Compositions based on chimeric proteins containing factor viii and options for their application

Abstract

FIELD: pharmaceuticals.SUBSTANCE: pharmaceutical composition containing a chimeric protein containing a first polypeptide chain that contains an amino acid sequence that is at least 95% identical to the amino acid sequence under SEQ ID NO: 1, and a second polypeptide chain that contains an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2, sucrose, histidine, arginine, calcium chloride and polysorbate 80, and to a pharmaceutical kit comprising a first container containing a lyophilized pharmaceutical composition containing a chimeric protein, sucrose, histidine, arginine, calcium chloride and polysorbate 80, and a second container containing sterile water.EFFECT: stability of the protein in the composition.17 cl, 28 dwg, 20 tbl, 7 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Serial Nos. 63 / 214,245, filed June 23, 2021, 63 / 214,246, filed June 23, 2021, 63 / 214,752, filed June 24, 2021, and 63 / 231,909, filed August 11, 2021, each of which is incorporated herein by reference in its entirety.REFERENCE TO ELECTRONICALLY FILED SEQUENCE LISTING

[0002] The content of the sequence listing, filed electronically as an ASCII text file (name: SA9-480PC_ST25.txt; size 158 KB; generated on June 22, 2022), is incorporated herein by reference in its entirety.BACKGROUND OF THE INVENTION

[0003] Hemophilia A is a bleeding disorder caused by defects in the gene encoding coagulation factor VIII (FVIII) and affects 1–2 in 10,000 male births. Grawet al., Nat. Rev. Genet. 6(6): 488–501 (2005). Patients with hemophilia A can be treated with infusions of plasma-purified FVIII or recombinantly produced FVIII. Many commercially available FVIII products are known to have a half-life of approximately 8–12 hours, requiring frequent intravenous administration to patients. See Weiner MA and Cairo MS, Pediatric Hematology Secrets, Lee MT, 12. Disorders of Coagulation, Elsevier Health Sciences, 2001; Lillicrap D. Thromb. Res. 122 Suppl 4:S2-8 (2008) In addition, a number of approaches have been tried to extend the half-life of FVIII.For example, approaches under development to extend the half-life of coagulation factors include PEGylation, glycopegylation, and albumin conjugation. See Dumont et al., Blood. 119(13): 3024–3030 (2012). Significant results have been demonstrated in humans, with the rFVIII-Fc fusion protein reported to provide up to a 1.7-fold improvement in half-life compared to ADVATE. ® in patients with hemophilia A. See Powell et al., Blood. 119(13): 3031–3037 (2012). Therefore, increases in half-life, despite minor improvements, indicate the presence of other half-life-limiting factors, such as clearance by VWF. Pipe et al., Blood. 128(16): 2007–2016 (2016).

[0004] Ephanesoctocog alfa (also known as Efa and BIVV001) is a fusion protein designed to separate recombinant coagulation factor VIII from VWF in the circulation. The chimeric protein contains one protein representing recombinant factor VIII fused to a dimeric Fc, the D'D3 domain of VWF, and two ELNN polypeptides. Chhabra et al. Blood 135(17): 1484–1496 (2020). In one early study in patients with severe hemophilia A, a single intravenous injection of ephanesoctocog alfa resulted in sustained high levels of factor VIII activity, with a half-life up to fourfold longer than that associated with recombinant factor VIII. See Konkle et al., NEJM. 383:1018-27 (2020).

[0005] However, there remains a need for improved pharmaceutical compositions. SUMMARY OF THE INVENTION

[0006] The present invention relates to, among other things, pharmaceutical compositions containing a protein that is factor VIII ("FVIII"), kits containing such pharmaceutical compositions, as well as therapeutic methods and uses of such pharmaceutical compositions.

[0007] In some embodiments, a pharmaceutical composition comprises a chimeric protein that comprises a first polypeptide chain that comprises a FVIII protein or a portion thereof and a first immunoglobulin ("Ig") constant region or a portion thereof, and a second polypeptide chain that comprises 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 that comprises a FVIII polypeptide, an ELNN polypeptide inserted into a B domain (e.g., replacing at least a portion of the B domain) of a FVIII polypeptide, and a first Fc region; and (ii) a VWF protein that comprises a VWF fragment (e.g., a fragment comprising the D'D3 domains of VWF, wherein the fragment may comprise mutations), a second ELNN polypeptide, a thrombin-cleavable linker (such as 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.

[0008] In some embodiments, the pharmaceutical composition comprises: (a) FVIII protein; (b) sucrose; (c) histidine; (d) arginine; (e) calcium chloride, and (f) polysorbate. In some embodiments, the pharmaceutical composition comprises: (a) FVIII protein; (b) sucrose; (c) L-histidine; (d) L-arginine; (e) calcium chloride, and (f) polysorbate. In some embodiments, the pharmaceutical composition comprises: (a) a protein that is factor VIII (“FVIII”); (b) sucrose; (c) L-histidine; (d) L-arginine HCl; (e) calcium chloride dihydrate, and (f) polysorbate.

[0009] In some embodiments, the pharmaceutical composition comprises: (a) FVIII protein; (b) about 1% (w / v) to about 4% (w / v) sucrose; (c) about 5 mM to about 15 mM histidine; (d) about 150 mM to about 300 mM arginine; (e) about 2.5 mM to about 10 mM calcium chloride; and (f) about 0.008% (w / v) to about 0.1% (w / v) polysorbate. In some embodiments, the pharmaceutical composition comprises: (a) FVIII protein; (b) about 1% (w / v) to about 4% (w / v) sucrose; (c) about 5 mM to about 15 mM histidine; (d) from about 200 mM to about 300 mM arginine; (e) from about 2.5 mM to about 10 mM calcium chloride and (f) from about 0.008% (w / v) to about 0.1% (w / v) polysorbate.In some embodiments, the pharmaceutical composition comprises: (a) a FVIII protein; (b) about 1% (w / v) to about 4% (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 0.008% (w / v) to about 0.1% (w / v) polysorbate. In some embodiments, the pharmaceutical composition comprises: (a) a protein that is factor VIII ("FVIII"); (b) about 1% (w / v) to about 4% (w / v) sucrose; (c) about 5 mM to about 15 mM L-histidine; (d) from about 200 mM to about 300 mM L-arginine HCl; (e) from about 2.5 mM to about 10 mM calcium chloride dihydrate and (f) from about 0.008% (w / v) to about 0.1% (w / v) polysorbate.

[0010] In some embodiments, the pharmaceutical composition comprises: (a) FVIII protein; (b) about 5% (w / v) to about 7.5% (w / v) sucrose; (c) about 5 mM to about 15 mM histidine; (d) about 150 mM to about 300 mM arginine; (e) about 2.5 mM to about 10 mM calcium chloride; and (f) about 0.008% (w / v) to about 0.1% (w / v) polysorbate. In some embodiments, the pharmaceutical composition comprises: (a) FVIII protein; (b) about 5% (w / v) to about 7.5% (w / v) sucrose; (c) about 5 mM to about 15 mM histidine; (d) from about 200 mM to about 300 mM arginine; (e) from about 2.5 mM to about 10 mM calcium chloride and (f) from about 0.008% (w / v) to about 0.1% (w / v) polysorbate.In some embodiments, the pharmaceutical composition comprises: (a) a FVIII protein; (b) about 5% (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 0.008% (w / v) to about 0.1% (w / v) polysorbate. In some embodiments, the pharmaceutical composition comprises: (a) a protein that is factor VIII ("FVIII"); (b) about 5% (w / v) to about 7.5% (w / v) sucrose; (c) about 5 mM to about 15 mM L-histidine; (d) from about 200 mM to about 300 mM L-arginine HCl; (e) from about 2.5 mM to about 10 mM calcium chloride dihydrate and (f) from about 0.008% (w / v) to about 0.1% (w / v) polysorbate.

[0011] In some embodiments, the pharmaceutical composition comprises from about 1% (w / v) to about 4% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises from about 5% (w / v) to about 7.5% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises from about 5 mM to about 15 mM histidine. In some embodiments, the pharmaceutical composition comprises at least 150 mM arginine. In some embodiments, the pharmaceutical composition comprises from about 150 mM to about 300 mM arginine. In some embodiments, the pharmaceutical composition comprises at least 250 mM arginine. In some embodiments, the pharmaceutical composition comprises from about 200 mM to about 300 mM arginine.In some embodiments, the pharmaceutical composition comprises from about 2.5 mM to about 10 mM calcium chloride. In some embodiments, the pharmaceutical composition comprises polysorbate 20. In some embodiments, the pharmaceutical composition comprises polysorbate 80. In some embodiments, the pharmaceutical composition comprises from about 0.008% (w / v) to about 0.1% (w / v) polysorbate 20 or polysorbate 80.

[0012] In some embodiments, the composition comprises at least about 250 mM L-arginine. In some embodiments, the composition comprises at least about 250 mM L-arginine-HCl. 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 polysorbate is polysorbate 80. In some embodiments, the polysorbate is polysorbate 20.

[0013] In some embodiments, disclosed herein is a pharmaceutical composition comprising: (a) a chimeric protein comprising a first polypeptide chain that comprises a protein that is factor VIII ("FVIII") and a first immunoglobulin ("Ig") constant region or a portion thereof, and a second polypeptide chain that comprises a protein that is von Willebrand factor ("VWF") and a second Ig constant region or a portion thereof; (b) sucrose; (c) histidine; (d) arginine; (e) calcium chloride; and (f) polysorbate.

[0014] In some embodiments, disclosed herein is a pharmaceutical composition comprising: (a) a chimeric protein comprising a first polypeptide chain that comprises a protein that is factor VIII ("FVIII") and a first immunoglobulin ("Ig") constant region or a portion thereof, and a second polypeptide chain that comprises a protein that is von Willebrand factor ("VWF") and a second Ig constant region or a portion thereof; (b) from about 1% (w / v) to about 4% (w / v) sucrose; (c) from about 5 mM to about 15 mM histidine; (d) from about 150 mM to about 300 mM arginine; (e) from about 2.5 mM to about 10 mM calcium chloride, and (f) from about 0.008% (w / v) to about 0.1% (w / v) polysorbate.

[0015] In some embodiments, disclosed herein is a pharmaceutical composition comprising: (a) from about 5% (w / v) to about 7.5% (w / v) sucrose; (b) from about 5 mM to about 15 mM histidine; (c) from about 150 mM to about 300 mM arginine; (d) from about 2.5 mM to about 10 mM calcium chloride, and (e) from about 0.008% (w / v) to about 0.1% (w / v) polysorbate 20 or polysorbate 80.

[0016] In some embodiments, disclosed herein is a pharmaceutical composition comprising: (a) a chimeric protein comprising a first polypeptide chain that comprises a protein that is factor VIII ("FVIII") and a first immunoglobulin ("Ig") constant region or a portion thereof, and a second polypeptide chain that comprises a protein that is von Willebrand factor ("VWF") and a second Ig constant region or a portion thereof; (b) from about 1% (w / v) to about 4% (w / v) sucrose; (c) from about 5 mM to about 15 mM histidine; (d) from about 200 mM to about 300 mM arginine; (e) from about 2.5 mM to about 10 mM calcium chloride, and (f) from about 0.008% (w / v) to about 0.1% (w / v) polysorbate.

[0017] In some embodiments, disclosed herein is a pharmaceutical composition comprising: (a) from about 5% (w / v) to about 7.5% (w / v) sucrose; (b) from about 5 mM to about 15 mM histidine; (c) from about 200 mM to about 300 mM arginine; (d) from about 2.5 mM to about 10 mM calcium chloride, and (e) from about 0.008% (w / v) to about 0.1% (w / v) polysorbate 20 or polysorbate 80.

[0018] 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 first polypeptide chain comprises the amino acid sequence presented under SEQ ID NO: 1, and the second polypeptide chain comprises the amino acid sequence presented under SEQ ID NO: 2, wherein the first polypeptide chain and the second polypeptide chain are covalently linked by two disulfide bonds between the Fc domains in the first and second polypeptide chains. In some embodiments, the chimeric protein is ephanesoctocog-alpha.

[0019] In some embodiments, the pharmaceutical composition comprises from about 1% (w / v) to about 4% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises from about 5 mM to about 15 mM histidine. In some embodiments, the pharmaceutical composition comprises at least 150 mM arginine. In some embodiments, the pharmaceutical composition comprises from about 150 mM to about 300 mM arginine. In some embodiments, the pharmaceutical composition comprises at least 250 mM arginine. In some embodiments, the pharmaceutical composition comprises from about 200 mM to about 300 mM arginine. In some embodiments, the pharmaceutical composition comprises from about 2.5 mM to about 10 mM calcium chloride. In some embodiments, the pharmaceutical composition comprises polysorbate 20.In some embodiments, the pharmaceutical composition comprises polysorbate 80. In some embodiments, the pharmaceutical composition comprises from about 0.008% (w / v) to about 0.1% (w / v) polysorbate 20 or polysorbate 80.

[0020] In some embodiments, disclosed herein is a pharmaceutical composition comprising: (a) from about 1% (w / v) to about 4% (w / v) sucrose; (b) from about 5 mM to about 15 mM histidine; (c) from about 150 mM to about 300 mM arginine; (d) from about 2.5 mM to about 10 mM calcium chloride, and (e) from about 0.008% (w / v) to about 0.1% (w / v) polysorbate 20 or polysorbate 80.

[0021] In some embodiments, disclosed herein is a pharmaceutical composition comprising: (a) from about 1% (w / v) to about 4% (w / v) sucrose; (b) from about 5 mM to about 15 mM histidine; (c) from about 200 mM to about 300 mM arginine; (d) from about 2.5 mM to about 10 mM calcium chloride, and (e) from about 0.008% (w / v) to about 0.1% (w / v) polysorbate 20 or polysorbate 80.

[0022] In some embodiments, disclosed herein is a pharmaceutical composition comprising: (a) about 2% (w / v) sucrose; (b) about 10 mM histidine; (c) about 250 mM arginine; (d) about 5 mM calcium chloride, and (e) about 0.05% polysorbate 20 or polysorbate 80.

[0023] In some embodiments, disclosed herein is a pharmaceutical composition comprising: (a) about 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) about 0.05% polysorbate 80.

[0024] In some embodiments, disclosed herein is a pharmaceutical composition comprising: (a) 10 mg / ml to 40 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) from 0.4 mg / ml to 0.9 mg / ml calcium chloride and (e) 0.4 mg / ml to 0.7 mg / ml polysorbate 80.

[0025] In some embodiments, disclosed herein is a pharmaceutical composition comprising: (a) 22.45 mg / mL sucrose; (b) 1.74 mg / mL L-histidine; (c) 59.11 mg / mL L-arginine-HCl; (d) 0.82 mg / mL calcium chloride dihydrate; and (e) 0.56 mg / ml polysorbate 80.

[0026] In some embodiments, disclosed herein is a pharmaceutical composition comprising: (a) 22.45 mg / mL sucrose; (b) 1.74 mg / mL L-histidine; (c) 59.11 mg / mL L-arginine-HCl; (d) 0.62 mg / mL calcium chloride; and (e) 0.56 mg / ml polysorbate 80.

[0027] In some embodiments, disclosed herein is a pharmaceutical composition comprising: (a) 10 mg / ml to 40 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) from 0.5 mg / ml to 0.9 mg / ml calcium chloride dihydrate and (e) 0.4 mg / ml to 0.7 mg / ml polysorbate 80.

[0028] In some embodiments, disclosed herein is a pharmaceutical composition comprising: (a) about 20 mg / mL sucrose; (b) about 1.6 mg / mL L-histidine; (c) about 52.7 mg / mL L-arginine HCl; (d) about 0.7 mg / mL calcium chloride dihydrate, and (e) about 0.5 mg / mL polysorbate 80.

[0029] In some embodiments, disclosed herein is a pharmaceutical composition comprising: (a) about 20 mg / mL sucrose; (b) about 1.6 mg / mL L-histidine; (c) about 52.7 mg / mL L-arginine HCl; (d) about 0.6 mg / mL calcium chloride, and (e) about 0.5 mg / mL polysorbate 80.

[0030] In some embodiments, disclosed herein is a pharmaceutical composition comprising: (a) about 20 mg / mL sucrose; (b) about 1.6 mg / mL L-histidine; (c) about 43.6 mg / mL L-arginine; (d) about 0.7 mg / mL calcium chloride dihydrate, and (e) about 0.5 mg / mL polysorbate 80.

[0031] In some embodiments, disclosed herein is a pharmaceutical composition comprising: (a) about 20 mg / mL sucrose; (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) about 0.5 mg / mL polysorbate 80.

[0032] In some embodiments, a pharmaceutical composition is disclosed herein comprising:(a) from 10 mg / mL to 40 mg / mL sucrose;(b) from 1.5 mg / mL to 2.0 mg / mL L-histidine;(c) from 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) 0.4 mg / mL to 0.7 mg / mL polysorbate 80.

[0033] In some embodiments, a pharmaceutical composition is disclosed herein comprising:(a) from 10 mg / mL to 40 mg / mL sucrose;(b) from 1.5 mg / mL to 2.0 mg / mL L-histidine;(c) from 50 mg / mL to 70 mg / mL; L-arginine-HCl;(d) 0.4 mg / mL to 0.8 mg / mL calcium chloride and(e) 0.4 mg / mL to 0.7 mg / mL polysorbate 80.

[0034] In some embodiments, a pharmaceutical composition is disclosed herein comprising:(a) from 10 mg / mL to 40 mg / mL sucrose;(b) from 1.5 mg / mL to 2.0 mg / mL L-histidine;(c) from 40 mg / mL to 60 mg / mL; L-arginine;(d) 0.7 mg / mL to 0.9 mg / mL calcium chloride and(e) 0.4 mg / mL to 0.7 mg / mL polysorbate 80.

[0035] In some embodiments, a pharmaceutical composition is disclosed herein comprising:(a) from 10 mg / mL to 40 mg / mL sucrose;(b) from 1.5 mg / mL to 2.0 mg / mL L-histidine;(c) from 40 mg / mL to 60 mg / mL; L-arginine;(d) 0.4 mg / mL to 0.7 mg / mL calcium chloride dihydrate and(e) 0.4 mg / mL to 0.7 mg / mL polysorbate 80.

[0036] In some embodiments, a pharmaceutical composition is disclosed herein comprising:(a) 22.45 mg / mL sucrose;(b) 1.74 mg / mL L-histidine;(c) 59.11 mg / mL L-arginine; calcium chloride dihydrate and(e) 0.56 mg / ml polysorbate 80.

[0037] In some embodiments, a pharmaceutical composition is disclosed herein comprising:(a) 22.45 mg / mL sucrose;(b) 1.74 mg / mL L-histidine;(c) 48.88 mg / mL L-arginine;8.8 mg / mL didragine; calcium chloride and(e) 0.56 mg / ml polysorbate 80.

[0038] In some embodiments, a pharmaceutical composition is disclosed herein comprising:(a) 22.45 mg / mL sucrose;(b) 1.74 mg / mL L-histidine;(c) 59.11 mg / mL L-arginine; calcium chloride and(e) 0.56 mg / ml polysorbate 80.

[0039] In some embodiments, a pharmaceutical composition is disclosed herein comprising:(a) 22.45 mg / mL sucrose;(b) 1.74 mg / mL L-histidine;(c) 48.88 mg / mL L-arginine chloride; calcium and(e) 0.56 mg / ml polysorbate 80.

[0040] In some embodiments, the pharmaceutical composition comprises from about 5% (w / v) to about 7.5% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises from about 5 mM to about 15 mM histidine. In some embodiments, the pharmaceutical composition comprises at least 150 mM arginine. In some embodiments, the pharmaceutical composition comprises from about 150 mM to about 300 mM arginine. In some embodiments, the pharmaceutical composition comprises at least 250 mM arginine. In some embodiments, the pharmaceutical composition comprises from about 200 mM to about 300 mM arginine. In some embodiments, the pharmaceutical composition comprises from about 2.5 mM to about 10 mM calcium chloride. In some embodiments, the pharmaceutical composition comprises polysorbate 20.In some embodiments, the pharmaceutical composition comprises polysorbate 80. In some embodiments, the pharmaceutical composition comprises from about 0.008% (w / v) to about 0.1% (w / v) polysorbate 20 or polysorbate 80.

[0041] In some embodiments, disclosed herein is a pharmaceutical composition comprising: (a) from about 5% (w / v) to about 7.5% (w / v) sucrose; (b) from about 5 mM to about 15 mM histidine; (c) from about 150 mM to about 300 mM arginine; (d) from about 2.5 mM to about 10 mM calcium chloride, and (e) from about 0.008% (w / v) to about 0.1% (w / v) polysorbate 20 or polysorbate 80.

[0042] In some embodiments, disclosed herein is a pharmaceutical composition comprising: (a) from about 5% (w / v) to about 7.5% (w / v) sucrose; (b) from about 5 mM to about 15 mM histidine; (c) from about 200 mM to about 300 mM arginine; (d) from about 2.5 mM to about 10 mM calcium chloride, and (e) from about 0.008% (w / v) to about 0.1% (w / v) polysorbate 20 or polysorbate 80.

[0043] In some embodiments, disclosed herein is a pharmaceutical composition comprising: (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 0.05% polysorbate 20 or polysorbate 80.

[0044] In some embodiments, disclosed herein is a pharmaceutical composition comprising: (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) about 0.05% polysorbate 80.

[0045] In some embodiments, disclosed herein is a pharmaceutical composition comprising: (a) 45 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) from 0.4 mg / ml to 0.9 mg / ml calcium chloride and (e) 0.4 mg / ml to 0.7 mg / ml polysorbate 80.

[0046] In some embodiments, disclosed herein is a pharmaceutical composition comprising: (a) 56.12 mg / mL sucrose; (b) 1.74 mg / mL L-histidine; (c) 59.11 mg / mL L-arginine-HCl; (d) 0.82 mg / mL calcium chloride dihydrate; and (e) 0.56 mg / ml polysorbate 80.

[0047] In some embodiments, disclosed herein is a pharmaceutical composition comprising: (a) 56.12 mg / mL sucrose; (b) 1.74 mg / mL L-histidine; (c) 59.11 mg / mL L-arginine-HCl; (d) 0.62 mg / mL calcium chloride; and (e) 0.56 mg / ml polysorbate 80.

[0048] In some embodiments, disclosed herein is a pharmaceutical composition comprising: (a) 45 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) from 0.5 mg / ml to 0.9 mg / ml calcium chloride dihydrate and (e) 0.4 mg / ml to 0.7 mg / ml polysorbate 80.

[0049] In some embodiments, disclosed herein is a pharmaceutical composition comprising: (a) about 50 mg / mL sucrose; (b) about 1.6 mg / mL L-histidine; (c) about 52.7 mg / mL L-arginine HCl; (d) about 0.7 mg / mL calcium chloride dihydrate, and (e) about 0.5 mg / mL polysorbate 80.

[0050] In some embodiments, disclosed herein is a pharmaceutical composition comprising: (a) about 50 mg / mL sucrose; (b) about 1.6 mg / mL L-histidine; (c) about 52.7 mg / mL L-arginine HCl; (d) about 0.6 mg / mL calcium chloride, and (e) about 0.5 mg / mL polysorbate 80.

[0051] In some embodiments, disclosed herein is a pharmaceutical composition comprising: (a) about 50 mg / mL sucrose; (b) about 1.6 mg / mL L-histidine; (c) about 43.6 mg / mL L-arginine; (d) about 0.7 mg / mL calcium chloride dihydrate, and (e) about 0.5 mg / mL polysorbate 80.

[0052] In some embodiments, disclosed herein is a pharmaceutical composition comprising: (a) about 50 mg / mL sucrose; (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) about 0.5 mg / mL polysorbate 80.

[0053] In some embodiments, a pharmaceutical composition is disclosed herein comprising:(a) from 45 mg / mL to 60 mg / mL sucrose;(b) from 1.5 mg / mL to 2.0 mg / mL L-histidine;(c) from 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) 0.4 mg / mL to 0.7 mg / mL polysorbate 80.

[0054] In some embodiments, a pharmaceutical composition is disclosed herein comprising:(a) from 45 mg / mL to 60 mg / mL sucrose;(b) from 1.5 mg / mL to 2.0 mg / mL L-histidine;(c) from 50 mg / mL to 70 mg / mL; L-arginine-HCl;(d) 0.4 mg / mL to 0.8 mg / mL calcium chloride and(e) 0.4 mg / mL to 0.7 mg / mL polysorbate 80.

[0055] In some embodiments, a pharmaceutical composition is disclosed herein comprising:(a) from 45 mg / mL to 60 mg / mL sucrose;(b) from 1.5 mg / mL to 2.0 mg / mL L-histidine;(c) from 40 mg / mL to 60 mg / mL; L-arginine;(d) 0.7 mg / mL to 0.9 mg / mL calcium chloride and(e) 0.4 mg / mL to 0.7 mg / mL polysorbate 80.

[0056] In some embodiments, a pharmaceutical composition is disclosed herein comprising:(a) from 45 mg / mL to 60 mg / mL sucrose;(b) from 1.5 mg / mL to 2.0 mg / mL L-histidine;(c) from 40 mg / mL to 60 mg / mL; L-arginine;(d) 0.4 mg / mL to 0.7 mg / mL calcium chloride dihydrate and(e) 0.4 mg / mL to 0.7 mg / mL polysorbate 80.

