a half-life of up to four times that associated with recombinant factor VIII. (Evans et al., 2002, J. Immunol. 149:1311-1323, 2002). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Graw et al., Nat. Rev. Genet. 6(6): 488-501 (2005) [Non-Patent Document 2] Weiner MA and Cairo, MS, Pediatric Hematology Secrets, Lee, MT, 12. Disorders of Coagulation, Elsevier Health Sciences, 2001 [Non-Patent Document 3] Lillicrap, D. Thromb.Res. 122 Suppl 4:S2-8 (2008) [Non-Patent Document 4] Dumont et al., Blood. 119(13): 3024-3030 (2012) [Non-Patent Document 5] Powell et al., Blood. 119(13): 3031-3037 (2012) [Non-Patent Document 6] Pipe et al., Blood. 128(16):2007-2016 (2016) [Non-Patent Document 7] Chhabra et al. Blood 135(17): 1484-1496 (2020) [Non-Patent Document 8] Konkle et al., NEJM. 383:1018-27 (2020) Summary of the Invention [Problem to be solved by the invention]

[0006] However, there remains a need for improved pharmaceutical compositions. [Means for solving the problem]

[0007] The present disclosure is directed, inter alia, to pharmaceutical compositions comprising factor VIII ("FVIII") proteins, kits comprising such pharmaceutical compositions, and methods of treatment with and use of the pharmaceutical compositions.

[0008] In some embodiments, the pharmaceutical composition comprises a chimeric protein or protein comprising a first polypeptide chain comprising a FVIII protein or a portion thereof and a first immunoglobulin ("Ig") constant region or a portion thereof, and a second polypeptide chain comprising a von Willebrand factor ("VWF") protein and a second Ig constant region or a portion thereof. In some embodiments, the chimeric protein comprises (i) a FVIII protein comprising a FVIII polypeptide, an ELNN polypeptide inserted into the B domain of the FVIII polypeptide (e.g., replacing at least a portion of the B domain), and a first Fc region; and (ii) a VWF protein comprising a VWF fragment (e.g., a fragment that may include a mutation, comprising the D'D3 domain of VWF), a second ELNN polypeptide, a thrombin-cleavable linker (e.g., a2 linker, etc.), and a second Fc region. In some embodiments, the chimeric protein disclosed herein is a FVIII-ELNN-Fc / D'D3-ELNN-Fc heterodimer.

[0009] In some embodiments, the pharmaceutical composition comprises: (a) a FVIII protein; (b) about 1% (w / v) to about 7.5% (w / v) sucrose; (c) about 5 mM to about 15 mM histidine; (d) about 200 mM to about 300 mM arginine; (e) about 2.5 mM to about 10 mM calcium chloride; and (f) about 0.01% (w / v) to about 1.0% (w / v) poloxamer. In some embodiments, the pharmaceutical composition comprises: (a) a FVIII protein; (b) about 1% (w / v) to about 7.5% (w / v) sucrose; (c) about 5 mM to about 15 mM L-histidine; (d) about 200 mM to about 300 mM L-arginine; (e) about 2.5 mM to about 10 mM calcium chloride; and (f) about 0.01% (w / v) to about 1.0% (w / v) poloxamer. In some embodiments, the pharmaceutical composition comprises: (a) factor VIII ("FVIII") protein; (b) about 1% (w / v) to about 7.5% (w / v) sucrose; (c) about 5 mM to about 15 mM L-histidine; (d) about 200 mM to about 300 mM L-arginine-HCl; (e) about 2.5 mM to about 10 mM calcium chloride dihydrate; and (f) about 0.008% (w / v) to about 0.1% (w / v) poloxamer.

[0010] In some embodiments, the composition comprises about 250 mM L-arginine. In some embodiments, the composition comprises about 250 mM L-arginine-HCl. In some embodiments, the poloxamer is poloxamer 188 (also known as poloxamer 188, P188). Poloxamer 188 is well known in the art.

[0011] In some embodiments, (a) a chimeric protein comprising a first polypeptide chain comprising a factor VIII ("FVIII") protein and a first immunoglobulin ("Ig") constant region or a portion thereof, and a second polypeptide chain comprising a von Willebrand factor ("VWF") protein and a second Ig constant region or a portion thereof; (b) about 1% (w / v) to about 7.5% (w / v) sucrose; (c) about 5 mM to about 15 mM histidine; (d) about 200 mM to about 300 mM arginine; (e) about 2.5 mM to about 10 mM calcium chloride; and (f) about 0.01% (w / v) to about 1.0% (w / v) poloxamer 188 Disclosed herein is a pharmaceutical composition comprising:

[0012] In some embodiments, the pharmaceutical composition comprises about 250 IU, 500 IU, 1000 IU, 2000 IU, 3000 IU, or 4000 IU of the chimeric protein. In some embodiments, the pharmaceutical composition comprises more than about 250 IU of the chimeric protein. In some embodiments, the pharmaceutical composition comprises more than about 300 IU of the chimeric protein. In some embodiments, the pharmaceutical composition comprises more than about 500 IU of the chimeric protein. In some embodiments, the pharmaceutical composition comprises at least about 500 IU of the chimeric protein. In some embodiments, the pharmaceutical composition comprises more than about 85 IU / ml of the chimeric protein. In some embodiments, the pharmaceutical composition comprises more than about 100 IU / ml of the chimeric protein. In some embodiments, the pharmaceutical composition comprises more than about 200 IU / ml of the chimeric protein. In some embodiments, the pharmaceutical composition comprises at least about 200 IU / ml of the chimeric protein.

[0013] In some embodiments, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:1, and the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:2, and the first and second polypeptide chains are covalently linked by two disulfide bonds between the Fc domains of the first and second polypeptide chains. In some embodiments, the chimeric protein is efanesectocog alfa.

[0014] In some embodiments, the pharmaceutical composition comprises about 1% (w / v) to about 5% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 5 mM to about 15 mM histidine. In some embodiments, the pharmaceutical composition comprises about 200 mM to about 300 mM arginine. In some embodiments, the pharmaceutical composition comprises about 2.5 mM to about 10 mM calcium chloride. In some embodiments, the pharmaceutical composition comprises poloxamer 188 (P188). In some embodiments, the pharmaceutical composition comprises about 0.01% (w / v) to about 1.0% (w / v) poloxamer 188.

[0015] In some embodiments, (a) about 1% (w / v) to about 5% (w / v) sucrose; (b) about 5 mM to about 15 mM histidine; (c) about 200 mM to about 300 mM arginine; (d) about 2.5 mM to about 10 mM calcium chloride; and (e) about 0.01% (w / v) to about 1.0% (w / v) poloxamer 188 Disclosed herein is a pharmaceutical composition comprising:

[0016] In some embodiments, (a) about 1%, 2%, or 5% (w / v) sucrose; (b) about 10 mM histidine; (c) about 250 mM arginine; (d) about 5 mM calcium chloride; and (e) Poloxamer 188 at about 0.1% (w / v) Disclosed herein is a pharmaceutical composition comprising:

[0017] In some embodiments, (a) about 5% (w / v) sucrose; (b) about 10 mM L-histidine; (c) about 250 mM L-arginine-HCl; (d) about 5 mM calcium chloride; and (e) Poloxamer 188 at about 0.1% (w / v) Disclosed herein is a pharmaceutical composition comprising:

[0018] In some embodiments, (a) approximately 2% (w / v) sucrose; (b) about 10 mM L-histidine; (c) about 250 mM L-arginine-HCl; (d) about 5 mM calcium chloride; and (e) Poloxamer 188 at about 0.1% (w / v) Disclosed herein is a pharmaceutical composition comprising:

[0019] In some embodiments, (a) about 1% (w / v) sucrose; (b) about 10 mM L-histidine; (c) about 250 mM L-arginine-HCl; (d) about 5 mM calcium chloride; and (e) Poloxamer 188 at about 0.1% (w / v) Disclosed herein is a pharmaceutical composition comprising:

[0020] In some embodiments, (a) 10 mg / mL to 60 mg / mL sucrose; (b) 1.5 mg / mL to 2.0 mg / mL L-histidine; (c) 40 mg / mL to 70 mg / mL L-arginine-HCl; (d) 0.4 mg / mL to 0.9 mg / mL calcium chloride; and (e) 0.6 mg / mL to 1.6 mg / mL of poloxamer 188 Disclosed herein is a pharmaceutical composition comprising:

[0021] In some embodiments, (a) 56.12 mg / ml sucrose; (b) 1.74 mg / ml L-histidine; (c) L-arginine-HCl at 59.11 mg / ml; (d) 0.82 mg / ml calcium chloride dihydrate; and (e) 1.12 mg / ml of Poloxamer 188 Disclosed herein is a pharmaceutical composition comprising:

[0022] In some embodiments, (a) 22.45 mg / ml sucrose; (b) 1.74 mg / ml L-histidine; (c) L-arginine-HCl at 59.11 mg / ml; (d) 0.82 mg / ml calcium chloride dihydrate; and (e) 1.12 mg / ml of Poloxamer 188 Disclosed herein is a pharmaceutical composition comprising:

[0023] In some embodiments, (a) 22.45 mg / ml sucrose; (b) 1.74 mg / ml L-histidine; (c) L-arginine-HCl at 59.11 mg / ml; (d) 0.62 mg / ml calcium chloride; and (e) 1.12 mg / ml of Poloxamer 188 Disclosed herein is a pharmaceutical composition comprising:

[0024] In some embodiments, (a) 11.23 mg / ml sucrose; (b) 1.74 mg / ml L-histidine; (c) L-arginine-HCl at 59.11 mg / ml; (d) 0.82 mg / ml calcium chloride dihydrate; and (e) 1.12 mg / ml of Poloxamer 188 Disclosed herein is a pharmaceutical composition comprising:

[0025] In some embodiments, (a) 11.23 mg / ml sucrose; (b) 1.74 mg / ml L-histidine; (c) L-arginine-HCl at 59.11 mg / ml; (d) 0.62 mg / ml calcium chloride; and (e) 1.12 mg / ml of Poloxamer 188 Disclosed herein is a pharmaceutical composition comprising:

[0026] In some embodiments, (a) 10 mg / mL to 60 mg / mL sucrose; (b) 1.5 mg / mL to 2.0 mg / mL L-histidine; (c) 40 mg / mL to 70 mg / mL L-arginine-HCl; (d) 0.5 mg / mL to 0.9 mg / mL calcium chloride dihydrate; and (e) 0.6 mg / mL to 1.6 mg / mL of poloxamer 188 Disclosed herein is a pharmaceutical composition comprising:

[0027] In some embodiments, (a) sucrose at approximately 50 mg / mL; (b) about 1.6 mg / mL L-histidine; (c) L-arginine-HCl at about 52.7 mg / mL; (d) calcium chloride dihydrate at about 0.7 mg / mL; and (e) Poloxamer 188 at approximately 1 mg / mL Disclosed herein is a pharmaceutical composition comprising:

[0028] In some embodiments, (a) sucrose at approximately 20 mg / mL; (b) about 1.6 mg / mL L-histidine; (c) L-arginine-HCl at about 52.7 mg / mL; (d) calcium chloride dihydrate at about 0.7 mg / mL; and (e) Poloxamer 188 at approximately 1 mg / mL Disclosed herein is a pharmaceutical composition comprising:

[0029] In some embodiments, (a) sucrose at approximately 20 mg / mL; (b) about 1.6 mg / mL L-histidine; (c) L-arginine-HCl at about 52.7 mg / mL; (d) about 0.6 mg / mL calcium chloride; and (e) Poloxamer 188 at approximately 1 mg / mL Disclosed herein is a pharmaceutical composition comprising:

[0030] In some embodiments, (a) sucrose at approximately 20 mg / mL; (b) about 1.6 mg / mL L-histidine; (c) about 43.6 mg / mL L-arginine; (d) calcium chloride dihydrate at about 0.7 mg / mL; and (e) Poloxamer 188 at approximately 1 mg / mL Disclosed herein is a pharmaceutical composition comprising:

[0031] In some embodiments, (a) sucrose at approximately 10 mg / mL; (b) about 1.6 mg / mL L-histidine; (c) about 43.6 mg / mL L-arginine; (d) about 0.6 mg / mL calcium chloride; and (e) Poloxamer 188 at approximately 1 mg / mL Disclosed herein is a pharmaceutical composition comprising:

[0032] In some embodiments, (a) sucrose at approximately 10 mg / mL; (b) about 1.6 mg / mL L-histidine; (c) L-arginine-HCl at about 52.7 mg / mL; (d) calcium chloride dihydrate at about 0.7 mg / mL; and (e) Poloxamer 188 at approximately 1 mg / mL Disclosed herein is a pharmaceutical composition comprising:

[0033] In some embodiments, (a) sucrose at approximately 10 mg / mL; (b) about 1.6 mg / mL L-histidine; (c) L-arginine-HCl at about 52.7 mg / mL; (d) about 0.6 mg / mL calcium chloride; and (e) Poloxamer 188 at approximately 1 mg / mL Disclosed herein is a pharmaceutical composition comprising:

[0034] In some embodiments, (a) sucrose at approximately 10 mg / mL; (b) about 1.6 mg / mL L-histidine; (c) about 43.6 mg / mL L-arginine; (d) calcium chloride dihydrate at about 0.7 mg / mL; and (e) Poloxamer 188 at approximately 1 mg / mL Disclosed herein is a pharmaceutical composition comprising:

[0035] In some embodiments, (a) sucrose at approximately 10 mg / mL; (b) about 1.6 mg / mL L-histidine; (c) about 43.6 mg / mL L-arginine; (d) about 0.6 mg / mL calcium chloride; and (e) Poloxamer 188 at approximately 1 mg / mL Disclosed herein is a pharmaceutical composition comprising:

[0036] In some embodiments, (a) 10 mg / mL to 60 mg / mL sucrose; (b) 1.5 mg / mL to 2.0 mg / mL L-histidine; (c) 50 mg / mL to 70 mg / mL L-arginine-HCl; (d) 0.7 mg / mL to 0.9 mg / mL calcium chloride dihydrate; and (e) 0.6 mg / mL to 1.6 mg / mL of poloxamer 188 Disclosed herein is a pharmaceutical composition comprising:

[0037] In some embodiments, (a) 10 mg / mL to 60 mg / mL sucrose; (b) 1.5 mg / mL to 2.0 mg / mL L-histidine; (c) 50 mg / mL to 70 mg / mL L-arginine-HCl; (d) 0.4 mg / mL to 0.8 mg / mL calcium chloride; and (e) 0.6 mg / mL to 1.6 mg / mL of poloxamer 188 Disclosed herein is a pharmaceutical composition comprising:

[0038] In some embodiments, (a) 10 mg / mL to 60 mg / mL sucrose; (b) 1.5 mg / mL to 2.0 mg / mL L-histidine; (c) 40 mg / mL to 60 mg / mL L-arginine; (d) 0.7 mg / mL to 0.9 mg / mL calcium chloride; and (e) 0.6 mg / mL to 1.6 mg / mL of poloxamer 188 Disclosed herein is a pharmaceutical composition comprising:

[0039] In some embodiments, (a) 10 mg / mL to 60 mg / mL sucrose; (b) 1.5 mg / mL to 2.0 mg / mL L-histidine; (c) 40 mg / mL to 60 mg / mL L-arginine; (d) 0.4 mg / mL to 0.7 mg / mL calcium chloride dihydrate; and (e) 0.6 mg / mL to 1.6 mg / mL of poloxamer 188 Disclosed herein is a pharmaceutical composition comprising:

[0040] In some embodiments, (a) 56.12 mg / ml sucrose; (b) 1.74 mg / ml L-histidine; (c) L-arginine-HCl at 59.11 mg / ml; (d) 0.82 mg / ml calcium chloride dihydrate; and (e) 1.12 mg / ml of Poloxamer 188 Disclosed herein is a pharmaceutical composition comprising:

[0041] In some embodiments, (a) 22.45 mg / ml sucrose; (b) 1.74 mg / ml L-histidine; (c) L-arginine-HCl at 59.11 mg / ml; (d) 0.82 mg / ml calcium chloride dihydrate; and (e) 1.12 mg / ml of Poloxamer 188 Disclosed herein is a pharmaceutical composition comprising:

[0042] In some embodiments, (a) 22.45 mg / ml sucrose; (b) 1.74 mg / ml L-histidine; (c) 48.88 mg / mL L-arginine; (d) 0.82 mg / ml calcium chloride dihydrate; and (e) 1.12 mg / ml of Poloxamer 188 Disclosed herein is a pharmaceutical composition comprising:

[0043] In some embodiments, (a) 22.45 mg / ml sucrose; (b) 1.74 mg / ml L-histidine; (c) L-arginine-HCl at 59.11 mg / ml; (d) 0.62 mg / ml calcium chloride; and (e) 1.12 mg / ml of Poloxamer 188 Disclosed herein is a pharmaceutical composition comprising:

[0044] In some embodiments, (a) 22.45 mg / ml sucrose; (b) 1.74 mg / ml L-histidine; (c) 48.88 mg / mL L-arginine; (d) 0.62 mg / ml calcium chloride; and (e) 1.12 mg / ml of Poloxamer 188 Disclosed herein is a pharmaceutical composition comprising:

[0045] In some embodiments, (a) 11.23 mg / ml sucrose; (b) 1.74 mg / ml L-histidine; (c) L-arginine-HCl at 59.11 mg / ml; (d) 0.82 mg / ml calcium chloride dihydrate; and (e) 1.12 mg / ml of Poloxamer 188 Disclosed herein is a pharmaceutical composition comprising:

[0046] In some embodiments, (a) 11.23 mg / ml sucrose; (b) 1.74 mg / ml L-histidine; (c) 48.88 mg / mL L-arginine; (d) 0.82 mg / ml calcium chloride dihydrate; and (e) 1.12 mg / ml of Poloxamer 188 Disclosed herein is a pharmaceutical composition comprising:

[0047] In some embodiments, (a) 11.23 mg / ml sucrose; (b) 1.74 mg / ml L-histidine; (c) L-arginine-HCl at 59.11 mg / ml; (d) 0.62 mg / ml calcium chloride; and (e) 1.12 mg / ml of Poloxamer 188 Disclosed herein is a pharmaceutical composition comprising:

[0048] In some embodiments, (a) 11.23 mg / ml sucrose; (b) 1.74 mg / ml L-histidine; (c) 48.88 mg / mL L-arginine; (d) 0.62 mg / ml calcium chloride; and (e) 1.12 mg / ml of Poloxamer 188 Disclosed herein is a pharmaceutical composition comprising:

[0049] In some embodiments, disclosed herein is a pharmaceutical composition according to any of the disclosed embodiments, further comprising a pH of about 6.5 to about 7.5. In some embodiments, disclosed herein is a pharmaceutical composition having a pH of about 7.0. In some embodiments, disclosed herein is a pharmaceutical composition having a pH of about 6.8. In some embodiments, disclosed herein is a pharmaceutical composition that does not contain NaCl. In some embodiments, disclosed herein is a pharmaceutical composition that contains less than 8.8 mg / mL sodium chloride (NaCl). In some embodiments, disclosed herein is a pharmaceutical composition that contains L-histidine. In some embodiments, disclosed herein is a pharmaceutical composition that contains L-arginine. In some embodiments, disclosed herein is a pharmaceutical composition that contains arginine-HCl. In some embodiments, disclosed herein is a pharmaceutical composition that contains L-arginine-HCl. In some embodiments, disclosed herein is a pharmaceutical composition that contains calcium chloride dihydrate.

[0050] In some embodiments, disclosed herein is a pharmaceutical composition according to any of the disclosed embodiments, having a chimeric protein concentration of about 0.8 to about 1.2 mg / mL. In some embodiments, disclosed herein is a pharmaceutical composition comprising 75 IU / mL to 2000 IU / mL of chimeric protein. In some embodiments, disclosed herein is a pharmaceutical composition having an osmolality of about 525 mOs to about 725 mOsm / kg. In some embodiments, disclosed herein is a pharmaceutical composition having an osmolality of about 500 mOsm / kg to about 650 mOsm / kg. In some embodiments, disclosed herein is a pharmaceutical composition having an osmolality of about 600 mOsm / kg to about 650 mOsm / kg. In some embodiments, disclosed herein is a pharmaceutical composition having a turbidity of about 7 Nephelometric Turbidity Units (NTU).

[0051] In some embodiments, disclosed herein is a method of storing a pharmaceutical composition, comprising maintaining a pharmaceutical composition disclosed herein at a temperature of about -20°C to about -40°C for at least about 1 month to about 36 months. In some embodiments, the pharmaceutical composition is stored at a temperature of about -20°C, about -25°C, about -30°C, about -35°C, or about -40°C for at least about 1 month to about 36 months. In some embodiments, the pharmaceutical composition is stored at a temperature of about -20°C, about -25°C, about -30°C, about -35°C, or about -40°C for at least about 3, 6, 9, 12, 18, 21, 24, 27, 30, 33, or 36 months. In some embodiments, the pharmaceutical composition is stored at a temperature of about -30°C for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, or 12 months.

[0052] In some embodiments, the pharmaceutical composition is stored at a temperature of about -20°C to about -40°C for at least about 1 month to about 36 months. In some embodiments, the pharmaceutical composition is stored at a temperature of about -20°C, about -25°C, about -30°C, about -35°C, or about -40°C for at least about 1 month to about 36 months. In some embodiments, the pharmaceutical composition is stored at a temperature of about -20°C, about -25°C, about -30°C, about -35°C, or about -40°C for at least about 3, 6, 9, 12, 18, 21, 24, 27, 30, 33, or 36 months. In some embodiments, the pharmaceutical composition is stored at a temperature of about -30°C for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, or 12 months.

[0053] In some embodiments, the pharmaceutical composition is a liquid pharmaceutical composition that has been frozen and thawed 2 to 5 times. In some embodiments, the pharmaceutical composition is a liquid pharmaceutical composition that has been frozen and thawed twice. In some embodiments, the pharmaceutical composition is a liquid pharmaceutical composition that has been frozen and thawed three times. In some embodiments, the pharmaceutical composition is a liquid pharmaceutical composition that has been frozen and thawed four times. In some embodiments, the pharmaceutical composition is a liquid pharmaceutical composition that has been frozen and thawed five times.

[0054] In some embodiments, the pharmaceutical composition comprises about 0.5 mg / ml to about 10 mg / ml of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 0.5 mg / ml to about 5 mg / ml of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 0.5 mg / ml to about 2 mg / ml of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 0.5 mg / ml of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 1 mg / ml of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 1.5 mg / ml of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 2 mg / ml of the chimeric protein.