[0057] In some embodiments, a pharmaceutical composition is disclosed herein comprising:(a) 56.12 mg / mL sucrose;(b) 1.74 mg / mL L-histidine;(c) 59.11 mg / mL L-arginine HCl;8.2 mg / mL; calcium chloride dihydrate and(e) 0.56 mg / ml polysorbate 80.

[0058] In some embodiments, a pharmaceutical composition is disclosed herein comprising:(a) 56.12 mg / mL sucrose;(b) 1.74 mg / mL L-histidine;(c) 48.88 mg / mL L-arginine;8.8 mg / mL didragine; calcium chloride and(e) 0.56 mg / ml polysorbate 80.

[0059] In some embodiments, a pharmaceutical composition is disclosed herein comprising:(a) 56.12 mg / mL sucrose;(b) 1.74 mg / mL L-histidine;(c) 59.11 mg / mL L-arginine; calcium chloride and(e) 0.56 mg / ml polysorbate 80.

[0060] In some embodiments, a pharmaceutical composition is disclosed herein comprising:(a) 56.12 mg / mL sucrose;(b) 1.74 mg / mL L-histidine;(c) 48.88 mg / mL L-arginine chloride; calcium and(e) 0.56 mg / ml polysorbate 80.

[0061] In some embodiments, disclosed herein is a pharmaceutical composition according to any of the disclosed embodiments, which is further characterized by a pH of from about 6.5 to about 7.5. In some embodiments, the pharmaceutical composition disclosed herein is characterized by a pH of from about 6.8 to about 7.3. In some embodiments, the pharmaceutical composition disclosed herein is characterized by a pH of about 7.0. In some embodiments, the pharmaceutical composition disclosed herein is characterized by a pH of about 6.8. In some embodiments, the pharmaceutical composition disclosed herein does not contain NaCl. In some embodiments, the pharmaceutical composition does not contain NaOH.In some embodiments, the pharmaceutical composition does not contain sodium ions. In some embodiments, the pharmaceutical composition disclosed herein contains less than 8.8 mg / mL of sodium chloride (NaCl). In some embodiments, the pharmaceutical composition disclosed herein contains L-histidine. In some embodiments, the pharmaceutical composition disclosed herein contains L-arginine. In some embodiments, the pharmaceutical composition disclosed herein contains arginine-HCl. In some embodiments, the pharmaceutical composition disclosed herein contains L-arginine-HCl. In some embodiments, the pharmaceutical composition disclosed herein contains calcium chloride dihydrate.

[0062] In some embodiments, disclosed herein is a pharmaceutical composition according to any of the disclosed embodiments, wherein the pharmaceutical composition is characterized by a chimeric protein concentration of from about 0.8 to about 1.2 mg / mL. In some embodiments, the pharmaceutical composition disclosed herein comprises from 75 IU / mL to 2000 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition disclosed herein is characterized by an osmolality of from about 525 to about 725 mOsm / kg. In some embodiments, the pharmaceutical composition disclosed herein is characterized by an osmolality of from about 600 to about 650 mOsm / kg.In some embodiments, the pharmaceutical composition disclosed herein has a turbidity of less than about 7 nephelometric turbidity units (NTU).

[0063] 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 from 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.

[0064] Also disclosed in this document is a pharmaceutical kit comprising: (i) a first container that contains a lyophilized pharmaceutical composition comprising: (a) a chimeric protein comprising a first polypeptide chain that contains a protein that is factor VIII ("FVIII"), or a portion thereof, and a first immunoglobulin ("Ig") constant region, or a portion thereof, and a second polypeptide chain that contains a protein that is von Willebrand factor ("VWF"), and a second Ig constant region, or a portion thereof; (b) sucrose; (c) histidine; (d) arginine; (e) calcium chloride, and (f) polysorbate 20 or polysorbate 80, and (ii) a second container containing sterile water.

[0065] In some embodiments, the chimeric protein comprises a first polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 1 and a second polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 2, wherein the first polypeptide chain and the second polypeptide chain are covalently linked by two disulfide bonds between the Fc domains in the first and second polypeptide chains.

[0066] Also disclosed herein is a pharmaceutical kit comprising: (i) a first container that contains a lyophilized pharmaceutical composition comprising: (a) a chimeric protein comprising a first polypeptide chain that comprises a protein that is factor VIII ("FVIII"), or a portion thereof, and a first immunoglobulin ("Ig") constant region, or a portion thereof, and a second polypeptide chain that comprises a protein that is von Willebrand factor ("VWF"), and a second Ig constant region, or a portion thereof; (b) from about 30 mg to about 135 mg of sucrose; (c) from about 2.5 mg to about 7.5 mg of histidine; (d) from about 140 mg to about 200 mg of arginine; (e) from about 1.5 mg to about 5 mg of calcium chloride, and (f) from about 1 mg to about 5 mg of polysorbate 20 or polysorbate 80; and(ii) a second container containing sterile water.In some embodiments, the chimeric protein comprises a first polypeptide chain comprising the amino acid sequence presented under SEQ ID NO: 1 and a second polypeptide chain comprising the amino acid sequence presented under SEQ ID NO: 2, wherein the first polypeptide chain and the second polypeptide chain are covalently linked by two disulfide bonds between the Fc domains in the first and second polypeptide chains.

[0067] In some embodiments, the pharmaceutical kit disclosed herein comprises a lyophilized pharmaceutical composition comprising: (a) from about 30 mg to about 135 mg of sucrose; (b) from about 2.5 mg to about 7.5 mg of histidine; (c) from about 140 mg to about 200 mg of arginine; (d) from about 1.5 mg to about 5 mg of calcium chloride, and (e) from about 1 mg to about 5 mg of polysorbate 20 or polysorbate 80.

[0068] In some embodiments, the pharmaceutical kit comprises a lyophilized pharmaceutical composition that does not contain NaCl. In some embodiments, the lyophilized pharmaceutical composition contains less than 8.8 mg / mL of sodium chloride (NaCl). In some embodiments, the pharmaceutical composition does not contain NaOH. In some embodiments, the pharmaceutical composition does not contain sodium ions. In some embodiments, the lyophilized pharmaceutical composition contains L-histidine. In some embodiments, the lyophilized pharmaceutical composition contains L-arginine. In some embodiments, the lyophilized pharmaceutical composition contains arginine-HCl. In some embodiments, the lyophilized pharmaceutical composition contains L-arginine-HCl.In some embodiments, the lyophilized pharmaceutical composition disclosed herein comprises calcium chloride dihydrate.

[0069] In some embodiments, the pharmaceutical kit comprises a lyophilized pharmaceutical composition comprising: (a) about 67.3 mg sucrose; (b) about 5.2 mg L-histidine; (c) about 177.3 mg L-arginine HCl; (d) about 2.5 mg calcium chloride, and (e) about 1.7 mg polysorbate 20 or polysorbate 80.

[0070] In some embodiments, the pharmaceutical kit comprises a lyophilized pharmaceutical composition comprising: (a) about 67.3 mg sucrose; (b) about 5.2 mg L-histidine; (c) about 146.6 mg L-arginine HCl; (d) about 2.5 mg calcium chloride, and (e) about 1.7 mg polysorbate 20 or polysorbate 80.

[0071] In some embodiments, the pharmaceutical kit disclosed herein comprises a lyophilized pharmaceutical composition comprising: (a) from about 160 mg to about 200 mg sucrose; (b) from about 2.5 mg to about 7.5 mg histidine; (c) from about 140 mg to about 200 mg arginine; (d) from about 1.5 mg to about 5 mg calcium chloride, and (e) from about 1 mg to about 5 mg polysorbate 20 or polysorbate 80.

[0072] In some embodiments, the pharmaceutical kit comprises a lyophilized pharmaceutical composition that does not contain NaCl. In some embodiments, the lyophilized pharmaceutical composition contains less than 8.8 mg / mL of sodium chloride (NaCl). In some embodiments, the pharmaceutical composition does not contain NaOH. In some embodiments, the pharmaceutical composition does not contain sodium ions. In some embodiments, the lyophilized pharmaceutical composition contains L-histidine. In some embodiments, the lyophilized pharmaceutical composition contains L-arginine. In some embodiments, the lyophilized pharmaceutical composition contains arginine-HCl. In some embodiments, the lyophilized pharmaceutical composition contains L-arginine-HCl.In some embodiments, the lyophilized pharmaceutical composition disclosed herein comprises calcium chloride dihydrate.

[0073] In some embodiments, the pharmaceutical kit comprises a lyophilized pharmaceutical composition comprising: (a) about 168.3 mg sucrose; (b) about 5.2 mg L-histidine; (c) about 177.3 mg L-arginine HCl; (d) about 2.5 mg calcium chloride, and (e) about 1.7 mg polysorbate 20 or polysorbate 80.

[0074] In some embodiments, the pharmaceutical kit comprises a lyophilized pharmaceutical composition comprising: (a) about 168.3 mg sucrose; (b) about 5.2 mg L-histidine; (c) about 146.6 mg L-arginine HCl; (d) about 2.5 mg calcium chloride, and (e) about 1.7 mg polysorbate 20 or polysorbate 80.

[0075] In some embodiments, the pharmaceutical kit comprises a lyophilized pharmaceutical composition characterized by a moisture content of less than 2%. In some embodiments, the lyophilized pharmaceutical composition is characterized by a moisture content of less than 1.8%. In some embodiments, the lyophilized pharmaceutical composition is characterized by a moisture content of less than 1.6%. In some embodiments, the lyophilized pharmaceutical composition is in the form of a lyophilized mass. In some embodiments, the lyophilized mass is white. In some embodiments, the lyophilized mass is characterized by a value of less than Y4 on the color scale of the European Pharmacopoeia. In some embodiments, the lyophilized pharmaceutical composition is a powder.

[0076] In some embodiments, the pharmaceutical kit comprises a first container containing from 100 IU to 10,000 IU of the chimeric protein. In some embodiments, the first container contains 250 IU, 500 IU, 1000 IU, 2000 IU, 3000 IU, or 4000 IU of the chimeric protein.

[0077] In some embodiments, the pharmaceutical kit further comprises instructions for combining the lyophilized pharmaceutical composition and sterile water. In some embodiments, upon combining the lyophilized pharmaceutical composition and sterile water, the lyophilized pharmaceutical composition is reconstituted within 7-12 seconds.

[0078] In some embodiments, the pharmaceutical composition comprises a second container containing sterilized water in a volume sufficient to produce, when combined with the lyophilized powder of the first container, the injection solution disclosed herein.

[0079] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the osmolality of the resulting solution is from about 525 to about 725 mOsm / kg. In some embodiments, the osmolality of the resulting solution is from about 600 to about 650 mOsm / kg. In some embodiments, the pH of the resulting solution is from 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 in the resulting solution is from about 0.8 to about 1.2 mg / mL. In some embodiments, less than 3% of the protein is aggregated.

[0080] In some embodiments, the pharmaceutical kit comprises a second container containing sterilized water in a volume sufficient to produce, when combined with the lyophilized pharmaceutical composition of the first container, a solution containing: (a) from 10 mg / ml to 40 mg / ml sucrose; (b) from 1.5 mg / ml to 2.0 mg / ml L-histidine; (c) from 40 mg / ml to 70 mg / ml L-arginine HCl; (d) from 0.5 mg / ml to 0.9 mg / ml calcium chloride and (e) from 0.4 mg / ml to 0.7 mg / ml polysorbate 80.

[0081] In some embodiments of the pharmaceutical kit disclosed herein, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) 10 mg / mL to 40 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) 0.4 mg / mL to 0.7 mg / mL polysorbate 80.

[0082] In some embodiments of the pharmaceutical kit 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) 59.11 mg / mL L-arginine HCl; (d) 0.62 mg / mL calcium chloride; and (e) 0.56 mg / mL polysorbate 80.

[0083] In some embodiments of the pharmaceutical kit 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) 0.56 mg / mL polysorbate 80.

[0084] In some embodiments, the pharmaceutical kit comprises a second container containing sterilized water in a volume sufficient to produce, when combined with the lyophilized pharmaceutical composition of the first container, a solution containing: (a) from 45 mg / ml to 60 mg / ml sucrose; (b) from 1.5 mg / ml to 2.0 mg / ml L-histidine; (c) from 40 mg / ml to 70 mg / ml L-arginine HCl; (d) from 0.5 mg / ml to 0.9 mg / ml calcium chloride and (e) from 0.4 mg / ml to 0.7 mg / ml polysorbate 80.

[0085] In some embodiments of the pharmaceutical kit disclosed herein, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) 45 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) 0.4 mg / mL to 0.7 mg / mL polysorbate 80.

[0086] In some embodiments of the pharmaceutical kit 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) 59.11 mg / mL L-arginine HCl; (d) 0.62 mg / mL calcium chloride; and (e) 0.56 mg / mL polysorbate 80.

[0087] In some embodiments of the pharmaceutical kit 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) 48.88 mg / mL L-arginine; (d) 0.62 mg / mL calcium chloride; and (e) 0.56 mg / mL polysorbate 80.

[0088] In some embodiments, the pharmaceutical kit comprises a second container containing from about 2 to about 5 ml of sterile water. In some embodiments, the pharmaceutical kit comprises a second container containing about 3 ml of sterile water. In some embodiments, the pharmaceutical kit comprises a second container containing about 3.3 ml of sterile water.

[0089] In some embodiments, the pharmaceutical kit comprises a first container, which is a glass vial containing a rubber stopper. In some embodiments, the pharmaceutical kit comprises a second container, which is a syringe body. In some embodiments, sterile water is contained in the syringe body. In some embodiments, the syringe body is associated with a plunger. In some embodiments, the pharmaceutical kit further comprises an adapter for attaching the glass vial to the syringe body. In some embodiments, the pharmaceutical kit further comprises an infusion system associated with a needle designed to be attached to the body of a syringe suitable for intravenous infusion.

[0090] Also disclosed herein is a method of treating hemophilia A in a subject in need thereof, comprising combining a lyophilized pharmaceutical composition and sterile water from a pharmaceutical kit according to any of the embodiments disclosed herein, and administering to the subject an effective amount of the resulting combination (i.e., solution). In some embodiments, the subject combines the lyophilized pharmaceutical composition and sterile water from the kit. In some embodiments, the combination is self-administered by the subject. BRIEF DESCRIPTION OF THE DRAWINGS / FIGURES

[0091] FIG. 1 is a schematic diagram of ephanesoctocog-alpha, an exemplary FVIII-ELNN-Fc / D'D3-ELNN-Fc heterodimer. FVIII: factor VIII; VWF: von Willebrand factor; A1, A2, A3, C1, C2: FVIII domains; D'D3: VWF domains; Fc: Fc region of immunoglobulin constant region.

[0092] FIG. 2 shows the pH of pharmaceutical compositions containing different concentrations of L-histidine (10 mM, 20 mM, 50 mM) at ephanesoctocoga alfa concentrations of 1 mg / mL, 0.67 mg / mL (4000 IU / mL), and 0.045 mg / mL (250 IU / mL). Controls are compositions without DS, without excipients, or compositions containing only excipients. Abbreviations: DP: drug product; BDP: bulk drug product; DS: drug substance; IU: international units.

[0093] FIG. 3 shows the measured turbidity (NTU) for pharmaceutical compositions containing different concentrations of L-histidine (10 mM, 20 mM, 50 mM) at ephanesoctocoga alfa concentrations of 1 mg / mL, 0.67 mg / mL (4000 IU / mL), and 0.045 mg / mL (250 IU / mL). The results of the compositions at 5, 25, and 40°C are shown. The time points were T0 and week 1.

[0094] FIG. 4 shows the aggregation levels (% HMWS) for pharmaceutical compositions containing different concentrations of L-histidine (10 mM, 20 mM, 50 mM) at ephanesoctocoga alfa concentrations of 1 mg / mL, 0.67 mg / mL (4000 IU / mL), and 0.045 mg / mL (250 IU / mL). The results are shown for the compositions at 5 and 25°C. The time points were T0 and week 1.

[0095] FIG. 5 shows the concentration (μg / mL) of ephanesoctocog alpha protein measured for each of the 13 tested buffer formulations. The formulations were tested at both 2-8°C and 30°C. The time points at which the measurements were taken were the beginning of the experiment (T0), one month (T1), 3 months (T3), and 6 months (T6). Abbreviations: DP: drug product; DDS: diluted drug substance; DoE: experimental design.

[0096] FIG. 6 shows the specific activity (IU / mg) of ephanesoctocog alpha protein measured for each of the 13 tested buffer formulations. The formulations were tested at both 2-8°C and 30°C. The time points at which the measurements were taken were the beginning of the experiment (T0), one month (T1), and 3 months (T3). Abbreviations: DP: drug product; DDS: diluted drug substance; DoE: experimental design.

[0097] FIG. 7 shows the aggregation levels (% HMWS) of ephanesoctocog alpha protein measured for each of the 13 tested buffer formulations. The formulations were tested at both 2-8°C and 30°C. The time points at which measurements were taken were the start of the experiment (T0), one month (T1), 3 months (T3), and 6 months (T6). The specification limit of 5% is indicated on the graph. Abbreviations: DP: drug product; DDS: diluted drug substance; DoE: experimental design.

[0098] FIG. 8 shows the glass transition temperature (Tg) measured using DSC for each of the 13 tested buffer formulations. The formulations were tested at both 2-8°C and 30°C. The time points at which the measurements were taken were: the beginning of the experiment (T0), one month (T1), 3 months (T3), 6 months (T6), and 12 months (T12). Abbreviations: DP: drug product; DDS: diluted drug substance; DoE: experimental design.

[0099] FIG. 9 shows the aggregation levels (% HMWS) over time (hours) for formulations of the drug substance (DS) ephanesoctocog alfa (1 mg / mL) containing 5% sucrose (dashed line) or 1% sucrose (solid line) under room temperature (RT) / room light (RL) conditions.

[0100] FIG. 10 shows the aggregation levels (% HMWS) over time (hours) for formulations of the drug substance (DS) ephanesoctocog alfa (1 mg / mL) containing 5% sucrose (dashed line) or 1% sucrose (solid line) at 2-8°C.

[0101] FIG. 11 shows the aggregation levels (% HMWS) over time (hours) for formulations containing 250 IU of ephanesoctocog alfa bulk drug product (BDP) containing sucrose at concentrations of 0, 1, 2, and 5% w / v under room temperature (RT) / room light (RL) conditions.

[0102] FIG. 12 shows the aggregation levels (% HMWS) over time (hours) of formulations containing 250 IU of bulk drug product (BDP) of ephanesoctocog alfa containing sucrose at concentrations of 0, 1, 2, and 5% w / v at 2-8°C.

[0103] FIG. 13 shows the aggregation levels (% HMWS) of formulations containing 250 IU ephanesoctocog alfa-based lyophilized drug product (Lyo DP) at 5°C. Lyo DP-based formulations containing 0, 1, 2, and 5% w / v sucrose were tested. Samples were tested at T0, 1 month, 2 months, 3 months, and 6 months.

[0104] FIG. 14 shows the aggregation levels (% HMWS) of formulations containing 250 IU ephanesoctocog alfa-based lyophilized drug product (Lyo DP) at 30°C. Lyo DP-based formulations containing 0, 1, 2, and 5% w / v sucrose were tested. Samples were tested at T0, 1 month, 2 months, 3 months, and 6 months.

[0105] FIG. 15 shows the aggregation levels (% HMWS) of formulations containing 250 IU ephanesoctocog alfa-based lyophilized drug product (Lyo DP) at 40°C. Lyo DP-based formulations containing 0, 1, 2, and 5% w / v sucrose were tested. Samples were tested at T0, 1 month, 2 months, 3 months, and 6 months.

[0106] FIG. 16 shows the aggregation levels (% HMWS) of formulations containing 4000 IU ephanesoctocog alfa-based lyophilized drug product (Lyo DP) at 5°C. Lyo DP-based formulations containing 0, 1, 2, and 5% w / v sucrose were tested. Samples were tested at T0, 1 month, 2 months, 3 months, and 6 months.

[0107] FIG. 17 shows the aggregation levels (% HMWS) of formulations containing 4000 IU ephanesoctocog alfa-based lyophilized drug product (Lyo DP) at 30°C. Lyo DP-based formulations containing 0, 1, 2, and 5% w / v sucrose were tested. Samples were tested at T0, 1 month, 2 months, 3 months, and 6 months.

[0108] FIG. 18 shows the aggregation levels (% HMWS) of formulations containing 4000 IU ephanesoctocog alfa-based lyophilized drug product (Lyo DP) at 40°C. Lyo DP-based formulations containing 0, 1, 2, and 5% w / v sucrose were tested. Samples were tested at T0, 1 month, 2 months, 3 months, and 6 months.

[0109] FIG. 19 shows the aggregation levels (% HMWS) over time (hours) of DS, which is ephanesoctocog alfa, and liquid formulations containing 4000 IU of bulk drug product (BDP), which contain either 2% w / v sucrose (solid line) or 5% w / v sucrose (dashed line). The samples were stored at room temperature or 5°C and tested at 0, 5, 7, 25, 43, and 55 hours.

[0110] FIG. 20 shows the glass transition temperature (Tg) measured using DSC for compositions containing 250 IU or 4000 IU of ephanesoctocog-alpha-based DP at 5°C. Compositions containing 0, 1, 2, and 5% sucrose (w / v) were tested at both activity levels. The time points at which the measurements were taken were: the beginning of the experiment (T0), one month (T1), 3 months (T3), and 6 months (T6).

[0111] FIG. 21 shows the glass transition temperature (Tg) measured using DSC for compositions containing 250 IU or 4000 IU of ephanesoctocog-alpha-based DP at 30°C. Compositions containing 0, 1, 2, and 5% sucrose (w / v) were tested at both activity levels. The time points at which the measurements were taken were: the beginning of the experiment (T0), one month (T1), 3 months (T3), and 6 months (T6).

[0112] FIG. 22 shows the glass transition temperature (Tg) measured using DSC for compositions containing 250 IU or 4000 IU of ephanesoctocog-alpha-based DP at 40°C. Compositions containing 0, 1, 2, and 5% sucrose (w / v) were tested at both activity levels. The time points at which the measurements were taken were: the beginning of the experiment (T0), one month (T1), 3 months (T3), and 6 months (T6).

[0113] FIG. 23 shows the glass transition temperature (Tg), measured using DSC, for liquid BDPs containing ephanesoctocog-alpha at 250 IU or 4000 IU. Formulations containing 0, 1, 2, and 5% w / v sucrose were tested at both activity levels.

[0114] FIG. 24 shows the residual moisture content of formulations containing lyophilized drug products (Lyo DP) based on ephanesoctocog alfa in an amount of 250 IU or 4000 IU at 5°C. Lyo DP-based formulations containing 0, 1, 2, and 5% w / v sucrose were tested. The time points at which measurements were taken were: the beginning of the experiment (T0), one month (T1), 3 months (T3), and 6 months (T6).

[0115] FIG. 25 shows the residual moisture content of formulations containing lyophilized drug products (Lyo DP) based on ephanesoctocog alfa in an amount of 250 IU or 4000 IU at 30°C. Lyo DP-based formulations containing 0, 1, 2, and 5% w / v sucrose were tested. The time points at which measurements were taken were: the beginning of the experiment (T0), one month (T1), 3 months (T3), and 6 months (T6).

[0116] FIG. 26 shows the residual moisture content of formulations containing lyophilized drug products (Lyo DP) based on ephanesoctocog alfa in an amount of 250 IU or 4000 IU at 40°C. Lyo DP-based formulations containing 0, 1, 2, and 5% w / v sucrose were tested. The time points at which measurements were taken were: the beginning of the experiment (T0), one month (T1), 3 months (T3), and 6 months (T6).

[0117] FIG. 27 shows a graphical representation of the percentage of high-molecular-weight (HMW) aggregates of ephanesoctocoga-alpha (BIVV001) analyzed by size-exclusion chromatography (SEC) with buffers at different arginine concentrations (Experiment 1). Data were collected at 0 min, 40 min, 80 min, 120 min, and 160 min after thawing.

[0118] FIG. 28 is a graphical representation of the percentage of high molecular weight (HMW) aggregates of ephanesoctocoga-alpha (BIVV001) analyzed by size exclusion chromatography (SEC) with buffers at different arginine concentrations (Experiment 2). Data were collected at 0 minutes, 40 minutes, 80 minutes, 120 minutes, and 160 minutes after thawing. DETAILED DESCRIPTION OF THE INVENTION

[0119] The present invention is directed to, among other things, formulations (including aqueous and lyophilized formulations, as well as related kits) comprising FVIII proteins. In some embodiments, the FVIII protein is a chimeric FVIII protein, such as ephanesoctocog-alpha, comprising two polypeptides, i.e., a first polypeptide comprising a FVIII protein that comprises an insert of a first ELNN polypeptide sequence fused to a first Fc region, and a second polypeptide comprising a VWF protein fused to a second Ig constant region via a second ELNN polypeptide sequence, wherein the first ELNN polypeptide sequence comprises approximately 288 amino acids, and the second ELNN polypeptide sequence comprises approximately 144 amino acids, and wherein the first Ig constant region and the second Ig constant region are covalently linked to each other by disulfide bonds.

[0120] The present invention provides formulations (including aqueous and lyophilized formulations, as well as kits thereof) for a chimeric protein comprising: (i) a polypeptide that is factor VIII (FVIII), and (ii) a fragment of von Willebrand factor (VWF) comprising the D' domain of VWF and the D3 domain of VWF. Compositions that can be lyophilized are included herein, as well as compositions formed upon reconstitution of lyophilized formulations with a diluent. Therapeutic methods and uses are also provided.I. Definitions

[0121] It should be noted that the singular form of an object refers to one or more such objects; for example, "a nucleotide sequence" refers to one or more nucleotide sequences. Therefore, the singular forms, the terms "one or more" and "at least one" may be used interchangeably herein.