[0055] Also disclosed herein is a method of treating hemophilia A in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical composition according to any of the embodiments disclosed herein. In some embodiments, the pharmaceutical composition is self-administered. In some embodiments, the pharmaceutical composition is administered intravenously. In some embodiments, the pharmaceutical composition is administered intravenously at a dose of about 20 IU / kg to about 70 IU / kg. In some embodiments, the pharmaceutical composition is administered intravenously at a dose of about 50 IU / kg. In some embodiments, the pharmaceutical composition is administered intravenously once every 7-10 days. In some embodiments, the pharmaceutical composition is administered intravenously once a week.

[0056] (i) (a) a chimeric protein comprising a first polypeptide chain comprising a factor VIII ("FVIII") protein or a portion thereof and a first immunoglobulin ("Ig") constant region or a portion thereof, and a second polypeptide chain comprising a von Willebrand factor ("VWF") protein and a second Ig constant region or a portion thereof; (b) about 10 mg to about 200 mg of sucrose; (c) about 2.5 mg to about 7.5 mg of histidine; (d) about 140 mg to about 200 mg arginine; (e) about 1.5 mg to about 5 mg of calcium chloride; and (f) about 1 mg to about 10 mg of poloxamer 188 a first container containing a lyophilized pharmaceutical composition comprising: (ii) a second container containing sterile water; Also disclosed herein is a pharmaceutical kit comprising:

[0057] In some embodiments, the chimeric protein comprises a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:1 and a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:2, wherein the first and second polypeptide chains are covalently linked by two disulfide bonds between the Fc domains of the first and second polypeptide chains.

[0058] In some embodiments, the pharmaceutical kit disclosed herein comprises: (a) about 10 mg to about 200 mg of sucrose; (b) about 2.5 mg to about 7.5 mg of histidine; (c) about 140 mg to about 200 mg arginine; (d) about 1.5 mg to about 5 mg of calcium chloride; and (e) about 1 mg to about 10 mg of poloxamer 188 The present invention includes a lyophilized pharmaceutical composition comprising:

[0059] In some embodiments, the pharmaceutical kit comprises a lyophilized pharmaceutical composition that does not contain NaCl. In some embodiments, the lyophilized pharmaceutical composition comprises less than 8.8 mg / mL sodium chloride (NaCl). In some embodiments, the lyophilized pharmaceutical composition comprises L-histidine. In some embodiments, the lyophilized pharmaceutical composition comprises L-arginine. In some embodiments, the lyophilized pharmaceutical composition comprises arginine-HCl. In some embodiments, the lyophilized pharmaceutical composition comprises L-arginine-HCl. In some embodiments, the lyophilized pharmaceutical composition disclosed herein comprises calcium chloride dihydrate.

[0060] In some embodiments, the pharmaceutical kit comprises: (a) about 33.7 mg of sucrose; (b) approximately 5.2 mg of L-histidine; (c) about 177.3 mg L-arginine-HCl; (d) about 2.5 mg of calcium chloride; and (e) approximately 3.4 mg of poloxamer 188 The present invention includes a lyophilized pharmaceutical composition comprising:

[0061] In some embodiments, the pharmaceutical kit comprises: (a) about 67.3 mg of sucrose; (b) approximately 5.2 mg of L-histidine; (c) about 177.3 mg L-arginine-HCl; (d) about 2.5 mg of calcium chloride; and (e) approximately 3.4 mg of poloxamer 188 The present invention includes a lyophilized pharmaceutical composition comprising:

[0062] In some embodiments, the pharmaceutical kit comprises: (a) about 67.3 mg of sucrose; (b) approximately 5.2 mg of L-histidine; (c) about 146.6 mg L-arginine-HCl; (d) about 2.5 mg of calcium chloride; and (e) approximately 3.4 mg of poloxamer 188 The present invention includes a lyophilized pharmaceutical composition comprising:

[0063] In some embodiments, the pharmaceutical kit comprises: (a) about 168.3 mg of sucrose; (b) approximately 5.2 mg of L-histidine; (c) about 177.3 mg L-arginine-HCl; (d) about 2.5 mg of calcium chloride; and (e) approximately 3.4 mg of poloxamer 188 The present invention includes a lyophilized pharmaceutical composition comprising:

[0064] In some embodiments, the pharmaceutical kit comprises a lyophilized pharmaceutical composition having a moisture content of less than 2%. In some embodiments, the lyophilized pharmaceutical composition has a moisture content of less than 1.8%. In some embodiments, the lyophilized pharmaceutical composition has a moisture content of less than 1.6%. In some embodiments, the lyophilized pharmaceutical composition is in a lyophilized cake. In some embodiments, the lyophilized cake is white in color. In some embodiments, the lyophilized cake is less than Y4 on the European Pharmacopoeia color scale. In some embodiments, the lyophilized pharmaceutical composition is a powder.

[0065] In some embodiments, the pharmaceutical kit includes a first container containing 100 IU to 10,000 IU of chimeric protein. In some embodiments, the first container contains 250 IU, 500 IU, 1000 IU, 2000 IU, 3000 IU, or 4000 IU of chimeric protein. In some embodiments, the first container contains more than about 250 IU of chimeric protein. In some embodiments, the first container contains more than about 300 IU of chimeric protein. In some embodiments, the first container contains more than about 500 IU of chimeric protein. In some embodiments, the first container contains at least about 500 IU of chimeric protein.

[0066] In some embodiments, the pharmaceutical kit further comprises instructions for combining the lyophilized pharmaceutical composition and sterile water. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the lyophilized pharmaceutical composition is reconstituted within 7 to 12 seconds.

[0067] In some embodiments, the pharmaceutical composition includes a second container containing sterilized water in a quantity sufficient to create a solution when the sterile water is combined with the lyophilized powder in the first container.

[0068] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the osmolality of the resulting solution is about 525 to about 725 mOsm / kg. In some embodiments, the pharmaceutical compositions disclosed herein have an osmolality of about 500 mOsm / kg to about 650 mOsm / kg. In some embodiments, the osmolality of the resulting solution is about 600 to about 650 mOsm / kg. In some embodiments, the pH of the resulting solution is about 6.5 to about 7.5. In some embodiments, the pH of the resulting solution is about 7.0. In some embodiments, the pH of the resulting solution is about 6.8. In some embodiments, the turbidity of the resulting solution is less than about 7 Nephelometric Turbidity Units (NTU). In some embodiments, the protein concentration of the resulting solution is about 0.8 to about 1.2 mg / mL. In some embodiments, less than 3% of the protein aggregates.

[0069] In some embodiments, the pharmaceutical kit, when combined with the lyophilized pharmaceutical composition in the first container, comprises: (a) 10 mg / mL to 60 mg / mL sucrose; (b) 1.5 mg / mL to 2.0 mg / mL L-histidine; (c) 40 mg / mL to 70 mg / mL L-arginine-HCl; (d) 0.5 mg / mL to 0.9 mg / mL calcium chloride; and (e) 0.6 mg / mL to 1.6 mg / mL of poloxamer 188 A second container containing sterilized water in a quantity sufficient to make a solution containing:

[0070] In some embodiments of the pharmaceutical kits disclosed herein, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) 10 mg / mL to 60 mg / mL sucrose; (b) 1.5 mg / mL to 2.0 mg / mL L-histidine; (c) 40 mg / mL to 70 mg / mL L-arginine-HCl; (d) 0.5 mg / mL to 0.9 mg / mL calcium chloride; and (e) 0.6 mg / mL to 1.6 mg / mL of poloxamer 188 Includes.

[0071] In some embodiments of the pharmaceutical kits disclosed herein, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) 11.23 mg / ml sucrose; (b) 1.74 mg / ml L-histidine; (c) L-arginine-HCl at 59.11 mg / ml; (d) 0.62 mg / ml calcium chloride; and (e) 1.12 mg / ml of Poloxamer 188 Includes.

[0072] In some embodiments of the pharmaceutical kits disclosed herein, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) 22.45 mg / ml sucrose; (b) 1.74 mg / ml L-histidine; (c) L-arginine-HCl at 59.11 mg / ml; (d) 0.62 mg / ml calcium chloride; and (e) 1.12 mg / ml of Poloxamer 188 Includes.

[0073] In some embodiments of the pharmaceutical kits disclosed herein, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) 22.45 mg / ml sucrose; (b) 1.74 mg / ml L-histidine; (c) 48.88 mg / mL L-arginine; (d) 0.62 mg / ml calcium chloride; and (e) 1.12 mg / ml of Poloxamer 188 Includes.

[0074] In some embodiments of the pharmaceutical kits disclosed herein, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) 56.12 mg / ml sucrose; (b) 1.74 mg / ml L-histidine; (c) L-arginine-HCl at 59.11 mg / ml; (d) 0.62 mg / ml calcium chloride; and (e) 1.12 mg / ml of Poloxamer 188 Includes.

[0075] In some embodiments, the pharmaceutical kit includes a second container containing about 2 mL to about 5 mL of sterile water. In some embodiments, the pharmaceutical kit includes a second container containing about 3 mL of sterile water. In some embodiments, the pharmaceutical kit includes a second container containing about 3.3 mL of sterile water.

[0076] In some embodiments, the pharmaceutical kit is stored at a temperature of about -20°C to about -40°C for at least about 1 month to about 36 months. In some embodiments, the pharmaceutical kit is stored at a temperature of about -20°C, about -25°C, about -30°C, about -35°C, or about -40°C for at least about 1 month to about 36 months. In some embodiments, the pharmaceutical kit is stored at a temperature of about -20°C, about -25°C, about -30°C, about -35°C, or about -40°C for at least about 3, 6, 9, 12, 18, 21, 24, 27, 30, 33, or 36 months. In some embodiments, the pharmaceutical kit is stored at a temperature of about -30°C for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, or 12 months.

[0077] In some embodiments, the pharmaceutical kit is stored at a temperature of about -20°C to about -40°C for at least about 1 month to about 36 months. In some embodiments, the pharmaceutical kit is stored at a temperature of about -20°C, about -25°C, about -30°C, about -35°C, or about -40°C for at least about 1 month to about 36 months. In some embodiments, the pharmaceutical kit is stored at a temperature of about -20°C, about -25°C, about -30°C, about -35°C, or about -40°C for at least about 3, 6, 9, 12, 18, 21, 24, 27, 30, 33, or 36 months. In some embodiments, the pharmaceutical kit is stored at a temperature of about -30°C for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, or 12 months.

[0078] In some embodiments, the pharmaceutical kit includes a first container that is a glass vial including a rubber stopper. In some embodiments, the pharmaceutical kit includes a second container that is a syringe body. In some embodiments, sterile water is present in the syringe body. In some embodiments, the syringe body is associated with a plunger. In some embodiments, the pharmaceutical kit further includes an adapter to connect the glass vial to the syringe body. In some embodiments, the pharmaceutical kit further includes an infusion tube associated with a needle that is connected to the syringe body, suitable for intravenous infusion.

[0079] Also disclosed herein is a method of treating hemophilia A in a subject in need thereof, comprising combining a lyophilized pharmaceutical composition of a pharmaceutical kit according to any of the embodiments disclosed herein and sterile water, and administering a therapeutically effective amount of the resulting combination (i.e., solution) to the subject. In some embodiments, the subject combines the lyophilized pharmaceutical composition of the kit and sterile water. In some embodiments, the combination is self-administered by the subject. [Brief description of the drawings]

[0080] [Figure 1]Schematic diagram of an exemplary FVIII-ELNN-Fc / D'D3-ELNN-Fc heterodimer, efanesectocog alfa. FVIII: Factor VIII; VWF: von Willebrand factor; A1, A2, A3, C1, C2: domains of FVIII; D'D3: domains of VWF; Fc: Fc region of immunoglobulin constant region. [Diagram 2] FIG. 1 shows the aggregation level (%HMWS) over time (hours) at room temperature (RT) / room light (RL) conditions for efanesectocog alfa drug substance (DS) compositions (1 mg / mL) containing either 0.05% (w / v) PS80 or 0.1% (w / v) poloxamer 188 as surfactants and either 1% or 5% (w / v) sucrose. The rate of change (slope) of aggregation level is also provided (% per hour). [Diagram 3] Figure 1 shows the aggregation level (%HMWS) over time (hours) at 2-8°C for efanesectocog alfa drug substance (DS) compositions (1 mg / mL) containing either 0.05% (w / v) PS80 or 0.1% (w / v) poloxamer 188 as surfactants and either 1% or 5% (w / v) sucrose. The rate of change (slope) of aggregation level is also provided (% per hour). [Figure 4] Figure 1 shows aggregation levels (%HMWS) of efanesectocog alfa drug substance (DS) compositions containing either 0.05% (w / v) PS80 or 0.1% (w / v) Poloxamer 188 as surfactants and 1% or 5% (w / v) sucrose, diluted to 4000 IU bulk drug product after 7 hours. Samples were kept at room temperature and tested after 0, 5, 7, 25, 43, and 55 hours. Samples were also tested after lyophilization (Post-Lyo). [Diagram 5]Figure 1 shows a frozen storage stability study measuring aggregation levels (%HMWS) of efanesectocog alfa DS compositions (1 mg / mL) containing 0.05% (w / v) PS80 as a surfactant. Compositions containing 0, 1, 2, or 5% (w / v) sucrose were kept at freezing temperatures (-80°C or -30°C). Time points measured were before freezing, at the start of the experiment (T0), 1 month (T1M), 3 months (T3M), and 6 months (T6M). [Figure 6] Figure 1 shows a frozen storage stability study measuring the aggregation level (%HMWS) of efanesectocog alfa DS composition (1 mg / mL) containing 0.1% (w / v) poloxamer 188 as surfactant. Compositions containing 0, 1, 2, or 5% (w / v) sucrose were kept at freezing temperatures (-80°C or -30°C). Time points measured were before freezing, at the start of the experiment (T0), 1 month (T1M), 3 months (T3M), and 6 months (T6M). [Figure 7] Figure 7 shows the results of analysis of efanesectocog alfa DS composition (1 mg / mL) by particle testing using a high accuracy liquid particle counter (HIAC). DS compositions containing 0.1% (w / v) poloxamer 188 and 0, 1, 2, or 5% (w / v) sucrose were kept at -80°C and evaluated at the following time points: before freezing, at the start of the experiment (T0), 1 month (T1M), 3 months (T3M), and 6 months (T6M). Results for particles > 10 μm are shown in Figure 7A. Results for particles > 25 μm are shown in Figure 7B. [Figure 8A] Figure 8 shows aggregation levels (%HMWS) of 250 IU efanesectocog alfa lyophilized drug product (Lyo DP) compositions containing 0.1% (w / v) poloxamer 188 as surfactant. Lyo DP compositions containing 1% or 5% (w / v) sucrose were tested. Samples were kept at 5°C, 30°C, or 40°C and tested at TO, 1 month, 2 months, 3 months, and 6 months. Results are shown in Figure 8A (5°C), Figure 8B (30°C), and Figure 8C (40°C). [Figure 8B]Figure 8 shows aggregation levels (%HMWS) of 250 IU efanesectocog alfa lyophilized drug product (Lyo DP) compositions containing 0.1% (w / v) poloxamer 188 as surfactant. Lyo DP compositions containing 1% or 5% (w / v) sucrose were tested. Samples were kept at 5°C, 30°C, or 40°C and tested at TO, 1 month, 2 months, 3 months, and 6 months. Results are shown in Figure 8A (5°C), Figure 8B (30°C), and Figure 8C (40°C). [Figure 8C] Figure 8 shows aggregation levels (%HMWS) of 250 IU efanesectocog alfa lyophilized drug product (Lyo DP) compositions containing 0.1% (w / v) poloxamer 188 as surfactant. Lyo DP compositions containing 1% or 5% (w / v) sucrose were tested. Samples were kept at 5°C, 30°C, or 40°C and tested at TO, 1 month, 2 months, 3 months, and 6 months. Results are shown in Figure 8A (5°C), Figure 8B (30°C), and Figure 8C (40°C). [Figure 9A] Figure 9 shows aggregation levels (%HMWS) of 4000 IU efanesectocog alfa lyophilized drug product (Lyo DP) compositions containing 0.1% (w / v) poloxamer 188 as surfactant. Lyo DP compositions containing 1% or 5% (w / v) sucrose were tested. Samples were kept at 5°C, 30°C, or 40°C and tested at TO, 1 month, 2 months, 3 months, and 6 months. Results are shown in Figure 9A (5°C), Figure 9B (30°C), and Figure 9C (40°C). [Figure 9B] Figure 9 shows aggregation levels (%HMWS) of 4000 IU efanesectocog alfa lyophilized drug product (Lyo DP) compositions containing 0.1% (w / v) poloxamer 188 as surfactant. Lyo DP compositions containing 1% or 5% (w / v) sucrose were tested. Samples were kept at 5°C, 30°C, or 40°C and tested at TO, 1 month, 2 months, 3 months, and 6 months. Results are shown in Figure 9A (5°C), Figure 9B (30°C), and Figure 9C (40°C). [Figure 9C]Figure 9 shows aggregation levels (%HMWS) of 4000 IU efanesectocog alfa lyophilized drug product (Lyo DP) compositions containing 0.1% (w / v) poloxamer 188 as surfactant. Lyo DP compositions containing 1% or 5% (w / v) sucrose were tested. Samples were kept at 5°C, 30°C, or 40°C and tested at TO, 1 month, 2 months, 3 months, and 6 months. Results are shown in Figure 9A (5°C), Figure 9B (30°C), and Figure 9C (40°C). [Figure 10] Figure 10 shows aggregation levels (%HMWS) of compositions of efanesectocog alfa DS (1 mg / mL) before (pre-freeze) and after stress by freeze / thaw (F / T) at either -80° C. or -30° C. for 5 cycles (5x) (thawed at room temperature for at least 24 hours). Figure 10 shows results for DS compositions containing 0.05% (w / v) PS80 and 0, 1, 2, or 5% (w / v) sucrose. [Figure 11] Figure 11 shows aggregation levels (%HMWS) of compositions of efanesectocog alfa DS (1 mg / mL) before (pre-freeze) and after stress by freeze / thaw (F / T) at either -80° C. or -30° C. for 5 cycles (5X) (thawing at room temperature for at least 24 hours). Figure 11 shows results for DS compositions containing 0.1% (w / v) poloxamer 188 and 0, 1, 2, or 5% (w / v) sucrose. [Figure 12] Figure 12 shows protein concentration analysis by A280 (absorbance at 280 nm) of different sucrose concentrations in efanesectocog alfa DS (1 mg / mL) compositions after F / T stress. Samples were analyzed before (pre-freezing) and after 1, 3, or 5 F / T cycles. Results for DS compositions containing 0.05% (w / v) PS80 and 0, 1, 2, or 5% (w / v) sucrose are shown in Figure 12A. Results for DS compositions containing 0.1% (w / v) poloxamer 188 and 0, 1, 2, or 5% (w / v) sucrose are shown in Figure 12B. [Figure 13]Figure 1 shows an analysis of pH after F / T stress of efanesectocog alfa DS compositions containing either 0.05% (w / v) PS80 or 0.1% (w / v) poloxamer 188 as surfactants and 0, 1, 2, or 5% (w / v) sucrose. pH was measured after 0, 1, 3, or 5 F / T cycles. [Figure 14] Figure 1 shows the glass transition temperature (Tg) and residual water content (%) at the start of the experiment (T0) of lyophilized drug product (Lyo DP) compositions of 250 IU efanesectocog alfa. Lyo DP containing either 0.05% (w / v) PS80 or 0.1% (w / v) poloxamer 188 and 0, 1, 2, or 5% (w / v) sucrose were tested. [Figure 15] Figure 1 shows the glass transition temperature (Tg) and residual water content (%) at the start of the experiment (T0) of 4000 IU efanesectocog alfa lyophilized drug product (Lyo DP) compositions. Lyo DP containing either 0.05% (w / v) PS80 or 0.1% (w / v) poloxamer 188 and 0, 1, 2, or 5% (w / v) sucrose were tested. [Figure 16A] FIG. 16 shows the glass transition temperature (Tg) of lyophilized pharmaceutical product (Lyo DP) compositions of 4000 IU or 250 IU efanesectocog alfa with 0.1% (w / v) poloxamer 188 and either 5% or 1% (w / v) sucrose as surfactant. Samples were kept at 5° C., 30° C., or 40° C. and tested at TO, 1 month, 2 months, 3 months, and 6 months. Results are shown in FIG. 16A (5° C.), FIG. 16B (30° C.), and FIG. 16C (40° C.). [Figure 16B]Figure 16 shows the glass transition temperature (Tg) of lyophilized pharmaceutical product (Lyo DP) compositions of 4000 IU or 250 IU efanesectocog alfa with 0.1% (w / v) poloxamer 188 and either 5% or 1% (w / v) sucrose as surfactants. Samples were kept at 5°C, 30°C, or 40°C and tested at TO, 1 month, 2 months, 3 months, and 6 months. Results are shown in Figure 16A (5°C), Figure 16B (30°C), and Figure 16C (40°C). [Figure 16C] Figure 16 shows the glass transition temperature (Tg) of lyophilized pharmaceutical product (Lyo DP) compositions of 4000 IU or 250 IU efanesectocog alfa with 0.1% (w / v) poloxamer 188 and either 5% or 1% (w / v) sucrose as surfactants. Samples were kept at 5°C, 30°C, or 40°C and tested at TO, 1 month, 2 months, 3 months, and 6 months. Results are shown in Figure 16A (5°C), Figure 16B (30°C), and Figure 16C (40°C). [Figure 17] FIG. 1 shows the glass transition temperature (Tg) of liquid bulk drug product (BDP) compositions of 250 IU or 4000 IU efanesectocog alfa containing either 0.05% (w / v) PS80 or 0.1% (w / v) poloxamer 188 as surfactants and 0, 1, 2, or 5% (w / v) sucrose. [Figure 18A] Figure 18 shows the residual moisture content (%) of lyophilized drug product (Lyo DP) compositions of 4000 IU or 250 IU efanesectocog alfa. Lyo DP with 0.1% (w / v) poloxamer 188 and 1% or 5% (w / v) sucrose as surfactant were tested. Samples were kept at 5°C, 30°C, or 40°C and tested at TO, 1 month, 2 months, 3 months, and 6 months. Results are shown in Figure 18A (5°C), Figure 18B (30°C), and Figure 18C (40°C). [Figure 18B]Figure 18 shows the residual moisture content (%) of lyophilized drug product (Lyo DP) compositions of 4000 IU or 250 IU efanesectocog alfa. Lyo DP with 0.1% (w / v) poloxamer 188 and 1% or 5% (w / v) sucrose as surfactant were tested. Samples were kept at 5°C, 30°C, or 40°C and tested at TO, 1 month, 2 months, 3 months, and 6 months. Results are shown in Figure 18A (5°C), Figure 18B (30°C), and Figure 18C (40°C). [Figure 18C] Figure 18 shows the residual moisture content (%) of lyophilized drug product (Lyo DP) compositions of 4000 IU or 250 IU efanesectocog alfa. Lyo DP with 0.1% (w / v) poloxamer 188 and 1% or 5% (w / v) sucrose as surfactant were tested. Samples were kept at 5°C, 30°C, or 40°C and tested at TO, 1 month, 2 months, 3 months, and 6 months. Results are shown in Figure 18A (5°C), Figure 18B (30°C), and Figure 18C (40°C). [Figure 19] Figure 1 shows aggregation levels (%HMWS) after freeze / thaw (F / T) stress in compositions of efanesectocog alfa DS (concentrations: >2mg / mL) containing increasing concentrations of arginine (Arg). The arginine concentrations tested in the compositions were 100, 125, 150, 175, 200, and 250mM. Each column represents a replicate injection from the same vial after thawing at room temperature for 30 minutes. Measurements were taken immediately after thawing and at 40, 80, 120, and 160 minutes after thawing. [Figure 20] Figure 1 shows the mean concentration of efanesectocog alfa (ng / mL) over time as determined by ELISA after a single IV bolus injection of compositions containing either PS80 or P188. Vehicle 1: 10 mM L-histidine, 250 mM arginine-HCl, 5 mM calcium chloride, 5% w / v sucrose, 0.05% polysorbate 80. Vehicle 2: 10 mM L-histidine, 250 mM arginine-HCl, 5 mM calcium chloride, 5% w / v sucrose, 0.1% poloxamer 188. [Figure 21] Figure 1 shows the mean FVIII activity (mIU / mL) of efanesectocog alfa over time as determined by chromogenic assay after a single IV bolus injection of compositions containing either PS80 or P188. Vehicle 1: 10 mM L-histidine, 250 mM arginine-HCl, 5 mM calcium chloride, 5% w / v sucrose, 0.05% polysorbate 80. Vehicle 2: 10 mM L-histidine, 250 mM arginine-HCl, 5 mM calcium chloride, 5% w / v sucrose, 0.1% poloxamer 188. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0081] The present disclosure is directed, inter alia, to formulations (including aqueous and lyophilized formulations, and related kits) comprising FVIII protein.In some embodiments, the FVIII protein is a chimeric FVIII protein, such as efanesectocog alfa, comprising two polypeptides, a first polypeptide comprising a FVIII protein comprising a first ELNN polypeptide sequence insert fused to a first Fc region, and a second polypeptide comprising a VWF protein fused to a second Ig constant region by a second ELNN polypeptide sequence, wherein the first ELNN polypeptide sequence comprises about 288 amino acids, and the second ELNN polypeptide sequence comprises about 144 amino acids, and the first Ig constant region and the second Ig constant region are covalently linked to each other by disulfide bonds.