[0122] Furthermore, "and / or" when used herein shall be understood to mean a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and / or" used herein in an expression such as "A and / or B" is intended to include "A and B", "A or B", "A" (alone) and "B" (alone). Similarly, the term "and / or" used in an expression such as "A, B and / or C" is intended to cover each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone) and C (alone).

[0123] It should be understood that whenever aspects are described in this document with the wording "comprising," other similar aspects are also contemplated when described with the terms "consisting of" and / or "consisting essentially of."

[0124] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention 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 Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press can provide one skilled in the art with a general dictionary containing many of the terms used in this invention.

[0125] Units of measurement, prefixes, and symbols are indicated in their SI form. Numerical ranges include the numbers defining the range. Unless otherwise indicated, amino acid sequences are written from left to right in the direction from the amino to the carboxy terminus. The headings provided herein are not intended to limit the various aspects of the present invention. Accordingly, terms defined immediately below are more fully defined by reference to this specification in its entirety.

[0126] The term "approximately" is used herein to mean approximately, on the order of, around, or roughly. When used in conjunction with a numerical range, the term "approximately" modifies the range, extending the boundaries above and below the stated numerical values. In general, the term "approximately" can modify a numerical value, making it greater or less than the stated value, with a deviation of, for example, 10 percent, up or down (higher or lower). In some embodiments, the term denotes a deviation from the stated numerical value by ±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%. In some embodiments, "approximately" denotes a deviation from the stated numerical value by ±10%. In some embodiments, "approximately" denotes a deviation from the stated numerical value by ±5%.In some embodiments, "about" means a deviation from the stated numerical value by ±4%. In some embodiments, "about" means a deviation from the stated numerical value by ±3%. In some embodiments, "about" means a deviation from the stated numerical value by ±2%. In some embodiments, "about" means a deviation from the stated numerical value by ±1%. In some embodiments, "about" means a deviation from the stated numerical value by ±0.9%. In some embodiments, "about" means a deviation from the stated numerical value by ±0.8%. In some embodiments, "about" means a deviation from the stated numerical value by ±0.7%. In some embodiments, "about" means a deviation from the stated numerical value by ±0.6%.In some embodiments, "about" means a deviation from the stated numerical value by ±0.5%. In some embodiments, "about" means a deviation from the stated numerical value by ±0.4%. In some embodiments, "about" means a deviation from the stated numerical value by ±0.3%. In some embodiments, "about" means a deviation from the stated numerical value by ±0.1%. In some embodiments, "about" means a deviation from the stated numerical value by ±0.05%. In some embodiments, "about" means a deviation from the stated numerical value by ±0.01%.

[0127] Depending on the context, the terms "polynucleotide" or "nucleotide" can encompass the singular, nucleic acid, as well as the plural, nucleic acids. In some embodiments, a polynucleotide is an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA) or plasmid DNA (pDNA). In some embodiments, a polynucleotide comprises a conventional phosphodiester linkage. In some embodiments, a polynucleotide comprises a non-conventional linkage (e.g., an amide linkage, such as that found in peptide nucleic acids (PNA)). 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 meant a nucleic acid molecule, DNA, or RNA, that has been removed from its native environment.For example, a recombinant polynucleotide encoding a factor VIII polypeptide contained in a vector is considered isolated within the framework of the present invention. Additional examples of isolated polynucleotides include recombinant polynucleotides contained in heterologous host cells or purified (partially or substantially) from other polynucleotides in solution. Isolated RNA molecules include RNA transcripts of the polynucleotides of the present invention produced in vivo or in vitro. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced by synthesis. In addition, a polynucleotide or nucleic acid may contain regulatory elements such as promoters, enhancers, ribosome binding sites, or transcription termination signals.

[0128] Certain proteins secreted by mammalian cells are associated with a secretory signal peptide that is cleaved from the mature protein after export of the nascent protein chain through the rough endoplasmic reticulum begins. Those of ordinary skill in the art recognize that signal polypeptides are typically fused to the N-terminus of a polypeptide and are cleaved from the complete or "full-length" polypeptide to form the secreted or "mature" form of the polypeptide. In some embodiments, a native signal peptide or a functional derivative of such a sequence that retains the ability to direct the secretion of the polypeptide functionally associated with it is used. Alternatively, a heterologous mammalian signal peptide can be used, such as human tissue plasminogen activator (TPA) or mouse ß-glucuronidase signal peptide or a functional derivative thereof.

[0129] The term "polypeptide" as used herein is intended to encompass the singular "polypeptide" and the plural "polypeptides" and refers to a molecule composed of monomers (amino acids) linked linearly by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of "polypeptide," and the term "polypeptide" may be used in place of any of these terms or interchangeably with any of them.The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with amino acids not found in nature. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but does not necessarily have to be obtained by translation of a specific nucleic acid sequence. It may be produced by any method, including chemical synthesis.

[0130] An "isolated" polypeptide or fragment, variant, or derivative thereof refers to a polypeptide that is not in its natural environment. No specific level of purification is required. For example, an isolated polypeptide may 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 the present invention, as are native or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0131] Also included in the present invention are fragments or variants of polypeptides and any combination thereof. The terms "fragment" or "variant" when referring to the binding domains of polypeptides or binding molecules of the present invention include any polypeptide that retains at least some of the properties of the reference polypeptide (e.g., FcRn binding affinity for an FcRn-binding domain or Fc variant, coagulation activity for an FVIII variant, or FVIII binding activity for a VWF fragment). Polypeptide fragments include fragments produced by proteolysis as well as fragments produced by deletion, in addition to the specific antibody fragments discussed elsewhere herein, but do not include a naturally occurring full-length polypeptide (or mature polypeptide).Variants of the binding domains of the polypeptides or binding molecules of the present invention include the fragments described above, as well as polypeptides with amino acid sequences altered by amino acid substitutions, deletions, or insertions. Variants may be naturally occurring or non-naturally occurring. Non-naturally occurring variants can be obtained using mutagenesis techniques known in the art. Polypeptide variants may contain conservative or non-conservative amino acid substitutions, deletions, or additions.

[0132] The term "VWF protein" as used herein means any fragment of VWF that interacts with FVIII and retains one or more properties that full-length VWF typically imparts to FVIII, such as preventing premature activation of FVIIIa, preventing premature proteolysis, preventing clearance, preventing association with phospholipid membranes that could lead to premature clearance, preventing binding to receptors that mediate FVIII clearance that can bind naked FVIII but not FVIII bound to VWF, and / or stabilizing interactions between the heavy chain and light chain of FVIII. A VWF fragment referred to herein is a VWF polypeptide that is smaller in size than full-length VWF protein, wherein the VWF fragment retains the ability to interact with and / or bind to FVIII.In some embodiments, the VWF protein is a fragment (which may be mutated) of full-length VWF that binds to the FVIII protein such that the FVIII protein has reduced or no binding to full-length VWF (e.g., endogenous VWF in the subject's body).

[0133] "Conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, if an amino acid in a polypeptide is replaced by another amino acid from the same side chain family, the substitution is considered conservative.In some embodiments, a strand of amino acids may be conservatively substituted with a structurally similar strand that differs in the order and / or composition of the side chain family members.

[0134] As is 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 terms "% identical," "% identity," or similar terms are intended to specifically refer to the percentage of nucleotides or amino acids (as the case may be) that are identical when optimally aligned in the compared sequences. The percentage indicated is purely statistical, and differences between the two sequences may necessarily be randomly distributed over the entire length of the compared sequences.Comparison of two sequences is typically performed by comparing the sequences after optimal alignment relative to a segment or "comparison window" to identify local regions of the corresponding sequences. For example, optimal alignment for comparison can be performed manually or by using the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by using the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by using the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or by using computer programs that use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI, USA).In some embodiments, the percent identity of two sequences is determined using the BLASTN or BLASTP algorithm available on the U.S. 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) an expected threshold set to 10; (ii) a word length set to 28; (iii) a maximum number of matches in a query range set to 0; (iv) a hit / mismatch score set to 1, -2; (v) a gap penalty set to "Linear"; and (vi) a filter to use for low complexity regions.In some embodiments, the algorithm parameters used for the BLASTP algorithm on the NCBI website include: (i) an expected threshold set to 10; (ii) a word length set to 3; (iii) a maximum number of matches in a query range set to 0; (iv) a matrix set to BLOSUM62; (v) gap penalties set to "Presence": 11, "Extension": 1; and (vi) a conditional compositional adjustment of the substitution matrix. As discussed herein, it can be determined whether any particular polypeptide is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to another polypeptide 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 Smith-Waterman local homology search algorithm (Advances in Applied Mathematics 2:482–489 (1981)) to find the best segment of homology between two sequences. When using BESTFIT or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the present invention, the parameters are, of course, set such that the percent identity is calculated over the full length of the reference polypeptide sequence, and gaps in homology of up to 5% of the total number of amino acids in the reference sequence are allowed.

[0135] As used herein, the "corresponding amino acid" or "equivalent amino acid" in the VWF sequence or FVIII protein sequence are identified by alignment to maximize the identity or similarity between the first VWF or FVIII sequence and the second VWF or FVIII sequence. The number used to identify the equivalent amino acid in the second VWF or FVIII sequence corresponds to the number used to identify the corresponding amino acid in the first VWF or FVIII sequence.

[0136] 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 a position into which a half-life extending component or a heterologous component can be inserted. The "insertion site" is indicated as a number, wherein the number is the amino acid number in mature native FVIII (SEQ ID NO: 8) to which the insertion site corresponds, which is located immediately downstream of the insertion position in the C-terminal direction. For example, the phrase "comprises an ELNN polypeptide at an insertion site that corresponds to amino acid 1656 of the sequence of SEQ ID NO: 8" indicates that the heterologous component is located between two amino acids corresponding to amino acid 1656 and amino acid 1657 in SEQ ID NO: 8.

[0137] As used herein, the term "immediately downstream of an amino acid" refers to a position immediately adjacent to the terminal carboxyl group of an amino acid. For example, an insertion site immediately downstream of amino acid 745, corresponding to that of the mature wild-type FVIII protein (SEQ ID NO: 8), means that the insertion site is located between amino acids 745 and 746, corresponding to those of the mature wild-type FVIII protein. Similarly, the term "immediately upstream of an amino acid" refers to a position immediately after the terminal amino group of an amino acid.

[0138] As used herein, the term "between two amino acids of the insertion site" refers to the position at which the ELNN polypeptide or any other polypeptide is inserted between two adjacent amino acids. Thus, the terms "inserted immediately downstream of an amino acid" and "inserted between two amino acids of the insertion site" are used as synonyms for the term "inserted into the insertion site."

[0139] The terms "inserted," "inserted," "inserted into," or grammatically related terms, as used herein with respect to insertions of the ELNN polypeptide into FVIII, refer to the position of the ELNN polypeptide in the chimeric protein relative to the analogous position in native mature human FVIII. The terms used herein refer to characteristics of the recombinant FVIII polypeptide relative to native mature human FVIII and do not indicate, imply, or suggest any methods or process by which the chimeric protein was produced.For example, with respect to the chimeric protein provided herein, the phrase "an ELNN polypeptide is inserted immediately downstream of residue 745 of a FVIII polypeptide" means that the chimeric protein comprises an ELNN polypeptide immediately downstream of an amino acid that corresponds to amino acid 745 in native mature human FVIII, e.g., limited 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 amino acid 746 of native mature human FVIII), and does not indicate the order or production method by which the chimeric protein was constructed.

[0140] As used herein, the terms "ELNN polypeptide" and "ELNN" are synonymous and refer to extended-length polypeptides with non-naturally occurring, substantially non-repetitive sequences that are composed primarily of small, hydrophilic amino acids, wherein the sequence is characterized by a low or no degree of secondary or tertiary structure formation under physiological conditions. ELNNs can impart certain desired pharmacokinetic, physicochemical, and pharmaceutical properties when linked to the VWF protein or FVIII sequence of the present invention to form a chimeric polypeptide. Such desired properties include, but are not limited to, improved pharmacokinetic parameters and solubility characteristics.As used herein, the terms "ELNN polypeptide" and "ELNN" specifically exclude antibodies or antibody fragments, such as single-chain antibodies or Fc fragments of the light chain or heavy chain. ELNN polypeptides are known in the art, and non-limiting descriptions and examples of ELNN polypeptides are known as XTEN polypeptides. ® , 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 full contents of each of which are incorporated herein by reference.

[0141] A "fusion" or "chimeric" protein comprises a first amino acid sequence linked to a second amino acid sequence to which it is not naturally linked. Amino acid sequences that normally exist in separate proteins may be combined in a fusion polypeptide, or amino acid sequences that normally exist in the same protein may be arranged in a new order in a fusion polypeptide, for example, by fusing the Factor VIII domain of the present invention with the Fc domain of Ig. The fusion protein is created, for example, by chemical synthesis or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired mutual arrangement. The chimeric protein may further comprise a second amino acid sequence associated with the first amino acid sequence via a covalent non-peptide bond or a non-covalent bond.

[0142] With respect to sequences, the term "linked" as used herein refers to a first amino acid sequence or nucleotide sequence covalently or non-covalently attached to a second amino acid sequence or nucleotide sequence, respectively. The first amino acid or nucleotide sequence may be directly attached to or combined with the second amino acid or nucleotide sequence, or, alternatively, an intervening sequence may covalently attach the first sequence to the second sequence.Regardless of the context, the term "linked" means not only the fusion of a first amino acid sequence to a 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) between any two amino acids in the second amino acid sequence (or, respectively, in the first amino acid sequence). In some embodiments, the first amino acid sequence may be linked to the second amino acid sequence via a peptide bond or a linker. The first nucleotide sequence may be linked to the second nucleotide sequence via a phosphodiester bond or a linker.A linker may be a peptide or polypeptide (in the case of polypeptide chains), a nucleotide or nucleotide chain (in the case of nucleotide chains), or any chemical component (in the case of both polypeptide and polynucleotide chains). The term "linked" may also be denoted by a hyphen (-).

[0143] When used with two polypeptides, the term "associated with" refers to one or more covalent or non-covalent bonds formed between a first polypeptide and a second polypeptide. In some embodiments, the term "associated with" means a covalent non-peptide bond or a non-covalent bond. This bond may be indicated by a colon, i.e. (:). In some embodiments, this means any covalent bond except a peptide bond. For example, the amino acid cysteine ​​contains a thiol group that can form a disulfide bond or bridge with the thiol group at the second cysteine ​​residue. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by a disulfide bond, and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 according to the Kabat numbering system (positions 226 or 229, EU numbering system).Examples of covalent bonds include, but are not limited to, a peptide bond, a metallic bond, a hydrogen bond, a disulfide bond, a sigma bond, a pi bond, a delta bond, a glycosidic bond, an agostic bond, a banana bond, a dipolar bond, a reverse donor-acceptor pi bond, a double bond, a triple bond, a quadruple bond, a quinary bond, a sextuple bond, conjugation, hyperconjugation, aromaticity, hapticity, or antibonding. Non-limiting examples of non-covalent bonding include ionic bonding (e.g., cationic pi bonding or salt bonding), metallic bonding, hydrogen bonding (e.g., dihydrogen bonding, dihydrogen complex, low-barrier hydrogen bonding, or symmetric hydrogen bonding), van der Waals force, London dispersion force, mechanical bonding, halogen bonding, aurophilicity, intercalation, stacking, entropic force, or chemical polarity.In some embodiments, one or more covalent bonds between the first amino acid chain and the second amino acid chain are two disulfide bonds. In some embodiments, one or more covalent bonds between the first amino acid chain and the second amino acid chain are two disulfide bonds between the first Fc portion of the first amino acid chain and the second Fc portion of the second amino acid chain, wherein the two disulfide bonds occur in the hinge region of the two Fc portions.

[0144] In other embodiments, the polypeptide comprises an enzymatic cleavage site cleaved by an enzyme that is activated during the blood coagulation cascade, such that cleavage of such sites occurs at the site of clot formation. Examples of such sites include, for example, sites recognized by thrombin, factor XIa, or factor Xa. Other enzymatic cleavage sites are known in the art and are described elsewhere herein. It should be understood that in constructs that contain 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 expressed as the time required for half of the amount administered to the subject to be eliminated from the bloodstream and / or other tissues of an animal.In some embodiments, when the elimination curve of a given polypeptide is plotted as a function of time, the curve is typically biphasic with a rapid α-phase and a longer β-phase. Typically, the α-phase reflects the equilibration of the administered Fc polypeptide between the intravascular and extravascular spaces and is determined in part by the size of the polypeptide. Typically, the β-phase reflects the catabolism of the polypeptide in the intravascular space. In some embodiments, FVIII and chimeric proteins containing FVIII are monophasic and, thus, are characterized by the absence of an alpha phase and the presence of only a distinct beta phase. Therefore, in some embodiments, the term "half-life" as used herein refers to the half-life of the polypeptide in the β-phase. In humans, the typical beta-phase half-life for a human antibody is 21 days. In some embodiments, the half-life is expressed as the terminal phase half-life.

[0145] In some embodiments, the subject has hemophilia A. In some embodiments, hemophilia A represents a severe form of hemophilia A.

[0146] As used herein, "administer" or "administering" refers to delivering a composition described herein, such as a chimeric protein, to a subject. The composition, such as 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 through any mucosal surface, such as orally, sublingually, buccally, nasally, rectally, vaginally, or pulmonarily. In some embodiments, the administration is intravenous. In some embodiments, the administration is subcutaneous. In some embodiments, the 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 the subject by a healthcare professional, such as a physician, a healthcare professional, or a nurse.

[0147] As used herein, the term "dose" refers to a single administration of a composition to a subject. A single dose may be administered all at once, such as by bolus injection, or over a period of time, such as by intravenous infusion. The term "multiple doses" refers to more than one dose, such as more than one administration.

[0148] If more than one composition is administered together, a dose of composition A may be administered simultaneously with a dose of composition B. Alternatively, a dose of composition A may be administered before or after a dose of composition B. In some embodiments, composition A and composition B are combined into a single formulation.

[0149] As used herein, the term "interval" or "interval between doses" refers to the amount of time that elapses between the first dose of Composition A and the subsequent dose of the same composition administered to a subject. The interval between doses may refer to the time that elapses between the first dose and the second dose, or the interval between doses may refer to the amount of time that elapses between multiple doses.

[0150] The term "dosing frequency" as used in this document refers to the number of doses administered per specific dosing interval. For example, dosing frequency may be written as once a week, once every two weeks, etc. Therefore, a dosing interval of 7 days may also be written as a dosing interval of once every 7 days, or every week, or once a week.

[0151] 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 episodes of spontaneous bleeding, 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 progression of joint disease, patients with hemophilia A may receive regular infusions of clotting factor as part of a prophylactic treatment regimen.The basis for such prophylactic treatment is the observation that patients with hemophilia with clotting factor levels, such as FVIII, of 1% or greater, rarely experience spontaneous bleeding episodes and have fewer hemophilia-related comorbidities than patients with severe hemophilia. See, e.g., Coppola A. et al, Semin. Thromb. Hemost. 38(1): 79–94 (2012). Clinicians treating such patients with hemophilia have suggested that maintaining factor levels at around 1% with regular infusions could potentially reduce the risk of hemophilia symptoms, including bleeding episodes and joint damage. See ibid. Subsequent studies have confirmed these benefits in pediatric patients with hemophilia receiving prophylactic treatment with clotting factor, making prophylactic treatment a goal for people with severe hemophilia. See ibid.

[0152] "Prophylactic" treatment may also refer to the preventive administration to a subject of a composition described herein, such as a protein (such as a chimeric protein), for the purpose of controlling, managing, preventing, or reducing the incidence or severity of one or more symptoms of hemophilia A, such as bleeding episodes. In some embodiments, prophylactic treatment with a clotting factor, such as FVIII, is used to treat subjects with severe hemophilia A. In some embodiments, prophylactic treatment refers to administering a composition disclosed herein to a subject in need thereof to reduce the incidence of one or more symptoms of hemophilia A. Prophylactic treatment may involve the administration of multiple doses. Multiple doses used in prophylactic treatment are typically administered with specific intervals between dose administrations.In some embodiments, the average annual bleeding frequency may 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.

[0153] The term "on-demand treatment" or "episodic treatment" refers to the "as-needed" administration of a chimeric molecule in response to symptoms of hemophilia A, such as a bleeding episode, or before the start of an activity that may cause bleeding. In some aspects, on-demand treatment may be administered to a subject when bleeding has begun, such as after an injury, or when bleeding is expected, such as before surgery. In some aspects, on-demand treatment may be administered before the start of activities that increase the risk of bleeding, such as contact sports. In some embodiments, on-demand treatment is administered as a single dose. In some embodiments, on-demand treatment is administered as a first dose, followed by one or more additional doses.If the chimeric protein is administered on-demand, one or more additional doses may 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 first dose. However, it should be noted that the dosing interval associated with on-demand treatment is not the same as the dosing interval used for prophylactic treatment.

[0154] ​​In some embodiments, a subject in need of a general hemostatic agent is undergoing or is about to undergo surgery. The chimeric protein of the present invention can be administered before or after surgery. The chimeric protein of the present invention can also be administered during or after surgery to control an acute bleeding episode. If the chimeric protein is administered before surgery, the administration can be performed 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.If the chimeric protein is administered after surgery, the administration can be performed 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. Surgery may include, but is not limited to, liver transplantation, liver resection, dental procedures, or stem cell transplantation.

[0155] "Treat," "treatment," "treating," as used herein, refers to, for example, reducing the severity of a disease or condition; reducing the duration of the disease; reducing the intensity of one or more symptoms associated with the disease or condition; providing beneficial effects in a subject with the disease or condition, without necessarily curing the disease or condition, or preventing one or more symptoms associated with the disease or condition. In some embodiments, performing the treatment or treating comprises maintaining in the subject a residual FVIII level of 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 the chimeric protein of the present invention.As used herein, a "trough level" in a patient with hemophilia A is a measured value of the lowest concentration achieved with coagulation factor therapy, such as FVIII therapy, before the next dose. In some embodiments, treating or treating means maintaining a trough level of FVIII of at least about 1 IU / dL between doses. In some embodiments, treating or treating means maintaining a trough level of FVIII of at least about 3 IU / dL between doses. In some embodiments, treating or treating means maintaining a trough level of FVIII of at least about 5 IU / dL between doses.In some embodiments, performing treatment or treating means maintaining a residual FVIII level of from about 1 to about 20 IU / dL, from about 2 to about 20 IU / dL, from about 3 to about 20 IU / dL, from about 4 to about 20 IU / dL, from about 5 to about 20 IU / dL, from about 6 to about 20 IU / dL, from about 7 to about 20 IU / dL, from about 8 to about 20 IU / dL, from about 9 to about 20 IU / dL, or from about 10 to about 20 IU / dL during the interval between dose administrations.

[0156] In some embodiments, treating or treating a disease or condition comprises 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 between doses. In some embodiments, treating or treating means maintaining a FVIII activity level of at least about 1% between doses. In some embodiments, treating or treating means maintaining a FVIII activity level of at least about 2% between doses. In some embodiments, performing treatment or treating means maintaining a FVIII activity level of at least about 3% between doses.In some embodiments, treating or treating means maintaining a FVIII activity level of at least about 4% between doses. In some embodiments, treating or treating means maintaining a FVIII activity level of at least about 5% between doses. In some embodiments, treating or treating means maintaining a FVIII activity level of at least about 6% between doses. In some embodiments, treating or treating means maintaining a FVIII activity level of at least about 7% between doses. In some embodiments, treating or treating means maintaining a FVIII activity level of at least about 8% between doses.In some embodiments, treating or treating means maintaining a FVIII activity level of at least about 9% between doses. In some embodiments, treating or treating means maintaining a FVIII activity level of at least about 10% between doses. In some embodiments, the trough level required for treatment can be measured by one or more known methods (e.g., activated partial thromboplastin time (aPTT) assays or chromogenic assays, 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. Publication No. 20190375822, which is incorporated herein by reference in its entirety. II.Chimeric proteins.

[0157] In one aspect, the present invention is directed to pharmaceutical compositions comprising a chimeric protein or a protein that comprises a first polypeptide chain that comprises a protein that is factor VIII ("FVIII"), or a portion thereof, and a first immunoglobulin ("Ig") constant region or a portion thereof, and a second polypeptide chain that comprises a protein that is von Willebrand factor ("VWF"), 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 a 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.Non-limiting examples of chimeric proteins that can be used in various embodiments are described in U.S. Patent No. 10,138,291 and U.S. Patent No. 11,192,936 B2, the entire contents of each of which are incorporated herein by reference.

[0158] In some embodiments, the chimeric protein is ephanesoctocog-alpha. Ephanesoctocog-alpha, also known as "BIVV001," "ephanesoctocogum-alpha," 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 representation of ephanesoctocog-alpha as an exemplary FVIII-ELNN-Fc / D'D3-ELNN-Fc heterodimer is shown in FIG. 1.

[0159] Ephanesoctocog-alpha is a large protein (over 300 kDa) consisting of multiple moieties in each of its two polypeptide chains, which are associated through covalent bonds and non-covalent interactions. The protein exhibits a tendency to aggregate under certain conditions, which may reduce the stability of formulations unless an excipient, such as L-arginine, is selected and present in an amount sufficient to reduce aggregation. For example, aggregation can be reduced by adding high levels of L-arginine (e.g., approximately 250 mM).