[0082] The present disclosure provides formulations, including aqueous and lyophilized formulations, and associated kits, for a chimeric protein comprising (i) a factor VIII (FVIII) polypeptide and (ii) a von Willebrand factor (vWF) fragment comprising the D' domain of VWF and the D3 domain of VWF. Included herein are compositions that can be lyophilized, as well as compositions formed upon reconstitution of the lyophilized formulation with a diluent. Methods of treatment and uses are also provided.

[0083] I. Definition It should be noted that a noun with the term "a" or "an" means one or more of that noun; for example, a "nucleotide sequence" is understood to refer to one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0084] Furthermore, "and / or" as used herein is understood as a specific disclosure of each of the two specified features or components with or without the other. Thus, when the term "and / or" is used herein in a phrase such as "A and / or B", it is intended to include "A and B", "A or B", "A" (only), and "B" (only). Similarly, when the term "and / or" is used in a phrase such as "A, B, and / or C", it is intended to include each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (only); B (only); and C (only).

[0085] Where an embodiment is described herein by the language "comprising," it should be understood that other similar embodiments described in the terms "consisting of" and / or "consisting essentially of" are also provided.

[0086] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains.For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press;The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press;and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press can provide one of the skills in many general dictionaries of the terms used in this disclosure.

[0087] Units, prefixes, and symbols are designated in their International System of Units (SI) accepted form. Numeric ranges include the numbers that define the range. Unless otherwise stated, amino acid sequences are written from left to right in the amino to carboxy direction. The headings provided herein are not limitations of the various aspects of the disclosure. Thus, the terms defined immediately below are more fully defined by reference to the entire specification.

[0088] The term "about" is used herein to mean approximately, roughly, around, or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the upper and lower limits of the stated numerical values. In general, the term "about" can modify a numerical value above and below the stated value, for example, by a variation of 10 percent above or below (higher or lower) the stated value. In some embodiments, the term indicates a variation of ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, or ±0.01% from the stated numerical value. In some embodiments, "about" indicates a variation of ±10% from the stated numerical value. In some embodiments, "about" indicates a ±5% variation from the indicated numerical value. In some embodiments, "about" indicates a ±4% variation from the indicated numerical value. In some embodiments, "about" indicates a ±3% variation from the indicated numerical value. In some embodiments, "about" indicates a ±2% variation from the indicated numerical value. In some embodiments, "about" indicates a ±1% variation from the indicated numerical value. In some embodiments, "about" indicates a ±0.9% variation from the indicated numerical value. In some embodiments, "about" indicates a ±0.8% variation from the indicated numerical value. In some embodiments, "about" indicates a ±0.7% variation from the indicated numerical value. In some embodiments, "about" indicates a ±0.6% variation from the indicated numerical value. In some embodiments, "about" indicates a ±0.5% variation from the indicated numerical value. In some embodiments, "about" indicates a ±0.4% variation from the indicated numerical value. In some embodiments, "about" indicates a ±0.3% variation from the indicated numerical value. In some embodiments, "about" indicates a ±0.1% variation from the indicated numerical value. In some embodiments, "about" indicates a ±0.05% variation from the indicated numerical value.In some embodiments, "about" indicates a ±0.01% variation from the indicated numerical value.

[0089] Depending on the context, the term "polynucleotide" or "nucleotide" can encompass a single nucleic acid as well as multiple nucleic acids. In some embodiments, a polynucleotide is an isolated nucleic acid molecule or construct, such as a messenger RNA (mRNA) or a plasmid DNA (pDNA). In some embodiments, a polynucleotide comprises a conventional phosphodiester bond. In some embodiments, a polynucleotide comprises a non-conventional bond (e.g., an amide bond, such as those found in peptide nucleic acids (PNAs)). The term "nucleic acid" can refer to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide. By "isolated" nucleic acid or polynucleotide is intended a nucleic acid molecule, DNA, or RNA, that has been removed from its natural environment. For example, a recombinant polynucleotide encoding a Factor VIII polypeptide contained in a vector is considered isolated for the purposes of this disclosure. Further examples of isolated polynucleotides include recombinant polynucleotides that are maintained in a heterologous host cell or that have been purified (partially or substantially) from other polynucleotides in solution. The isolated RNA molecule includes the in vivo or in vitro RNA transcript of the polynucleotide of the present disclosure.The isolated polynucleotide or nucleic acid according to the present invention further includes such synthetically produced molecules.The polynucleotide or nucleic acid may further include regulatory elements, such as promoters, enhancers, ribosome binding sites, or transcription termination signals.

[0090] Certain proteins secreted by mammalian cells are accompanied by secretory signal peptides that are cleaved from the mature protein after the export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those skilled in the art are aware that signal peptides are generally fused to the N-terminus of a polypeptide and cleaved from the complete or "full-length" polypeptide to produce a secreted or "mature" form of the polypeptide. In some embodiments, the native signal peptide or a functional derivative of its sequence maintains the ability to direct the secretion of a polypeptide operably associated therewith. Alternatively, a heterologous mammalian signal peptide can be used, such as human tissue plasminogen activator (TPA) or mouse β-glucuronidase signal peptide or a functional derivative thereof.

[0091] As used herein, the term "polypeptide" is intended to encompass the singular form "polypeptide" and the plural form "polypeptides" and refers to a molecule composed of monomers (amino acids) linked in a linear chain by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain(s) of two or more amino acids and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein", "amino acid chain", or any other term used to refer to a chain(s) of two or more amino acids are included within the definition of "polypeptide", and the term "polypeptide" can be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to post-expression modifications of the polypeptide, such as, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. A polypeptide can be obtained from a natural biological source or can be produced by recombinant technology, and is not necessarily translated from a designated nucleic acid sequence. It can be produced in any manner, including chemical synthesis.

[0092] An "isolated" polypeptide or a fragment, variant, or derivative thereof refers to a polypeptide that is not present in its natural environment. A particular level of purification is not required. For example, an isolated polypeptide can simply be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purposes of this disclosure if they are natural or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0093] Polypeptide fragments or variants, and any combination thereof, are also included in the present disclosure. The term "fragment" or "variant", when referring to a polypeptide binding domain or binding molecule of the present disclosure, encompasses any polypeptide that retains at least some properties of the reference polypeptide (e.g., FcRn binding affinity for FcRn binding domain or Fc variant, coagulation activity for FVIII variant, or FVIII binding activity for VWF fragment). A fragment of a polypeptide includes proteolytic fragments, as well as deletion fragments, in addition to the specific antibody fragments described elsewhere herein, but does not include naturally occurring full-length polypeptides (or mature polypeptides). A variant of a polypeptide binding domain or binding molecule of the present disclosure also includes fragments as described above, as well as polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. A variant may be naturally occurring or non-naturally occurring. Non-naturally occurring variants can be generated using mutagenesis techniques known in the art. A variant polypeptide may include conservative or non-conservative amino acid substitutions, deletions, or additions.

[0094] The term "VWF protein" as used herein means that it interacts with FVIII and maintains at least one or more properties normally provided to FVIII by full-length VWF, such as, for example, preventing premature activation to FVIIIa, preventing premature proteolysis, preventing clearance, preventing association with phospholipid membranes that may result in premature clearance, preventing binding to FVIII clearance receptors that can bind naked FVIII but not VWF-bound FVIII, and / or stabilizing the interaction of FVIII heavy and light chains. A VWF fragment as referred to herein is a VWF polypeptide that is less than a full-length VWF protein, and the VWF fragment maintains the ability to interact with and / or bind to FVIII. In some embodiments, the VWF protein is a fragment of full-length VWF (which may be mutated) that binds to the FVIII protein, such that the FVIII protein has reduced binding to full-length VWF (e.g., endogenous VWF in a subject) or does not bind to full-length VWF.

[0095] "Conservative amino acid substitution" refers to the substitution of an amino acid residue with a similar side chain.Families of amino acid residues with similar side chains have been defined in the art, including, for example, amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).Thus, if an amino acid of a polypeptide is replaced with another amino acid from the same side chain family, the substitution is considered to be conservative. In some embodiments, amino acid sequences can be conservatively substituted with structurally similar sequences that differ in the order and / or position of side chain family members.

[0096] As known in the art, "sequence identity" between two polypeptides is determined by comparing the amino acid sequence of one polypeptide with the sequence of a second polypeptide. Similarly, "sequence identity" between two polynucleotides is determined by comparing the nucleotide sequence of one polynucleotide with the sequence of a second polynucleotide. The term "% identical", "% identity", or similar terms is intended to mean, in particular, the percentage of nucleotides or amino acids (where appropriate) that are identical in optimal alignment between the sequences being compared. Said percentage is purely statistical, and the differences between the two sequences may, but are not necessarily, randomly distributed over the entire length of the sequences being compared. Usually, the comparison of two sequences is performed by comparing the sequences after optimal alignment for a segment or "window of comparison" to identify local regions of corresponding sequences. For example, optimal alignment for comparison can be performed manually or with the aid of the local homology algorithm of Smith and Waterman, 1981, Ads App.Math.2, 482, with the aid of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol.Biol.48, 443, with the aid of the similarity search algorithm of Pearson and Lipman, 1988, Proc.Natl Acad.Sci.USA 88, 2444, or with the aid of computer programs that use said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).In some embodiments, the percent identity of two sequences is determined using the BLASTN or BLASTP algorithm available at the National Center for Biotechnology Information (NCBI) website (e.g., at blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK _LOC=align2seq). In some embodiments, the algorithm parameters used for the BLASTN algorithm on the NCBI website include: (i) setting the prediction threshold to 10; (ii) setting the word size to 28; (iii) setting the maximum matches in the query range to 0; (iv) setting the match / mismatch score to 1, -2; (v) setting the gap cost to linear; and (vi) using a filter for low complexity regions. In some embodiments, the algorithm parameters used for the BLASTP algorithm of the NCBI website include: (i) prediction threshold set to 10; (ii) word size set to 3; (iii) maximum match in query range set to 0; (iv) matrix set to BLOSUM62; (v) gap cost set to presence: 11 extension: 1; and (vi) conditional compositional score matrix adjustment. As described herein, whether any particular polypeptide is at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to another polypeptide can be identified using methods and computer programs / software known in the art, such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711).BESTFIT uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981) to find the best segment of homology between two sequences. When using BESTFIT or other sequence alignment programs to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the present disclosure, it is understood that parameters are set such that the percentage of identity is calculated over the entire length of the reference polypeptide sequence and that gaps in identity of up to 5% of the total number of amino acids in the reference sequence are allowed.

[0097] As used herein, a "corresponding amino acid" or "equivalent amino acid" in a VWF or FVIII protein sequence is identified by an alignment that maximizes identity or similarity between a first VWF or FVIII sequence and a second VWF or FVIII sequence. The numerical value used to identify an equivalent amino acid in a second VWF or FVIII sequence is based on the numerical value used to identify the corresponding amino acid in the first VWF or FVIII sequence.

[0098] As used herein, the term "insertion site" refers to a position in a FVIII polypeptide, or a fragment, variant, or derivative thereof, that is immediately downstream of the position where a half-life extending moiety or a heterologous moiety can be inserted. An "insertion site" is designated as a numerical value, which is the number of amino acids in mature native FVIII (SEQ ID NO: 9) to which the insertion site corresponds, which is the C-terminus immediately adjacent to the insertion site. For example, the phrase "comprises an ELNN polypeptide at an insertion position corresponding to amino acid 1656 of SEQ ID NO: 9" indicates that the heterologous moiety is located between two amino acids corresponding to amino acid 1656 and amino acid 1657 of SEQ ID NO: 9.

[0099] The phrase "immediately downstream of an amino acid" as used herein means a position immediately adjacent to the terminal carboxyl group of an amino acid. For example, an insertion position immediately downstream of amino acid 745 corresponding to the mature wild-type FVIII protein (SEQ ID NO: 9) means that the insertion site is between amino acids 745 and 746 corresponding to the mature wild-type FVIII. Similarly, the phrase "immediately upstream of an amino acid" means a position immediately adjacent to the terminal amino group of an amino acid.

[0100] The phrase "between two amino acids at the insertion site" as used herein refers to the position where an ELNN polypeptide or any other polypeptide is inserted between two adjacent amino acids. Thus, the phrases "inserted immediately downstream of an amino acid" and "inserted between two amino acids at the insertion site" are used interchangeably with "inserted at the insertion site."

[0101] The term "inserted", "inserted", "inserted into", or grammatically related terms, when used herein with respect to the insertion of an ELNN polypeptide into FVIII, refers to the position of the ELNN polypeptide in the chimeric protein relative to the analogous position in native mature human FVIII. As used herein, the term refers to the properties of the recombinant FVIII polypeptide relative to native mature human FVIII, and does not indicate, imply, or infer any method or process of making the chimeric protein. For example, with respect to the chimeric protein provided herein, the phrase "the ELNN polypeptide is inserted immediately downstream of residue 745 of the FVIII polypeptide" means that the chimeric protein comprises an ELNN polypeptide immediately downstream of the amino acid corresponding to amino acid 745 in native mature human FVIII, for example, linked to amino acids corresponding to amino acids 745 and 746 of native mature human FVIII (without requiring the presence of an amino acid corresponding to 746 of native mature human FVIII), and does not imply the order or method of making the chimeric protein.

[0102] As used herein, the terms "ELNN polypeptide" and "ELNN" are synonymous and refer to a polypeptide that is extended in length by a non-naturally occurring, substantially non-repetitive sequence that is composed mainly of small hydrophilic amino acids, which have no or only a low degree of secondary or tertiary structure under physiological conditions. ELNNs can confer certain desirable pharmacokinetic, physicochemical, and pharmaceutical properties when linked to the VWF protein or FVIII sequence of the present disclosure to create chimeric polypeptides. Such desirable properties include, but are not limited to, enhanced pharmacokinetic parameters and solubility properties. As used herein, the terms "ELNN polypeptide" and "ELNN" specifically exclude antibodies or antibody fragments, such as single chain antibodies or Fc fragments of light or heavy chains. ELNN polypeptides are known in the art, and non-limiting descriptions and examples related to ELNN polypeptides known as XTEN® polypeptides are available in Schellenberger et al., (2009) Nat Biotechnol 27(12):1186-90; Brandl et al., (2020) Journal of Controlled Release 327:186-197; and Radon et al., (2021) Advanced Functional Materials 31, 2101633 (pages 1-33), the entire contents of each of which are incorporated herein by reference.

[0103] A "fusion" or "chimeric" protein comprises a first amino acid sequence linked to a second amino acid sequence that is not linked in nature. Amino acid sequences that are normally present in separate proteins can be brought together in a fusion polypeptide, or amino acid sequences that are normally present in the same protein can be placed in a new arrangement in a fusion polypeptide, such as the fusion of the factor VIII domain and the Ig Fc domain of the present disclosure. Fusion proteins are produced, for example, by chemical synthesis, or by creating or translating a polynucleotide in which the peptide regions are encoded in the desired relationship. Chimeric proteins can further comprise a second amino acid sequence associated with the first amino acid sequence by a covalent non-peptide bond or a non-covalent bond.

[0104] The term "linked" as used herein with respect to sequences means that a first amino acid sequence or nucleotide sequence is covalently or non-covalently linked to a second amino acid sequence or nucleotide sequence, respectively. The first amino acid sequence or nucleotide sequence can be directly joined or juxtaposed to the second amino acid sequence or nucleotide sequence, or alternatively, the first sequence can be covalently joined to the second sequence by an intervening sequence. Depending on the context, the term "linked" does not only mean the fusion of the first amino acid sequence with the second amino acid sequence at the C-terminus or N-terminus, but also includes the insertion of the entire first amino acid sequence (or second amino acid sequence) into any two amino acids in the second amino acid sequence (or first amino acid sequence, respectively). In some embodiments, the first amino acid sequence can be linked to the second amino acid sequence by a peptide bond or a linker. The first nucleotide sequence can be linked to the second nucleotide sequence by a phosphodiester bond or a linker. The linker may be a peptide or polypeptide (in the case of a polypeptide chain), or a nucleotide or a chain of nucleotides (in the case of a nucleotide chain), or any chemical moiety (in the case of both polypeptide and polynucleotide chains). The term "linked" may also be indicated by a hyphen (-).

[0105] With respect to two polypeptides, the term "associated with" refers to one or more covalent or non-covalent bonds formed between the first and second polypeptides. In some embodiments, the term "associated with" refers to a covalent non-peptide bond or non-covalent bond. This association can be indicated by a colon, i.e., (:). In some embodiments, it refers to a covalent bond excluding a peptide bond. For example, the amino acid cysteine ​​contains a thiol group that can form a disulfide bond or bridge with a thiol group on a second cysteine ​​residue. In most naturally occurring IgG molecules, the CH1 and CL regions are associated by disulfide bonds, and the two heavy chains are associated by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (positions 226 or 229, EU numbering system). Examples of covalent bonds include, but are not limited to, peptide bonds, metal bonds, hydrogen bonds, disulfide bonds, σ bonds, π bonds, δ bonds, glycosidic bonds, an agnostic bond, a bent bond, a dipolar bond, a π back bond, a double bond, a triple bond, a quadruple bond, a quintuple bond, a sextuple bond, conjugation, hyperconjugation, aromaticity, hapticity, or an antibond. Non-limiting examples of non-covalent bonds include ionic bonds (e.g., cation-π bonds or salt bonds), metal bonds, hydrogen bonds (e.g., dihydrogen bonds, dihydrogen complexes, low barrier hydrogen bonds, or symmetric hydrogen bonds), van der Waals forces, London dispersion forces, mechanical bonds, halogen bonds, aurophilic, intercalation, stacking, entropic forces, or chemical polarity. In some embodiments, the one or more covalent bonds between the first amino acid chain and the second amino acid chain are two disulfide bonds. In some embodiments, the one or more covalent bonds between the first amino acid chain and the second amino acid chain are two disulfide bonds between a first Fc moiety on the first amino acid chain and a second Fc moiety on the second amino acid chain, the two disulfide bonds occurring in the hinge region of the two Fc moieties.

[0106] In some embodiments, the polypeptide has an enzymatic cleavage site that is cleaved by an enzyme activated during the coagulation cascade, such that cleavage of such a site occurs at the site of thrombus formation. Examples of such sites include those recognized by, for example, thrombin, factor XIa, or factor Xa. Other enzymatic cleavage sites of SEQ ID NO:9, SEQ ID NO:15 are known in the art and are described elsewhere herein. It will be understood that in constructs that include more than one processing or cleavage site, such sites may be the same or different. As used herein, the term "half-life" refers to the biological half-life of a particular polypeptide in vivo. Half-life can be represented by the time required for an amount administered to a subject to be cleared from the blood circulation and / or other tissues of the animal to become half its amount. In some embodiments, when a clearance curve of a given polypeptide is constructed as a function of time, the curve is usually biphasic with a rapid alpha phase and a longer beta phase. The α phase typically represents the equilibrium between intravascular and extravascular spaces of the administered Fc polypeptide, and is determined in part by the size of the polypeptide. The β phase typically represents the catabolism of the polypeptide in the intravascular space. In some embodiments, FVIII and chimeric proteins comprising FVIII are monophasic, and therefore have no α phase, but only a single β phase. Thus, in some embodiments, the term half-life as used herein refers to the half-life of a polypeptide in β phase. The typical β phase half-life of a human antibody in humans is 21 days. In some embodiments, the half-life is expressed as the half-life of the terminal phase.