[0160] Additional information on ephanesoctocog alfa can be found in the 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) the first polypeptide and (ii) the second polypeptide, which are 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, ephanesoctocog-alpha has a potency of at least 1600 IU / mg. In some embodiments, ephanesoctocog-alpha has a potency of at least 1700 IU / mg. In some embodiments, ephanesoctocog-alpha has a potency of at least 1800 IU / mg. In some embodiments, ephanesoctocog-alpha has a potency of at least 1900 IU / mg. In some embodiments, ephanesoctocog-alpha has a potency of between 1600 IU / mg and 2000 IU / mg.

[0161] 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 locations: 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 locations: residues 153-179, 248-329, 528-554, 630-711, 1220-1246, 1287-1291, 1409-1557, 1562-1714, 1761-1821 and 1867-1925 of the sequence under 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 the sequence under SEQ ID NO: 1.In some embodiments, the chimeric protein comprises a FVIII protein comprising Cys-SH residues at each of residues 310, 692, and / or 1388 of SEQ ID NO: 1.

[0162] In some embodiments, the chimeric protein comprises a FVIII protein comprising one or more N-glycosylation sites at residues N41, N239, N1198, N1506 and / or N1797 of the sequence of SEQ ID NO: 1. In some embodiments, the chimeric protein comprises a FVIII protein comprising one or more O-glycosylation sites at residues 746-1036 of the sequence of SEQ ID NO: 1 and / or Ser and Thr residues in linker peptides. In some embodiments, the chimeric protein comprises a FVIII protein comprising one or more Tyr-sulfation sites at residues 346, 718, 719, 723, 729, 1052 and / or 1068 of the sequence of SEQ ID NO: 1.

[0163] 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 locations: 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 from the sequence under 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 locations: 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 from the sequence under SEQ ID NO: 2.

[0164] In some embodiments, the chimeric protein comprises a VWF protein comprising one or more N-glycosylation sites at residues N94, N384, N734 of the sequence 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 the sequence of SEQ ID NO: 2 and / or Ser and Thr residues in linker peptides. 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 the sequence of SEQ ID NO: 2. In some embodiments, the VWF protein comprises a VWF fragment comprising a VWF domain D1, D2, D' and / or D3. 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 that comprises the amino acid sequence of SEQ ID NO: 19, a VWF D1D2 region that comprises the amino acid sequence of SEQ ID NO: 20, a VWF D' domain that comprises the amino acid sequence of SEQ ID NO: 21, a VWF D3 domain that comprises the amino acid sequence of SEQ ID NO: 22; an ELNN polypeptide sequence that comprises the amino acid sequence of SEQ ID NO: 14 (AE144_5A); an a2 linker that comprises the amino acid sequence of SEQ ID NO: 15; and / or an Fc region that comprises the amino acid sequence of SEQ ID NO: 23.

[0165] In some embodiments, a chimeric protein of the present invention 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, an FVIII a2 linker, and a second Fc region; wherein: the FVIII protein is characterized by having a deletion of amino acids 746-1648 corresponding to mature FVIII; the first ELNN polypeptide sequence is inserted into the FVIII polypeptide immediately downstream of amino acid 745 corresponding to mature FVIII;the first 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 AE288 (SEQ ID NO: 9); the first Fc region is fused to the C-terminus of the FVIII polypeptide; the second ELNN polypeptide sequence is fused to the C-terminus of the VWF fragment;the second ELNN polypeptide sequence comprises an amino acid sequence characterized by 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 linker a2 is fused to the C-terminus of the ELNN polypeptide;linker a2 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: 15; a second Fc region is fused to the C-terminus of linker a2; and the first Fc region is covalently linked to the second Fc region via a disulfide bond (e.g., two disulfide bonds).

[0166] In some embodiments, a chimeric protein of the present invention comprises two polypeptide sequences - a first polypeptide sequence comprising an amino acid sequence that is 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 comprising a VWF fragment comprising the VWF D' domain and the VWF D3 domain, and an Fc region. In some embodiments, a chimeric protein of the present invention comprises two polypeptide sequences - a first polypeptide sequence comprising a FVIII polypeptide and an Fc region; and a second polypeptide sequence comprising an amino acid sequence that is 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 invention comprises two polypeptide sequences - a first polypeptide sequence comprising an amino acid sequence that is 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 comprising an amino acid sequence that is 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 invention comprises two polypeptide sequences - a first polypeptide sequence comprising the amino acid sequence set forth in SEQ ID NO: 7, and a second polypeptide sequence comprising the amino acid sequence set forth in SEQ ID NO: 2.In some embodiments, the chimeric protein of the present invention 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 and the second polypeptide sequence are linked to each other via a disulfide bond.In some embodiments, the chimeric protein of the present invention 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 and the second polypeptide sequence are linked to each other via two disulfide bonds.In some embodiments, the chimeric protein of the present invention 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, wherein the second polypeptide sequence comprises a second Fc portion, wherein the first Fc portion and the second Fc portion are linked to each other via two disulfide bonds in the hinge region.

[0167] In some embodiments, the chimeric protein of the present invention 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.

[0168] In some embodiments, the chimeric protein of the present invention comprises: (i) a FVIII protein comprising a first FVIII polypeptide fragment that comprises the amino acid sequence of SEQ ID NO: 17; a first ELNN polypeptide sequence that comprises the amino acid sequence of SEQ ID NO: 9 (AE288); a second FVIII polypeptide fragment that comprises the amino acid sequence of SEQ ID NO: 18; and a first Fc region that comprises the amino acid sequence of SEQ ID NO: 23; and (ii) a VWF protein comprising: a VWF D' domain that comprises the amino acid sequence of SEQ ID NO: 21; a VWF D3 domain that comprises the amino acid sequence of SEQ ID NO: 22; a second ELNN polypeptide sequence that comprises the amino acid sequence of SEQ ID NO: 14 (AE144_5A); linker a2, which contains the amino acid sequence of SEQ ID NO: 15;and a second Fc region that comprises the amino acid sequence of SEQ ID NO: 23, and wherein the first Fc region is covalently linked to the second Fc region via a disulfide bond (e.g., two disulfide bonds).;

[0169] In some embodiments, a chimeric protein of the present invention 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, an 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; the first Fc region comprises the amino acid sequence of SEQ ID NO: 23 and is fused to the C-terminus of SEQ ID NO: 18; the VWF D' domain comprises the amino acid sequence of SEQ ID NO: 21;the VWF D3 domain comprises the amino acid sequence of SEQ ID NO: 21, 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 VWF D3 domain; 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 sequence; 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; and wherein the first Fc region is covalently linked to the second Fc region via a disulfide bond.

[0170] In some embodiments, the chimeric protein of the present invention comprises a FVIII protein comprising a FVIII signal peptide that comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the chimeric protein comprises a VWF protein comprising a VWF signal peptide that comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, the chimeric protein comprises a VWF protein comprising a VWF D1D2 domain that comprises the amino acid sequence of SEQ ID NO: 20.

[0171] In some embodiments, the chimeric protein comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 3; and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, the chimeric protein comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 7; and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 2. 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.

[0172] In some embodiments, the chimeric protein comprises one or more disulfide bridges between the first polypeptide and the second polypeptide. In some embodiments, the chimeric protein comprises two disulfide bridges between the first polypeptide and the second polypeptide. 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, wherein the chimeric protein comprises a disulfide bridge between residue 1726 of SEQ ID NO: 1 and residue 663 of SEQ ID NO: 2 and a disulfide bridge between residue 1729 of SEQ ID NO: 1 and residue 666 of SEQ ID NO: 2.IV. Pharmaceutical Compositions

[0173] In one aspect, the present invention is directed to chimeric protein-based pharmaceutical compositions that are formulated to improve protein stability. In some embodiments, the disclosed pharmaceutical compositions exhibit increased stability, as determined by visual inspection, protein concentration, pH stability, high molecular weight species (HMWS) formation, and / or turbidity change. Analysis of these stability properties can be performed using conventional techniques, including size exclusion chromatography (SEC), reversed-phase high-performance liquid chromatography (RP-HPLC), and many others.

[0174] The pharmaceutical compositions disclosed herein comprise a certain amount of a chimeric protein. In some embodiments, the pharmaceutical composition is characterized by a chimeric protein concentration of from about 0.8 to about 1.2 mg / mL. In some embodiments, the pharmaceutical composition is characterized by a chimeric protein concentration of about 0.8 mg / mL. In some embodiments, the pharmaceutical composition is characterized by a chimeric protein concentration of about 0.9 mg / mL. In some embodiments, the pharmaceutical composition is characterized by a chimeric protein concentration of about 1.0 mg / mL. In some embodiments, the pharmaceutical composition is characterized by a chimeric protein concentration of about 1.1 mg / mL.In some embodiments, the pharmaceutical composition has a chimeric protein concentration of approximately 1.2 mg / mL.

[0175] In some embodiments, the pharmaceutical composition comprises from 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.

[0176] 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 approximately 1900 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises approximately 1950 IU / mL of the chimeric protein. In some embodiments, the pharmaceutical composition comprises approximately 2000 IU / mL of the chimeric protein.

[0177] Pharmaceutical compositions containing the chimeric protein of the present invention also comprise a suitable pharmaceutically acceptable carrier. For example, they may contain excipients and / or auxiliary agents that provide increased stability of the chimeric protein or facilitate the processing of the active compounds into preparations intended for delivery to the site of action.

[0178] In one aspect, pharmaceutical compositions comprising a certain amount of a chimeric protein together with the disclosed excipients are disclosed herein. The pharmaceutical compositions disclosed herein contain, as disclosed, various concentrations of said excipients, and these 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 (mg / ml). The pharmaceutical compositions provided herein can contain certain amounts of various excipients at a level of accuracy ranging from approximate, for example, concentrations expressed only to one significant digit (e.g., about 0.1% (w / v))), or with greater accuracy, such as to 2, 3, 4, 5, or 6 significant digits (e.g., approximately 3.88 mg / mL to three significant digits). The required level of accuracy may vary depending on, for example, the requirements of a particular regulatory agency or manufacturing process.

[0179] The pharmaceutical compositions disclosed herein may contain a stabilizing agent.

[0180] In some embodiments, the pharmaceutical compositions disclosed herein comprise certain amounts or concentrations of sucrose.

[0181] In some embodiments, the pharmaceutical composition comprises from 4.5% (w / v) to 8% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises from about 5% (w / v) to about 7.5% (w / v) sucrose.

[0182] 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 5.0% (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 6.0% (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 7.0% (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.

[0183] In some embodiments, the pharmaceutical composition comprises 168.3 mg of sucrose. In some embodiments, the pharmaceutical composition comprises approximately 168.3 mg of sucrose. In some embodiments, the amount of sucrose may vary by no more than 10% of the specified amount. In some embodiments, the specific amount of sucrose is 168.3 mg. In some embodiments, the amount of sucrose may vary by no more than 5% of the specified amount. In some embodiments, the specific amount of sucrose is 168.3 mg. In some embodiments, the amount of sucrose may vary by no more than 1% of the specified amount. In some embodiments, the specific amount of sucrose is 168.3 mg.

[0184] In some embodiments, the pharmaceutical composition comprises from 1% (w / v) to 4% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises from about 1.5% (w / v) to about 2.5% (w / v) sucrose.

[0185] 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.

[0186] In some embodiments, the pharmaceutical composition comprises 67.34 mg of sucrose. In some embodiments, the pharmaceutical composition comprises approximately 67.34 mg of sucrose. In some embodiments, the amount of sucrose may vary by no more than 10% of the specified amount. In some embodiments, the specific amount of sucrose is 67.34 mg. In some embodiments, the amount of sucrose may vary by no more than 5% of the specified amount. In some embodiments, the specific amount of sucrose is 67.34 mg. In some embodiments, the amount of sucrose may vary by no more than 1% of the specified amount. In some embodiments, the specific amount of sucrose is 67.34 mg.

[0187] The pharmaceutical compositions disclosed herein may include a buffer. In some embodiments, the pharmaceutical compositions disclosed herein include certain amounts or concentrations of histidine. In some embodiments, the histidine included in the pharmaceutical composition is L-histidine. In some embodiments, the pharmaceutical composition comprises from about 5 mM to about 15 mM histidine.

[0188] 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.

[0189] In some embodiments, the pharmaceutical compositions disclosed herein include certain amounts or concentrations of arginine. In some embodiments, the pharmaceutical composition comprises arginine hydrochloride (HCl). In some embodiments, the arginine is L-arginine. In some embodiments, the composition comprises L-arginine-HCl.

[0190] In some embodiments, the pharmaceutical composition comprises at least 150 mM arginine. In some embodiments, the pharmaceutical composition comprises at least 200 mM arginine. In some embodiments, the pharmaceutical composition comprises at least 250 mM arginine. In some embodiments, the pharmaceutical composition comprises from about 150 mM to about 300 mM arginine.

[0191] In some embodiments, the pharmaceutical composition comprises from about 200 mM to about 300 mM arginine.

[0192] In some embodiments, the pharmaceutical composition comprises about 150 mM arginine. In some embodiments, the pharmaceutical composition comprises about 160 mM arginine. In some embodiments, the pharmaceutical composition comprises about 170 mM arginine. In some embodiments, the pharmaceutical composition comprises about 180 mM arginine. In some embodiments, the pharmaceutical composition comprises about 190 mM arginine. 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.

[0193] The pharmaceutical compositions disclosed herein may comprise a bulking agent. In some embodiments, the pharmaceutical compositions disclosed herein comprise certain amounts or concentrations of calcium chloride (CaCl2). In some embodiments, the composition comprises CaCl2⋅2H2O, CaCl2 (anhydrous), CaCl2⋅4H2O, or CaCl2⋅6H2O. In some embodiments, the composition comprises calcium chloride dihydrate. In some embodiments, the pharmaceutical composition comprises from about 2.5 mM to about 10 mM calcium chloride. In some embodiments, the composition comprises calcium chloride dihydrate.

[0194] 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.

[0195] In some embodiments, the pharmaceutical compositions disclosed herein do not contain bulking agents other than calcium chloride. In some embodiments, the only bulking agent is calcium chloride. In some embodiments, the pharmaceutical composition contains less than 8.8 mg / mL sodium chloride (NaCl). In some embodiments, the pharmaceutical composition is substantially free of sodium chloride. In some embodiments, the pharmaceutical composition does not contain sodium chloride.

[0196] In some embodiments, the pharmaceutical compositions disclosed herein comprise certain amounts or concentrations of polysorbate 20 (PS20) or polysorbate 80 (PS80). In some embodiments, the pharmaceutical composition comprises from about 0.008% (w / v) to about 0.1% (w / v) PS80 or PS20. In some embodiments, the pharmaceutical composition comprises at least about 0.03% PS20 or PS80. In some embodiments, the pharmaceutical composition comprises about 0.05% PS20 or PS80. In some embodiments, the pharmaceutical composition comprises PS20. In some embodiments, the pharmaceutical composition comprises PS80.

[0197] In some embodiments, the pharmaceutical composition comprises about 0.008% (w / v) polysorbate 20. In some embodiments, the pharmaceutical composition comprises about 0.01% (w / v) polysorbate 20. In some embodiments, the pharmaceutical composition comprises about 0.02% (w / v) polysorbate 20. In some embodiments, the pharmaceutical composition comprises about 0.03% (w / v) polysorbate 20. In some embodiments, the pharmaceutical composition comprises about 0.04% (w / v) polysorbate 20. In some embodiments, the pharmaceutical composition comprises about 0.05% (w / v) polysorbate 20. In some embodiments, the pharmaceutical composition comprises about 0.06% (w / v) polysorbate 20. In some embodiments, the pharmaceutical composition comprises about 0.07% (w / v) polysorbate 20.In some embodiments, the pharmaceutical composition comprises about 0.08% (w / v) polysorbate 20. In some embodiments, the pharmaceutical composition comprises about 0.09% (w / v) polysorbate 20. In some embodiments, the pharmaceutical composition comprises about 0.1% (w / v) polysorbate 20.

[0198] In some embodiments, the pharmaceutical composition comprises about 0.008% (w / v) polysorbate 80. In some embodiments, the pharmaceutical composition comprises about 0.01% (w / v) polysorbate 80. In some embodiments, the pharmaceutical composition comprises about 0.02% (w / v) polysorbate 80. In some embodiments, the pharmaceutical composition comprises about 0.03% (w / v) polysorbate 80. In some embodiments, the pharmaceutical composition comprises about 0.04% (w / v) polysorbate 80. In some embodiments, the pharmaceutical composition comprises about 0.05% (w / v) polysorbate 80. In some embodiments, the pharmaceutical composition comprises about 0.06% (w / v) polysorbate 80. In some embodiments, the pharmaceutical composition comprises about 0.07% (w / v) polysorbate 80.In some embodiments, the pharmaceutical composition comprises about 0.08% (w / v) polysorbate 80. In some embodiments, the pharmaceutical composition comprises about 0.09% (w / v) polysorbate 80. In some embodiments, the pharmaceutical composition comprises about 0.1% (w / v) polysorbate 80.

[0199] In some embodiments, the pharmaceutical composition is a solution intended for lyophilization. In some embodiments, the solution intended for lyophilization does not contain NaCl. In some embodiments, the solution intended for lyophilization does not contain NaOH. In some embodiments, the solution intended for lyophilization does not contain sodium ions.

[0200] In some embodiments, the pharmaceutical composition comprises: (a) from about 1% (w / v) to about 4% (w / v) sucrose; (b) from about 5 mM to about 15 mM histidine; (c) from about 150 mM to about 300 mM arginine; (d) from about 2.5 mM to about 10 mM calcium chloride, and (e) from about 0.008% (w / v) to about 0.1% (w / v) polysorbate 20 or polysorbate 80.

[0201] In some embodiments, the pharmaceutical composition comprises: (a) from about 1% (w / v) to about 4% (w / v) sucrose; (b) from about 5 mM to about 15 mM histidine; (c) from about 200 mM to about 300 mM arginine; (d) from about 2.5 mM to about 10 mM calcium chloride, and (e) from about 0.008% (w / v) to about 0.1% (w / v) polysorbate 20 or polysorbate 80. In some embodiments, the composition comprises polysorbate 80. 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 solution intended for lyophilization.

[0202] In some embodiments, the pharmaceutical composition comprises: (a) 2% (w / v) to 3% (w / v) sucrose; (b) 7.5 mM to 12.5 mM histidine; (c) 225 mM to about 300 mM arginine; (d) 5 mM to 6 mM calcium chloride, and (e) 0.01% (w / v) to about 0.075% (w / v) polysorbate 20 or polysorbate 80. In some embodiments, the composition comprises polysorbate 80. In some embodiments, the histidine is L-histidine. In some embodiments, the arginine is L-arginine. In some embodiments, the composition comprises 225 mM to about 300 mM L-arginine-HCl. In some embodiments, the composition comprises 5 mM to 6 mM calcium chloride dihydrate. In some embodiments, the pharmaceutical composition is a solution intended for lyophilization.

[0203] In some embodiments, the pharmaceutical composition comprises: (a) 2% (w / v) to 3% (w / v) sucrose; (b) 7.5 mM to 12.5 mM L-histidine; (c) 225 mM to about 300 mM L-arginine; (d) 5 mM to 6 mM calcium chloride, and (e) 0.01% (w / v) to about 0.075% (w / v) polysorbate 80.

[0204] In some embodiments, the pharmaceutical composition comprises: (a) 2% (w / v) to 3% (w / v) sucrose; (b) 7.5 mM to 12.5 mM L-histidine; (c) 225 mM to about 300 mM L-arginine HCl; (d) 5 mM to 6 mM calcium chloride, and (e) 0.01% (w / v) to about 0.075% (w / v) polysorbate 80.

[0205] In some embodiments, the pharmaceutical composition comprises: (a) 2% (w / v) to 3% (w / v) sucrose; (b) 7.5 mM to 12.5 mM L-histidine; (c) 225 mM to about 300 mM L-arginine HCl; (d) 5 mM to 6 mM calcium chloride dihydrate, and (e) 0.01% (w / v) to about 0.075% (w / v) polysorbate 80.

[0206] In some embodiments, the pharmaceutical composition comprises: (a) about 2.25% (w / v) sucrose; (b) about 11.2 mM L-histidine; (c) about 280.58 mM L-arginine HCl; (d) about 5.61 mM calcium chloride dihydrate, and (e) 0.056% (w / v) polysorbate 80.

[0207] In some embodiments, the pharmaceutical composition comprises: (a) about 2.25% (w / v) sucrose; (b) about 11.2 mM L-histidine; (c) about 280.58 mM L-arginine; (d) about 5.61 mM calcium chloride, and (e) about 0.056% (w / v) polysorbate 80.

[0208] In some embodiments, the pharmaceutical composition comprises: (a) 2.25% (w / v) sucrose; (b) 11.2 mM L-histidine; (c) 280.58 mM L-arginine HCl; (d) about 5.61 mM calcium chloride dihydrate, and (e) 0.056% (w / v) polysorbate 80.

[0209] In some embodiments, the pharmaceutical composition comprises: (a) 2.25% (w / v) sucrose; (b) 11.2 mM L-histidine; (c) 280.58 mM L-arginine; (d) 5.61 mM calcium chloride, and (e) 0.056% (w / v) polysorbate 80.

[0210] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 2.25% (w / v) sucrose; (b) about 11.2 mM L-histidine; (c) about 280.58 mM L-arginine HCl; (d) about 5.61 mM calcium chloride dihydrate, and (e) 0.056% (w / v) polysorbate 80.

[0211] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 2.25% (w / v) sucrose; (b) about 11.2 mM L-histidine; (c) about 280.58 mM L-arginine; (d) about 5.61 mM calcium chloride, and (e) about 0.056% (w / v) polysorbate 80.

[0212] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) 2.25% (w / v) sucrose; (b) 11.2 mM L-histidine; (c) 280.58 mM L-arginine HCl; (d) approximately 5.61 mM calcium chloride dihydrate (5.61 mM calcium chloride); and (e) 0.056% (w / v) polysorbate 80.

[0213] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) 2.25% (w / v) sucrose; (b) 11.2 mM L-histidine; (c) 280.58 mM L-arginine; (d) 5.61 mM calcium chloride, and (e) 0.056% (w / v) polysorbate 80.

[0214] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) 2% (w / v) to 3% (w / v) sucrose; (b) 11.2 mM L-histidine; (c) 280.58 mM L-arginine; (d) 5.61 mM calcium chloride; and (e) 0.056% (w / v) polysorbate 80.

[0215] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) 2.25% (w / v) sucrose; (b) 7.5 mM to 12.5 mM L-histidine; (c) 280.58 mM L-arginine; (d) 5.61 mM calcium chloride, and (e) 0.056% (w / v) polysorbate 80.

[0216] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) 2.25% (w / v) sucrose; (b) 11.2 mM L-histidine; (c) 225 mM to about 300 mM L-arginine; (d) 5.61 mM calcium chloride, and (e) 0.056% (w / v) polysorbate 80.

[0217] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) 2.25% (w / v) sucrose; (b) 11.2 mM L-histidine; (c) 280.58 mM L-arginine; (d) 5 mM to 6 mM calcium chloride, and (e) 0.056% (w / v) polysorbate 80.

[0218] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) 2.25% (w / v) sucrose; (b) 11.2 mM L-histidine; (c) 280.58 mM L-arginine; (d) 5.61 mM calcium chloride, and (e) from 0.01% (w / v) to about 0.075% (w / v) polysorbate 80.

[0219] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution does not contain NaCl. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution does not contain NaOH. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution does not contain sodium ions.

[0220] In some embodiments, the pharmaceutical composition comprises: (a) about 2% (w / v) sucrose; (b) about 10 mM histidine; (c) about 250 mM arginine; (d) about 5 mM calcium chloride, and (e) about 0.05% polysorbate 20 or polysorbate 80. In some embodiments, the composition comprises polysorbate 80. 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 solution intended for lyophilization.

[0221] In some embodiments, the pharmaceutical composition comprises: (a) about 2% (w / v) to about 3% (w / v) sucrose; (b) about 10 mM histidine; (c) about 250 mM arginine; (d) about 5 mM calcium chloride, and (e) about 0.05% polysorbate 20 or polysorbate 80. In some embodiments, the composition comprises polysorbate 80. 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 solution intended for lyophilization.

[0222] In some embodiments, the pharmaceutical composition comprises: (a) about 2% (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 0.05% polysorbate 80. 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 solution intended for lyophilization.

[0223] In some embodiments, the pharmaceutical composition comprises: (a) about 2% (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 0.05% polysorbate 80. 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 solution intended for lyophilization.

[0224] In some embodiments, the pharmaceutical composition comprises: (a) about 2% (w / v) sucrose; (b) about 10 mM histidine; (c) about 250 mM arginine; (d) from about 2.5 mM to about 10 mM calcium chloride, and (e) about 0.05% polysorbate 80. 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 solution intended for lyophilization.

[0225] In some embodiments, the pharmaceutical composition comprises: (a) about 2% (w / v) sucrose; (b) about 10 mM histidine; (c) about 250 mM arginine; (d) about 5 mM calcium chloride, and

[0226] (e) from about 0.008% (w / v) to about 0.1% (w / v) polysorbate 80. 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 solution intended for lyophilization.