[0107] In some embodiments, the subject has hemophilia A. In some embodiments, the hemophilia A is severe hemophilia A.

[0108] "Administer" or "administering" as used herein means delivering a composition, e.g., a chimeric protein, described herein, to a subject. The composition, e.g., a chimeric protein, can be administered to a subject using methods known in the art. In particular, the composition can be administered intravenously, subcutaneously, intramuscularly, intradermally, or via any mucosal surface, e.g., orally, sublingually, bucally, nasally, rectally, vaginally, or via a pulmonary route. In some embodiments, administration is intravenous. In some embodiments, administration is subcutaneous. In some embodiments, administration is self-administration. In some embodiments, a parent administers the chimeric protein to a child. In some embodiments, the chimeric protein is administered to a subject by a medical professional, such as a doctor, a medical professional, or a nurse.

[0109] As used herein, the term "dose" refers to a single administration of a composition to a subject. A single dose can be administered all at once, e.g., as a bolus, or over a period of time, e.g., by intravenous infusion. The term "multiple doses" refers to two or more doses, e.g., two or more administrations.

[0110] When co-administration of two or more compositions is referred to, a dose of composition A can be administered simultaneously with a dose of composition B. Alternatively, a dose of composition A can be administered before or after a dose of composition B. In some embodiments, composition A and composition B are combined into a single formulation.

[0111] As used herein, the term "interval" or "dosing interval" refers to the amount of time that elapses between an initial dose of Composition A and subsequent doses of the same composition are administered to a subject. The dosing interval can refer to the time that elapses between a first dose and a second dose, or the dosing interval can refer to the amount of time that elapses between multiple doses.

[0112] The term "dosing frequency" as used herein refers to the number of doses administered per particular dosing interval. For example, dosing frequency can be described as once per week, once every two weeks, etc. Thus, a seven day dosing interval can also be described as once per seven days, or once per week, or a weekly dosing interval.

[0113] As used herein, the term "prophylactic treatment" refers to the administration of a therapy for the treatment of hemophilia A, where such treatment is intended to prevent or reduce the severity of one or more symptoms of hemophilia A, such as bleeding episodes, such as one or more spontaneous bleeding episodes, and / or joint damage. See Jimenez-Yuste et al., Blood Transfus. 12(3):314-19 (2014). To prevent or reduce the severity of such symptoms, such as bleeding episodes and joint disease, patients with hemophilia A may receive regular infusions of clotting factors as part of a prophylactic treatment regimen. The basis for such prophylactic treatment is the observation that hemophilia patients with clotting factors, such as FVIII, at levels of 1% or higher rarely experience spontaneous bleeding episodes and have fewer hemophilia-related complications compared to severe hemophilia patients. See, e.g., Coppola A. et al, Semin.Thromb.Hemost.38(1):79-94 (2012). Medical personnel treating these hemophilia patients speculated that maintaining factor levels at approximately 1% with regular infusions could potentially reduce the risk of hemophilia symptoms, such as bleeding episodes and joint damage. See ibid. Subsequent studies confirmed these benefits in pediatric hemophilia patients receiving prophylactic treatment with clotting factors, making prophylactic treatment a goal for patients with severe hemophilia. See ibid.

[0114] "Prophylactic" treatment can also mean preemptive administration of a composition, e.g., a protein (e.g., a chimeric protein, etc.) described herein to a subject to control, manage, prevent, or reduce the onset or severity of one or more symptoms, e.g., bleeding symptoms, of hemophilia A. In some embodiments, prophylactic treatment with a clotting factor, e.g., FVIII, is used to treat a subject with severe hemophilia A. In some embodiments, prophylactic treatment means administering a composition disclosed herein to a subject in need thereof to reduce the onset of one or more symptoms of hemophilia A. Prophylactic treatment can include administration of multiple doses. Multiple doses used in prophylactic treatment are typically administered at a specific dosing interval. In some embodiments, the annualized bleeding rate can be reduced to less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1.

[0115] The term "on-demand treatment" or "episodic treatment" refers to administration of the chimeric molecule "on demand" in response to a symptom of hemophilia A, e.g., a bleeding episode, or prior to activation that may cause bleeding. In some embodiments, the on-demand treatment can be given to a subject when bleeding begins, e.g., after an injury, or when bleeding is expected, e.g., prior to surgery. In some embodiments, the on-demand treatment can be given prior to an activity that increases the risk of bleeding, e.g., contact sports, etc. In some embodiments, the on-demand treatment is given as a single dose. In some embodiments, the on-demand treatment is given as an initial dose, followed by one or more additional doses. When the chimeric protein is administered on demand, the one or more additional doses can be administered at least about 12 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, at least about 108 hours, or at least about 120 hours after the initial dose. It should be noted, however, that the dosing intervals associated with on-demand replacement therapy are not the same as those used for prophylactic treatment.

[0116] In some embodiments, the subject in need of a general hemostatic agent is undergoing or about to undergo surgery. The chimeric protein of the present disclosure can be administered before or after surgery. The chimeric protein of the present disclosure can also be administered during or after surgery to control acute bleeding episodes. When the chimeric protein is administered before surgery, the administration can be at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, or at least about 72 hours before surgery. When the chimeric protein is administered after surgery, the administration can be at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, or at least about 72 hours after surgery. The surgery can include, but is not limited to, liver transplantation, liver resection, dental procedure, or stem cell transplantation.

[0117] "Treat", "treatment", "treating" as used herein means, for example, reducing the severity of a disease or condition; reducing the duration of the course of a disease; ameliorating one or more symptoms associated with a disease or condition; providing a beneficial effect to a subject having a disease or condition, but not necessarily curing the disease or condition; or preventing one or more symptoms associated with a disease or condition. In some embodiments, treating or treatment includes maintaining FVIII trough levels in a subject at least about 1 IU / dL, 2 IU / dL, 3 IU / dL, 4 IU / dL, 5 IU / dL, 6 IU / dL, 7 IU / dL, 8 IU / dL, 9 IU / dL, 10 IU / dL, 11 IU / dL, 12 IU / dL, 13 IU / dL, 14 IU / dL, 15 IU / dL, 16 IU / dL, 17 IU / dL, 18 IU / dL, 19 IU / dL, or 20 IU / dL by administering a chimeric protein of the present disclosure. As used herein, a "trough level" in a hemophilia A patient is a measurement of the lowest concentration reached by factor therapy, e.g., FVIII therapy, before the next dose is administered. In some embodiments, treating or treatment means maintaining a FVIII trough level at least about 1 IU / dL during a dosing interval. In some embodiments, treating or treatment means maintaining a FVIII trough level at least about 3 IU / dL during a dosing interval. In some embodiments, treating or treatment means maintaining a FVIII trough level at least about 5 IU / dL during a dosing interval. In some embodiments, treating or treatment means maintaining a FVIII trough level at about 1 to about 20 IU / dL, about 2 to about 20 IU / dL, about 3 to about 20 IU / dL, about 4 to about 20 IU / dL, about 5 to about 20 IU / dL, about 6 to about 20 IU / dL, about 7 to about 20 IU / dL, about 8 to about 20 IU / dL, about 9 to about 20 IU / dL, or about 10 to about 20 IU / dL during a dosing interval.

[0118] In some embodiments, treating or treating a disease or condition includes maintaining FVIII activity in a subject at a level comparable to at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the FVIII activity in a non-hemophilic subject during a dosing interval. In some embodiments, treating or treating means maintaining FVIII activity level at at least about 1% during a dosing interval. In some embodiments, treating or treating means maintaining FVIII activity level at at least about 2% during a dosing interval. In some embodiments, treating or treating means maintaining FVIII activity level at at least about 3% during a dosing interval. In some embodiments, treating or treating means maintaining FVIII activity level at at least about 4% during a dosing interval. In some embodiments, treating or treatment means maintaining a FVIII activity level of at least about 5% during a dosing interval. In some embodiments, treating or treatment means maintaining a FVIII activity level of at least about 6% during a dosing interval. In some embodiments, treating or treatment means maintaining a FVIII activity level of at least about 7% during a dosing interval. In some embodiments, treating or treatment means maintaining a FVIII activity level of at least about 8% during a dosing interval. In some embodiments, treating or treatment means maintaining a FVIII activity level of at least about 9% during a dosing interval. In some embodiments, treating or treatment means maintaining a FVIII activity level of at least about 10% during a dosing interval. In some embodiments, the minimum trough level required for treatment can be measured by one or more known methods (e.g., activated partial thromboplastin time (aPTT) assay or chromogenic assay, which are well known in the art) and can be adjusted (increased or decreased) for each individual.Non-limiting examples of assays for measuring trough levels are disclosed in U.S. Patent Application Publication No. 20190375822, which is incorporated by reference in its entirety.

[0119] II. Chimeric Proteins In some embodiments, the present disclosure is directed to a chimeric protein or pharmaceutical composition comprising a protein comprising a first polypeptide chain comprising a factor VIII ("FVIII") protein or a portion thereof and a first immunoglobulin ("Ig") constant region or a portion thereof, and a second polypeptide chain comprising a von Willebrand factor ("VWF") protein and a second Ig constant region or a portion thereof. In some embodiments, the chimeric protein comprises: (i) a FVIII protein comprising a FVIII polypeptide, an ELNN polypeptide inserted in the B domain of the FVIII polypeptide, and a first Fc region; and (ii) a VWF protein comprising a VWF fragment, a second ELNN polypeptide sequence, an a2 linker, and a second Fc region. In some embodiments, the chimeric protein disclosed herein is a FVIII-ELNN-Fc / D'D3-ELNN-Fc heterodimer.

[0120] In some embodiments, the chimeric protein is efanesoctocog alfa. Efanesoctocog alfa, also known as "BIVV001", "efanesoctocogum alfa", "rFVIIIFc-VWF-ELNN" and "rFVIIIFc-VWF-XTEN", is described in Chhabra et al. Blood 135(17):1484-1496 (2020), the entire contents of which are incorporated herein by reference in their entirety. A schematic diagram of efanesoctocog alfa as an exemplary FVIII-ELNN-Fc / D'D3-ELNN-Fc heterodimer is shown in FIG. 1.

[0121] Evanesoctocog alfa is a large protein (over 300 kDa) that contains multiple moieties on each of two polypeptide chains that are associated by covalent and non-covalent interactions. The protein has a tendency to aggregate under certain conditions, which can reduce the stability of the formulation unless an excipient, such as L-arginine, is selected and present in sufficient amounts to reduce aggregation. For example, aggregation can be reduced by adding high levels (e.g., about 250 mM) of L-arginine.

[0122] Additional information regarding efanesectocog alfa can be found in International Nonproprietary Names for Pharmaceutical Substances (INN) WHO Drug Information, 2019, Vol. 33, No. 4, p. 828-30. In some embodiments, the chimeric protein is a FVIII-ELNN-Fc / D'D3-ELNN-Fc heterodimer comprising (i) a first polypeptide comprising the amino acid sequence of SEQ ID NO:1, and (ii) a second polypeptide comprising the amino acid sequence of SEQ ID NO:2. In some embodiments, the chimeric protein comprises (i) a first polypeptide, and (ii) a second polypeptide covalently linked via one or more disulfide bonds (e.g., two disulfide bonds). In some embodiments, the chimeric protein comprises a FVIII protein encoded by the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the chimeric protein comprises a VWF protein encoded by the nucleic acid sequence of SEQ ID NO:6. In some embodiments, efanesectocog alfa has an activity of at least 1600 IU / mg. In some embodiments, efanesectocog alfa has an activity of at least 1700 IU / mg. In some embodiments, efanesectocog alfa has an activity of at least 1800 IU / mg. In some embodiments, efanesectocog alfa has an activity of at least 1900 IU / mg. In some embodiments, efanesectocog alfa has an activity of 1600 IU / mg to 2000 IU / mg.

[0123] In some embodiments, the chimeric protein comprises a FVIII protein comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the chimeric protein comprises a FVIII protein comprising one or more disulfide bridges at one or more of the following positions: residues 153-179, 248-329, 528-554, 630-711, 1220-1246, 1287-1291, 1409-1557, 1562-1714, 1761-1821, and / or 1867-1925 of SEQ ID NO:1. In some embodiments, the chimeric protein comprises a FVIII protein comprising one or more disulfide bridges at each of the following positions: residues 153-179, 248-329, 528-554, 630-711, 1220-1246, 1287-1291, 1409-1557, 1562-1714, 1761-1821, and 1867-1925 of SEQ ID NO:1. In some embodiments, the chimeric protein comprises a FVIII protein comprising one or more Cys-SH residues at residues 310, 692, and / or 1388 of SEQ ID NO:1. In some embodiments, the chimeric protein comprises a FVIII protein comprising a Cys-SH residue at each of residues 310, 692, and / or 1388 of SEQ ID NO:1.

[0124] In some embodiments, the chimeric protein comprises a FVIII protein that comprises one or more N-glycosylation sites at residues N41, N239, N1198, N1506, and / or N1797 of SEQ ID NO: 1. In some embodiments, the chimeric protein comprises a FVIII protein that comprises one or more O-glycosylation sites at residues 746-1036 of SEQ ID NO: 1 and / or Ser and Thr residues in the linker peptide. In some embodiments, the chimeric protein comprises a FVIII protein that comprises one or more Tyr-sulfation sites at residues 346, 718, 719, 723, 729, 1052, and / or 1068 of SEQ ID NO: 1.

[0125] In some embodiments, the chimeric protein comprises a VWF protein comprising the amino acid sequence of SEQ ID NO:2. In some embodiments, the chimeric protein comprises a VWF protein comprising one or more disulfide bridges at one or more of the following positions: residues 4-45, 13-41, 25-36, 29-64, 47-58, 66-88, 83-100, 86-95, 104-233, 126-268, 135-230, 151-158, 283-326, 297-321, 308-348, 328-334, 338-363, 367-410, 386-406, 390-402, 394-433, 414-427, 436-464, 459-474, 462-471, 698-758, and / or 804-862 of SEQ ID NO:2. In some embodiments, the chimeric protein comprises a VWF protein comprising one or more disulfide bridges at each of the following positions: residues 4-45, 13-41, 25-36, 29-64, 47-58, 66-88, 83-100, 86-95, 104-233, 126-268, 135-230, 151-158, 283-326, 297-321, 308-348, 328-334, 338-363, 367-410, 386-406, 390-402, 394-433, 414-427, 436-464, 459-474, 462-471, 698-758, and / or 804-862 of SEQ ID NO:2.

[0126] In some embodiments, the chimeric protein comprises a VWF protein comprising one or more N-glycosylation sites at residues N94, N384, N734 of SEQ ID NO:2. In some embodiments, the chimeric protein comprises a VWF protein comprising one or more O-glycosylation sites at residues 478-625 of SEQ ID NO:2 and / or Ser and Thr residues in the linker peptide. In some embodiments, the chimeric protein comprises a VWF protein comprising one or more Tyr-sulfation sites at residues 632, 633, 637, and / or 643 of SEQ ID NO:2. In some embodiments, the VWF protein comprises a VWF fragment comprising the D1, D2, D', and / or D3 domains of VWF. In one embodiment, the VWF fragment comprises the D1D2 region of VWF comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, the VWF protein further comprises a VWF signal peptide sequence. In one embodiment, the VWF signal peptide comprises the amino acid sequence of SEQ ID NO:19. In one specific embodiment, the VWF protein comprises a VWF signal peptide comprising the amino acid sequence of SEQ ID NO: 19, a VWF D1D2 region comprising the amino acid sequence of SEQ ID NO: 20, a VWF D' domain comprising the amino acid sequence of SEQ ID NO: 21, a VWF D3 domain comprising the amino acid sequence of SEQ ID NO: 22, an ELNN polypeptide sequence (AE144_5A) comprising the amino acid sequence of SEQ ID NO: 14, an a2 linker comprising the amino acid sequence of SEQ ID NO: 15, and / or an Fc region comprising the amino acid sequence of SEQ ID NO: 23.

[0127] In some embodiments, the chimeric protein of the disclosure comprises: (i) a FVIII protein comprising a FVIII polypeptide, a first ELNN polypeptide sequence, and a first Fc region; and (ii) a VWF fragment comprising a VWF D' domain and a VWF D3 domain, a second ELNN polypeptide sequence, a FVIII a2 linker, and a second Fc region, wherein the FVIII protein has a deletion of amino acids 746 to 1648 corresponding to mature FVIII; and the first ELNN polypeptide sequence has a deletion of amino acids 746 to 1648 corresponding to mature FVIII. a first ELNN polypeptide sequence having an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of AE288 (SEQ ID NO: 9); a first Fc region is fused to the C-terminus of the FVIII polypeptide; a2 linker is fused to the C-terminus of the ELNN polypeptide; the second ELNN polypeptide sequence comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of AE144_5A (SEQ ID NO: 14); The mosquito comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO:9; the second Fc region is fused to the C-terminus of the a2 linker; and the first Fc region is covalently linked to the second Fc region by a disulfide bond (e.g., two disulfide bonds).

[0128] In some embodiments, a chimeric protein of the present disclosure comprises two polypeptide sequences, a first polypeptide sequence comprises an amino acid sequence at least about 80%, 90%, 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 1; and a second polypeptide sequence comprises a VWF fragment comprising a VWF D' domain, a VWF D3 domain, and an Fc region. In some embodiments, a chimeric protein of the present disclosure comprises two polypeptide sequences, a first polypeptide sequence comprises a FVIII polypeptide and an Fc region; and a second polypeptide sequence comprises an amino acid sequence at least about 80%, 90%, 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, a chimeric protein of the present disclosure comprises two polypeptide sequences, a first polypeptide sequence comprises an amino acid sequence at least about 80%, 90%, 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:1; and a second polypeptide sequence comprises an amino acid sequence at least about 80%, 90%, 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, a chimeric protein of the present disclosure comprises two polypeptide sequences, a first polypeptide sequence comprises an amino acid sequence set forth in SEQ ID NO:7, and a second polypeptide sequence comprises an amino acid sequence set forth in SEQ ID NO:2. In some embodiments, a chimeric protein of the present disclosure comprises two polypeptide sequences, a first polypeptide sequence comprises an amino acid sequence set forth in SEQ ID NO:1, and a second polypeptide sequence comprises an amino acid sequence set forth in SEQ ID NO:2, and the first and second polypeptide sequences are linked to each other by a disulfide bond. In some embodiments, a chimeric protein of the present disclosure comprises two polypeptide sequences, a first polypeptide sequence comprises the amino acid sequence set forth in SEQ ID NO:1, and a second polypeptide sequence comprises the amino acid sequence set forth in SEQ ID NO:2, and the first polypeptide sequence and the second polypeptide sequence are linked to each other by two disulfide bonds.In some embodiments, a chimeric protein of the present disclosure comprises two polypeptide sequences, a first polypeptide sequence comprising the amino acid sequence set forth in SEQ ID NO:1 and a second polypeptide sequence comprising the amino acid sequence set forth in SEQ ID NO:2, wherein the first polypeptide sequence comprises a first Fc portion and the second polypeptide sequence comprises a second Fc portion, and wherein the first Fc portion and the second Fc portion are linked to each other by two disulfide bonds in the hinge region.

[0129] In some embodiments, a chimeric protein of the present disclosure comprises a FVIII protein comprising an amino acid sequence that is at least about 80%, 90%, 95%, or 100% identical to SEQ ID NO:7, SEQ ID NO:3, or SEQ ID NO:1, and a VWF protein comprising an amino acid sequence that is at least about 80%, 90%, 95%, or 100% identical to SEQ ID NO:2 or SEQ ID NO:5.

[0130] In some embodiments, the chimeric protein of the present disclosure comprises: (i) a FVIII protein comprising a first FVIII polypeptide fragment comprising the amino acid sequence of SEQ ID NO: 17; a first ELNN polypeptide sequence comprising the amino acid sequence of SEQ ID NO: 9 (AE288); a second FVIII polypeptide fragment comprising the amino acid sequence of SEQ ID NO: 18; and a first Fc region comprising the amino acid sequence of SEQ ID NO: 23; and (ii) a VWF protein comprising a D' domain of VWF comprising the amino acid sequence of SEQ ID NO: 21; a D3 domain of VWF comprising the amino acid sequence of SEQ ID NO: 22; a second ELNN polypeptide sequence comprising the amino acid sequence of SEQ ID NO: 14 (AE144_5A); an a2 linker comprising the amino acid sequence of SEQ ID NO: 15; and a second Fc region comprising the amino acid sequence of SEQ ID NO: 23, wherein the first Fc region is covalently linked to the second Fc region by a disulfide bond (e.g., two disulfide bonds).

[0131] In some embodiments, a chimeric protein of the disclosure comprises a FVIII protein comprising a FVIII polypeptide, a first ELNN polypeptide sequence, a first Fc region, and a VWF protein comprising a VWF D' domain, a VWF D3 domain, a second ELNN polypeptide sequence, a FVIII a2 linker, and a second Fc region, wherein the FVIII polypeptide comprises the amino acid sequence of SEQ ID NO: 17, the first ELNN polypeptide sequence comprises the amino acid sequence of AE288 (SEQ ID NO: 9) and is fused to the C-terminus of SEQ ID NO: 17, the FVIII polypeptide further comprises the amino acid sequence of SEQ ID NO: 18, and the first Fc region comprises the amino acid sequence of SEQ ID NO: 20. the D' domain of VWF comprises the amino acid sequence of SEQ ID NO:21; the D3 domain of VWF comprises the amino acid sequence of SEQ ID NO:214, the second ELNN polypeptide sequence comprises the amino acid sequence of AE144_5A (SEQ ID NO:14) and is fused to the C-terminus of the D3 domain of VWF; the a2 linker comprises the amino acid sequence of SEQ ID NO:15 and is fused to the C-terminus of the second ELNN polypeptide; the second Fc region comprises the amino acid sequence of SEQ ID NO:23 and is fused to the C-terminus of the a2 linker; the first Fc region is covalently linked to the second Fc region by a disulfide bond.

[0132] In some embodiments, a chimeric protein of the present disclosure comprises a FVIII protein comprising a FVIII signal peptide comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, a chimeric protein comprises a VWF protein comprising a VWF signal peptide comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, a chimeric protein comprises a VWF protein comprising the D1D2 domain of VWF comprising the amino acid sequence of SEQ ID NO: 20.

[0133] In some embodiments, a chimeric protein comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:3, as well as a second polypeptide comprising the amino acid sequence of SEQ ID NO:5. In some embodiments, a chimeric protein comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:7, as well as a second polypeptide comprising the amino acid sequence of SEQ ID NO:2. In some embodiments, a chimeric protein comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:1, as well as a second polypeptide comprising the amino acid sequence of SEQ ID NO:2.