[0227] In some embodiments, the pharmaceutical composition comprises: (a) about 20 mg / mL sucrose; (b) about 1.552 mg / mL L-histidine; (c) about 52.665 mg / mL L-arginine HCl; (d) about 0.735 mg / mL calcium chloride, and (e) about 0.5 mg / mL polysorbate 80.

[0228] In some embodiments, the pharmaceutical composition comprises: (a) about 20 mg / mL sucrose; (b) about 1.552 mg / mL L-histidine; (c) about 52.665 mg / mL L-arginine HCl; (d) about 0.735 mg / mL calcium chloride dihydrate, and (e) about 0.5 mg / mL polysorbate 80.

[0229] In some embodiments, the pharmaceutical composition comprises: (a) about 20 mg / mL sucrose; (b) about 1.552 mg / mL L-histidine; (c) about 52.665 mg / mL L-arginine HCl; (d) about 0.555 mg / mL calcium chloride; and (e) about 0.5 mg / mL polysorbate 80. In some embodiments, the composition comprises calcium chloride dihydrate. In some embodiments, the pharmaceutical composition is a solution intended for lyophilization.

[0230] In some embodiments, the pharmaceutical composition comprises: (a) about 20 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 dihydrate; and (e) about 0.5 mg / mL polysorbate 80. 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 solution intended for lyophilization.

[0231] In some embodiments, the pharmaceutical composition comprises: (a) about 20 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) about 0.5 mg / mL polysorbate 80. 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 solution intended for lyophilization.

[0232] In some embodiments, the pharmaceutical composition comprises: (a) about 20 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) about 0.5 mg / mL polysorbate 80. 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 solution intended for lyophilization.

[0233] In some embodiments, the pharmaceutical composition contains: (a) 22.45 mg / ml sucrose; (b) 1.74 mg / ml L-histidine; (c) 59.11 mg / ml L-arginine-HCl; (d) 0.82 mg / ml calcium chloride dihydrate; and (e) 0.56 mg / ml polysorbate 80.

[0234] In some embodiments, the pharmaceutical composition comprises:(a) 22.45 mg / mL sucrose;(b) 1.74 mg / mL L-histidine;(c) 59.11 mg / mL L-arginine-HCl;(d) 0.62 mg / mL calcium chloride and(e) 0.56 mg / mL polysorbate 80. In some embodiments, the composition comprises calcium chloride dihydrate.

[0235] In some embodiments, the pharmaceutical composition comprises:(a) 22.45 mg / mL sucrose;(b) 1.74 mg / mL L-histidine;(c) 48.88 mg / mL L-arginine;(d) 0.8 mg / mL dihydrate chloride and(e) 0.56 mg / ml polysorbate 80.

[0236] In some embodiments, the pharmaceutical composition comprises:(a) 22.45 mg / mL of sucrose;(b) 1.74 mg / mL of L-histidine;(c) 48.88 mg / mL of L-arginine;(d) 0.62 mg / mL of calcium chloride (0.06) mg / mL polysorbate 80. In some embodiments, the composition comprises L-arginine-HCl. In some embodiments, the composition comprises calcium chloride dihydrate.

[0237] In some embodiments, the pharmaceutical composition comprises: (a) from about 5% (w / v) to about 7.5% (w / v) sucrose; (b) from about 5 mM to about 15 mM histidine; (c) from about 150 mM to about 300 mM arginine; (d) from about 2.5 mM to about 10 mM calcium chloride and (e) from about 0.008% (w / v) to about 0.1% (w / v) polysorbate 20 or polysorbate 80.

[0238] In some embodiments, the pharmaceutical composition comprises: (a) from about 5% (w / v) to about 7.5% (w / v) sucrose; (b) from about 5 mM to about 15 mM histidine; (c) from about 200 mM to about 300 mM arginine; (d) from about 2.5 mM to about 10 mM calcium chloride, and (e) from about 0.008% (w / v) to about 0.1% (w / v) polysorbate 20 or polysorbate 80. In some embodiments, the composition comprises polysorbate 80. 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 solution intended for lyophilization.

[0239] In some embodiments, the pharmaceutical composition comprises: (a) 5% (w / v) to 6% (w / v) sucrose; (b) 7.5 mM to 12.5 mM histidine; (c) 225 mM to about 300 mM arginine; (d) 5 mM to 6 mM calcium chloride, and (e) 0.01% (w / v) to about 0.075% (w / v) polysorbate 20 or polysorbate 80. In some embodiments, the composition comprises polysorbate 80. In some embodiments, the histidine is L-histidine. In some embodiments, the arginine is L-arginine. In some embodiments, the composition comprises 225 mM to about 300 mM L-arginine-HCl. In some embodiments, the composition comprises 5 mM to 6 mM calcium chloride dihydrate. In some embodiments, the pharmaceutical composition is a solution intended for lyophilization.

[0240] In some embodiments, the pharmaceutical composition comprises: (a) from 5% (w / v) to 6% (w / v) sucrose; (b) from 7.5 mM to 12.5 mM L-histidine; (c) from 225 mM to about 300 mM L-arginine; (d) from 5 mM to 6 mM calcium chloride and (e) from 0.01% (w / v) to about 0.075% (w / v) polysorbate 80.

[0241] In some embodiments, the pharmaceutical composition comprises: (a) 5% (w / v) to 6% (w / v) sucrose; (b) 7.5 mM to 12.5 mM L-histidine; (c) 225 mM to about 300 mM L-arginine HCl; (d) 5 mM to 6 mM calcium chloride, and (e) 0.01% (w / v) to about 0.075% (w / v) polysorbate 80.

[0242] In some embodiments, the pharmaceutical composition comprises: (a) 5% (w / v) to 6% (w / v) sucrose; (b) 7.5 mM to 12.5 mM L-histidine; (c) 225 mM to about 300 mM L-arginine HCl; (d) 5 mM to 6 mM calcium chloride dihydrate, and (e) 0.01% (w / v) to about 0.075% (w / v) polysorbate 80.

[0243] In some embodiments, the pharmaceutical composition comprises: (a) about 5.61% (w / v) sucrose; (b) about 11.2 mM L-histidine; (c) about 280.58 mM L-arginine HCl; (d) about 5.61 mM calcium chloride dihydrate, and (e) 0.056% (w / v) polysorbate 80.

[0244] In some embodiments, the pharmaceutical composition comprises: (a) about 5.61% (w / v) sucrose; (b) about 11.2 mM L-histidine; (c) about 280.58 mM L-arginine; (d) about 5.61 mM calcium chloride, and (e) about 0.056% (w / v) polysorbate 80.

[0245] In some embodiments, the pharmaceutical composition comprises: (a) 5.61% (w / v) sucrose; (b) 11.2 mM L-histidine; (c) 280.58 mM L-arginine HCl; (d) about 5.61 mM calcium chloride dihydrate, and (e) 0.056% (w / v) polysorbate 80.

[0246] In some embodiments, the pharmaceutical composition comprises: (a) 5.61% (w / v) sucrose; (b) 11.2 mM L-histidine; (c) 280.58 mM L-arginine; (d) 5.61 mM calcium chloride, and (e) 0.056% (w / v) polysorbate 80.

[0247] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 5.61% (w / v) sucrose; (b) about 11.2 mM L-histidine; (c) about 280.58 mM L-arginine HCl; (d) about 5.61 mM calcium chloride dihydrate, and (e) 0.056% (w / v) polysorbate 80.

[0248] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 5.61% (w / v) sucrose; (b) about 11.2 mM L-histidine; (c) about 280.58 mM L-arginine; (d) about 5.61 mM calcium chloride, and (e) about 0.056% (w / v) polysorbate 80.

[0249] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) 5.61% (w / v) sucrose; (b) 11.2 mM L-histidine; (c) 280.58 mM L-arginine HCl; (d) approximately 5.61 mM calcium chloride dihydrate (5.61 mM calcium chloride); and (e) 0.056% (w / v) polysorbate 80.

[0250] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) 5.61% (w / v) sucrose; (b) 11.2 mM L-histidine; (c) 280.58 mM L-arginine; (d) 5.61 mM calcium chloride, and (e) 0.056% (w / v) polysorbate 80.

[0251] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) 5% (w / v) to 6% (w / v) sucrose; (b) 11.2 mM L-histidine; (c) 280.58 mM L-arginine; (d) 5.61 mM calcium chloride, and (e) 0.056% (w / v) polysorbate 80.

[0252] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) 5.61% (w / v) sucrose; (b) 7.5 mM to 12.5 mM L-histidine; (c) 280.58 mM L-arginine; (d) 5.61 mM calcium chloride, and (e) 0.056% (w / v) polysorbate 80.

[0253] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) 5.61% (w / v) sucrose; (b) 11.2 mM L-histidine; (c) 225 mM to about 300 mM L-arginine; (d) 5.61 mM calcium chloride, and (e) 0.056% (w / v) polysorbate 80.

[0254] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) 5.61% (w / v) sucrose; (b) 11.2 mM L-histidine; (c) 280.58 mM L-arginine; (d) 5 mM to 6 mM calcium chloride, and (e) 0.056% (w / v) polysorbate 80.

[0255] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) 5.61% (w / v) sucrose; (b) 11.2 mM L-histidine; (c) 280.58 mM L-arginine; (d) 5.61 mM calcium chloride, and (e) from 0.01% (w / v) to about 0.075% (w / v) polysorbate 80.

[0256] In some embodiments, when combining the lyophilized pharmaceutical composition and sterile water, the resulting solution does not contain NaCl. In some embodiments, when combining the lyophilized pharmaceutical composition and sterile water, the resulting solution does not contain NaOH. In some embodiments, when combining the lyophilized pharmaceutical composition and sterile water, the resulting solution does not contain sodium ions.

[0257] 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 0.05% polysorbate 20 or polysorbate 80. In some embodiments, the composition comprises polysorbate 80. 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 solution intended for lyophilization.

[0258] In some embodiments, the pharmaceutical composition comprises: (a) about 5% (w / v) to about 7.5% (w / v) sucrose; (b) about 10 mM histidine; (c) about 250 mM arginine; (d) about 5 mM calcium chloride, and (e) about 0.05% polysorbate 20 or polysorbate 80. In some embodiments, the composition comprises polysorbate 80. 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 solution intended for lyophilization.

[0259] In some embodiments, the pharmaceutical composition comprises: (a) about 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 0.05% polysorbate 80. 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 solution intended for lyophilization.

[0260] In some embodiments, the pharmaceutical composition comprises: (a) about 5% (w / v) sucrose; (b) about 10 mM histidine; (c) from about 200 mM to about 300 mM arginine; (d) about 5 mM calcium chloride, and (e) about 0.05% polysorbate 80. 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 solution intended for lyophilization.

[0261] In some embodiments, the pharmaceutical composition comprises: (a) about 5% (w / v) sucrose; (b) about 10 mM histidine; (c) about 250 mM arginine; (d) from about 2.5 mM to about 10 mM calcium chloride, and (e) about 0.05% polysorbate 80. 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 solution intended for lyophilization.

[0262] 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) from about 0.008% (w / v) to about 0.1% (w / v) polysorbate 80. 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 solution intended for lyophilization.

[0263] In some embodiments, the pharmaceutical composition comprises: (a) about 50 mg / mL sucrose; (b) about 1.552 mg / mL L-histidine; (c) about 52.665 mg / mL L-arginine HCl; (d) about 0.735 mg / mL calcium chloride, and (e) about 0.5 mg / mL polysorbate 80.

[0264] In some embodiments, the pharmaceutical composition comprises: (a) about 50 mg / mL sucrose; (b) about 1.552 mg / mL L-histidine; (c) about 52.665 mg / mL L-arginine HCl; (d) about 0.735 mg / mL calcium chloride dihydrate, and (e) about 0.5 mg / mL polysorbate 80.

[0265] In some embodiments, the pharmaceutical composition comprises: (a) about 50 mg / mL sucrose; (b) about 1.552 mg / mL L-histidine; (c) about 52.665 mg / mL L-arginine HCl; (d) about 0.555 mg / mL calcium chloride; and (e) about 0.5 mg / mL polysorbate 80. In some embodiments, the composition comprises calcium chloride dihydrate. In some embodiments, the pharmaceutical composition is a solution intended for lyophilization.

[0266] In some embodiments, the pharmaceutical composition comprises: (a) about 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 dihydrate; and (e) about 0.5 mg / mL polysorbate 80. 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 solution intended for lyophilization.

[0267] In some embodiments, the pharmaceutical composition comprises: (a) about 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) about 0.5 mg / mL polysorbate 80. 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 solution intended for lyophilization.

[0268] In some embodiments, the pharmaceutical composition comprises: (a) about 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) about 0.5 mg / mL polysorbate 80. 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 solution intended for lyophilization.

[0269] In some embodiments, the pharmaceutical composition contains: (a) 56.12 mg / ml sucrose; (b) 1.74 mg / ml L-histidine; (c) 59.11 mg / ml L-arginine-HCl; (d) 0.82 mg / ml calcium chloride dihydrate; and (e) 0.56 mg / ml polysorbate 80.

[0270] In some embodiments, the pharmaceutical composition comprises:(a) 56.12 mg / mL sucrose;(b) 1.74 mg / mL L-histidine;(c) 59.11 mg / mL L-arginine-HCl;(d) 0.62 mg / mL calcium chloride and(e) 0.56 mg / mL polysorbate 80. In some embodiments, the composition comprises calcium chloride dihydrate.

[0271] In some embodiments, the pharmaceutical composition comprises:(a) 56.12 mg / mL of sucrose;(b) 1.74 mg / mL of L-histidine;(c) 48.88 mg / mL of L-arginine;(d) 0.8 mg / mL of dihydrate chloride (e) 0.56 mg / ml polysorbate 80.

[0272] In some embodiments, the pharmaceutical composition comprises:(a) 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 mg / mL polysorbate 80. In some embodiments, the composition comprises L-arginine-HCl. In some embodiments, the composition comprises calcium chloride dihydrate.

[0273] In some embodiments, the pharmaceutical composition is characterized by a pH of from about 6.5 to about 7.5. In some embodiments, the pharmaceutical composition is characterized by a pH of about 7.0. In some embodiments, the pharmaceutical composition is characterized by a pH of about 6.8.

[0274] In some embodiments, the pharmaceutical composition is characterized by a pH of about 6.5. In some embodiments, the pharmaceutical composition is characterized by a pH of about 6.6. In some embodiments, the pharmaceutical composition is characterized by a pH of about 6.7. In some embodiments, the pharmaceutical composition is characterized by a pH of about 6.8. In some embodiments, the pharmaceutical composition is characterized by a pH of about 6.9. In some embodiments, the pharmaceutical composition is characterized by a pH of about 7.0. In some embodiments, the pharmaceutical composition is characterized by a pH of about 7.1. In some embodiments, the pharmaceutical composition is characterized by a pH of about 7.2.In some embodiments, the pharmaceutical composition is characterized by a pH of about 7.3. In some embodiments, the pharmaceutical composition is characterized by a pH of about 7.4. In some embodiments, the pharmaceutical composition is characterized by a pH of about 7.5.

[0275] In some embodiments, the pharmaceutical composition is characterized by a pH of 6.5. In some embodiments, the pharmaceutical composition is characterized by a pH of 6.6. In some embodiments, the pharmaceutical composition is characterized by a pH of 6.7. In some embodiments, the pharmaceutical composition is characterized by a pH of 6.8. In some embodiments, the pharmaceutical composition is characterized by a pH of 6.9. In some embodiments, the pharmaceutical composition is characterized by a pH of 7.0. In some embodiments, the pharmaceutical composition is characterized by a pH of 7.1. In some embodiments, the pharmaceutical composition is characterized by a pH of 7.2. In some embodiments, the pharmaceutical composition is characterized by a pH of 7.3.In some embodiments, the pharmaceutical composition is characterized by a pH of 7.4. In some embodiments, the pharmaceutical composition is characterized by a pH of 7.5.

[0276] In some embodiments, a volume of 3.367 mL of a solution to be lyophilized is added to a container or vial. In some embodiments, the solution to be lyophilized is lyophilized, resulting in a lyophilized pharmaceutical composition.

[0277] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) from about 30 mg to about 135 mg sucrose; (b) from about 2.5 mg to about 7.5 mg histidine; (c) from about 140 mg to about 200 mg arginine; (d) from about 1.5 mg to about 5 mg calcium chloride, and (e) from about 1 mg to about 5 mg polysorbate 20 or polysorbate 80.

[0278] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) from about 30 mg to about 135 mg sucrose; (b) from about 2.5 mg to about 7.5 mg L-histidine; (c) from about 140 mg to about 200 mg L-arginine; (d) from about 1.5 mg to about 5 mg calcium chloride, and (e) from about 1 mg to about 5 mg polysorbate 80. In some embodiments, the composition comprises L-arginine HCl. In some embodiments, the composition comprises calcium chloride dihydrate.

[0279] In some embodiments, the lyophilized pharmaceutical composition contains: (a) 30 mg to 135 mg sucrose; (b) 2.5 mg to 7.5 mg histidine; (c) 140 mg to 200 mg arginine; (d) 1.5 mg to 5 mg calcium chloride; and (e) 1 mg to 5 mg polysorbate 20 or polysorbate 80.

[0280] In some embodiments, the lyophilized pharmaceutical composition comprises:(a) from 30 mg to 135 mg of sucrose;(b) from 2.5 mg to 7.5 mg of L-histidine;(c) from 140 mg to 20 mg of L-HCl; 1.5 mg to 5 mg of calcium chloride and(e) from 1 mg to 5 mg of polysorbate 80. In some embodiments, the composition comprises calcium chloride dihydrate.

[0281] In some embodiments, the lyophilized pharmaceutical composition comprises:(a) from 30 mg to 135 mg of sucrose;(b) from 2.5 mg to 7.5 mg of L-histidine;(c) from 140 mg to 20 mg of arginine; mg to 5 mg calcium chloride and(e) from 1 mg to 5 mg polysorbate 80. In some embodiments, the composition comprises L-arginine-HCl. In some embodiments, the composition comprises calcium chloride dihydrate.

[0282] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) about 67.34 mg sucrose; (b) about 5.2 mg L-histidine; (c) about 177.3 mg L-arginine HCl; (d) about 2.5 mg calcium chloride, and (e) about 1.7 mg polysorbate 20 or polysorbate 80.

[0283] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) about 67.34 mg sucrose; (b) about 5.2 mg L-histidine; (c) about 146.6 mg L-arginine; (d) about 2.5 mg calcium chloride, and (e) about 1.7 mg polysorbate 20 or polysorbate 80.

[0284] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 67.34 mg sucrose; (b) 5.2 mg L-histidine; (c) 177.3 mg L-arginine HCl; (d) 2.5 mg calcium chloride, and (e) 1.7 mg polysorbate 80.

[0285] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 67.34 mg sucrose; (b) 5.2 mg L-histidine; (c) 146.6 mg L-arginine; (d) 2.5 mg calcium chloride, and (e) 1.7 mg polysorbate 80.

[0286] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 67.34 mg sucrose; (b) 5.23 mg L-histidine; (c) 177.32 mg L-arginine HCl; (d) 2.47 mg calcium chloride dihydrate and

[0287] (e) 1.68 mg polysorbate 80. In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 67.34 mg sucrose; (b) 5.23 mg L-histidine; (c) 177.32 mg L-arginine HCl; (d) 1.87 mg calcium chloride, and (e) 1.68 mg polysorbate 80.

[0288] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 67.34 mg sucrose; (b) 5.23 mg L-histidine; (c) 146.63 mg L-arginine; (d) 2.47 mg calcium chloride dihydrate, and (e) 1.68 mg polysorbate 80.

[0289] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 67.34 mg sucrose; (b) 5.23 mg L-histidine; (c) 146.63 mg L-arginine; (d) 1.87 mg calcium chloride, and (e) 1.68 mg polysorbate 80.

[0290] In some embodiments, the lyophilized pharmaceutical composition contains: (a) 30 mg to 135 mg sucrose; (b) 5.2 mg L-histidine; (c) 177.3 mg L-arginine-HCl; (d) 2.5 mg calcium chloride dihydrate; and (e) 1.7 mg polysorbate 80.

[0291] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 30 mg to 135 mg sucrose; (b) 5.2 mg L-histidine; (c) 177.3 mg L-arginine HCl; (d) 1.9 mg calcium chloride, and (e) 1.7 mg polysorbate 80.

[0292] In some embodiments, the lyophilized pharmaceutical composition contains: (a) 30 mg to 135 mg sucrose; (b) 5.2 mg L-histidine; (c) 146.6 mg L-arginine; (d) 2.5 mg calcium chloride dihydrate; and (e) 1.7 mg polysorbate 80.

[0293] In some embodiments, the lyophilized pharmaceutical composition comprises:(a) from 30 mg to 135 mg of sucrose;(b) 5.2 mg of L-histidine;(c) 146.6 mg of L-arginine;(d) 1.9 mg of calcium chloride, and (e) calcium chloride. mg polysorbate 80.

[0294] In some embodiments, the freeze-dried pharmaceutical composition comprises:(a) 67.34 mg of sucrose;(b) from 2.5 mg to 7.5 mg of L-histidine;(c) 177.3 mg of L-arginine-HCl; calcium and(e) 1.7 mg polysorbate 80.

[0295] In some embodiments, the lyophilized pharmaceutical composition comprises:(a) 67.34 mg of sucrose;(b) from 2.5 mg to 7.5 mg of L-histidine;(c) 177.3 mg of L-arginine-HCl;(9.9 mg) calcium chloride; and(e) 1.7 mg of polysorbate 80.

[0296] In some embodiments, the lyophilized pharmaceutical composition comprises:(a) 67.34 mg of sucrose;(b) from 2.5 mg to 7.5 mg of L-histidine;(c) 146.6 mg of L-arginine;(d) 2.5 mg of chloride dihydrate. and(e) 1.7 mg of polysorbate 80.

[0297] In some embodiments, the lyophilized pharmaceutical composition comprises:(a) 67.34 mg of sucrose;(b) from 2.5 mg to 7.5 mg of L-histidine;(c) 146.6 mg of L-arginine;(d) 1.9 mg of calcium chloride; 1.7 mg polysorbate 80.

[0298] In some embodiments, the lyophilized pharmaceutical composition comprises:(a) 67.34 mg of sucrose;(b) 5.23 mg of L-histidine;(c) from 140 mg to 200 mg of L-arginine;(d) from 1.5 mg of calcium chloride to 5 mg. and(e) 1.7 mg of polysorbate 80. In some embodiments, the composition comprises from 140 mg to 200 mg of L-arginine-HCl.

[0299] In some embodiments, the lyophilized pharmaceutical composition comprises:(a) 67.34 mg of sucrose;(b) 5.23 mg of L-histidine;(c) 177.32 mg of L-arginine-HCl;(d) from 1.5 mg of calcium chloride to 5 mg. and(e) 1.7 mg of polysorbate 80.

[0300] In some embodiments, the lyophilized pharmaceutical composition comprises:(a) 67.34 mg of sucrose;(b) 5.23 mg of L-histidine;(c) 146.63 mg of L-arginine;(d) from 1.5 to 15 mg of calcium chloride and( 1.7 mg polysorbate 80.

[0301] In some embodiments, the lyophilized pharmaceutical composition comprises:(a) 67.34 mg of sucrose;(b) 5.23 mg of L-histidine;(c) 177.32 mg of L-arginine-HCl;(d) 2.7 mg of dihydride calcium chloride; and(e) from 1 mg to 5 mg of polysorbate 80.

[0302] In some embodiments, the lyophilized pharmaceutical composition comprises:(a) 67.34 mg of sucrose;(b) 5.23 mg of L-histidine;(c) 177.32 mg of L-arginine-HCl;(d) 1.8 mg of calcium chloride and 1(e) mg to 5 mg polysorbate 80.

[0303] In some embodiments, the lyophilized pharmaceutical composition comprises:(a) 67.34 mg of sucrose;(b) 5.23 mg of L-histidine;(c) 146.63 mg of L-arginine;(d) 2.47 mg of chloride dihydrate and chloride. 1 mg to 5 mg polysorbate 80.

[0304] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 67.34 mg sucrose; (b) 5.23 mg L-histidine; (c) 146.63 mg L-arginine; (d) 1.87 mg calcium chloride, and (e) 1 mg to 5 mg polysorbate 80.

[0305] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) from about 160 mg to about 200 mg sucrose; (b) from about 2.5 mg to about 7.5 mg histidine; (c) from about 140 mg to about 200 mg arginine; (d) from about 1.5 mg to about 5 mg calcium chloride, and (e) from about 1 mg to about 5 mg polysorbate 20 or polysorbate 80.

[0306] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) from about 160 mg to about 200 mg sucrose; (b) from about 2.5 mg to about 7.5 mg L-histidine; (c) from about 140 mg to about 200 mg L-arginine; (d) from about 1.5 mg to about 5 mg calcium chloride, and (e) from about 1 mg to about 5 mg polysorbate 80. In some embodiments, the composition comprises L-arginine HCl. In some embodiments, the composition comprises calcium chloride dihydrate.

[0307] In some embodiments, the lyophilized pharmaceutical composition contains: (a) 160 mg to 200 mg sucrose; (b) 2.5 mg to 7.5 mg histidine; (c) 140 mg to 200 mg arginine; (d) 1.5 mg to 5 mg calcium chloride; and (e) 1 mg to 5 mg polysorbate 20 or polysorbate 80.