[0134] In some embodiments, the chimeric protein comprises one or more disulfide bridges between the first and second polypeptides. In some embodiments, the chimeric protein comprises two disulfide bridges between the first and second polypeptides. In some embodiments, the chimeric protein comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:1 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:2, and the chimeric protein comprises a disulfide bridge between residue 1726 of SEQ ID NO:1 and residue 663 of SEQ ID NO:2 as well as a disulfide bridge between residue 1729 of SEQ ID NO:1 and residue 666 of SEQ ID NO:2.

[0135] IV. Pharmaceutical Compositions In some aspects, the present disclosure is directed to pharmaceutical compositions of chimeric proteins that are formulated to improve protein stability. In some embodiments, the disclosed pharmaceutical compositions demonstrate increased stability based on analysis by visual inspection, protein concentration, pH stability, formation of high molecular weight species (HMWS), and / or changes in turbidity. Analysis of these characteristics of stability can be performed using conventional techniques, such as size exclusion chromatography (SEC), reversed-phase high performance liquid chromatography (RP-HPLC), as well as many others.

[0136] The pharmaceutical compositions disclosed herein comprise a specified amount of chimeric protein. In some embodiments, the pharmaceutical composition has a chimeric protein concentration of about 0.8 to about 1.2 mg / mL. In some embodiments, the pharmaceutical composition has a chimeric protein concentration of about 0.8 mg / mL. In some embodiments, the pharmaceutical composition has a chimeric protein concentration of about 0.9 mg / mL. In some embodiments, the pharmaceutical composition has a chimeric protein concentration of about 1.0 mg / mL. In some embodiments, the pharmaceutical composition has a chimeric protein concentration of about 1.1 mg / mL. In some embodiments, the pharmaceutical composition has a chimeric protein concentration of about 1.2 mg / mL.

[0137] In some embodiments, the pharmaceutical composition comprises about 75 IU / mL to about 2000 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 75 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 100 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 150 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 200 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 250 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 300 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 350 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 400 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 450 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 500 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 550 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 600 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 650 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 700 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 750 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 800 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 850 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 900 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 950 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 1000 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises more than about 250 IU of the chimeric protein. In some embodiments, the pharmaceutical composition comprises more than about 300 IU of the chimeric protein.In some embodiments, the pharmaceutical composition comprises more than about 500 IU of the chimeric protein. In some embodiments, the pharmaceutical composition comprises at least about 500 IU of the chimeric protein. In some embodiments, the pharmaceutical composition comprises more than about 85 IU / ml of the chimeric protein. In some embodiments, the pharmaceutical composition comprises more than about 100 IU / ml of the chimeric protein. In some embodiments, the pharmaceutical composition comprises more than about 200 IU / ml of the chimeric protein. In some embodiments, the pharmaceutical composition comprises at least about 200 IU / ml of the chimeric protein.

[0138] In some embodiments, the pharmaceutical composition comprises about 1100 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 1150 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 1200 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 1250 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 1300 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 1350 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 1400 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 1450 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 1500 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 1550 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 1600 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 1650 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 1700 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 1750 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 1800 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 1850 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 1900 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 1950 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 2000 IU / mL of the chimeric protein.

[0139] Pharmaceutical compositions containing the chimeric proteins of the present disclosure also contain suitable pharma- ceutically acceptable carriers, e.g., they can contain excipients and / or auxiliary agents that provide enhanced stability of the chimeric protein or facilitate processing of the active compound into preparations designed for delivery to the site of action.

[0140] In some embodiments, pharmaceutical compositions are disclosed herein that include a specified amount of chimeric protein with the disclosed excipients. The pharmaceutical compositions disclosed herein include various concentrations of the disclosed excipients, and the concentrations can be expressed in various ways. For example, the concentration of a given excipient can be expressed as a molar concentration (e.g., M or mM), as a weight / volume percentage (e.g., grams per 100 ml of diluent), or as milligrams per milliliter. The pharmaceutical compositions provided herein can contain the specified amount of various excipients at an approximation, e.g., a level of precision ranging from the expressed concentration to one significant digit (e.g., about 0.1% (w / v)), or more precisely, e.g., to two, three, four, five, or six significant digits (e.g., about 3.88 mg / ml with a precision of three significant digits). The level of precision required can vary, e.g., depending on the requirements of a given regulatory agency or manufacturing process.

[0141] In some embodiments, the pharmaceutical composition comprises 1% (w / v) to 4% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises 2% (w / v) to 5% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises 4% (w / v) to 8% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 1.5% (w / v) to about 2.5% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 5% (w / v) to about 7.5% (w / v) sucrose.

[0142] In some embodiments, the pharmaceutical composition comprises about 1.0% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 1.1% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 1.2% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 1.3% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 1.4% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 1.5% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 1.6% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 1.7% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 1.8% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 1.9% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 2.0% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 2.1% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 2.2% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 2.3% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 2.4% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 2.5% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 2.6% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 2.7% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 2.8% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 2.9% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 3.0% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 3.1% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 3.2% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 3.3% (w / v) sucrose.In some embodiments, the pharmaceutical composition comprises about 3.4% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 3.5% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 3.6% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 3.7% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 3.8% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 3.9% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 4.0% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 4.1% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 4.2% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 4.3% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 4.4% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 4.5% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 4.6% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 4.7% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 4.8% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 4.9% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 5% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 5.1% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 5.2% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 5.3% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 5.4% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 5.5% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 5.6% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 5.7% (w / v) sucrose.In some embodiments, the pharmaceutical composition comprises about 5.8% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 5.9% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 6% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 6.1% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 6.2% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 6.3% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 6.4% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 6.5% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 6.6% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 6.7% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 6.8% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 6.9% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 7% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 7.1% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 7.2% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 7.3% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 7.4% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 7.5% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 7.6% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 7.7% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 7.8% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 7.9% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 8% (w / v) sucrose.

[0143] In some embodiments, the pharmaceutical composition comprises about 33.67 mg of sucrose. In some embodiments, the pharmaceutical composition comprises about 67.34 mg of sucrose. In some embodiments, the pharmaceutical composition comprises about 168.35 mg of sucrose. In some embodiments, the amount of sucrose may vary up to 10% of the specified amount. In some embodiments, the amount of sucrose may vary up to 5% of the specified amount. In some embodiments, the amount of sucrose may vary up to 1% of the specified amount. In some embodiments, the specified amount of sucrose is 33.67 mg, 67.34 mg, or 168.35 mg.

[0144] The pharmaceutical composition disclosed herein may include a buffer.In some embodiments, the pharmaceutical composition disclosed herein includes a specified amount or concentration of histidine.In some embodiments, the histidine included in the pharmaceutical composition is L-histidine.In some embodiments, the pharmaceutical composition includes about 5mM to about 15mM of histidine.

[0145] In some embodiments, the pharmaceutical composition comprises about 5 mM histidine. In some embodiments, the pharmaceutical composition comprises about 5.5 mM histidine. In some embodiments, the pharmaceutical composition comprises about 6 mM histidine. In some embodiments, the pharmaceutical composition comprises about 6.5 mM histidine. In some embodiments, the pharmaceutical composition comprises about 7 mM histidine. In some embodiments, the pharmaceutical composition comprises about 7.5 mM histidine. In some embodiments, the pharmaceutical composition comprises about 8 mM histidine. In some embodiments, the pharmaceutical composition comprises about 8.5 mM histidine. In some embodiments, the pharmaceutical composition comprises about 9 mM histidine. In some embodiments, the pharmaceutical composition comprises about 9.5 mM histidine. In some embodiments, the pharmaceutical composition comprises about 10 mM histidine. In some embodiments, the pharmaceutical composition comprises about 10.5 mM histidine. In some embodiments, the pharmaceutical composition comprises about 11 mM histidine. In some embodiments, the pharmaceutical composition comprises about 11.5 mM histidine. In some embodiments, the pharmaceutical composition comprises about 12 mM histidine. In some embodiments, the pharmaceutical composition comprises about 12.5 mM histidine. In some embodiments, the pharmaceutical composition comprises about 13 mM histidine. In some embodiments, the pharmaceutical composition comprises about 13.5 mM histidine. In some embodiments, the pharmaceutical composition comprises about 14 mM histidine. In some embodiments, the pharmaceutical composition comprises about 14.5 mM histidine. In some embodiments, the pharmaceutical composition comprises about 15 mM histidine. In some embodiments, the histidine is L-histidine.

[0146] In some embodiments, the pharmaceutical compositions disclosed herein contain a specified amount or concentration of arginine. In some embodiments, the pharmaceutical compositions contain arginine hydrochloride (HCl). In some embodiments, the arginine is L-arginine. In some embodiments, the compositions contain L-arginine-HCl.

[0147] In some embodiments, the pharmaceutical composition comprises about 200 mM to about 300 mM arginine.

[0148] In some embodiments, the pharmaceutical composition comprises about 200 mM arginine. In some embodiments, the pharmaceutical composition comprises about 210 mM arginine. In some embodiments, the pharmaceutical composition comprises about 220 mM arginine. In some embodiments, the pharmaceutical composition comprises about 230 mM arginine. In some embodiments, the pharmaceutical composition comprises about 240 mM arginine. In some embodiments, the pharmaceutical composition comprises about 250 mM arginine. In some embodiments, the pharmaceutical composition comprises about 260 mM arginine. In some embodiments, the pharmaceutical composition comprises about 270 mM arginine. In some embodiments, the pharmaceutical composition comprises about 280 mM arginine. In some embodiments, the pharmaceutical composition comprises about 290 mM arginine. In some embodiments, the pharmaceutical composition comprises about 300 mM arginine. In some embodiments, the arginine is L-arginine. In some embodiments, the composition comprises L-arginine-HCl.

[0149] The pharmaceutical compositions disclosed herein may include a bulking agent. In some embodiments, the pharmaceutical compositions disclosed herein include a specified amount or concentration of calcium chloride (CaCl2). In some embodiments, the compositions include CaCl2·2H2O, CaCl2 (anhydrous), CaCl2·4H2O, or CaCl2·6H2O. In some embodiments, the compositions include calcium chloride dihydrate. In some embodiments, the pharmaceutical compositions include about 2.5 mM to about 10 mM calcium chloride. In some embodiments, the compositions include calcium chloride dihydrate.

[0150] In some embodiments, the pharmaceutical composition comprises about 2.5 mM calcium chloride. In some embodiments, the pharmaceutical composition comprises about 3 mM calcium chloride. In some embodiments, the pharmaceutical composition comprises about 3.5 mM calcium chloride. In some embodiments, the pharmaceutical composition comprises about 4 mM calcium chloride. In some embodiments, the pharmaceutical composition comprises about 4.5 mM calcium chloride. In some embodiments, the pharmaceutical composition comprises about 5 mM calcium chloride. In some embodiments, the pharmaceutical composition comprises about 5.5 mM calcium chloride. In some embodiments, the pharmaceutical composition comprises about 6 mM calcium chloride. In some embodiments, the pharmaceutical composition comprises about 6.5 mM calcium chloride. In some embodiments, the pharmaceutical composition comprises about 7 mM calcium chloride. In some embodiments, the pharmaceutical composition comprises about 7.5 mM calcium chloride. In some embodiments, the pharmaceutical composition comprises about 8 mM calcium chloride. In some embodiments, the pharmaceutical composition comprises about 8.5 mM calcium chloride. In some embodiments, the pharmaceutical composition comprises about 9 mM calcium chloride. In some embodiments, the pharmaceutical composition comprises about 9.5 mM calcium chloride. In some embodiments, the pharmaceutical composition comprises about 10 mM calcium chloride. In some embodiments, the composition comprises calcium chloride dihydrate.

[0151] In some embodiments, the pharmaceutical compositions disclosed herein do not contain any filler other than calcium chloride. In some embodiments, calcium chloride is the only filler. In some embodiments, the pharmaceutical compositions contain less than 8.8 mg / mL sodium chloride (NaCl). In some embodiments, the pharmaceutical compositions are substantially free of sodium chloride. In some embodiments, the pharmaceutical compositions are free of sodium chloride.

[0152] In some embodiments, the pharmaceutical compositions disclosed herein contain a specified amount or concentration of poloxamer 188 (P188). In some embodiments, the poloxamer is poloxamer 101, 105, 108, 122, 123, 124, 181, 182, 183, 184, 185, 188, 212, 215, 217, 231, 234, 235, 237, 238, 282, 284, 288, 331, 333, 334, 335, 338, 401, 402, 403, or 407. In some embodiments, the poloxamer is poloxamer 407. In some embodiments, the pharmaceutical compositions contain about 0.01 mg / ml to about 10 mg / ml of the poloxamer. In some embodiments, the pharmaceutical compositions contain at least about 1 mg / ml of the poloxamer.

[0153] In some embodiments, the pharmaceutical compositions disclosed herein include a specified amount or concentration of poloxamer 188 (P188). A non-limiting disclosure regarding P188 can be found in Strickley and Lambert (2021) Journal of Pharmaceutical Sciences 110 2590-2608, the entire contents of each of which are incorporated herein by reference. In some embodiments, the pharmaceutical composition includes about 0.01% (w / v) to about 1.0% (w / v) poloxamer 188. In some embodiments, the pharmaceutical composition includes at least about 0.1% poloxamer 188. In some embodiments, the pharmaceutical composition includes about 0.1% poloxamer 188.

[0154] In some embodiments, the pharmaceutical composition comprises about 0.01% (w / v) poloxamer 188. In some embodiments, the pharmaceutical composition comprises about 0.02% (w / v) poloxamer 188. In some embodiments, the pharmaceutical composition comprises about 0.03% (w / v) poloxamer 188. In some embodiments, the pharmaceutical composition comprises about 0.04% (w / v) poloxamer 188. In some embodiments, the pharmaceutical composition comprises about 0.05% (w / v) poloxamer 188. In some embodiments, the pharmaceutical composition comprises about 0.06% (w / v) poloxamer 188. In some embodiments, the pharmaceutical composition comprises about 0.07% (w / v) poloxamer 188. In some embodiments, the pharmaceutical composition comprises about 0.08% (w / v) poloxamer 188. In some embodiments, the pharmaceutical composition comprises about 0.09% (w / v) poloxamer 188. In some embodiments, the pharmaceutical composition comprises about 0.1% (w / v) poloxamer 188.

[0155] In some embodiments, the pharmaceutical composition comprises about 0.11% (w / v) poloxamer 188. In some embodiments, the pharmaceutical composition comprises about 0.12% (w / v) poloxamer 188. In some embodiments, the pharmaceutical composition comprises about 0.13% (w / v) poloxamer 188. In some embodiments, the pharmaceutical composition comprises about 0.14% (w / v) poloxamer 188. In some embodiments, the pharmaceutical composition comprises about 0.15% (w / v) poloxamer 188. In some embodiments, the pharmaceutical composition comprises about 0.16% (w / v) poloxamer 188. In some embodiments, the pharmaceutical composition comprises about 0.17% (w / v) poloxamer 188. In some embodiments, the pharmaceutical composition comprises about 0.18% (w / v) poloxamer 188. In some embodiments, the pharmaceutical composition comprises about 0.19% (w / v) poloxamer 188. In some embodiments, the pharmaceutical composition comprises about 0.2% (w / v) poloxamer 188.

[0156] In some embodiments, the pharmaceutical composition comprises about 0.3% (w / v) poloxamer 188. In some embodiments, the pharmaceutical composition comprises about 0.4% (w / v) poloxamer 188. In some embodiments, the pharmaceutical composition comprises about 0.5% (w / v) poloxamer 188. In some embodiments, the pharmaceutical composition comprises about 0.6% (w / v) poloxamer 188. In some embodiments, the pharmaceutical composition comprises about 0.7% (w / v) poloxamer 188. In some embodiments, the pharmaceutical composition comprises about 0.8% (w / v) poloxamer 188. In some embodiments, the pharmaceutical composition comprises about 0.9% (w / v) poloxamer 188. In some embodiments, the pharmaceutical composition comprises about 1.0% (w / v) poloxamer 188.

[0157] In some embodiments, the pharmaceutical composition is a pre-lyophilization solution. In some embodiments, the pre-lyophilization solution does not contain NaCl.

[0158] In some embodiments, the pharmaceutical composition comprises: (a) about 1% (w / v) to about 8% (w / v) sucrose; (b) about 5 mM to about 15 mM histidine; (c) about 200 mM to about 300 mM arginine; (d) about 2.5 mM to about 10 mM calcium chloride; and (e) about 0.1% (w / v) to about 1.0% (w / v) poloxamer In some embodiments, the poloxamer is poloxamer 188 (P188). In some embodiments, the composition comprises poloxamer 188. In some embodiments, the histidine is L-histidine. In some embodiments, the arginine is L-arginine. In some embodiments, the composition comprises L-arginine-HCl. In some embodiments, the composition comprises calcium chloride dihydrate. In some embodiments, the pharmaceutical composition is a pre-lyophilized solution.

[0159] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution does not contain NaCl.

[0160] In some embodiments, the pharmaceutical composition comprises: (a) about 1% (w / v) sucrose; (b) about 10 mM histidine; (c) about 250 mM arginine; (d) about 5 mM calcium chloride; and (e) about 0.1% poloxamer 188 In some embodiments, the histidine is L-histidine. In some embodiments, the arginine is L-arginine. In some embodiments, the composition comprises L-arginine-HCl. In some embodiments, the composition comprises calcium chloride dihydrate. In some embodiments, the pharmaceutical composition is a pre-lyophilized solution.

[0161] In some embodiments, the pharmaceutical composition comprises: (a) approximately 2% (w / v) sucrose; (b) about 10 mM histidine; (c) about 250 mM arginine; (d) about 5 mM calcium chloride; and (e) about 0.1% poloxamer 188 In some embodiments, the histidine is L-histidine. In some embodiments, the arginine is L-arginine. In some embodiments, the composition comprises L-arginine-HCl. In some embodiments, the composition comprises calcium chloride dihydrate. In some embodiments, the pharmaceutical composition is a pre-lyophilized solution.

[0162] In some embodiments, the pharmaceutical composition comprises: (a) about 5% (w / v) sucrose; (b) about 10 mM histidine; (c) about 250 mM arginine; (d) about 5 mM calcium chloride; and (e) about 0.1% poloxamer 188 Includes.

[0163] In some embodiments, the histidine is L-histidine. In some embodiments, the arginine is L-arginine. In some embodiments, the composition comprises L-arginine-HCl. In some embodiments, the composition comprises calcium chloride dihydrate. In some embodiments, the pharmaceutical composition is a pre-lyophilized solution. In some embodiments, the pharmaceutical composition comprises (a) about 1% (w / v) to about 5% (w / v) sucrose; (b) about 10 mM histidine; (c) about 250 mM arginine; (d) about 5 mM calcium chloride; and (e) about 0.1% poloxamer 188 In some embodiments, the histidine is L-histidine. In some embodiments, the arginine is L-arginine. In some embodiments, the composition comprises L-arginine-HCl. In some embodiments, the composition comprises calcium chloride dihydrate. In some embodiments, the pharmaceutical composition is a pre-lyophilized solution.

[0164] In some embodiments, the pharmaceutical composition comprises: (a) about 1%, 2% or 5% (w / v) sucrose; (b) about 5 mM to about 15 mM histidine; (c) about 250 mM arginine; (d) about 5 mM calcium chloride; and (e) about 0.1% poloxamer 188 In some embodiments, the histidine is L-histidine. In some embodiments, the arginine is L-arginine. In some embodiments, the composition comprises L-arginine-HCl. In some embodiments, the composition comprises calcium chloride dihydrate. In some embodiments, the pharmaceutical composition is a pre-lyophilized solution.

[0165] In some embodiments, the pharmaceutical composition comprises: (a) about 1%, 2% or 5% (w / v) sucrose; (b) about 10 mM histidine; (c) about 200 mM to about 300 mM arginine; (d) about 5 mM calcium chloride; and (e) about 0.1% poloxamer 188 In some embodiments, the histidine is L-histidine. In some embodiments, the arginine is L-arginine. In some embodiments, the composition comprises L-arginine-HCl. In some embodiments, the composition comprises calcium chloride dihydrate. In some embodiments, the pharmaceutical composition is a pre-lyophilized solution.

[0166] In some embodiments, the pharmaceutical composition comprises: (a) about 1%, 2% or 5% (w / v) sucrose; (b) about 10 mM histidine; (c) about 250 mM arginine; (d) about 2.5 mM to about 10 mM calcium chloride; and (e) about 0.1% poloxamer 188 In some embodiments, the histidine is L-histidine. In some embodiments, the arginine is L-arginine. In some embodiments, the composition comprises L-arginine-HCl. In some embodiments, the composition comprises calcium chloride dihydrate. In some embodiments, the pharmaceutical composition is a pre-lyophilized solution.

[0167] In some embodiments, the pharmaceutical composition comprises: (a) about 1%, 2% or 5% (w / v) sucrose; (b) about 10 mM histidine; (c) about 250 mM arginine; (d) about 5 mM calcium chloride; and (e) about 0.008% (w / v) to about 0.1% (w / v) poloxamer 188 Includes.

[0168] In some embodiments, the histidine is L-histidine. In some embodiments, the arginine is L-arginine. In some embodiments, the composition comprises L-arginine-HCl. In some embodiments, the composition comprises calcium chloride dihydrate. In some embodiments, the pharmaceutical composition is a pre-lyophilized solution.

[0169] In some embodiments, the pharmaceutical composition comprises: (a) about 10, 20 or 50 mg / ml sucrose; (b) about 1.552 mg / ml L-histidine; (c) L-arginine-HCl at about 52.665 mg / ml; (d) about 0.735 mg / ml calcium chloride; and (e) Poloxamer 188 at approximately 1 mg / ml Includes.

[0170] In some embodiments, the pharmaceutical composition comprises: (a) about 10, 20 or 50 mg / ml sucrose; (b) about 1.552 mg / ml L-histidine; (c) L-arginine-HCl at about 52.665 mg / ml; (d) calcium chloride dihydrate at about 0.735 mg / ml; and (e) Poloxamer 188 at approximately 1 mg / ml Includes.

[0171] In some embodiments, the pharmaceutical composition comprises: (a) about 10, 20 or 50 mg / ml sucrose; (b) about 1.552 mg / ml L-histidine; (c) L-arginine-HCl at about 52.665 mg / ml; (d) about 0.555 mg / ml calcium chloride; and (e) Poloxamer 188 at approximately 1 mg / ml In some embodiments, the composition comprises calcium chloride dihydrate. In some embodiments, the pharmaceutical composition is a pre-lyophilized solution.