[0308] In some embodiments, the lyophilized pharmaceutical composition comprises:(a) from 160 mg to 200 mg of sucrose;(b) from 2.5 mg to 7.5 mg of L-histidine;(c) from 140 mg to 200 mg of L-HCl; 1.5 mg to 5 mg of calcium chloride and(e) from 1 mg to 5 mg of polysorbate 80. In some embodiments, the composition comprises calcium chloride dihydrate.

[0309] In some embodiments, the freeze-dried pharmaceutical composition comprises:(a) from 160 mg to 200 mg of sucrose;(b) from 2.5 mg to 7.5 mg of L-histidine;(c) from 140 mg to 200 mg of L-histidine;(c) from 140 mg to 200 mg of arginine; mg to 5 mg calcium chloride and(e) from 1 mg to 5 mg polysorbate 80. In some embodiments, the composition comprises L-arginine-HCl. In some embodiments, the composition comprises calcium chloride dihydrate.

[0310] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) about 168.3 mg sucrose; (b) about 5.2 mg L-histidine; (c) about 177.3 mg L-arginine HCl; (d) about 2.5 mg calcium chloride, and (e) about 1.7 mg polysorbate 20 or polysorbate 80.

[0311] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) about 168.3 mg sucrose; (b) about 5.2 mg L-histidine; (c) about 146.6 mg L-arginine; (d) about 2.5 mg calcium chloride, and (e) about 1.7 mg polysorbate 20 or polysorbate 80.

[0312] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 168.3 mg sucrose; (b) 5.2 mg L-histidine; (c) 177.3 mg L-arginine HCl; (d) 2.5 mg calcium chloride, and (e) 1.7 mg polysorbate 80.

[0313] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 168.3 mg sucrose; (b) 5.2 mg L-histidine; (c) 146.6 mg L-arginine; (d) 2.5 mg calcium chloride, and (e) 1.7 mg polysorbate 80.

[0314] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 168.35 mg sucrose; (b) 5.23 mg L-histidine; (c) 177.32 mg L-arginine HCl; (d) 2.47 mg calcium chloride dihydrate, and (e) 1.68 mg polysorbate 80.

[0315] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 168.35 mg sucrose; (b) 5.23 mg L-histidine; (c) 177.32 mg L-arginine HCl; (d) 1.87 mg calcium chloride, and (e) 1.68 mg polysorbate 80.

[0316] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 168.35 mg sucrose; (b) 5.23 mg L-histidine; (c) 146.63 mg L-arginine; (d) 2.47 mg calcium chloride dihydrate, and (e) 1.68 mg polysorbate 80.

[0317] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 168.35 mg sucrose; (b) 5.23 mg L-histidine; (c) 146.63 mg L-arginine; (d) 1.87 mg calcium chloride, and (e) 1.68 mg polysorbate 80.

[0318] In some embodiments, the lyophilized pharmaceutical composition contains: (a) 160 mg to 200 mg sucrose; (b) 5.2 mg L-histidine; (c) 177.3 mg L-arginine-HCl; (d) 2.5 mg calcium chloride dihydrate; and (e) 1.7 mg polysorbate 80.

[0319] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 160 mg to 200 mg sucrose; (b) 5.2 mg L-histidine; (c) 177.3 mg L-arginine HCl; (d) 1.9 mg calcium chloride, and (e) 1.7 mg polysorbate 80.

[0320] In some embodiments, the lyophilized pharmaceutical composition contains: (a) 160 mg to 200 mg sucrose; (b) 5.2 mg L-histidine; (c) 146.6 mg L-arginine; (d) 2.5 mg calcium chloride dihydrate; and (e) 1.7 mg polysorbate 80.

[0321] In some embodiments, the freeze-dried pharmaceutical composition comprises:(a) from 160 mg to 200 mg of sucrose;(b) 5.2 mg of L-histidine;(c) 146.6 mg of L-arginine;(d) 1.9 mg of calcium chloride and (e) 1.7 mg polysorbate 80.

[0322] In some embodiments, the freeze-dried pharmaceutical composition comprises:(a) 168.3 mg of sucrose;(b) from 2.5 mg to 7.5 mg of L-histidine;(c) 177.3 mg of L-arginine-HCl;(5) mg of dihydride chloride; calcium and(e) 1.7 mg polysorbate 80.

[0323] In some embodiments, the lyophilized pharmaceutical composition comprises:(a) 168.3 mg of sucrose;(b) from 2.5 mg to 7.5 mg of L-histidine;(c) 177.3 mg of L-arginine-HCl;(9.9 mg) calcium chloride; and(e) 1.7 mg of polysorbate 80.

[0324] In some embodiments, the freeze-dried pharmaceutical composition comprises:(a) 168.3 mg of sucrose;(b) from 2.5 mg to 7.5 mg of L-histidine;(c) 146.6 mg of L-arginine;(d) 2.5 mg of chloride dihydrate. and(e) 1.7 mg of polysorbate 80.

[0325] In some embodiments, the lyophilized pharmaceutical composition comprises:(a) 168.3 mg of sucrose;(b) from 2.5 mg to 7.5 mg of L-histidine;(c) 146.6 mg of L-arginine;(d) 1.9 mg of calcium chloride; 1.7 mg polysorbate 80.

[0326] In some embodiments, the freeze-dried pharmaceutical composition comprises:(a) 168.35 mg of sucrose;(b) 5.23 mg of L-histidine;(c) from 140 mg to 200 mg of L-arginine;(d) from 15.5 mg of chloride; calcium and(e) 1.7 mg of polysorbate 80. In some embodiments, the composition comprises from 140 mg to 200 mg of L-arginine-HCl.

[0327] In some embodiments, the lyophilized pharmaceutical composition contains:(a) 168.35 mg of sucrose;(b) 5.23 mg of L-histidine;(c) 177.32 mg of L-arginine-HCl;(d) from 1.5 mg of calcium chloride to 1.5 mg. and(e) 1.7 mg of polysorbate 80.

[0328] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 168.35 mg sucrose; (b) 5.23 mg L-histidine; (c) 146.63 mg L-arginine; (d) 1.5 mg to 5 mg calcium chloride, and (e) 1.7 mg polysorbate 80.

[0329] In some embodiments, the lyophilized pharmaceutical composition contains: (a) 168.35 mg of sucrose; (b) 5.23 mg of L-histidine; (c) 177.32 mg of L-arginine-HCl; (d) 2.47 mg of calcium chloride dihydrate; and (e) 1 mg to 5 mg of polysorbate 80.

[0330] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 168.35 mg sucrose; (b) 5.23 mg L-histidine; (c) 177.32 mg L-arginine HCl; (d) 1.87 mg calcium chloride, and (e) 1 mg to 5 mg polysorbate 80.

[0331] In some embodiments, the lyophilized pharmaceutical composition contains: (a) 168.35 mg of sucrose; (b) 5.23 mg of L-histidine; (c) 146.63 mg of L-arginine; (d) 2.47 mg of calcium chloride dihydrate; and (e) 1 mg to 5 mg of polysorbate 80.

[0332] In some embodiments, the lyophilized pharmaceutical composition comprises: (a) 168.35 mg sucrose; (b) 5.23 mg L-histidine; (c) 146.63 mg L-arginine; (d) 1.87 mg calcium chloride, and (e) 1 mg to 5 mg polysorbate 80.

[0333] In some embodiments, the lyophilized pharmaceutical composition is characterized by a moisture content of less than 2%. In some embodiments, the lyophilized pharmaceutical composition is characterized by a moisture content of less than 1.8%. In some embodiments, the lyophilized pharmaceutical composition is characterized by a moisture content of less than 1.6%.

[0334] In some embodiments, the lyophilized pharmaceutical composition is in the form of a lyophilized mass. In some embodiments, the lyophilized mass is white. In some embodiments, the lyophilized mass is characterized by a value of less than Y4 on the color scale of the European Pharmacopoeia. See Coloration of Liquids (Method 2.2.2), European Pharmacopoeia, 10 th Ed. (2021).

[0335] In some embodiments, a lyophilized pharmaceutical composition and sterile water are combined to form an injectable solution. In some embodiments, the lyophilized pharmaceutical composition is combined with sterile water in an amount of from about 2 to about 5 ml. In some embodiments, the lyophilized pharmaceutical composition is combined with sterile water in an amount of about 3 ml. In some embodiments, the lyophilized pharmaceutical composition is combined with sterile water in an amount of 3 ml. 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 pyrogen-free. In some embodiments, the sterile water does not contain a bacteriostatic or antimicrobial agent. In some embodiments, the sterile water contains a bacteriostatic or antimicrobial agent. In some embodiments, the sterile water is sterilized using a filter. In some embodiments, the sterile water is sterilized using a filter with a pore size of 0.1 μm. In some embodiments, the sterile water is distilled water. In some embodiments, the sterile water is sterile, pyrogen-free, distilled water, hypotonic, with an osmolarity of zero mOsmol / L, and does not contain a bacteriostatic or antimicrobial agent.

[0336] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) 10 mg / mL to 40 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

[0337] (e) 0.4 mg / ml to 0.7 mg / ml polysorbate 80. In some embodiments, the composition contains L-arginine-HCl. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution contains: (a) 10 mg / ml to 40 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) 0.4 mg / ml to 0.7 mg / ml polysorbate 80. In some embodiments, the composition contains L-arginine-HCl. In some embodiments, the composition contains calcium chloride dihydrate.

[0338] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 22.45 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) about 59.11 mg / mL L-arginine HCl; (d) about 0.82 mg / mL calcium chloride dihydrate; and (e) about 0.56 mg / mL polysorbate 80.

[0339] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 22.45 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) about 59.11 mg / mL L-arginine HCl; (d) about 0.62 mg / mL calcium chloride; and (e) about 0.56 mg / mL polysorbate 80. In some embodiments, the composition comprises calcium chloride dihydrate.

[0340] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 22.45 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) about 48.88 mg / mL L-arginine; (d) about 0.82 mg / mL calcium chloride dihydrate, and (e) about 0.56 mg / mL polysorbate 80.

[0341] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 22.45 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 0.56 mg / mL polysorbate 80. In some embodiments, the composition comprises L-arginine HCl. In some embodiments, the composition comprises calcium chloride dihydrate.

[0342] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 10 mg / mL to about 40 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) about 59.11 mg / mL L-arginine HCl; (d) about 0.82 mg / mL calcium chloride dihydrate; and (e) about 0.56 mg / mL polysorbate 80.

[0343] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 10 mg / mL to about 40 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) about 59.11 mg / mL L-arginine HCl; (d) about 0.62 mg / mL calcium chloride; and (e) about 0.56 mg / mL polysorbate 80. In some embodiments, the composition comprises calcium chloride dihydrate.

[0344] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 10 mg / mL to about 40 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) about 44.88 mg / mL L-arginine; (d) about 0.82 mg / mL calcium chloride dihydrate; and (e) about 0.56 mg / mL polysorbate 80.

[0345] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 10 mg / mL to about 40 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) about 0.56 mg / mL polysorbate 80. In some embodiments, the composition comprises L-arginine HCl. In some embodiments, the composition comprises calcium chloride dihydrate.

[0346] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 22.45 mg / mL sucrose; (b) from about 1.5 mg / mL to about 2.0 mg / mL L-histidine; (c) about 59.11 mg / mL L-arginine HCl; (d) about 0.82 mg / mL calcium chloride dihydrate; and (e) about 0.56 mg / mL polysorbate 80.

[0347] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 22.45 mg / mL sucrose; (b) from about 1.5 mg / mL to about 2.0 mg / mL L-histidine; (c) about 59.11 mg / mL L-arginine HCl; (d) about 0.62 mg / mL calcium chloride; and (e) about 0.56 mg / mL polysorbate 80. In some embodiments, the composition comprises calcium chloride dihydrate.

[0348] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 22.45 mg / mL sucrose; (b) from about 1.5 mg / mL to about 2.0 mg / mL L-histidine; (c) about 48.88 mg / mL L-arginine; (d) about 0.82 mg / mL calcium chloride dihydrate; and (e) about 0.56 mg / mL polysorbate 80.

[0349] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 22.45 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) about 0.62 mg / mL calcium chloride; and (e) about 0.56 mg / mL polysorbate 80. In some embodiments, the composition comprises L-arginine HCl. In some embodiments, the composition comprises calcium chloride dihydrate.

[0350] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 22.45 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) from about 50 mg / mL to about 70 mg / mL L-arginine HCl; (d) about 0.62 mg / mL calcium chloride dihydrate; and (e) about 0.56 mg / mL polysorbate 80.

[0351] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 22.45 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) from about 50 mg / mL to about 70 mg / mL L-arginine HCl; (d) about 0.62 mg / mL calcium chloride; and (e) about 0.56 mg / mL polysorbate 80. In some embodiments, the composition comprises calcium chloride dihydrate.

[0352] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 22.45 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) from about 40 mg / mL to about 60 mg / mL L-arginine; (d) about 0.82 mg / mL calcium chloride dihydrate; and (e) about 0.56 mg / mL polysorbate 80.

[0353] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 22.45 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) from about 40 mg / mL to about 60 mg / mL L-arginine; (d) about 0.62 mg / mL calcium chloride; and (e) about 0.56 mg / mL polysorbate 80. In some embodiments, the composition comprises L-arginine HCl. In some embodiments, the composition comprises calcium chloride dihydrate.

[0354] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 22.45 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) about 59.11 mg / mL L-arginine HCl; (d) from about 0.7 mg / mL to about 0.9 mg / mL calcium chloride dihydrate, and (e) about 0.56 mg / mL polysorbate 80.

[0355] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 22.45 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) about 59.11 mg / mL L-arginine HCl; (d) from about 0.5 mg / mL to about 0.9 mg / mL calcium chloride, and (e) about 0.56 mg / mL polysorbate 80. In some embodiments, the composition comprises calcium chloride dihydrate.

[0356] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 22.45 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) about 48.88 mg / mL L-arginine; (d) from about 0.7 mg / mL to about 0.9 mg / mL calcium chloride dihydrate, and (e) about 0.56 mg / mL polysorbate 80. In some embodiments, the composition comprises L-arginine HCl.

[0357] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 22.45 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) about 48.88 mg / mL L-arginine; (d) from about 0.5 mg / mL to about 0.7 mg / mL calcium chloride, and (e) about 0.56 mg / mL polysorbate 80. In some embodiments, the composition comprises L-arginine HCl. In some embodiments, the composition comprises calcium chloride dihydrate.

[0358] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 22.45 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) about 59.11 mg / mL L-arginine HCl; (d) about 0.82 mg / mL calcium chloride dihydrate, and (e) from about 0.4 mg / mL to about 0.7 mg / mL polysorbate 80.

[0359] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 22.45 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) about 59.11 mg / mL L-arginine HCl; (d) about 0.62 mg / mL calcium chloride, and (e) from about 0.4 mg / mL to about 0.7 mg / mL polysorbate 80. In some embodiments, the composition comprises L-arginine HCl. In some embodiments, the composition comprises calcium chloride dihydrate.

[0360] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 22.45 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) about 48.88 mg / mL L-arginine; (d) about 0.82 mg / mL calcium chloride dihydrate, and (e) from about 0.4 mg / mL to about 0.7 mg / mL polysorbate 80.

[0361] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 22.45 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) from about 0.4 mg / mL to about 0.7 mg / mL polysorbate 80. In some embodiments, the composition comprises L-arginine HCl. In some embodiments, the composition comprises calcium chloride dihydrate.

[0362] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 22.45 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) about 59.11 mg / mL L-arginine HCl; (d) about 0.82 mg / mL calcium chloride dihydrate; and (e) about 0.56 mg / mL polysorbate 80.

[0363] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 22.45 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) about 59.11 mg / mL L-arginine HCl; (d) about 0.62 mg / mL calcium chloride; and (e) about 0.56 mg / mL polysorbate 80.

[0364] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 22.45 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) about 48.88 mg / mL L-arginine; (d) about 0.82 mg / mL calcium chloride dihydrate; and (e) about 0.56 mg / mL polysorbate 80.

[0365] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 22.45 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 0.56 mg / mL polysorbate 80.

[0366] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 67.34 mg sucrose; (b) about 5.23 mg L-histidine; (c) about 177.32 mg L-arginine HCl; (d) about 2.47 mg calcium chloride, and (e) about 1.68 mg polysorbate 80 in 3 mL of sterile water.

[0367] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 67.34 mg sucrose; (b) about 5.23 mg L-histidine; (c) about 146.63 mg L-arginine; (d) about 2.47 mg calcium chloride dihydrate, and (e) about 1.68 mg polysorbate 80 in 3 ml of sterile water.

[0368] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 67.34 mg sucrose; (b) about 5.23 mg L-histidine; (c) about 146.63 mg L-arginine; (d) about 1.87 mg calcium chloride; and (e) about 1.68 mg polysorbate 80 in 3 ml of sterile water.

[0369] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) 67.34 mg sucrose; (b) 5.23 mg L-histidine; (c) 177.32 mg L-arginine HCl; (d) 2.47 mg calcium chloride dihydrate; and (e) 1.68 mg polysorbate 80 in 3 mL of sterile water.

[0370] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) 67.34 mg sucrose; (b) 5.23 mg L-histidine; (c) 177.32 mg L-arginine HCl; (d) 1.87 mg calcium chloride; and (e) 1.68 mg polysorbate 80 in 3 mL of sterile water.

[0371] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) 67.34 mg sucrose; (b) 5.23 mg L-histidine; (c) 146.63 mg L-arginine; (d) 2.47 mg calcium chloride dihydrate; and (e) 1.68 mg polysorbate 80 in 3 ml sterile water.

[0372] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) 67.34 mg sucrose; (b) 5.23 mg L-histidine; (c) 146.63 mg L-arginine; (d) 1.87 mg calcium chloride; and (e) 1.68 mg polysorbate 80 in 3 ml sterile water.

[0373] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) 45 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

[0374] (e) 0.4 mg / ml to 0.7 mg / ml polysorbate 80. In some embodiments, the composition contains L-arginine-HCl. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution contains: (a) 45 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) 0.4 mg / ml to 0.7 mg / ml polysorbate 80. In some embodiments, the composition contains L-arginine-HCl. In some embodiments, the composition contains calcium chloride dihydrate.

[0375] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 56.12 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) about 59.11 mg / mL L-arginine HCl; (d) about 0.82 mg / mL calcium chloride dihydrate, and (e) about 0.56 mg / mL polysorbate 80.

[0376] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 56.12 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) about 59.11 mg / mL L-arginine HCl; (d) about 0.62 mg / mL calcium chloride; and (e) about 0.56 mg / mL polysorbate 80. In some embodiments, the composition comprises calcium chloride dihydrate.

[0377] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 56.12 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) about 48.88 mg / mL L-arginine; (d) about 0.82 mg / mL calcium chloride dihydrate, and (e) about 0.56 mg / mL polysorbate 80.

[0378] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 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 0.56 mg / mL polysorbate 80. In some embodiments, the composition comprises L-arginine HCl. In some embodiments, the composition comprises calcium chloride dihydrate.

[0379] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 45 mg / mL to about 60 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) about 59.11 mg / mL L-arginine HCl; (d) about 0.82 mg / mL calcium chloride dihydrate; and (e) about 0.56 mg / mL polysorbate 80.

[0380] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 45 mg / mL to about 60 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) about 59.11 mg / mL L-arginine HCl; (d) about 0.62 mg / mL calcium chloride; and (e) about 0.56 mg / mL polysorbate 80. In some embodiments, the composition comprises calcium chloride dihydrate.

[0381] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 45 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 0.82 mg / mL calcium chloride dihydrate; and (e) about 0.56 mg / mL polysorbate 80.

[0382] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 45 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) about 0.56 mg / mL polysorbate 80. In some embodiments, the composition comprises L-arginine HCl. In some embodiments, the composition comprises calcium chloride dihydrate.

[0383] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 56.12 mg / mL sucrose; (b) from about 1.5 mg / mL to about 2.0 mg / mL L-histidine; (c) about 59.11 mg / mL L-arginine HCl; (d) about 0.82 mg / mL calcium chloride dihydrate; and (e) about 0.56 mg / mL polysorbate 80.

[0384] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 56.12 mg / mL sucrose; (b) from about 1.5 mg / mL to about 2.0 mg / mL L-histidine; (c) about 59.11 mg / mL L-arginine HCl; (d) about 0.62 mg / mL calcium chloride; and (e) about 0.56 mg / mL polysorbate 80. In some embodiments, the composition comprises calcium chloride dihydrate.

[0385] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 56.12 mg / mL sucrose; (b) from about 1.5 mg / mL to about 2.0 mg / mL L-histidine; (c) about 48.88 mg / mL L-arginine; (d) about 0.82 mg / mL calcium chloride dihydrate; and (e) about 0.56 mg / mL polysorbate 80.

[0386] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 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) about 0.62 mg / mL calcium chloride; and (e) about 0.56 mg / mL polysorbate 80. In some embodiments, the composition comprises L-arginine HCl. In some embodiments, the composition comprises calcium chloride dihydrate.

[0387] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 56.12 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) from about 50 mg / mL to about 70 mg / mL L-arginine HCl; (d) about 0.62 mg / mL calcium chloride dihydrate; and (e) about 0.56 mg / mL polysorbate 80.

[0388] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 56.12 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) from about 50 mg / mL to about 70 mg / mL L-arginine HCl; (d) about 0.62 mg / mL calcium chloride; and (e) about 0.56 mg / mL polysorbate 80. In some embodiments, the composition comprises calcium chloride dihydrate.

[0389] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 56.12 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) from about 40 mg / mL to about 60 mg / mL L-arginine; (d) about 0.82 mg / mL calcium chloride dihydrate; and (e) about 0.56 mg / mL polysorbate 80.

[0390] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 56.12 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) from about 40 mg / mL to about 60 mg / mL L-arginine; (d) about 0.62 mg / mL calcium chloride; and (e) about 0.56 mg / mL polysorbate 80. In some embodiments, the composition comprises L-arginine HCl. In some embodiments, the composition comprises calcium chloride dihydrate.

[0391] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 56.12 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) about 59.11 mg / mL L-arginine HCl; (d) from about 0.7 mg / mL to about 0.9 mg / mL calcium chloride dihydrate, and (e) about 0.56 mg / mL polysorbate 80.

[0392] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 56.12 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) about 59.11 mg / mL L-arginine HCl; (d) from about 0.5 mg / mL to about 0.9 mg / mL calcium chloride, and (e) about 0.56 mg / mL polysorbate 80. In some embodiments, the composition comprises calcium chloride dihydrate.

[0393] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 56.12 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) about 48.88 mg / mL L-arginine; (d) from about 0.7 mg / mL to about 0.9 mg / mL calcium chloride dihydrate, and (e) about 0.56 mg / mL polysorbate 80. In some embodiments, the composition comprises L-arginine HCl.

[0394] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 56.12 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) about 48.88 mg / mL L-arginine; (d) from about 0.5 mg / mL to about 0.7 mg / mL calcium chloride, and (e) about 0.56 mg / mL polysorbate 80. In some embodiments, the composition comprises L-arginine HCl. In some embodiments, the composition comprises calcium chloride dihydrate.

[0395] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 56.12 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) about 59.11 mg / mL L-arginine HCl; (d) about 0.82 mg / mL calcium chloride dihydrate, and (e) from about 0.4 mg / mL to about 0.7 mg / mL polysorbate 80.

[0396] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 56.12 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) about 59.11 mg / mL L-arginine HCl; (d) about 0.62 mg / mL calcium chloride, and (e) from about 0.4 mg / mL to about 0.7 mg / mL polysorbate 80. In some embodiments, the composition comprises L-arginine HCl. In some embodiments, the composition comprises calcium chloride dihydrate.

[0397] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 56.12 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) about 48.88 mg / mL L-arginine; (d) about 0.82 mg / mL calcium chloride dihydrate, and (e) from about 0.4 mg / mL to about 0.7 mg / mL polysorbate 80.

[0398] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 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) from about 0.4 mg / mL to about 0.7 mg / mL polysorbate 80. In some embodiments, the composition comprises L-arginine HCl. In some embodiments, the composition comprises calcium chloride dihydrate.

[0399] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 56.12 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) about 59.11 mg / mL L-arginine HCl; (d) about 0.82 mg / mL calcium chloride dihydrate; and (e) about 0.56 mg / mL polysorbate 80.

[0400] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 56.12 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) about 59.11 mg / mL L-arginine HCl; (d) about 0.62 mg / mL calcium chloride; and (e) about 0.56 mg / mL polysorbate 80.

[0401] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 56.12 mg / mL sucrose; (b) about 1.74 mg / mL L-histidine; (c) about 48.88 mg / mL L-arginine; (d) about 0.82 mg / mL calcium chloride dihydrate, and (e) about 0.56 mg / mL polysorbate 80.

[0402] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 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 0.56 mg / mL polysorbate 80.

[0403] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 168.4 mg sucrose; (b) about 5.23 mg L-histidine; (c) about 177.32 mg L-arginine HCl; (d) about 2.47 mg calcium chloride, and (e) about 1.68 mg polysorbate 80 in 3 mL of sterile water.

[0404] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 168.4 mg sucrose; (b) about 5.23 mg L-histidine; (c) about 146.63 mg L-arginine; (d) about 2.47 mg calcium chloride dihydrate, and (e) about 1.68 mg polysorbate 80 in 3 ml of sterile water.

[0405] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) about 168.4 mg sucrose; (b) about 5.23 mg L-histidine; (c) about 146.63 mg L-arginine; (d) about 1.87 mg calcium chloride, and (e) about 1.68 mg polysorbate 80 in 3 ml of sterile water.

[0406] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) 168.35 mg sucrose; (b) 5.23 mg L-histidine; (c) 177.32 mg L-arginine HCl; (d) 2.47 mg calcium chloride dihydrate; and (e) 1.68 mg polysorbate 80 in 3 mL sterile water.

[0407] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) 168.35 mg sucrose; (b) 5.23 mg L-histidine; (c) 177.32 mg L-arginine HCl; (d) 1.87 mg calcium chloride; and (e) 1.68 mg polysorbate 80 in 3 mL of sterile water.

[0408] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) 168.35 mg sucrose; (b) 5.23 mg L-histidine; (c) 146.63 mg L-arginine; (d) 2.47 mg calcium chloride dihydrate; and (e) 1.68 mg polysorbate 80 in 3 ml sterile water.

[0409] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the resulting solution comprises: (a) 168.35 mg sucrose; (b) 5.23 mg L-histidine; (c) 146.63 mg L-arginine; (d) 1.87 mg calcium chloride; and (e) 1.68 mg polysorbate 80 in 3 ml sterile water.

[0410] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the lyophilized pharmaceutical composition is reconstituted in 7-12 seconds.

[0411] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the osmolality of the resulting solution is from about 525 to about 725 mOsm / kg. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the osmolality of the resulting solution is from about 600 to about 650 mOsm / kg.

[0412] 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.

[0413] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the pH of the resulting solution is from 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.

[0414] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the pH of the resulting solution is approximately 6.5. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the pH of the resulting solution is approximately 6.6. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the pH of the resulting solution is approximately 6.7. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the pH of the resulting solution is approximately 6.8. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the pH of the resulting solution is approximately 6.9.In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the pH of the resulting solution is approximately 7.0. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the pH of the resulting solution is approximately 7.1. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the pH of the resulting solution is approximately 7.2. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the pH of the resulting solution is approximately 7.3. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the pH of the resulting solution is approximately 7.4.In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the pH of the resulting solution is approximately 7.5.

[0415] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the protein concentration in the resulting solution is from about 0.8 to about 1.2 mg / mL.

[0416] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the protein concentration in the resulting solution is approximately 0.8 mg / mL. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the protein concentration in the resulting solution is approximately 0.9 mg / mL. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the protein concentration in the resulting solution is approximately 1.0 mg / mL. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the protein concentration in the resulting solution is approximately 1.1 mg / mL.In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the protein concentration in the resulting solution is approximately 1.2 mg / mL.

[0417] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the protein concentration in the resulting solution is 0.8 mg / mL. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the protein concentration in the resulting solution is 0.9 mg / mL. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the protein concentration in the resulting solution is 1.0 mg / mL. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the protein concentration in the resulting solution is 1.1 mg / mL. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, the protein concentration in the resulting solution is 1.2 mg / mL.

[0418] 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.

[0419] In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, less than 1% of the chimeric protein is aggregated. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, less than 2% of the chimeric protein is aggregated. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, less than 2.5% of the chimeric protein is aggregated. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, less than 3% of the chimeric protein is aggregated. In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, less than 3.5% of the chimeric protein is aggregated.In some embodiments, when the lyophilized pharmaceutical composition and sterile water are combined, less than 4% of the chimeric protein is aggregated.V. Pharmaceutical kits.

[0420] In some embodiments, the pharmaceutical composition comprises a second container containing sterile water. Disclosed herein are pharmaceutical kits that comprise a first container containing the pharmaceutical composition and a second container containing sterile water.

[0421] This document discloses a pharmaceutical kit comprising: (i) a first container containing a lyophilized pharmaceutical composition comprising: (a) a chimeric protein comprising a first polypeptide chain that comprises a protein that is factor VIII ("FVIII") and a first immunoglobulin ("Ig") constant region or a portion thereof, and a second polypeptide chain that comprises a protein that is von Willebrand factor ("VWF") and a second Ig constant region or a portion thereof; (b) sucrose; (c) histidine; (d) arginine; (e) calcium chloride, and (f) polysorbate (such as polysorbate 20 or polysorbate 80), and (ii) a second container containing sterile water.

[0422] Also disclosed herein is a pharmaceutical kit comprising:(i) a first container comprising a lyophilized pharmaceutical composition comprising:(a) a chimeric protein comprising a first polypeptide chain that comprises a protein that is factor VIII ("FVIII") and a first immunoglobulin ("Ig") constant region or a portion thereof, and a second polypeptide chain that comprises a protein that is von Willebrand factor ("VWF") and a second Ig constant region or a portion thereof;(b) from about 30 mg to about 135 mg of sucrose;(c) from about 2.5 mg to about 7.5 mg of histidine;(d) from about 140 mg to about 200 mg of arginine;(e) from about 1.5 mg to about 5 mg of calcium chloride, and(f) from about 1 mg to about 5 mg of polysorbate (such as polysorbate 20 or polysorbate 80), and(ii) a second container containing sterile water.

[0423] Also disclosed herein is a pharmaceutical kit comprising:(i) a first container comprising a lyophilized pharmaceutical composition comprising:(a) a chimeric protein comprising a first polypeptide chain that comprises a protein that is factor VIII ("FVIII") and a first immunoglobulin ("Ig") constant region or a portion thereof, and a second polypeptide chain that comprises a protein that is von Willebrand factor ("VWF") and a second Ig constant region or a portion thereof;(b) from about 160 mg to about 200 mg sucrose;(c) from about 2.5 mg to about 7.5 mg histidine;(d) from about 140 mg to about 200 mg arginine;(e) from about 1.5 mg to about 5 mg calcium chloride, and(f) from about 1 mg to about 5 mg polysorbate (such as polysorbate 20 or polysorbate 80), and(ii) a second container containing sterile water.

[0424] In some embodiments, the first container comprises from 100 IU to 10,000 IU of the chimeric protein.

[0425] In some embodiments, the first container comprises 250 IU, 500 IU, 1000 IU, 2000 IU, 3000 IU, or 4000 IU of the chimeric protein. In some embodiments, the first container comprises about 250 IU of the chimeric protein. In some embodiments, the first container comprises about 500 IU of the chimeric protein. In some embodiments, the first container comprises about 1000 IU of the chimeric protein. In some embodiments, the first container comprises about 2000 IU of the chimeric protein. In some embodiments, the first container comprises about 3000 IU of the chimeric protein. In some embodiments, the first container comprises about 4000 IU of the chimeric protein.

[0426] In some embodiments, the second container contains from about 2 ml to about 5 ml of sterile water. In some embodiments, the second container contains from about 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.

[0427] In some embodiments, the second container contains approximately 2 ml of sterile water. In some embodiments, the second container contains approximately 2.1 ml of sterile water. In some embodiments, the second container contains approximately 2.2 ml of sterile water. In some embodiments, the second container contains approximately 2.3 ml of sterile water. In some embodiments, the second container contains approximately 2.4 ml of sterile water. In some embodiments, the second container contains approximately 2.5 ml of sterile water. In some embodiments, the second container contains approximately 2.6 ml of sterile water. In some embodiments, the second container contains approximately 2.7 ml of sterile water. In some embodiments, the second container contains approximately 2.8 ml of sterile water.In some embodiments, the second container contains approximately 2.9 ml of sterile water. In some embodiments, the second container contains approximately 3 ml of sterile water. In some embodiments, the second container contains approximately 3.1 ml of sterile water. In some embodiments, the second container contains approximately 3.2 ml of sterile water. In some embodiments, the second container contains approximately 3.3 ml of sterile water. In some embodiments, the second container contains approximately 3.4 ml of sterile water. In some embodiments, the second container contains approximately 3.5 ml of sterile water. In some embodiments, the second container contains approximately 3.6 ml of sterile water. In some embodiments, the second container contains approximately 3.7 ml of sterile water.In some embodiments, the second container contains approximately 3.8 ml of sterile water. In some embodiments, the second container contains approximately 3.9 ml of sterile water. In some embodiments, the second container contains approximately 4 ml of sterile water. In some embodiments, the second container contains approximately 4.1 ml of sterile water. In some embodiments, the second container contains approximately 4.2 ml of sterile water. In some embodiments, the second container contains approximately 4.3 ml of sterile water. In some embodiments, the second container contains approximately 4.4 ml of sterile water. In some embodiments, the second container contains approximately 4.5 ml of sterile water. In some embodiments, the second container contains approximately 4.6 ml of sterile water.In some embodiments, the second container contains approximately 4.7 ml of sterile water. In some embodiments, the second container contains approximately 4.8 ml of sterile water. In some embodiments, the second container contains approximately 4.9 ml of sterile water. In some embodiments, the second container contains approximately 5 ml of sterile water.

[0428] In some embodiments, the pharmaceutical kit further comprises instructions for combining the lyophilized pharmaceutical composition and sterile water.

[0429] In some embodiments, the first container is a glass vial containing a rubber stopper.

[0430] In some embodiments, the second container is a syringe body. In some embodiments, the syringe body is associated with a plunger. In some embodiments, the pharmaceutical kit further comprises an adapter for attaching a glass vial to the syringe body. In some embodiments, the pharmaceutical kit further comprises an infusion system associated with a needle designed to be attached to the body of a syringe suitable for intravenous infusion. In some embodiments, the second container is a pre-filled syringe. VI. Methods and embodiments of using the pharmaceutical composition

[0431] Also disclosed herein is a use of a pharmaceutical composition or a method for treating hemophilia A in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of the present invention. 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 bleeding episodes in a human subject in need thereof. In some embodiments, treating hemophilia A comprises reducing the occurrence or frequency of bleeding episodes in a human subject in need thereof.

[0432] In some embodiments, the composition is used to treat a disease or condition associated with bleeding in a subject in need thereof. The disease or condition associated with bleeding is selected from the group consisting of a coagulation disorder, hemarthrosis, muscle bleeding, oral bleeding, hemorrhage, muscle bleeding, oral bleeding, trauma, traumatic brain injury, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, bleeding in the central nervous system, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, bleeding in the iliopsoas sheath, and any combinations thereof. In still other embodiments, the subject is scheduled to undergo surgery.In some embodiments, the treatment is prophylactic or as needed.

[0433] In some embodiments, the use or method comprises combining the lyophilized pharmaceutical composition and sterile water from a kit of the present invention and administering to a subject an effective amount of the resulting combination. In some embodiments, the subject combines the lyophilized pharmaceutical composition and sterile water from the kit. In some embodiments, the combination is self-administered by the subject.

[0434] In some embodiments, the multiple doses comprise at least two doses, at least three doses, at least four doses, at least five doses, at least six doses, at least seven doses, at least eight doses, at least nine doses, at least ten doses, at least eleven doses, at least twelve doses, at least thirteen doses, at least fourteen doses, at least fifteen doses, at least sixteen doses, at least seventeen doses, at least eighteen doses, at least nineteen doses, at least twenty doses, or more.In some embodiments, multiple doses are administered over 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 for at least about 25 years.

[0435] The chimeric protein described herein can be administered by any method known in the art. In some embodiments, the chimeric protein is administered via a route selected from the group consisting of intravenous injection, intravenous infusion, subcutaneous administration, intramuscular administration, oral administration, nasal administration, and pulmonary administration. In some embodiments, the chimeric protein is administered intravenously. In some embodiments, the chimeric protein is administered subcutaneously.

[0436] Having now described the present invention in detail, the same 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 present invention. All patents, publications, and articles mentioned herein are expressly and specifically incorporated by reference. EXAMPLES Example 1. Evaluation of the Effect of Buffer Selection and Target pH on the Stability of a Chimeric Protein

[0437] The experiments in this example demonstrated favorable pH values ​​and buffer types among various buffers, such as acetate, succinate, histidine, potassium phosphate, and Tris, over a wide pH range from 4.0 to 8.0. Eleven buffers were tested in combination with ephanesoctocog alfa at a target concentration of 1 mg / mL. The tested buffers are presented in Table 1. No additional excipients were included in the pharmaceutical compositions in this study. Each pharmaceutical composition containing ephanesoctocog alfa and a specific buffer was evaluated by measuring the actual protein concentration, pH, visual inspection, high molecular weight species (HMWS), and turbidity. The test samples were stored for no more than 4 weeks at 5°C, 25°C, and 40°C.Table 1. Buffers under study Abbreviated name Full name A4.0 10 mM sodium acetate, pH 4.0 A5.0 10 mM sodium acetate, pH 5.0 S5.5 10 mM sodium succinate, pH 5.5 H5.5 10 mM histidine-HCl, pH 5.5 H6.0 10 mM histidine-HCl, pH 6.0 H6.5 10 mM histidine-HCl, pH 6.5 H7.0 10 mM histidine-HCl, pH 7.0 H7.5 10 mM histidine-HCl, pH 7.5 KP6.5 10 mM potassium phosphate, pH 6.5 KP7.0 10 mM potassium phosphate, pH 7.0 T8.0 10 mM Tris, pH 8.0

[0438] The results of the initial appearance examination, measured protein concentration and pH of the 1 mg / mL ephanesoctocoga-alpha preparations in the corresponding buffers are shown in Table 2. For H7.0, H7.5 and T8.0, the measured pH value was 6.88, 7.05 and 7.84, respectively, which was lower than the target (expected) pH value.Table 2. Buffer Appearance Measured concentration (mg / ml) Measured pH value A4.0 Besieged Didn't measure Didn't measure A5.0 Opaque, white, no visible particles Didn't measure Didn't measure S5.5 Transparent, white, no visible particles 1,06 5,48 H5.5 Transparent, white, no visible particles 1,06 5,46 H6.0 Transparent, white, no visible particles 1,04 5,99 H6.5 Transparent, white, no visible particles 1,05 6,40 H7.0 Transparent, white, no visible particles 1,04 6,88 H7.5 Transparent, white, no visible particles 1,06 7,05 KP6.5 Transparent, white, no visible particles 1,03 6,49 KP7.0 Transparent, white, no visible particles 1,07 6,94 T8.0 Opaque, white, no visible particles 1,04 7,84

[0439] Pharmaceutical formulations A4.0 and A5.0 were excluded from further study because they exhibited sedimentation and an undesirable appearance. The remaining pharmaceutical formulations were stored at 5°C, and the percentage of HMWS (% HMWS) was measured by size-exclusion chromatography (SEC) after 0, 7, and 30 days. The results are shown in Table 3. Table 3. Aggregation (% HMWS) determined by SEC at 5°C Sample 0 days 7 days 30 days S5.5 88,14 86,50 95,15 H5.5 58,21 60,79 87,86 H6.0 68,38 68,50 69,73 H6.5 28,39 27,64 28,64 H7.0 7,01 6,31 4,32 H7.5 6,63 7,07 4,29 KP6.5 24,00 22,15 23,92 KP7.0 18,48 17,34 16,86 T8.0 27,25 49,11 65,52 Standard 2,56 2,48 2,85

[0440] The pharmaceutical compositions containing histidine buffer at pH 6.88 (H7.0) and pH 7.05 (H7.5) showed the lowest % HMWS. A slight loss of HMWS was observed after 1 month (30 days). The pharmaceutical compositions containing histidine buffer at pH 6.5 (H6.5) or potassium phosphate buffer at pH 6.5 (KP6.5) and pH 7 (KP7.0) provided comparable % HMWS, but demonstrated significantly higher % HMWS than the pharmaceutical compositions containing histidine buffer at pH 6.88 (H7.0) and pH 7.05 (H7.5).

[0441] The pharmaceutical compositions were stored at 25°C and the % HMWS was measured by size exclusion chromatography (SEC) on days 0, 0.25, 4, and 7. The results are shown in Table 4. The pharmaceutical compositions containing histidine buffer at pH 6.88 (H7.0) and pH 7.05 (H7.5) showed the lowest % HMWS at the beginning of the experiment (0 days). Table 4. Aggregation (% HMWS) determined by SEC at 25°C Sample 0 days 0.25 days 4 days 7 days S5.5 88,14 87,53 94,28 95,30 H5.5 58,21 87,23 93,53 93,63 H6.0 68,38 78,07 91,17 93,00 H6.5 28,39 28,39 39,27 65,32 H7.0 7,01 7,47 14,07 16,72 H7.5 6,63 5,23 39,41 46,55 KP6.5 24,00 22,99 24,77 25,26 KP7.0 18,48 19,43 19,61 20,22 T8.0 27,25 27,45 25,17 46,22 Standard 2,56 2,36 2,85 2,27

[0442] The pharmaceutical compositions were stored at 40°C and the % HMWS was measured by size exclusion chromatography (SEC) on days 0, 0.25, 1, and 7. The results are shown in Table 5.Table 5. Aggregation (% HMWS) determined by SEC at 40°C Sample 0 days 0.25 days Day 1 7 days S5.5 88,14 97,11 97,25 97,21 H5.5 58,21 98,85 97,44 98,35 H6.0 68,38 72,70 82,80 96,48 H6.5 28,39 73,51 85,18 93,39 H7.0 7,01 22,06 42,36 68,71 H7.5 6,63 7,79 14,71 46,99 KP6.5 24,00 23,14 25,82 35,79 KP7.0 18,48 18,69 20,00 28,70 T8.0 27,25 30,33 24,96 58,07 Standard 2,56 2,28 2,79 2,79

[0443] Pharmaceutical formulations containing histidine buffer at pH 6.88 (H7.0) and pH 7.05 (H7.5) demonstrated the lowest % HMWS at baseline. Significant degradation of ephanesoctocog-alpha was observed at 40°C for all tested buffers.

[0444] The concentration stability of the pharmaceutical compositions was also evaluated at 5°C, 25°C, and 40°C at various time points. The results are shown in Table 6. All samples except one showed a slight increase in concentration (mg / mL) after 1 month at 5°C, demonstrating possible instability or aggregation; only potassium phosphate at pH 6.5 (KP6.5) did not show an increase in concentration. The samples did not show any significant change in concentration after 1 week at all temperatures. Table 6. Concentration Stability Results pH BUFFER TEST: concentration (mg / ml), time (days) 5°C Sample 0 7 30 25°C Sample 0 0,25 4 7 40°C Sample 0 0,25 1 7 S5.5 1,01 1,01 1,25 S5.5 1,01 1,06 0,95 1,04 S5.5 1,01 1,02 0,97 1,04 H5.5 1,05 1,04 1,68 H5.5 1,05 0,99 1,04 1,07 H5.5 1,05 1,06 0,96 1,06 H6.0 1,05 1,05 1,50 H6.0 1,05 1,07 1,04 1,09 H6.0 1,05 1,06 1,01 1,12 H6.5 1,06 1,06 1,62 H6.5 1,06 0,97 1,07 1,04 H6.5 1,06 1,11 1,08 1,11 H6.8 1,06 1,04 1,38 H6.8 1,06 0,96 1,01 1,04 H6.8 1,06 1,10 1,07 1,07 H7.3 1,07 1,06 1,41 H7.3 1,07 1,01 1,05 1,06 H7.3 1,07 1,03 1,05 1,03 KP6.5 1,06 1,06 1,06 KP6.5 1,06 1,08 0,99 1,00 KP6.5 1,06 1,11 0,92 1,04 KP7.0 1,07 1,01 1,36 KP7.0 1,07 1,07 1,04 1,04 KP7.0 1,07 1,08 1,08 1,02 T8.0 1,10 1,00 1,55 T8.0 1,10 1,05 1,07 1,08 T8.0 1,10 1,11 1,03 1,06 pH stability was also assessed for pharmaceutical compositions at 5°C, 25°C, and 40°C at various time points. The pH values ​​of the samples did not change significantly when stored for one month at 5°C, one week at 25°C, or one week at 40°C (data not shown). All measured values ​​were within + / - 0.5 units of the value at time zero at all time points.

[0446] The turbidity of the pharmaceutical compositions was also evaluated at 5°C, 25°C, and 40°C at various time points. A 2-3-fold increase in optical density was observed in compositions containing potassium phosphate 7.0 (KP7.0) and histidine 5.5 (H5.5) after 7 days at 25°C and 40°C, respectively. Overall, the absorbance values ​​did not change significantly when stored for one month at 5°C, one week at 25°C, or one week at 40°C (data not shown).

[0447] Summary

[0448] Pharmaceutical compositions containing histidine and potassium phosphate in the pH range of 6.8 to 7.3 demonstrated minimal changes in concentration, pH, and turbidity, as well as low aggregation (% HMWS). Ephanesoctocog-alpha appears to be the most stable in the pharmaceutical composition characterized by the pH range of 6.8 to 7.3.

[0449] The initial aggregation value was significantly lower in histidine buffer than in potassium phosphate buffer. The smallest increase in aggregation was observed in phosphate buffer at pH 6.5 and pH 7.0 over 7 days at stress temperatures of 25°C and 40°C. Example 2. Evaluation of the effect of histidine molarity and protein concentration on the stability of ephanesoctocoga-alpha

[0450] In this example, the effect of histidine molarity and protein concentration on achieving the target pH of 7.0 was determined. Studies were also conducted to determine the effect of buffer molarity on the stability of ephanesoctocog-alpha at protein concentrations of 1, 0.67, and 0.045 mg / mL.

[0451] Study design: Pharmaceutical formulations were studied, each containing one of three different molar concentrations of histidine (10 mM, 20 mM, and 50 mM) and one of three different concentrations of ephanesoctocog alfa (1 mg / mL, 0.67 mg / mL (4000 IU / mL), and 0.045 mg / mL (250 IU / mL)) in the presence of other excipients at constant concentrations. In addition to histidine and ephanesoctocog alfa, all pharmaceutical formulations were formulated with the following excipients: 250 mM arginine hydrochloride (ArgHCl), 5 mM CaCl2, 5% sucrose, and 0.05% polysorbate 80 (PS80). Liquid stability was assessed for one week at 5°C, 25°C, and 40°C. The stability of each pharmaceutical composition was assessed by visual inspection, turbidity, protein concentration (Lunatic, Unchained Labs, Pleasanton, CA, USA), pH, osmolality, % HMWS determined by SEC, and protein concentration determined by RP-HPLC.

[0452] The maximum pH of the pharmaceutical compositions containing histidine at 10, 20, and 50 mM concentrations at any protein concentration (1 mg / mL, 4000 IU / mL, or 250 IU / mL) was 6.8, 6.95, and 7.1, respectively. The target pH of 7.0 was achieved at 20 mM and 50 mM histidine. As the molarity of histidine increased, the pH of the formulation increased, possibly due to an increase in buffering capacity (see FIG. 2). There was no significant effect of protein concentration on pH, and there was no significant change in pH with time and temperature (data not shown).

[0453] At the beginning of the experiment (T0), higher osmolality was observed as the histidine molarity increased from 10 mM to 50 mM. Visual inspection revealed that nine formulations tested at T0 and week 1 at 5°C, 25°C, and 40°C were clear and colorless. However, the protein significantly aggregated at 40°C after 1 week, and a significant increase in % HMWS was observed after 1 week at 40°C. There was no effect of molarity on concentration and osmolality over 1 week at three different temperatures (data not shown).

[0454] The turbidity results are shown in Table 7. Turbidity (NTU) showed a direct relationship with protein concentration. At 5°C and 25°C, the samples were clear at T0 and remained clear after 1 week. Although turbidity increased significantly at 40°C for the 1 mg / mL and 4000 IU samples, the 1 mg / mL samples remained clear after 1 week. Additional turbidity test results are shown in FIG. 3. Table 7.

[0455] Samples containing 4000 IU / mL (0.67 mg / mL) of protein showed loss of aggregates after 1 week at 25°C. Samples containing 250 IU / mL (0.045 mg / mL) showed loss of aggregates after 1 week at both 25°C and 40°C. Generally, some degree of HMWS aggregation was observed at 25°C, but over time, HMWS appeared to degrade into smaller molecules and were present as LMW peaks after a short time (approximately 25 minutes). Unlike at 25°C, at 40°C, HMWS did not appear to degrade into LMW species. The 250 IU / mL (0.045 mg / mL) sample was unstable at 40°C.

[0456] Summary

[0457] A maximum pH of 6.8 was achieved for pharmaceutical formulations containing ephanesoctocog alfa at 1 mg / mL, 4000 IU / mL, or 250 IU / mL; 10 mM histidine; 250 mM ArgHCl; 5% sucrose; 5 mM CaCl2, and 0.05% PS80. Increasing the molarity of the histidine buffer had no significant effects on pH, osmolality, protein concentration, turbidity, % HMWS at T0, or % HMWS during a 1-week stability study at 5°C, 25°C, and 40°C. Ephanesoctocog alfa showed significant aggregation under the stress temperature of 40°C after 1 week for all protein concentrations studied. For pharmaceutical compositions containing 4000 IU / mL and 250 IU / mL protein, a significant loss of aggregate peaks was observed at 25°C and 40°C within 1 week. A significant increase in turbidity was observed after 1 week at 40°C for pharmaceutical compositions containing 1 mg / mL and 4000 IU / mL ephanesoctocoga alfa. Example 3. Evaluation of the effect of polysorbate 80 (PS80) concentration on protein stability

[0458] These studies determined the effect of PS80 concentration on the stability of ephanesoctocog-alpha formulations.