[0172] In some embodiments, the pharmaceutical composition comprises: (a) about 10, 20 or 50 mg / ml sucrose; (b) about 1.552 mg / ml L-histidine; (c) about 43.550 mg / ml L-arginine; (d) calcium chloride dihydrate at about 0.735 mg / ml; and (e) Poloxamer 188 at approximately 1 mg / ml In some embodiments, the composition comprises L-arginine-HCl. In some embodiments, the composition comprises calcium chloride dihydrate. In some embodiments, the pharmaceutical composition is a pre-lyophilized solution.

[0173] In some embodiments, the pharmaceutical composition comprises: (a) about 10, 20 or 50 mg / ml sucrose; (b) about 1.552 mg / ml L-histidine; (c) about 43.550 mg / ml L-arginine; (d) about 0.735 mg / ml calcium chloride; and (e) Poloxamer 188 at approximately 1 mg / ml In some embodiments, the composition comprises L-arginine-HCl. In some embodiments, the composition comprises calcium chloride dihydrate. In some embodiments, the pharmaceutical composition is a pre-lyophilized solution.

[0174] In some embodiments, the pharmaceutical composition comprises: (a) about 10, 20 or 50 mg / ml sucrose; (b) about 1.552 mg / ml L-histidine; (c) about 43.550 mg / ml L-arginine; (d) about 0.555 mg / ml calcium chloride; and (e) Poloxamer 188 at approximately 1 mg / ml In some embodiments, the composition comprises L-arginine-HCl. In some embodiments, the composition comprises calcium chloride dihydrate. In some embodiments, the pharmaceutical composition is a pre-lyophilized solution.

[0175] In some embodiments, the pharmaceutical composition comprises: (a) 11.23, 22.45 or 56.12 mg / ml sucrose; (b) 1.74 mg / ml L-histidine; (c) L-arginine-HCl at 59.11 mg / ml; (d) 0.82 mg / ml calcium chloride dihydrate; and (e) 1.12 mg / ml of Poloxamer 188 Includes.

[0176] In some embodiments, the pharmaceutical composition comprises: (a) 11.23, 22.45 or 56.12 mg / ml sucrose; (b) 1.74 mg / ml L-histidine; (c) L-arginine-HCl at 59.11 mg / ml; (d) 0.62 mg / ml calcium chloride; and (e) 1.12 mg / ml of Poloxamer 188 In some embodiments, the composition comprises calcium chloride dihydrate.

[0177] In some embodiments, the pharmaceutical composition comprises: (a) 11.23, 22.45 or 56.12 mg / ml sucrose; (b) 1.74 mg / ml L-histidine; (c) 48.88 mg / ml L-arginine; (d) 0.82 mg / ml calcium chloride dihydrate; and (e) 1.12 mg / ml of Poloxamer 188 Includes.

[0178] In some embodiments, the pharmaceutical composition comprises: (a) 11.23, 22.45 or 56.12 mg / ml sucrose; (b) 1.74 mg / ml L-histidine; (c) 48.88 mg / ml L-arginine; (d) 0.62 mg / ml calcium chloride; and (e) 1.12 mg / ml of Poloxamer 188 In some embodiments, the composition comprises L-arginine-HCl. In some embodiments, the composition comprises calcium chloride dihydrate.

[0179] In some embodiments, the pharmaceutical composition has a pH of about 6.5 to about 7.5. In some embodiments, the pharmaceutical composition has a pH of about 7.0. In some embodiments, the pharmaceutical composition has a pH of about 6.8.

[0180] In some embodiments, the pharmaceutical composition has a pH of about 6.5. In some embodiments, the pharmaceutical composition has a pH of about 6.6. In some embodiments, the pharmaceutical composition has a pH of about 6.7. In some embodiments, the pharmaceutical composition has a pH of about 6.8. In some embodiments, the pharmaceutical composition has a pH of about 6.9. In some embodiments, the pharmaceutical composition has a pH of about 7.0. In some embodiments, the pharmaceutical composition has a pH of about 7.1. In some embodiments, the pharmaceutical composition has a pH of about 7.2. In some embodiments, the pharmaceutical composition has a pH of about 7.3. In some embodiments, the pharmaceutical composition has a pH of about 7.4. In some embodiments, the pharmaceutical composition has a pH of about 7.5.

[0181] In some embodiments, the pharmaceutical composition has a pH of 6.5. In some embodiments, the pharmaceutical composition has a pH of 6.6. In some embodiments, the pharmaceutical composition has a pH of 6.7. In some embodiments, the pharmaceutical composition has a pH of 6.8. In some embodiments, the pharmaceutical composition has a pH of 6.9. In some embodiments, the pharmaceutical composition has a pH of 7.0. In some embodiments, the pharmaceutical composition has a pH of 7.1. In some embodiments, the pharmaceutical composition has a pH of 7.2. In some embodiments, the pharmaceutical composition has a pH of 7.3. In some embodiments, the pharmaceutical composition has a pH of 7.4. In some embodiments, the pharmaceutical composition has a pH of 7.5.

[0182] In some embodiments, a quantity of 3.367 mL of the pre-lyophilization solution is added to the container or vial. In some embodiments, the pre-lyophilization solution is subjected to lyophilization, resulting in a lyophilized pharmaceutical composition.

[0183] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) about 10 mg to about 200 mg of sucrose; (b) about 2.5 mg to about 7.5 mg of histidine; (c) about 140 mg to about 200 mg arginine; (d) about 1.5 mg to about 5 mg of calcium chloride; and (e) about 1 mg to about 10 mg of poloxamer 188 Includes.

[0184] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) about 10 mg to about 200 mg of sucrose; (b) about 2.5 mg to about 7.5 mg L-histidine; (c) about 140 mg to about 200 mg L-arginine; (d) about 1.5 mg to about 5 mg of calcium chloride; and (e) about 1 mg to about 10 mg of poloxamer 188 In some embodiments, the composition comprises L-arginine-HCl. In some embodiments, the composition comprises calcium chloride dihydrate.

[0185] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 10 mg to 200 mg of sucrose; (b) 2.5 mg to 7.5 mg of histidine; (c) 140 mg to 200 mg of arginine; (d) 1.5 mg to 5 mg of calcium chloride; and (e) 1 mg to 10 mg of poloxamer 188 Includes.

[0186] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 10 mg to 200 mg of sucrose; (b) 2.5 mg to 7.5 mg L-histidine; (c) 140 mg to 200 mg of L-arginine-HCl (d) 1.5 mg to 5 mg of calcium chloride; and (e) approximately 1 mg to 10 mg of poloxamer 188 In some embodiments, the composition comprises calcium chloride dihydrate.

[0187] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 10 mg to 200 mg of sucrose; (b) 2.5 mg to 7.5 mg L-histidine; (c) 140 mg to 200 mg of L-arginine; (d) 1.5 mg to 5 mg of calcium chloride; and (e) approximately 1 mg to 10 mg of poloxamer 188 In some embodiments, the composition comprises L-arginine-HCl. In some embodiments, the composition comprises calcium chloride dihydrate.

[0188] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) approximately 33.7 mg, 67.3 mg, or 168.4 mg of sucrose; (b) approximately 5.2 mg of L-histidine; (c) about 177.3 mg L-arginine-HCl; (d) about 2.5 mg of calcium chloride; and (e) approximately 3.4 mg of poloxamer 188 Includes.

[0189] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) approximately 33.7 mg, 67.3 mg, or 168.4 mg of sucrose; (b) approximately 5.2 mg of L-histidine; (c) approximately 146.6 mg L-arginine; (d) about 2.5 mg of calcium chloride; and (e) approximately 3.4 mg of poloxamer 188 Includes.

[0190] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 33.7, 67.3, or 168.4 mg of sucrose; (b) 5.2 mg L-histidine; (c) 177.3 mg L-arginine-HCl; (d) 2.5 mg of calcium chloride; and (e) approximately 3.4 mg of poloxamer 188 Includes.

[0191] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 33.7, 67.3, or 168.4 mg of sucrose; (b) 5.2 mg L-histidine; (c) 146.6 mg L-arginine; (d) 2.5 mg of calcium chloride; and (e) 3.4 mg of poloxamer 188 Includes.

[0192] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 33.67, 67.34, or 168.35 mg of sucrose; (b) 5.23 mg L-histidine; (c) 177.32 mg L-arginine-HCl; (d) 2.47 mg of calcium chloride dihydrate; and (e) 3.37 mg of poloxamer 188 Includes.

[0193] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 33.67, 67.34, or 168.35 mg of sucrose; (b) 5.23 mg L-histidine; (c) 177.32 mg L-arginine-HCl; (d) 1.87 mg of calcium chloride; and (e) 3.37 mg of poloxamer 188 Includes.

[0194] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 33.67, 67.34, or 168.35 mg of sucrose; (b) 5.23 mg L-histidine; (c) 146.63 mg L-arginine; (d) 2.47 mg of calcium chloride dihydrate; and (e) 3.37 mg of poloxamer 188 Includes.

[0195] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 33.67, 67.34, or 168.35 mg of sucrose; (b) 5.23 mg L-histidine; (c) 146.63 mg L-arginine; (d) 1.87 mg of calcium chloride; and (e) 3.37 mg of poloxamer 188 Includes.

[0196] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 10 mg to 200 mg of sucrose; (b) 5.2 mg L-histidine; (c) 177.3 mg L-arginine-HCl; (d) 2.5 mg of calcium chloride dihydrate; and (e) 3.4 mg of poloxamer 188 Includes.

[0197] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 10 mg to 200 mg of sucrose; (b) 5.2 mg L-histidine; (c) 177.3 mg L-arginine-HCl; (d) 1.9 mg of calcium chloride; and (e) 3.4 mg of poloxamer 188 Includes.

[0198] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 10 mg to 200 mg of sucrose; (b) 5.2 mg L-histidine; (c) 146.6 mg L-arginine; (d) 2.5 mg of calcium chloride dihydrate; and (e) 3.4 mg of poloxamer 188 Includes.

[0199] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 10 mg to 200 mg of sucrose; (b) 5.2 mg L-histidine; (c) 146.6 mg L-arginine; (d) 1.9 mg of calcium chloride; and (e) 3.4 mg of poloxamer 188 Includes.

[0200] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 33.7 mg, 67.3 mg, or 168.4 mg of sucrose; (b) 2.5 mg to 7.5 mg L-histidine; (c) 177.3 mg L-arginine-HCl; (d) 2.5 mg of calcium chloride dihydrate; and (e) 3.4 mg of poloxamer 188 Includes.

[0201] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 33.7 mg, 67.3 mg, or 168.4 mg of sucrose; (b) 2.5 mg to 7.5 mg L-histidine; (c) 177.3 mg L-arginine-HCl; (d) 1.9 mg of calcium chloride; and (e) 3.4 mg of poloxamer 188 Includes.

[0202] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 33.7 mg, 67.3 mg, or 168.4 mg of sucrose; (b) 2.5 mg to 7.5 mg L-histidine; (c) 146.6 mg L-arginine; (d) 2.5 mg of calcium chloride dihydrate; and (e) 3.4 mg of poloxamer 188 Includes.

[0203] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 33.7 mg, 67.3 mg, or 168.4 mg of sucrose; (b) 2.5 mg to 7.5 mg L-histidine; (c) 146.6 mg L-arginine; (d) 1.9 mg of calcium chloride; and (e) 3.4 mg of poloxamer 188 Includes.

[0204] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 33.67 mg, 67.34 mg, or 168.35 mg of sucrose; (b) 5.23 mg L-histidine; (c) 140 mg to 200 mg of L-arginine; (d) 1.5 mg to 5 mg of calcium chloride; and (e) 3.37 mg of poloxamer 188 In some embodiments, the composition comprises 140 mg to 200 mg of L-arginine-HCl.

[0205] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 33.67, 67.34, or 168.35 mg of sucrose; (b) 5.23 mg L-histidine; (c) 177.32 mg L-arginine-HCl; (d) 1.5 mg to 5 mg of calcium chloride; and (e) 3.37 mg of poloxamer 188 Includes.

[0206] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 33.67, 67.34, or 168.35 mg of sucrose; (b) 5.23 mg L-histidine; (c) 146.63 mg L-arginine; (d) 1.5 mg to 5 mg of calcium chloride; and (e) 3.37 mg of poloxamer 188 Includes.

[0207] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 33.67, 67.34, or 168.35 mg of sucrose; (b) 5.23 mg L-histidine; (c) 177.32 mg L-arginine-HCl; (d) 2.47 mg of calcium chloride dihydrate; and (e) 1 mg to 10 mg of poloxamer 188 Includes.

[0208] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 33.67, 67.34, or 168.35 mg of sucrose; (b) 5.23 mg L-histidine; (c) 177.32 mg L-arginine-HCl; (d) 1.87 mg of calcium chloride; and (e) 1 mg to 10 mg of poloxamer 188 Includes.

[0209] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 33.67, 67.34, or 168.35 mg of sucrose; (b) 5.23 mg L-histidine; (c) 146.63 mg L-arginine; (d) 2.47 mg of calcium chloride dihydrate; and (e) 1 mg to 10 mg of poloxamer 188 Includes.

[0210] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 33.67, 67.34, or 168.35 mg of sucrose; (b) 5.23 mg L-histidine; (c) 146.63 mg L-arginine; (d) 1.87 mg of calcium chloride; and (e) 1 mg to 10 mg of poloxamer 188 Includes.

[0211] In some embodiments, the lyophilized pharmaceutical composition has a moisture content of less than 2%. In some embodiments, the lyophilized pharmaceutical composition has a moisture content of less than 1.8%. In some embodiments, the lyophilized pharmaceutical composition has a moisture content of less than 1.6%.

[0212] In some embodiments, the lyophilized pharmaceutical composition is in a lyophilized cake. In some embodiments, the lyophilized cake is white. In some embodiments, the lyophilized cake is less than Y4 on the European Pharmacopoeia color scale. See Degree of Coloration of Liquids (Method 2.2.2), European Pharmacopoeia, 10th Ed. (2021).

[0213] In some embodiments, the lyophilized pharmaceutical composition and sterile water are combined to make an injection solution. In some embodiments, the lyophilized pharmaceutical composition is combined with about 2 mL to about 5 mL of sterile water. In some embodiments, the lyophilized pharmaceutical composition is combined with about 3 mL of sterile water. In some embodiments, the lyophilized pharmaceutical composition is combined with 3 mL of sterile water. In some embodiments, the sterile water is USP grade sterile water. In some embodiments, the sterile water is USP grade sterile water for injection. In some embodiments, the sterile water is pyrogen-free or non-pyrogenic. In some embodiments, the sterile water does not contain a bacteriostatic or antibacterial agent. In some embodiments, the sterile water includes a bacteriostatic or antibacterial agent. In some embodiments, the sterile water is sterilized using a filter. In some embodiments, the sterile water is sterilized using a 0.1 μm filter. In some embodiments, the sterile water is distilled water. In some embodiments, sterile water is distilled water that is sterile, non-pyrogenic, hypotonic with an osmolality of zero mOsmol / L, and does not contain bacteriostatic or antibacterial agents.

[0214] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) 10 mg / mL to 60 mg / mL sucrose; (b) 1.5 mg / mL to 2.0 mg / mL L-histidine; (c) 50 mg / mL to 70 mg / mL L-arginine; (d) 0.7 mg / mL to 0.9 mg / mL calcium chloride dihydrate; and (e) 0.6 mg / mL to 1.6 mg / mL of poloxamer 188 Includes.

[0215] In some embodiments, the composition comprises L-arginine-HCl. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises (a) 10 mg / mL to 60 mg / mL sucrose; (b) 1.5 mg / mL to 2.0 mg / mL L-histidine; (c) 50 mg / mL to 70 mg / mL L-arginine; (d) 0.5 mg / mL to 0.8 mg / mL calcium chloride; and (e) 0.6 mg / mL to 1.6 mg / mL of poloxamer 188 In some embodiments, the composition comprises L-arginine-HCl. In some embodiments, the composition comprises calcium chloride dihydrate.

[0216] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 11.23, 22.45 or 56.12 mg / ml sucrose; (b) about 1.74 mg / ml L-histidine; (c) L-arginine-HCl at about 59.11 mg / ml; (d) calcium chloride dihydrate at about 0.82 mg / ml; and (e) Poloxamer 188 at about 1.12 mg / ml Includes.

[0217] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 11.23, 22.45 or 56.12 mg / ml sucrose; (b) about 1.74 mg / ml L-histidine; (c) L-arginine-HCl at about 59.11 mg / ml; (d) about 0.62 mg / ml calcium chloride; and (e) Poloxamer 188 at about 1.12 mg / ml In some embodiments, the composition comprises calcium chloride dihydrate.

[0218] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 11.23, 22.45 or 56.12 mg / ml sucrose; (b) about 1.74 mg / ml L-histidine; (c) about 48.88 mg / ml L-arginine; (d) calcium chloride dihydrate at about 0.82 mg / ml; and (e) Poloxamer 188 at about 1.12 mg / ml Includes.

[0219] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 11.23 mg, 22.45 mg, or 56.12 mg / ml sucrose; (b) about 1.74 mg / ml L-histidine; (c) about 48.88 mg / mL L-arginine; (d) about 0.62 mg / ml calcium chloride; and (e) Poloxamer 188 at about 1.12 mg / ml In some embodiments, the composition comprises L-arginine-HCl. In some embodiments, the composition comprises calcium chloride dihydrate.

[0220] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 10 mg / mL to about 60 mg / mL sucrose; (b) about 1.74 mg / ml L-histidine; (c) L-arginine-HCl at about 59.11 mg / ml; (d) calcium chloride dihydrate at about 0.82 mg / ml; and (e) Poloxamer 188 at about 1.12 mg / ml Includes.

[0221] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 10 mg / mL to about 60 mg / mL sucrose; (b) about 1.74 mg / ml L-histidine; (c) L-arginine-HCl at about 59.11 mg / ml; (d) about 0.62 mg / ml calcium chloride; and (e) Poloxamer 188 at about 1.12 mg / ml In some embodiments, the composition comprises calcium chloride dihydrate.

[0222] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 10 mg / mL to about 60 mg / mL sucrose; (b) about 1.74 mg / ml L-histidine; (c) about 44.88 mg / ml L-arginine; (d) calcium chloride dihydrate at about 0.82 mg / ml; and (e) Poloxamer 188 at about 1.12 mg / ml Includes.

[0223] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 10 mg / mL to about 60 mg / mL sucrose; (b) about 1.74 mg / ml L-histidine; (c) about 44.88 mg / ml L-arginine; (d) about 0.62 mg / ml calcium chloride; and (e) Poloxamer 188 at about 1.12 mg / ml In some embodiments, the composition comprises L-arginine-HCl. In some embodiments, the composition comprises calcium chloride dihydrate.

[0224] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 11.23, 22.45 or 56.12 mg / ml sucrose; (b) about 1.5 mg / mL to about 2.0 mg / mL L-histidine; (c) L-arginine-HCl at about 59.11 mg / ml; (d) calcium chloride dihydrate at about 0.82 mg / ml; and (e) Poloxamer 188 at about 1.12 mg / ml Includes.

[0225] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 11.23, 22.45 or 56.12 mg / ml sucrose; (b) about 1.5 mg / mL to about 2.0 mg / mL L-histidine; (c) L-arginine-HCl at about 59.11 mg / ml; (d) about 0.62 mg / ml calcium chloride; and (e) Poloxamer 188 at about 1.12 mg / ml In some embodiments, the composition comprises calcium chloride dihydrate.

[0226] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 11.23, 22.45 or 56.12 mg / ml sucrose; (b) about 1.5 mg / mL to about 2.0 mg / mL L-histidine; (c) about 48.88 mg / mL L-arginine; (d) calcium chloride dihydrate at about 0.82 mg / ml; and (e) Poloxamer 188 at about 1.12 mg / ml Includes.

[0227] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) sucrose at about 22.45 mg / ml; (b) about 1.5 mg / mL to about 2.0 mg / mL L-histidine; (c) about 48.88 mg / ml L-arginine; (d) about 0.62 mg / ml calcium chloride; and (e) Poloxamer 188 at about 1.12 mg / ml In some embodiments, the composition comprises L-arginine-HCl. In some embodiments, the composition comprises calcium chloride dihydrate.

[0228] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 11.23, 22.45 or 56.12 mg / ml sucrose; (b) about 1.74 mg / ml L-histidine; (c) about 50 mg / mL to about 70 mg / mL L-arginine-HCl; (d) calcium chloride dihydrate at about 0.62 mg / ml; and (e) Poloxamer 188 at about 1.12 mg / ml Includes.

[0229] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 11.23, 22.45 or 56.12 mg / ml sucrose; (b) about 1.74 mg / ml L-histidine; (c) about 50 mg / mL to about 70 mg / mL L-arginine-HCl; (d) about 0.62 mg / ml calcium chloride; and (e) Poloxamer 188 at about 1.12 mg / ml In some embodiments, the composition comprises calcium chloride dihydrate.

[0230] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 11.23, 22.45 or 56.12 mg / ml sucrose; (b) about 1.74 mg / ml L-histidine; (c) about 40 mg / mL to about 60 mg / mL L-arginine; (d) calcium chloride dihydrate at about 0.82 mg / ml; and (e) Poloxamer 188 at about 1.12 mg / ml Includes.

[0231] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 11.23, 22.45 or 56.12 mg / ml sucrose; (b) about 1.74 mg / ml L-histidine; (c) about 40 mg / mL to about 60 mg / mL L-arginine; (d) about 0.62 mg / ml calcium chloride; and (e) Poloxamer 188 at about 1.12 mg / ml In some embodiments, the composition comprises L-arginine-HCl. In some embodiments, the composition comprises calcium chloride dihydrate.

[0232] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 11.23, 22.45 or 56.12 mg / ml sucrose; (b) about 1.74 mg / ml L-histidine; (c) L-arginine-HCl at about 59.11 mg / ml; (d) about 0.7 mg / mL to about 0.9 mg / mL of calcium chloride dihydrate; and (e) Poloxamer 188 at about 1.12 mg / ml Includes.

[0233] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 11.23, 22.45 or 56.12 mg / ml sucrose; (b) about 1.74 mg / ml L-histidine; (c) L-arginine-HCl at about 59.11 mg / ml; (d) about 0.5 mg / mL to about 0.9 mg / mL calcium chloride; and (e) Poloxamer 188 at about 1.12 mg / ml In some embodiments, the composition comprises calcium chloride dihydrate.

[0234] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 11.23, 22.45 or 56.12 mg / ml sucrose; (b) about 1.74 mg / ml L-histidine; (c) about 48.88 mg / mL L-arginine; (d) about 0.7 mg / mL to about 0.9 mg / mL of calcium chloride dihydrate; and (e) Poloxamer 188 at about 1.12 mg / ml In some embodiments, the composition comprises L-arginine-HCl.

[0235] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 11.23, 22.45 or 56.12 mg / ml sucrose; (b) about 1.74 mg / ml L-histidine; (c) about 48.88 mg / ml L-arginine; (d) about 0.5 mg / mL to about 0.7 mg / mL calcium chloride; and (e) Poloxamer 188 at about 1.12 mg / ml In some embodiments, the composition comprises L-arginine-HCl. In some embodiments, the composition comprises calcium chloride dihydrate.

[0236] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 11.23, 22.45 or 56.12 mg / ml sucrose; (b) about 1.74 mg / ml L-histidine; (c) L-arginine-HCl at about 59.11 mg / ml; (d) calcium chloride dihydrate at about 0.82 mg / ml; and (e) about 0.6 mg / mL to about 1.6 mg / mL of poloxamer 188 Includes.

[0237] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 11.23, 22.45 or 56.12 mg / ml sucrose; (b) about 1.74 mg / ml L-histidine; (c) L-arginine-HCl at about 59.11 mg / ml; (d) about 0.62 mg / ml calcium chloride; and (e) about 0.6 mg / mL to about 1.6 mg / mL of poloxamer 188 In some embodiments, the composition comprises L-arginine-HCl. In some embodiments, the composition comprises calcium chloride dihydrate.

[0238] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 11.23, 22.45 or 56.12 mg / ml sucrose; (b) about 1.74 mg / ml L-histidine; (c) about 48.88 mg / ml L-arginine; (d) calcium chloride dihydrate at about 0.82 mg / ml; and (e) about 0.6 mg / mL to about 1.6 mg / mL of poloxamer 188 Includes.

[0239] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 11.23, 22.45 or 56.12 mg / ml sucrose; (b) about 1.74 mg / ml L-histidine; (c) about 48.88 mg / mL L-arginine; (d) about 0.62 mg / ml calcium chloride; and (e) about 0.6 mg / mL to about 1.6 mg / mL of poloxamer 188 In some embodiments, the composition comprises L-arginine-HCl. In some embodiments, the composition comprises calcium chloride dihydrate.

[0240] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 11.23, 22.45 or 56.12 mg / ml sucrose; (b) about 1.74 mg / ml L-histidine; (c) L-arginine-HCl at about 59.11 mg / ml; (d) calcium chloride dihydrate at about 0.82 mg / ml; and (e) Poloxamer 188 at about 1.12 mg / ml Includes.

[0241] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 11.23, 22.45 or 56.12 mg / ml sucrose; (b) about 1.74 mg / ml L-histidine; (c) L-arginine-HCl at about 59.11 mg / ml; (d) about 0.62 mg / ml calcium chloride; and (e) Poloxamer 188 at about 1.12 mg / ml Includes.

[0242] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 11.23, 22.45 or 56.12 mg / ml sucrose; (b) about 1.74 mg / ml L-histidine; (c) about 48.88 mg / ml L-arginine; (d) calcium chloride dihydrate at about 0.82 mg / ml; and (e) Poloxamer 188 at about 1.12 mg / ml Includes.

[0243] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 11.23, 22.45 or 56.12 mg / ml sucrose; (b) about 1.74 mg / ml L-histidine; (c) about 48.88 mg / ml L-arginine; (d) about 0.62 mg / ml calcium chloride; and (e) Poloxamer 188 at about 1.12 mg / ml Includes.

[0244] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution is (a) about 33.67, 67.34, or 168.35 mg of sucrose; (b) approximately 5.23 mg of L-histidine; (c) about 177.32 mg L-arginine-HCl; (d) about 2.47 mg of calcium chloride; and (e) approximately 37 mg of poloxamer 188 Includes.

[0245] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution is (a) approximately 33.67, 67.34, or 168.35 mg of sucrose; (b) approximately 5.23 mg of L-histidine; (c) approximately 146.63 mg of L-arginine; (d) about 2.47 mg of calcium chloride dihydrate; and (e) approximately 3.37 mg of poloxamer 188 Includes.

[0246] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution is (a) approximately 33.67, 67.34, or 168.35 mg of sucrose; (b) approximately 5.23 mg of L-histidine; (c) approximately 146.63 mg of L-arginine; (d) about 1.87 mg of calcium chloride; and (e) approximately 3.37 mg of poloxamer 188 Includes.

[0247] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution is about 3 mL of sterile water. (a) 33.67 mg, 67.34 mg, or 168.35 mg of sucrose; (b) 5.23 mg L-histidine; (c) 177.32 mg L-arginine-HCl; (d) 2.47 mg of calcium chloride dihydrate; and (e) 3.37 mg of poloxamer 188 Includes.

[0248] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution is about 3 mL of sterile water. (a) 33.67 mg, 67.34 mg, or 168.35 mg of sucrose; (b) 5.23 mg L-histidine; (c) 177.32 mg L-arginine-HCl; (d) 1.87 mg of calcium chloride; and (e) 3.37 mg of poloxamer 188 Includes.

[0249] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution is about 3 mL of sterile water. (a) 33.67 mg, 67.34 mg, or 168.35 mg of sucrose; (b) 5.23 mg L-histidine; (c) 146.63 mg L-arginine; (d) 2.47 mg of calcium chloride dihydrate; and (e) 3.37 mg of poloxamer 188 Includes.

[0250] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution is about 3 mL of sterile water. (a) 33.67 mg, 67.34 mg, or 168.35 mg of sucrose; (b) 5.23 mg L-histidine; (c) 146.63 mg L-arginine; (d) 1.87 mg of calcium chloride; and (e) 3.37 mg of poloxamer 188 Includes.

[0251] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the lyophilized pharmaceutical composition is reconstituted within 7-12 seconds.

[0252] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the osmolality of the resulting solution is about 525 to about 725 mOsm / kg. In some embodiments, the pharmaceutical compositions disclosed herein have an osmolality of about 500 mOsm / kg to about 650 mOsm / kg. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the osmolality of the resulting solution is about 600 to about 650 mOsm / kg.

[0253] In some embodiments, the pharmaceutical composition has an osmolality of about 525 mOsm / kg. In some embodiments, the pharmaceutical composition has an osmolality of about 550 mOsm / kg. In some embodiments, the pharmaceutical composition has an osmolality of about 575 mOsm / kg. In some embodiments, the pharmaceutical composition has an osmolality of about 600 mOsm / kg. In some embodiments, the pharmaceutical composition has an osmolality of about 625 mOsm / kg. In some embodiments, the pharmaceutical composition has an osmolality of about 650 mOsm / kg. In some embodiments, the pharmaceutical composition has an osmolality of about 675 mOsm / kg. In some embodiments, the pharmaceutical composition has an osmolality of about 700 mOsm / kg. In some embodiments, the pharmaceutical composition has an osmolality of about 725 mOsm / kg.

[0254] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the pH of the resulting solution is about 6.5 to about 7.5. In some embodiments, the pharmaceutical composition has a pH of about 7.0. In some embodiments, the pharmaceutical composition has a pH of about 6.8. In some embodiments, the pharmaceutical composition has a pH of 6.8.

[0255] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the pH of the resulting solution is about 6.5. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the pH of the resulting solution is about 6.6. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the pH of the resulting solution is about 6.7. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the pH of the resulting solution is about 6.8. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the pH of the resulting solution is about 6.9. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the pH of the resulting solution is about 7.0. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the pH of the resulting solution is about 7.1. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the pH of the resulting solution is about 7.2. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the pH of the resulting solution is about 7.3. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the pH of the resulting solution is about 7.4. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the pH of the resulting solution is about 7.5.

[0256] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the protein concentration of the resulting solution is from about 0.8 to about 1.2 mg / mL.

[0257] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the protein concentration of the resulting solution is about 0.8 mg / mL. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the protein concentration of the resulting solution is about 0.9 mg / mL. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the protein concentration of the resulting solution is about 1.0 mg / mL. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the protein concentration of the resulting solution is about 1.1 mg / mL. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the protein concentration of the resulting solution is about 1.2 mg / mL.

[0258] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the protein concentration of the resulting solution is 0.8 mg / mL. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the protein concentration of the resulting solution is 0.9 mg / mL. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the protein concentration of the resulting solution is 1.0 mg / mL. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the protein concentration of the resulting solution is 1.1 mg / mL. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the protein concentration of the resulting solution is 1.2 mg / mL.

[0259] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the turbidity of the resulting solution is less than about 7 nephelometric turbidity units.

[0260] In some embodiments, less than 3% of the chimeric protein aggregates when the lyophilized pharmaceutical composition and sterile water are combined.

[0261] Disclosed herein is a method of storing a pharmaceutical composition, comprising maintaining the pharmaceutical composition disclosed herein at a temperature of about -20°C to about -40°C for at least about 1 month to about 36 months. In some embodiments, the pharmaceutical composition is stored at a temperature of about -20°C, about -25°C, about -30°C, about -35°C, or about -40°C for at least about 1 month to about 36 months. In some embodiments, the pharmaceutical composition is stored at a temperature of about -20°C, about -25°C, about -30°C, about -35°C, or about -40°C for at least about 3, 6, 9, 12, 18, 21, 24, 27, 30, 33, or 36 months. In some embodiments, the pharmaceutical composition is stored at a temperature of about -30°C for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, or 12 months.

[0262] In some embodiments, the pharmaceutical composition is stored at a temperature of about -20°C to about -40°C for at least about 1 month to about 36 months. In some embodiments, the pharmaceutical composition is stored at a temperature of about -20°C, about -25°C, about -30°C, about -35°C, or about -40°C for at least about 1 month to about 36 months. In some embodiments, the pharmaceutical composition is stored at a temperature of about -20°C, about -25°C, about -30°C, about -35°C, or about -40°C for at least about 3, 6, 9, 12, 18, 21, 24, 27, 30, 33, or 36 months. In some embodiments, the pharmaceutical composition is stored at a temperature of about -30°C for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, or 12 months.

[0263] In some embodiments, the pharmaceutical composition is a liquid pharmaceutical composition that has been frozen and thawed 2 to 5 times. In some embodiments, the pharmaceutical composition is a liquid pharmaceutical composition that has been frozen and thawed twice. In some embodiments, the pharmaceutical composition is a liquid pharmaceutical composition that has been frozen and thawed three times. In some embodiments, the pharmaceutical composition is a liquid pharmaceutical composition that has been frozen and thawed four times. In some embodiments, the pharmaceutical composition is a liquid pharmaceutical composition that has been frozen and thawed five times.

[0264] In some embodiments, the pharmaceutical composition comprises about 0.5 mg / ml to about 10 mg / ml of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 0.5 mg / ml to about 5 mg / ml of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 0.5 mg / ml to about 2 mg / ml of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 0.5 mg / ml of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 1 mg / ml of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 1.5 mg / ml of the chimeric protein. In some embodiments, the pharmaceutical composition comprises about 2 mg / ml of the chimeric protein.

[0265] V. Medical Kits In some embodiments, the pharmaceutical composition is provided with a second container comprising sterile water. Disclosed herein is a pharmaceutical kit comprising a first container comprising a pharmaceutical composition and a second container comprising sterile water.

[0266] (i) (a) a chimeric protein comprising a first polypeptide chain comprising a factor VIII ("FVIII") protein or a portion thereof and a first immunoglobulin ("Ig") constant region or a portion thereof, and a second polypeptide chain comprising a von Willebrand factor ("VWF") protein and a second Ig constant region or a portion thereof; (b) about 10 mg to about 200 mg of sucrose; (c) about 2.5 mg to about 7.5 mg of histidine; (d) about 140 mg to about 200 mg arginine; (e) about 1.5 mg to about 5 mg of calcium chloride; and (f) about 0.5 mg to about 10 mg of poloxamer 188 a first container containing a lyophilized pharmaceutical composition comprising: (ii) a second container containing sterile water; Also disclosed herein is a pharmaceutical kit comprising:

[0267] In some embodiments, the first container contains between 100 IU and 10,000 IU of the chimeric protein.

[0268] In some embodiments, the first container contains 250 IU, 500 IU, 1000 IU, 2000 IU, 3000 IU, or 4000 IU of the chimeric protein. In some embodiments, the first container contains 250 IU of the chimeric protein. In some embodiments, the first container contains 500 IU of the chimeric protein. In some embodiments, the first container contains 1000 IU of the chimeric protein. In some embodiments, the first container contains 2000 IU of the chimeric protein. In some embodiments, the first container contains 3000 IU of the chimeric protein. In some embodiments, the first container contains 4000 IU of the chimeric protein. In some embodiments, the first container contains more than about 250 IU of the chimeric protein. In some embodiments, the first container contains more than about 300 IU of the chimeric protein. In some embodiments, the first container contains more than about 500 IU of the chimeric protein. In some embodiments, the first container contains at least about 500 IU of the chimeric protein.

[0269] In some embodiments, the second container contains about 2 mL to about 5 mL of sterile water. In some embodiments, the second container contains 2 mL to 5 mL of sterile water. In some embodiments, the second container contains about 3 mL of sterile water. In some embodiments, the second container contains 3 mL of sterile water.

[0270] In some embodiments, the second container comprises about 2 mL of sterile water. In some embodiments, the second container comprises about 2.1 mL of sterile water. In some embodiments, the second container comprises about 2.2 mL of sterile water. In some embodiments, the second container comprises about 2.3 mL of sterile water. In some embodiments, the second container comprises about 2.4 mL of sterile water. In some embodiments, the second container comprises about 2.5 mL of sterile water. In some embodiments, the second container comprises about 2.6 mL of sterile water. In some embodiments, the second container comprises about 2.7 mL of sterile water. In some embodiments, the second container comprises about 2.8 mL of sterile water. In some embodiments, the second container comprises about 2.9 mL of sterile water. In some embodiments, the second container comprises about 3 mL of sterile water. In some embodiments, the second container comprises about 3.1 mL of sterile water. In some embodiments, the second container comprises about 3.2 mL of sterile water. In some embodiments, the second container comprises about 3.3 mL of sterile water. In some embodiments, the second container comprises about 3.4 mL of sterile water. In some embodiments, the second container comprises about 3.5 mL of sterile water. In some embodiments, the second container comprises about 3.6 mL of sterile water. In some embodiments, the second container comprises about 3.7 mL of sterile water. In some embodiments, the second container comprises about 3.8 mL of sterile water. In some embodiments, the second container comprises about 3.9 mL of sterile water. In some embodiments, the second container comprises about 4 mL of sterile water. In some embodiments, the second container comprises about 4.1 mL of sterile water. In some embodiments, the second container comprises about 4.2 mL of sterile water. In some embodiments, the second container comprises about 4.3 mL of sterile water. In some embodiments, the second container comprises about 4.4 mL of sterile water. In some embodiments, the second container comprises about 4.5 mL of sterile water. In some embodiments, the second container contains about 4.6 mL of sterile water. In some embodiments, the second container contains about 4.7 mL of sterile water.In some embodiments, the second container contains about 4.8 mL of sterile water. In some embodiments, the second container contains about 4.9 mL of sterile water. In some embodiments, the second container contains about 5 mL of sterile water.

[0271] In some embodiments, the pharmaceutical kit further comprises instructions for combining the lyophilized pharmaceutical composition with sterile water.

[0272] In some embodiments, the first container is a glass vial containing a rubber stopper.

[0273] In some embodiments, the second container is a syringe body. In some embodiments, the syringe body is accompanied by a plunger. In some embodiments, the pharmaceutical kit further comprises an adapter for connecting a glass vial to the syringe body. In some embodiments, the pharmaceutical kit further comprises an infusion tube accompanied by a needle that is connected to the syringe body, suitable for intravenous infusion. In some embodiments, the second container is a prefilled syringe.

[0274] Disclosed herein is a method of storing a pharmaceutical kit, comprising maintaining the pharmaceutical kit disclosed herein at a temperature of about -20°C to about -40°C for at least about 1 month to about 36 months. In some embodiments, the pharmaceutical kit is stored at a temperature of about -20°C, about -25°C, about -30°C, about -35°C, or about -40°C for at least about 1 month to about 36 months. In some embodiments, the pharmaceutical kit is stored at a temperature of about -20°C, about -25°C, about -30°C, about -35°C, or about -40°C for at least about 3, 6, 9, 12, 18, 21, 24, 27, 30, 33, or 36 months. In some embodiments, the pharmaceutical kit is stored at a temperature of about -30°C for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, or 12 months.

[0275] In some embodiments, the pharmaceutical kit is stored at a temperature of about -20°C to about -40°C for at least about 1 month to about 36 months. In some embodiments, the pharmaceutical kit is stored at a temperature of about -20°C, about -25°C, about -30°C, about -35°C, or about -40°C for at least about 1 month to about 36 months. In some embodiments, the pharmaceutical kit is stored at a temperature of about -20°C, about -25°C, about -30°C, about -35°C, or about -40°C for at least about 3, 6, 9, 12, 18, 21, 24, 27, 30, 33, or 36 months. In some embodiments, the pharmaceutical kit is stored at a temperature of about -30°C for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, or 12 months.

[0276] VI. Methods and Uses of Pharmaceutical Compositions Also disclosed herein are uses of pharmaceutical compositions or methods for treating hemophilia A in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical composition of the present disclosure. In some embodiments, treating hemophilia A comprises preventing a bleeding episode in a human subject in need thereof. In some embodiments, treating hemophilia A comprises treating a bleeding episode in a human subject in need thereof. In some embodiments, treating hemophilia A comprises controlling the occurrence or frequency of a bleeding episode in a human subject in need thereof. In some embodiments, treating hemophilia A comprises reducing the occurrence or frequency of a bleeding episode in a human subject in need thereof.

[0277] In some embodiments, the composition is used to treat a bleeding condition or condition in a subject in need thereof. The bleeding condition or condition is selected from the group consisting of bleeding coagulation disorders, hemorrhagic arthropathy, muscle bleed, oral bleed, hemorrhage, bleeding into muscle, oral hemorrhage, trauma, head trauma, gastrointestinal bleeding, intracerebral bleeding, intraperitoneal bleeding, intrathoracic bleeding, bone fracture, central nervous system bleeding, bleeding into the retropharyngeal space, bleeding into the retroperitoneal space, bleeding into the iliopsoas sheath, and any combination thereof. In still other embodiments, the subject is scheduled to undergo surgery. In some embodiments, the treatment is prophylactic or on-demand.

[0278] In some embodiments, the use or method comprises combining a lyophilized pharmaceutical composition of the kit of the present disclosure with sterile water and administering an effective amount of the resulting combination to a subject. In some embodiments, the subject combines the lyophilized pharmaceutical composition of the kit with sterile water. In some embodiments, the combination is self-administered by the subject.

[0279] In some embodiments, the multiple doses include at least 2 doses, at least 3 doses, at least 4 doses, at least 5 doses, at least 6 doses, at least 7 doses, at least 8 doses, at least 9 doses, at least 10 doses, at least 11 doses, at least 12 doses, at least 13 doses, at least 14 doses, at least 15 doses, at least 16 doses, at least 17 doses, at least 18 doses, at least 19 doses, at least 20 doses, or more. In some embodiments, the multiple doses are administered over a period of at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 10 years, at least about 15 years, at least about 20 years, or at least about 25 years.

[0280] The chimeric proteins described herein can be administered by any means known in the art. In some embodiments, the chimeric proteins are administered by a route selected from the group consisting of intravenous injection, intravenous infusion, subcutaneous administration, intramuscular administration, oral administration, intranasal administration, and intrapulmonary administration. In some embodiments, the chimeric proteins are administered intravenously. In some embodiments, the chimeric proteins are administered subcutaneously.

[0281] Having now described the present disclosure in detail, it will be more clearly understood by reference to the following examples, which are included herein for illustrative purposes only and are not intended to limit the disclosure. All patents, publications, and articles referred to herein are expressly and specifically incorporated herein by reference. EXAMPLES

[0282] Assessment of aggregation of efanesectocog alfa compositions containing different surfactants The experiment described in this example evaluates the stability of efanesectocog alfa in pharmaceutical compositions containing different surfactants (poloxamer 188) formulated with different concentrations of sucrose. In addition to sucrose, each composition also contained 10 mM L-histidine, 250 mM L-arginine-HCl, 5 mM CaCl2, and 0.1% (w / v) poloxamer 188 (poloxamer 188) at a pH of 6.8. To evaluate the effect of the selected surfactants, the stability attributes of the poloxamer 188 compositions were also compared to those of an otherwise identical PS80 composition.

[0283] To further analyze the effect of sucrose concentration on efanesectocog alfa compositions with poloxamer 188, pharmaceutical compositions of efanesectocog alfa at 0, 1, 2, and 5% w / v sucrose were subjected to different stability conditions and stability was evaluated using several methods. Compositions were tested at the following product stages: Drug Substance (DS, not lyophilized or adjusted to any specific IU strength) frozen storage stability, DS freeze / thaw (FT) stability, liquid DS post-thaw stability, diluted DS and drug product (DP) stability, DP lyophilization stability, and post-reconstitution DP.

[0284] Aggregation of efanesectocog alfa compositions was measured by SEC assay (% HMWS). The SEC assay uses an HPLC instrument equipped with a pump, a temperature-controlled autosampler, a column heater, and a fluorescence detector. Sample solutions are analyzed using the following instrument and method parameters: Mobile phase: Dulbecco's phosphate buffered saline (D-PBS) containing calcium and magnesium with 0.36 M sodium chloride (0.9 mM calcium chloride, 0.5 mM magnesium chloride, 2.7 mM potassium chloride, 1.5 mM potassium phosphate monobasic, 496 mM sodium chloride, 8.1 mM sodium phosphate dibasic, pH 7.0 ± 0.1) Column heater: 26℃ Run Time: 40 minutes FL detector: Ex / Em=280 / 350nm, PMT Gain=5 ·Injected mass load: 2μg Autosampler temperature: 5℃ Flow rate: 0.5mL / min, isocratic

[0285] Aggregation of efanesectocog alfa drug substance (DS) compositions (1 mg / mL) containing either 0.5% (w / v) PS80 or 0.1% (w / v) poloxamer 188 as surfactants and either 1% or 5% (w / v) sucrose was tested at either room temperature (RT) / room light (RL) conditions or at 2-8°C. Aggregation results at RT / RL conditions over time (hours) are shown in Figure 2. At RT / RL conditions, a reduction in sucrose from 5% to 1% increased aggregation by 2-fold in compositions containing either surfactant. At RT / RL conditions, the increase in aggregation rate was approximately the same for both the poloxamer 188 and PS80 compositions.

[0286] The aggregation results over time (hours) at 2-8°C are shown in Figure 3. At 2-8°C, decreasing sucrose from 5% to 1% increased aggregation 2-fold in the PS80 composition, which was similar to the results at RT / RL conditions. However, at 2-8°C, decreasing sucrose from 5% to 1% increased aggregation 10-fold in the PS80 composition. At 2-8°C, the increase in aggregation rate was lower in the Poloxamer 188 composition than in the PS80 composition.

[0287] 4000 IU efanesectocog alfa drug substance (DS) liquid compositions containing either 0.5% (w / v) PS80 or 0.1% (w / v) poloxamer 188 as surfactants were evaluated in a cumulative retention time assay. Efanesectocog alfa liquid compositions were held at room temperature and tested after 0, 5, 7, 25, 43, and 55 hours. Samples were also tested after lyophilization.

[0288] The results of the cumulative retention time assay are shown in Figure 4. The efanesectocog alfa drug substance (DS) liquid composition containing 1% sucrose and 0.05% PS80, which was subsequently diluted to 4000 IU, showed the highest increase in aggregation (see square plot in Figure 4). The results of this cumulative retention time assay also showed that the composition containing poloxamer 188 was moderately more stable in the liquid state at room temperature than the PS80 composition.

[0289] The frozen storage stability of 1 mg / mL efanesectocog alfa DS composition was evaluated in compositions containing either 0.5% (w / v) PS80 or 0.1% (w / v) poloxamer 188 as surfactant. Compositions containing 0, 1, 2, or 5% (w / v) sucrose were kept at freezing temperatures (-80°C or -30°C). Measurement time points were before freezing, at the start of the experiment (T0), 1 month (T1M), 3 months (T3M), and 6 months (T6M).

[0290] The results of the frozen storage stability assay of the composition containing 0.5% (w / v) PS80 are shown in FIG. 5. The results of the frozen storage stability assay of the composition containing 0.1% (w / v) Poloxamer 188 are shown in FIG. 6. At −80° C., no tendency to aggregation was observed for any of the compositions tested. However, at −30° C., the composition containing PS80 surfactant showed increased aggregation (see FIG. 5). This effect was most evident at lower sucrose concentrations (see, for example, the results for 0% sucrose in FIG. 5). In contrast, the composition containing Poloxamer 188 did not show increased aggregation at −30° C. (see FIG. 6). The composition containing Poloxamer 188 showed similar aggregation levels at both −80° C. and −30° C. Thus, the use of Poloxamer 188 as a surfactant may provide improved frozen storage stability for efanesectocog alfa compositions, especially when the composition is stored at −30° C.

[0291] The frozen storage stability of efanesectocog alfa DS compositions (1 mg / mL) containing 0.1% (w / v) poloxamer 188 was further analyzed by performing particle testing using a high accuracy liquid particle counter (HIAC). DS compositions containing 0.1% (w / v) poloxamer 188 and 0, 1, 2, or 5% (w / v) sucrose were kept at -80°C and evaluated at the following time points: before freezing, at the start of the experiment (T0), 1 month (T1M), 3 months (T3M), and 6 months (T6M). The results for particles ≧10 μm are shown in Figure 7A. The results for particles ≧25 μm are shown in Figure 7B. The particles ≧10 μm were less than 6000 particles, the particles ≧25 μm were less than 600 particles,<USP 787> No significant trends were observed in the particles upon storage.

[0292] Lyophilized drug product (Lyo DP) compositions of 250 IU or 4000 IU efanesectocog alfa containing 0.1% (w / v) poloxamer 188 were tested for aggregation at 5° C., 30° C., and 40° C. Lyo DP compositions containing 1% or 5% w / v sucrose were tested. Samples were tested at TO, 1 month, 2 months, 3 months, and 6 months.

[0293] The results for the 250 IU Lyo DP composition are shown in Figure 8A (5°C), Figure 8B (30°C), and Figure 8C (40°C). For the 250 IU compositions, generally higher aggregation was observed in the poloxamer 188 compositions than in the PS80 compositions (data not shown). The 250 IU composition with 0.1% (w / v) poloxamer 188 and 1% sucrose showed a significant increase in aggregation at all temperatures.

[0294] Results for the 4000 IU Lyo DP compositions are shown in Figure 9A (5°C), Figure 9B (30°C), and Figure 9C (40°C). No tendency to aggregate was observed at any temperature in all 4000 IU compositions containing 0.1% (w / v) poloxamer 188 at both sucrose concentrations. This result is consistent with the results for the 4000 IU Lyo DP compositions containing 0.5% (w / v) PS80, which also showed no notable tendency to aggregate based on sucrose concentration or temperature (data not shown).

[0295] Aggregation of efanesectocog alfa compositions after freeze / thaw (F / T) stress was also analyzed for each surfactant. Compositions of efanesectocog alfa DS at 1 mg / mL containing either 0.5% (w / v) PS80 or 0.1% (w / v) poloxamer 188 as surfactant and 0, 1, 2, or 5% (w / v) sucrose were stressed by 5 cycles (5X) of F / T (at least 24 hours thawed at room temperature) at either -80°C or -30°C. Samples were tested pre-freeze and after 5 F / T cycles.

[0296] The results for the composition containing 0.5% (w / v) PS80 are shown in FIG. 10. The results for the composition containing 0.1% (w / v) Poloxamer 188 are shown in FIG. 11. The DS composition containing 0.5% (w / v) PS80 showed a moderate increase in aggregation after five F / T cycles at −80° C. (see FIG. 10). However, the DS composition containing 0.1% (w / v) Poloxamer 188 showed no change in aggregation after five F / T cycles at −80° C. (see FIG. 11). Compositions containing both surfactants showed an increase in aggregation after five F / T cycles at −30° C. However, the increase in aggregation was significantly higher in the DS composition containing 0.5% (w / v) PS80 than in the composition containing 0.1% (w / v) Poloxamer 188 (compare FIG. 10 and FIG. 11). Therefore, it is believed that 0.1% (w / v) poloxamer 188 as a surfactant in an efanesectocog alfa composition may provide slightly improved F / T stability at -80°C, as well as significantly improved F / T stability over 0.5% (w / v) PS80 as a surfactant at -30°C.

[0297] A summary of the aggregation results testing efanesectocog alfa compositions containing 0.1% (w / v) poloxamer 188 is provided in Table 1.

[0298] [Table 1]

[0299] Overall, these aggregation studies suggest that efanesectocog alfa compositions may have improved stability when formulated with 0.1% (w / v) poloxamer 188 rather than 0.5% (w / v) PS80. Poloxamer 188 compositions showed significant improvement over PS80 compositions in F / T and frozen storage stability assays at -30°C at all sucrose concentrations. Thus, poloxamer 188 may be preferred over PS80 in all efanesectocog alfa compositions stored at -30°C. Efanesectocog alfa DS and BDP liquid compositions also showed improved stability with poloxamer 188 compositions over PS80 compositions. In the case of efanesectocog alfa lyophilized DP compositions, the poloxamer 188 composition at 4000 IU showed favorable stability over 6 months at 40°C. However, the PS80 lyophilized drug product showed significantly increased stability at 250 IU compared to poloxamer 188. EXAMPLES

[0300] Evaluation of additional stability attributes of efanesectocog alfa compositions containing different surfactants In addition to aggregation, other indicators of stability of efanesectocog alfa compositions containing different surfactants were evaluated.

[0301] The concentration of surfactants in efanesectocog alfa compositions after F / T stress was analyzed by high performance liquid chromatography (HPLC). Compositions of efanesectocog alfa DS at 1 mg / mL containing either 0.5% (w / v) PS80 or 0.1% (w / v) poloxamer 188 as surfactant and 0, 1, 2, or 5% (w / v) sucrose were stressed by 5 F / T cycles at either -80°C or -30°C (thawed at room temperature for more than 24 hours). Samples were tested pre-freeze and after 1, 3, or 5 F / T cycles. The results for DS compositions containing 0.5% (w / v) PS80 and 0, 1, 2, or 5% (w / v) sucrose are shown in Figure 12A. The results for DS compositions containing 0.1% (w / v) poloxamer 188 and 0, 1, 2, or 5% (w / v) sucrose are shown in Figure 12B. No change in concentration was observed upon surfactant activity at any F / T for any surfactant or sucrose concentration.

[0302] The pH change of efanesectocog alfa compositions after F / T stress was also analyzed. Compositions of efanesectocog alfa DS at 1 mg / mL containing either 0.5% (w / v) PS80 or 0.1% (w / v) Poloxamer 188 as surfactant and 0, 1, 2, or 5% (w / v) sucrose were stressed by 5 F / T cycles at either -80°C or -30°C (thawed at room temperature for more than 24 hours). Samples were tested pre-freeze and after 1, 3, or 5 F / T cycles. The results of the pH analysis are shown in Figure 13. No significant pH trends or changes were observed in all compositions tested.

[0303] The turbidity of compositions containing 1 mg / mL efanesectocog alfa DS and poloxamer 188 was tested. Compositions containing 1% or 5% sucrose were tested and the results were compared to the turbidity results of a 1 mg / mL efanesectocog alfa DS composition containing PS80. No trends or changes in turbidity were observed in any of the poloxamer 188 compositions tested (data not shown).

[0304] The glass transition temperature (Tg) of 250 IU or 4000 IU efanesectocog alfa lyophilized pharmaceutical (Lyo DP) compositions containing 0.1% (w / v) poloxamer 188 and 0, 1, 2, or 5% (w / v) sucrose as surfactants was measured. Tg was measured by differential scanning calorimetry (DSC) controlled according to the following steps: (i) equilibration at 15°C, (ii) adjustment at 1°C every 60 seconds, (iii) isothermal for 5 minutes, and (iv) ramp to 130°C at 3°C / min. Residual moisture content (%) was also measured for all samples. The results of Tg and residual moisture content for Lyo DP 250IU at TO are shown in Figure 14. The results of Tg and residual moisture content for Lyo DP 4000IU at TO are shown in Figure 15.

[0305] The Tg of 4000 IU or 250 IU efanesectocog alfa lyophilized pharmaceutical (Lyo DP) compositions containing 0.1% (w / v) poloxamer 188 and 5% or 1% (w / v) sucrose as surfactants was determined. Samples were kept at 5°C, 30°C, or 40°C and tested at TO, 1 month, 2 months, 3 months, and 6 months. Results are shown in Figure 16A (5°C), Figure 16B (30°C), and Figure 16C (40°C).

[0306] The glass transition temperature (Tg) of 250 IU or 4000 IU efanesectocog alfa liquid bulk drug product (BDP) compositions containing either 0.5% (w / v) PS80 or 0.1% (w / v) poloxamer 188 as surfactant and 0, 1, 2, or 5% (w / v) sucrose was also determined. The Tg results at all strengths of both surfactants and sucrose concentrations are shown in Figure 17.

[0307] In both 250 IU or 4000 IU compositions containing either surfactant, the Tg was generally observed to correlate with the sucrose concentration, with higher sucrose concentrations exhibiting lower Tg. No significant differences in Tg were observed based on the type of surfactant at either the 250 IU or 4000 IU concentrations.

[0308] The residual moisture content of 250 IU or 4000 IU efanesectocog alfa lyophilized drug product (Lyo DP) compositions containing 0.1% (w / v) poloxamer 188 and 1% or 5% (w / v) sucrose as surfactants was determined. Samples were held at 5°C, 30°C, or 40°C and tested at TO, 1 month, 2 months, 3 months, and 6 months. The results are shown in Figure 18A (5°C), Figure 18B (30°C), and Figure 18C (40°C). No significant change in residual moisture content was observed in any of the poloxamer 188 compositions at all temperatures. All residual moisture content dropped below the 3% specification after 6 months.

[0309] Upon visual inspection, the concentration of sucrose had no effect on the appearance of the poloxamer 188 formulation cakes at 2-8°C, 30°C, and 40°C for 6 months (data not shown).

[0310] The results of this study indicate that, with the exception of aggregation, compositions of efanesectocog alfa formulated with either 0.5% (w / v) PS80 or 0.1% (w / v) poloxamer 188 have similar stability attributes. Evaluation of other stability attributes such as glass transition temperature, turbidity, and residual moisture did not show any significant differences based on the choice of surfactant. EXAMPLES

[0311] Effect of arginine concentration on the stability of efanesectocog alfa compositions Efanesectocog alfa showed a tendency to undergo reversible self-association after freeze / thaw (F / T) stress. The use of arginine in the efanesectocog alfa composition may provide a preventative effect against this F / T-induced molecular self-association. To identify the optimal concentration of arginine in the efanesectocog alfa composition, several compositions containing various concentrations of arginine were prepared and tested for aggregation levels by SEC. Each composition tested contained concentrated efanesectocog alfa DS (>2mg / mL), approximately 10mM L-histidine, 5mM CaCl2, 5% (w / v) sucrose, 0.1% (w / v) poloxamer 188 (P188), and L-arginine-HCl at concentrations of 125, 150, 175, 200, or 250mM.

[0312] To prepare the compositions for testing, the efanesoctocog alfa compositions were lyophilized using a LyoStar2 development scale freeze dryer (Protein Pharmaceutical Development Laboratories, Cambridge, MA). To test each sample, the composition was thawed and immediately injected into the HPLC for SEC analysis. Samples were analyzed immediately after thawing and at 40, 80, 120, and 160 minutes after thawing.

[0313] The results of aggregation assessment (% HMWS) for all samples are shown in Figure 19. Ef anesoctocog alfa compositions containing levels of arginine HCl below 250 mM showed increased aggregation levels immediately after thawing, followed by a decrease in aggregation levels over time (Figure 18). This aggregation tendency suggests the formation and dissociation of HMWS formed by F / T-induced molecular self-association of ef anesoctocog alfa. However, ef anesoctocog alfa compositions containing a concentration of 250 mM arginine HCl showed relatively low aggregation levels immediately after thawing, and these levels remained consistent over time (see the right-most vertical bar in Figure 18).

[0314] The results of this study indicate that efanesectocog alfa compositions with a concentration of 250 mM arginine HCl may be better protected from F / T-induced molecular self-association. Therefore, 250 mM is considered to be the preferred concentration of arginine HCl for use in formulating efanesectocog alfa compositions. EXAMPLES

[0315] Pharmacokinetic evaluation of efanesectocog alfa formulations containing different surfactants in non-human primates The objective of this study was to compare the pharmacokinetic (PK) profiles of efanesectocog alfa compositions containing polysorbate 80 (PS80) or poloxamer 188 (P188) in macaque monkeys.

[0316] Two vehicles were tested:

[0317] Vehicle 1: 10 mM L-histidine, 250 mM arginine-HCl, 5 mM calcium chloride, 5% w / v sucrose, 0.05% polysorbate 80

[0318] Vehicle 2: 10 mM L-histidine, 250 mM arginine-HCl, 5 mM calcium chloride, 5% w / v sucrose, 0.1% poloxamer 188

[0319] The first group of subjects received a single intravenous (IV) bolus injection of 75 IU / kg / dose of efanesectocog alfa in Vehicle 1. The second group of subjects received a single intravenous (IV) bolus injection of 75 IU / kg / dose of efanesectocog alfa in Vehicle 2. Both compositions contained efanesectocog alfa at a concentration of 37.5 IU / mL. A volume of 2 mL was administered to each subject. Blood samples were taken for PK analysis at pre-dose, 0.25, 1, 3, 8, 24, 48, 96, 168, 240, and 336 hours.

[0320] For the dose formulations, the concentrations of the analyzed formulation samples were 88.6% and 92.0% of the respective theoretical concentrations for groups 1 and 2. The dye activity (after replicate analysis) of the analyzed formulation samples was in the lower range, being 70.5% and 64.9% of the respective theoretical concentrations for groups 1 and 2, respectively.

[0321] Ephenesoctocog alfa protein concentration was determined by ELISA. Ephenesoctocog alfa protein concentration over time is shown in Figure 20. Ephenesoctocog alfa protein activity was determined using a chromogenic assay. Ephenesoctocog alfa activity over time is shown in Figure 21. Both protein concentration and activity profiles were similar in compositions containing either PS80 or P188.

[0322] PK parameters for efanesectocog alfa protein concentration and activity were determined and are shown in Table 2. Values ​​are presented as mean (±SD) and were generated from 4 animals for each group. As can be seen from Table 2, PK parameters were similar for compositions containing either PS80 or P188.

[0323] Ef anesoctocog alfa protein concentration and activity parameters were directly compared between subjects receiving the P188 or PS80 compositions.max and AUC (0-t) The ratios were determined to be 91.8% and 89.8%, respectively. max and AUC (0-t) The ratios were determined to be 106% and 105%, respectively.

[0324] Efanesectocog alfa and efanesectocog alfa activity max were observed at the first time point after dosing (i.e., 0.25 hours after dosing). The mean C for efanesectocog alfa concentrations in Vehicle 1 and 2, respectively max The mean AUC for efanesectocog alfa concentrations in vehicles 1 and 2 were 841 and 772, and for efanesectocog alfa activity, 2110 mIU / mL and 2230 mIU / mL. (0-t) The mean t1 / 2 for efanesectocog alfa concentrations were 36.1 hours and 35.8 hours, and for efanesectocog alfa activity were 34.4 hours and 35.3 hours, in vehicles 1 and 2, respectively. The mean CL for efanesectocog alfa concentrations were 36.1 hours and 35.8 hours, and for efanesectocog alfa activity were 34.4 hours and 35.3 hours, in vehicles 1 and 2, respectively. 血漿 were 1.33 mL / hr / kg and 1.44 mL / hr / kg, and for efanesectocog alfa activity were 1.29 mL / hr / kg and 1.20 mL / hr / kg. Mean Vd for efanesectocog alfa concentrations were 69.0 mL / kg and 74.4 mL / kg, and for BIVV001 activity were 63.8 mL / kg and 61.4 mL / kg, in vehicle 1 and 2, respectively.

[0325] In conclusion, the PK parameters of the efanesectocog alfa compositions were relatively similar between PS80 and P188, regardless of the choice of vehicle. There were no test item-related clinical signs or any injection site irritation noted following administration to subjects. All toxicokinetic parameters for each group were generated from four animals. Values ​​were rounded to three significant figures (T max (Except.

[0326] [Table 2]

[0327] The foregoing description of the specific embodiments fully discloses the general nature of the present disclosure, so that others can easily modify and / or adapt such specific embodiments for various applications by applying knowledge within the art without undue experimentation and without departing from the general concept of the present disclosure. Such adaptations and modifications are therefore intended to be within the meaning and range of equivalents of the disclosed embodiments based on the teaching and guidance presented herein. It should be understood that the words or terminology used herein are for the purpose of description, not of limitation, and thus the words and phrases used herein should be interpreted by those skilled in the art in light of the teaching and guidance.

[0328] Other embodiments of the present disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the present disclosure being indicated by the following claims.

[0329] All patents and publications cited herein are hereby incorporated by reference in their entirety.

[0330] array

[0331]

Table 3-1

Table 3-2

Table 3-3

Table 3-4

Table 3-5

Table 3-6

Table 3-7

[0332]

Table 4-1

Table 4-2

Table 4-3

Table 4-4

Claims

1. 1. A pharmaceutical composition comprising: (a) a chimeric protein, a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 1; a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:2; Including, a chimeric protein, wherein the first polypeptide chain and the second polypeptide chain are covalently linked by two disulfide bonds between the Fc domains of the first and second polypeptide chains; (b) sucrose; (c) histidine; (d) about 200 mM to about 300 mM arginine; (e) calcium chloride; and (f) poloxamer Including, About means ±10% of the pharmaceutical composition.

2. 10. The pharmaceutical composition of claim 1, comprising 200 mM to 300 mM arginine.

3. 3. The pharmaceutical composition of claim 1, comprising 1% (w / v) to 7.5% (w / v) sucrose.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the poloxamer is poloxamer 188 (P188).

5. 5. The pharmaceutical composition of claim 1, comprising 5 mM to 15 mM histidine.

6. 6. The pharmaceutical composition of any one of claims 1 to 5, comprising about 250 mM arginine, where about means ±10%.

7. The pharmaceutical composition of any one of claims 1 to 6, comprising 250 mM arginine.

8. 8. The pharmaceutical composition of claim 1, comprising 2.5 mM to 10 mM calcium chloride.

9. 7. The pharmaceutical composition of any one of claims 1 to 6, comprising 0.01% (w / v) to 1.0% (w / v) of poloxamer 188 (P188).

10. (a) about 5% (w / v) sucrose; (b) about 10 mM histidine; (c) about 250 mM arginine; (d) about 5 mM calcium chloride; and (e) about 0.1% (w / v) poloxamer 188 Including, The pharmaceutical composition according to any one of claims 1 to 7, wherein about means ±10%.

11. (a) 5% (w / v) sucrose; (b) 10 mM histidine; (c) 250 mM arginine; (d) 5 mM calcium chloride; and (e) 0.1% (w / v) Poloxamer 188 The pharmaceutical composition according to any one of claims 1 to 10, comprising:

12. The pharmaceutical composition according to any one of claims 1 to 11, having a pH of 6.5 to 7.

5.

13. 13. The pharmaceutical composition of any one of claims 1 to 12, having a pH of about 7.0, where about means ±10%.

14. The pharmaceutical composition according to any one of claims 1 to 13, having a pH of 7.

0.

15. 15. The pharmaceutical composition of any one of claims 1 to 14, comprising less than 8.8 mg / mL sodium chloride (NaCl).

16. The pharmaceutical composition according to any one of claims 1 to 15, wherein the histidine is L-histidine.

17. The pharmaceutical composition according to any one of claims 1 to 16, wherein the arginine is L-arginine.

18. The pharmaceutical composition of any one of claims 1 to 17, wherein the chimeric protein is efanesoctocog alfa.

19. 19. The pharmaceutical composition of any one of claims 1 to 18, comprising 250 IU, 500 IU, 1000 IU, 2000 IU, 3000 IU, or 4,000 IU of the chimeric protein.

20. The pharmaceutical composition according to any one of claims 1 to 19, which is suitable for intravenous infusion.

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