[0459] Two concentrations of ephanesoctocog alfa (0.67 mg / mL (4000 IU / mL) and 0.045 mg / mL (250 IU / mL)) were studied in 10 mM histidine, 250 mM arginine hydrochloride (ArgHCl), 5 mM CaCl2, 5% sucrose, and polysorbate 80 (PS80). The PS80 concentration range for the 250 IU / mL samples was 0.002% to 0.07%. The PS80 concentration range for the 4000 IU / mL samples was 0.03% to 0.1%. Samples were stressed by orbital shaking (250 rpm for 0, 6, and 24 h) or freeze / thaw (-80°C for at least 24 h, thawed at room temperature) for 0, 1, 3, and 5 cycles. Analysis included determination of aggregation level by SEC, PS80 concentration by high-performance liquid chromatography (HPLC) with charged aerosol detection (CAD), particle testing by microflow imaging (MFI) and high-accuracy particle counter (HIAC), pH, turbidity, and protein concentration. The results of each analysis are shown in Table 8.

[0460] Summary

[0461] The results of this study showed that PS80 ≥ 0.03% stabilized the concentration of ephanesoctocog alfa at both 250 IU and 4000 IU. No change in PS80 content was observed for the samples after 24 h of agitation or 5 freeze / thaw cycles. No trend in particulate matter was observed throughout the PS80 range studied. No trends or changes in pH or turbidity were observed within this PS80 range. Therefore, a pharmaceutical composition containing PS80 ≥ 0.03% can provide increased stability of ephanesoctocog alfa at a minimum concentration of 250 IU and a maximum concentration of 4000 IU with minimal impact on other properties of the formulation.Table 8. Particles invisible to the naked eye determined by MFI and HIAC Activity Conc. PS80 (%) Mixed mixing study Freeze / thaw study T0 T6hrs T24hrs F / T 1X F / T 3X F / T 5X ≥ 10 µm ≥ 25 µm ≥ 10 µm ≥ 25 µm ≥ 10 µm ≥ 25 µm ≥ 10 µm ≥ 25 µm ≥ 10 µm ≥ 25 µm ≥ 10 µm ≥ 25 µm Number of particles determined by MFI 250 IU 0,002 44 11 14 5 23 2 14 7 7 2 28 5 0,005 11 5 11 7 9 5 9 0 11 0 9 2 0,010 14 2 11 5 28 5 14 0 11 5 28 9 0,030 39 7 39 7 14 7 2 0 21 0 5 2 0,050 18 0 9 5 41 11 50 7 57 0 32 9 0,070 14 7 57 18 25 5 16 5 9 0 0 0 4000 IU 0,030 7 2 9 2 257 64 18 5 5 0 21 5 0,050 7 2 11 0 18 5 41 14 16 5 7 5 0,070 9 0 7 0 156 48 41 5 32 7 9 0 0,100 11 2 9 5 119 32 7 0 16 2 11 2 Particle count determined by HIAC 250 IU 0,002 60 8 18 0 15 0 100 25 2 0 7 0 0,005 27 0 27 0 7 2 0 0 0 0 0 0 0,010 20 0 17 0 27 2 2 0 3 2 0 0 0,030 28 3 20 0 33 2 2 2 3 0 2 0 0,050 22 2 10 2 23 2 7 0 0 0 0 0 0,070 17 2 43 2 10 0 23 7 2 0 2 0 4000 IU 0,030 25 0 17 0 18 0 0 0 5 2 2 0 0,050 12 2 25 0 12 2 2 0 2 0 2 0 0,070 10 2 2 0 17 3 0 0 2 0 3 0 0,100 32 0 12 0 13 2 2 0 52 22 2 0 Example 4. Evaluation of the effect of excipient concentrations on protein stability

[0462] The experiments described in this example evaluate the stability of pharmaceutical compositions containing the drug product ephanesoctocog alfa (DP) at a dose of 1000 IU, formulated with dilution buffers containing different concentrations of excipients. The minimum and maximum ranges of excipient concentrations were intentionally designed to deviate from current manufacturing tolerances in order to test the stability of the formulation. A series of 13 different dilution buffers were evaluated (see Table 9). The design of experiment (DoE) model was based on five parameters (i.e., five excipients) at two levels (i.e., ±5% of target values). It included a central control point at the target amounts of excipients in the formulation (i.e., for a number of buffers of 13).

[0463] Ephanesoctocog-alpha drug substance (DS) was preformulated at a concentration of 1 mg / mL in 10 mM L-histidine, 250 mM L-arginine hydrochloride (HCl), 5 mM calcium chloride, 5% (w / v) sucrose, and 0.05% (w / v) polysorbate 80 (PS80) at pH 7.01. Ephanesoctocog-alpha DS was diluted to one of several potencies using one of 13 dilution buffers to yield ephanesoctocog-alpha diluted drug substance (DDS). Table 9. Dilution buffers tested Buffer number L-arginine HCl (g / L) Sucrose (g / l) L-histidine (g / L) Calcium Chloride Dihydrate (g / l) Polysorbate-80 (g / l) 1 50,032 52,500 1,630 0,698 0,475 2 55,298 47,500 1,474 0,698 0,525 3 55,298 52,500 1,474 0,698 0,475 4 55,298 47,500 1,630 0,772 0,525 5 50,032 47,500 1,630 0,772 0,475 6 55,298 52,500 1,630 0,772 0,525 7 50,032 52,500 1,474 0,772 0,525 8 50,032 52,500 1,630 0,698 0,525 9 55,298 47,500 1,630 0,698 0,475 10 50,032 47,500 1,474 0,698 0,525 11 55,298 52,500 1,474 0,772 0,475 12 50,032 47,500 1,474 0,772 0,475 13 52,665 50,000 1,552 0,735 0,500

[0464] Then, the ephanesoctocoga alfa-based DDS was lyophilized to obtain ephanesoctocoga alfa-based DP with a target activity value per vial of 1000 IU. The resulting pharmaceutical compositions containing ephanesoctocoga alfa-based DP at a concentration of 1000 IU were evaluated under various stability study conditions (2-8°C and 30°C) and stability study time points (T0, one month (T1), 3 months (T3), 6 months (T6), and 12 months (T12)). Samples of the pharmaceutical compositions were tested for specific activity, protein concentration, aggregation by visual inspection, recovery time, pH, residual moisture, product / excipient crystallization, and glass transition temperature.

[0465] Protein concentration was measured using RP-HPLC with fluorescence detection. The specification range for protein concentration in the stability study was set at 75-125% of the baseline value.

[0466] Table 10 and FIG. 11 show the concentration stability data up to 6 months (T6). Generally, the concentration values ​​were lower than the associated target value of 167 μg / mL, but remained within 75-125% of the initial DP concentration value, thus falling within the acceptable specification range.Table 10. Protein concentration results (μg / mL) Buffer number T0, DDS T0, DP, 2-8°C T1, DP, 30°C T3, DP, 2-8°C T3, DP, 30°C T6, DP, 2-8°C T6, DP, 30°C Reduction of DP content in % T0 → T6 1 159,7 156,1 151,6 160,6 148,1 138,5 138,6 11,21 2 165,2 158,2 148,9 154,5 149,5 139,7 142 10,24 3 151,5 158,5 144,7 152,5 151,7 138 139,2 12,18 4 160,4 160,6 144,7 160 154,5 139,6 139,9 12,89 5 155,6 161,8 150,1 159,7 153,2 144 140,3 13,29 6 159,6 156,0 152,8 162 156 140,2 138,9 10,96 7 165,8 163,8 154,7 153,6 Not applicable 138,9 144,5 11,78 8 155,4 161,8 156,3 150,7 154,2 134,9 140,7 13,04 9 157 162,2 160,8 153,1 155,2 134,1 141,8 12,58 10 157,7 169,5 161,9 154 157,6 140,6 141,8 16,34 11 154,1 165,8 160,6 155,4 151,1 135,4 138,2 16,65 12 161,2 170,2 170,4 160,6 159,9 141,8 141,2 17,04 13 145,4 167,9 162,7 152,6 153,1 142,5 137,6 18,05 Average 157,58 162,49 155,40 156,10 156,62 139,09 140,36 Not applicable Std. dev. 5,49 4,76 7,63 3,88 11,10 2,96 1,91 Not applicable

[0467] The specific activity values ​​of DP and DDS were calculated using the concentration data presented in Figure 11. The results are shown in Table 11 and Figure 13. The specified specific activity is > 1600 IU / mg for DDS and > 1280 IU / mg for DP for all activity values. For DP, the specific activity of each sample tested exceeded the specification at every time point.

[0468] Using DDS testing based on ephanesoctocog-alpha, the activity values ​​before lyophilization were found to be within the specification range of 80-125%. Table 11. Specific activity results (IU / mg) Buffer number T0, DDS, DDS, by specification. T0, DP (2-8°C) T1, DP (30°C) T3, DP (2-8°C) T3, DP (30°C) DP by spec. 1 1748,3 1600 2053,7 1528,5 Not applicable 1497,0 1280,0 2 1679,8 1600 2138,1 1409,6 Not applicable 1554,5 1280,0 3 2093,7 1600 2251,5 1361,5 Not applicable 1624,3 1280,0 4 1791,1 1600 2036,6 1563,5 Not applicable 1622,7 1280,0 5 1822,0 1600 2060,9 1416,6 Not applicable 1560,1 1280,0 6 1830,8 1600 1946,4 1550,0 Not applicable 1443,6 1280,0 7 1769,6 1600 2040,5 1473,7 Not applicable 1352,60 1280,0 8 1718,8 1600 2098,6 1542,0 Not applicable 1655,0 1280,0 9 1759,2 1600 1600,6 1496,3 Not applicable 1607,6 1280,0 10 1750,8 1600 1620,5 1423,6 Not applicable Not applicable 1280,0 11 1835,8 1600 1591,8 1504,8 Not applicable 1653,9 1280,0 12 1719,6 1600 1608,5 1426,6 Not applicable 1627,9 1280,0 13 Not applicable 1600 1626,2 1432,4 Not applicable 1659,0 1280,0

[0469] DP and DDS aggregation testing was performed by SEC-HPLC, and the results are shown in FIG. 7. At T0, DP aggregation was in the range of 1.8-1.9%. At T3 and T6, samples at 30°C showed reduced aggregation values ​​of only 1.1%, which may be due to column characteristics or system variability. When comparing the results at T0 and T6 at 2-8°C, no significant change in the degree of aggregation was observed. All the obtained aggregation values ​​for DP based on ephanesoctocoga-alpha are less than 5%. Aggregation analysis of samples at T1 at 30ºC and T3 at 2-8ºC is still in progress.

[0470] The residual moisture values ​​showed no significant change during the stability study, with all results remaining below 1.2%, which was within the stability specification limit of 3%.

[0471] Upon visual inspection, lyophilized ephanesoctocog-alpha-based DP masses were white to off-white in color. These masses completely dissolved upon reconstitution within ~60 seconds. Recovery values ​​at T0 and T1 were slightly lower (within 25 seconds). All ephanesoctocog-alpha-based DP samples reconstituted within 70 seconds (significantly lower than the specification's suggested value of 3.5 minutes), and no unexpected changes in appearance were observed.

[0472] The samples were tested using powder X-ray diffraction. The samples appeared amorphous at time points T1-T6, as evidenced by the pattern of amorphous halos in the diffraction pattern. No signs of product or excipient crystallization, such as crystallization peaks, were observed.

[0473] The glass transition temperature (Tg) was measured by differential scanning calorimetry (DSC) in duplicate for each sample; the averaged results are shown in FIG. 16. The highest and lowest Tg values ​​were 71.75°C (T6, 30°C, buffer #10) and 61.95°C (buffer #3, T6, 2-8°C), respectively. The highest Tg value (buffer #10, T6, 30°C) coincided with the lowest moisture content (0.9%) observed in the samples at a given time. The lowest Tg value (buffer #3, T6, 2-8°C) coincided with the higher relative moisture content (1.11%) observed for buffer #3 at a given time. The samples showed no obvious trend toward increasing or decreasing Tg values ​​under various storage conditions during stability studies. Measured enthalpy values ​​ranged from 0.19 to 0.46 J / g°C.

[0474] All the stability results of the samples shown in Figure 16 had Tg values ​​above 60°C. This supports the stable long-term storage of ephanesoctocoga-alpha at 2-8°C, including six months of storage at room temperature. Example 5. Evaluation of the effect of arginine HCl concentration on protein stability

[0475] The experiments described in this example are used to evaluate the stability of ephanesoctocog alfa in pharmaceutical compositions formulated with varying concentrations of arginine HCl.

[0476] Two pharmaceutical formulations were evaluated, the only difference being the concentration of L-arginine HCl. The first formulation contained 1.0 mg / mL ephanesoctocoga alfa in 10 mM histidine, pH 7.0, 250 mM L-arginine HCl, 5 mM CaCl2, 5% sucrose, and 0.05% polysorbate 80. The second formulation contained 1.0 mg / mL ephanesoctocoga alfa in 10 mM histidine, pH 7.0, 175 mM L-arginine HCl, 5 mM CaCl2, 5% sucrose, and 0.05% polysorbate 80. Each composition is diluted approximately 60, 120, and 240 minutes after thawing at room temperature in preparation for size-exclusion chromatography (SEC) analysis. For SEC, each sample is injected in duplicate, and the % HMWS is determined for each injected sample.

[0477] The lyophilized ephanesoctocog alfa-based medicinal product is dissolved in 3.0 mL of water for injection (WFI). An aliquot of the reconstituted solution is transferred to an HPLC vial for analysis.

[0478] The SEC method analysis uses an HPLC instrument equipped with a pump, an automatic sampler with temperature control, a column heater and a fluorescence detector. The sample solutions were analyzed using the following instrumentation and method parameters. Mobile phase: Dulbecco's phosphate-buffered saline (D-PBS) containing calcium and magnesium supplemented with 0.36 M sodium chloride (0.9 mM calcium chloride, 0.5 mM magnesium chloride, 2.7 mM potassium chloride, 1.5 mM monobasic potassium phosphate, 496 mM sodium chloride, 8.1 mM dibasic sodium phosphate, pH 7.0 ± 0.1). Column heater: 26°C. Analysis time: 40 minutes. FL detector: Ex / Em=280 / 350 nm, PMT gain=5. Injection mass loading: 2 μg. Autosampler temperature: 5°C. Flow rate: 0.5 mL / min, isocratic.

[0479] Ephanesoctocoga alfa-based pharmaceutical compositions containing 250 mM L-arginine HCl were found to be more stable than compositions containing 175 mM L-arginine HCl. Example 6. Further evaluation of the effect of L-arginine HCl concentration on protein stability

[0480] The effect of arginine concentration was also studied over a wider range. Eight concentrations of L-arginine HCl (50, 100, 125, 150, 175, 200, 250, and 300 mM) were added to a master formula consisting of 10 mM histidine, 5 mM calcium chloride, 5% sucrose, and 0.05% polysorbate 80. A summary of the study design is presented in Table 12. After thawing at room temperature (25°C), the formulas were prepared, placed in an HPLC vial, and stored in an autosampler at 5°C ±3°. HPLC injection samples for each time point were taken from the same vial. % aggregation for each formula was obtained. Aggregation was measured by SEC (% HMWS) for each sample.

[0481] The SEC method utilizes 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 instrumentation and method parameters.

[0482] Mobile phase: Dulbecco's phosphate-buffered saline (D-PBS) containing calcium and magnesium, supplemented with 0.36 M sodium chloride (0.9 mM calcium chloride, 0.5 mM magnesium chloride, 2.7 mM potassium chloride, 1.5 mM monobasic potassium phosphate, 496 mM sodium chloride, 8.1 mM dibasic sodium phosphate, pH 7.0 ± 0.1). Column heater: 26 °C. Analysis time: 40 minutes. FL detector: Ex / Em = 280 / 350 nm, PMT gain = 5. Injection load: 2 μg. Autosampler temperature: 5 °C. Flow rate: 0.5 mL / min, isocratic. Table 12. Overview of arginine concentration studies Study Ephanesoctocog-alpha concentration Arginine levels Research conditions Time points of the study Quality attributes Selection of L-arginine HCl as a stabilizing excipient 1 mg / ml 50, 100, 125, 150, 175, 200, 250 and 300 mM Freezing at -80°C and thawing at room temperature. After 40, 80, 120 and 160 minutes after thawing Aggregate content in % determined by SE-HPLC

[0483] Two experiments (each performed in duplicate) were conducted using the conditions presented in Table 12. The results of Experiment 1 are shown in Table 13 and FIG. 27. In Experiment 1, high-molecular-weight (HMW) aggregates were detected at higher percentages in formulations with lower arginine concentrations. Higher arginine concentrations (200 mM to 300 mM) showed a consistently low percentage of HMW aggregates for up to 160 minutes after thawing. Table 13. Percentage of High-Molecular-Weight Aggregates - Experiment 1 HMW AREA IN % Arginine 0 min 40 min 80 min 120 min 160 min 0 mm 4,24 4,22 4,25 4,24 4,26 50 mm 8,38 8,27 8,16 8,05 7,93 100 mM 7,36 7,13 6,94 6,83 6,60 125 mmol 7,35 6,92 6,56 6,12 5,85 150 mM 4,79 4,42 4,32 4,19 4,01 175 mm 5,73 5,13 4,46 4,26 3,97 200 mM 4,10 3,77 3,78 3,54 3,63 250 mM 3,72 3,71 3,70 3,71 3,70 300 mM 3,60 3,60 3,60 3,59 3,61

[0484] The results of Experiment 2 can be found in Table 14 and FIG. 28. In Experiment 2, HMW aggregates were found at a higher percentage in formulations with lower arginine concentrations. Consistent with the observations of Experiment 1, Experiment 2 also demonstrated that higher arginine concentrations (200 mM to 300 mM) exhibited a consistently low percentage of HMW aggregates for up to 160 minutes after thawing. Table 14. Percentage of High Molecular Weight Aggregates - Experiment 2 HMW AREA IN % Arginine 0 min 40 min 80 min 120 min 160 min 0 mm 4,24 4,22 4,25 4,24 4,26 50 mm 8,38 8,27 8,16 8,05 7,93 100 mM 7,36 7,13 6,94 6,83 6,60 125 mM 7,35 6,92 6,56 6,12 5,85 150 mM 4,79 4,42 4,32 4,19 4,01 175 mm 5,73 5,13 4,46 4,26 3,97 200 mM 4,10 3,77 3,78 3,54 3,63 250 mM 3,72 3,71 3,70 3,71 3,70 300 mM 3,60 3,60 3,60 3,59 3,61

[0485] The osmolality of the samples across a range of arginine concentrations was also studied in two separate experiments (each conducted in duplicate). Osmolality (mOsm / kg) of the formulations was shown to increase with increasing arginine concentrations. In Experiment 1, the first set of replicates was analyzed on an osmometer after being kept at room temperature for 24 hours. The second set of replicates was taken from archives stored at -80°C. The results of Experiment 1 are shown in Table 15. In Experiment 2, a single set of replicate samples was taken from archives stored at -80°C. The results of Experiment 2 are shown in Table 16. Table 15. Osmolality of Formulations with Increasing Arginine Concentrations - Experiment 1 Experiment 1 Rep 1 (RT) Repeat 2 (-80°C) Sample ID mOsm / kg mOsm / kg Standard 290 295 288 0 mm 218 Not analyzed 50 mM Arg 327 331 100 mM Arg 371 420 125 mM Arg 408 425 150 mM Arg 495 484 175 mM Arg 530 524 200 mM Arg 534 537 250 mM Arg 655 667 300 mM Arg 758 699 Table 16. Osmolality of formulations with increasing arginine concentrations - experiment 2 Experiment 2 (-80°C) Sample ID mOsm / kg Standard 290 288 0 mm 184 50 mM Arg 302 100 mM Arg 356 125 mM Arg 398 150 mM Arg 476 175 mM Arg 513 200 mM Arg 538 250 mM Arg Not analyzed 300 mM Arg 701

[0486] The pH value was not adjusted for the test samples, and the pH of formulations with a range of arginine concentrations was subsequently evaluated. The results are shown in Table 17.

[0487] Table 17. pH values ​​of formulations with increasing concentrations of arginine Repetition 1 Repetition 2 Repetition 3 Sample Required Arg concentration pH measured with a pH meter 1 pH measured with a pH meter 2 pH measured with a pH meter 3 Composition 1 0 7,00 6,99 6,95 Composition 2 50 6,74 6,74 6,76 Composition 3 100 6,74 6,70 6,75 Composition 4 125 6,74 6,69 6,70 Composition 5 150 6,69 6,65 6,67 Composition 6 175 6,69 6,62 6,67 Composition 7 200 6,67 6,60 6,70 Composition 8 250 6,54 6,54 6,65 Composition 9 300 6,50 6,51 6,62

[0488] ConclusionIn these experiments, it was unexpected that ephanesoctocog alpha-containing formulations containing at least 250 mM arginine demonstrated the lowest % HMW at time 0 (T0) and subsequent time points tested (see FIG. 27 and FIG. 28), while lower levels of arginine provided increased aggregation at T0. At the beginning of time 0, the 0% arginine control had an HMW area at T0 of ~4%, while 50 mM, 100 mM, 125 mM, and 150 mM had significantly higher % HMW at the initial time points, demonstrating that arginine at low concentrations from 50 mM to 200 mM initiated aggregation at higher levels than the 0 mM arginine control. A decrease in HMW area in % was observed over time at concentrations of 50 mM, 100 mM, 125 mM, and 150 mM.

[0489] Thus, the use of at least 250 mM arginine in the formulation appears to stabilize ephanesoctocog-alpha over time with minimal aggregation. Example 7. Evaluation of the effect of low sucrose concentration on protein stability

[0490] The experiments described in this example evaluate the stability of ephanesoctocog-alpha in pharmaceutical compositions formulated with varying sucrose concentrations. In addition to sucrose, each composition also contained 10 mM L-histidine, 250 mM L-arginine HCl, 5 mM CaCl2, and 0.05% (w / v) polysorbate 80 at pH 6.8.

[0491] The drug substance (DS) which is ephanesoctocog-alpha provides 1 mg / mL ephanesoctocog-alpha, 10 mM L-histidine, 250 mM L-arginine HCl, 5 mM CaCl2 and 0.05% (w / v) polysorbate 80 and sucrose at pH 6.8. The drug product (DP) based on ephanesoctocog-alpha is DS diluted to the required activity in international units (IU), such as IU per vial (e.g., IU per 3.367 mL if the vial is filled with 3.367 mL of the drug product). DP can be lyophilized (e.g., in a vial) to yield lyophilized DP, which can then be reconstituted before injection.

[0492] The aggregation of ephanesoctocog alfa (1 mg / mL) drug substance (DS) formulations containing 5% or 1% sucrose was tested either under room temperature (RT) / room light (RL) conditions or at 2-8°C. As shown in FIG. 9 and FIG. 10, the DS formulations containing 5% sucrose exhibited a lower degree of aggregation (% HMWS) over time than the DS formulations containing 1% sucrose.

[0493] To further analyze the effect of sucrose concentration on ephanesoctocog alfa-based formulations, ephanesoctocog alfa-based pharmaceutical formulations with sucrose contents of 0, 1, 2, and 5% w / v were subjected to various stability test conditions, and stability was assessed using several methods. The formulations were tested at the following stages of product preparation: stability of the drug substance (DS, which was not lyophilized or adjusted to any specific IU potency value) during frozen storage, freeze / thaw stability of DS (FT), stability of liquid DS after thawing, stability of diluted DS and drug product (DP), stability of DP during lyophilization, and DP after reconstitution. The drug substance was tested at freezing temperatures (-80°C or -30°C). Samples of the drug product were tested at room temperature (RT), 5°C, 30°C and 40°C.Lyophilized DP samples were tested at two different DP concentrations (4000 IU and 250 IU).

[0494] Aggregation of formulations containing 250 IU ephanesoctocog alfa bulk drug product (BDP) was tested under either room temperature (RT) / room light (RL) or 2-8°C conditions. BDP formulations containing 0, 1, 2, and 5% sucrose (w / v) were tested. The RT / RL results are shown in FIG. 11, and the 2-8°C results are shown in FIG. 12. BDP formulations containing 0% sucrose demonstrated the highest levels of aggregation for both conditions. However, none of the 250 IU BDP formulations demonstrated a significant increase in % HMWS over the tested time period of 24 hours. For all formulations and both conditions, aggregation levels were observed to begin to decrease over time after approximately 7 days (data not shown).

[0495] The aggregation of lyophilized drug product (Lyo DP) formulations containing 250 IU or 4000 IU of ephanesoctocog alfa was tested at 5°C, 30°C, and 40°C. The aggregation was measured by SEC (% HMWS) for each sample. Lyo DP formulations containing 0, 1, 2, and 5% w / v sucrose were tested. The samples were tested at T0, 1 month, 2 months, 3 months, and 6 months. The results for the 250 IU Lyo DP formulations are shown in FIG. 13 (5°C), FIG. 14 (30°C), and FIG. 15 (40°C). The results for the 4000 IU Lyo DP formulations are shown in FIG. 16 (5°C), FIG. 17 (30°C), and FIG. 18 (40°C). Overall, all sucrose concentrations failed to show a significant increase in % HMW over 6 months at 5°C, 30°C, and 40°C. None of the tested formulations showed significant aggregation tendencies.

[0496] Aggregation of DS, which is ephanesoctocog-alpha, and bulk drug product (BDP) liquid formulations at a concentration of 4000 IU containing either 2% or 5% sucrose was tested by SEC. Samples were stored at room temperature or 5°C and tested at 0, 5, 7, 25, 43, and 55 hours. Samples were also tested after lyophilization. The results are shown in FIG. 19. A lower degree of aggregation over time was observed for formulations containing 5% sucrose. However, formulations containing 2% sucrose did not show significant increases in aggregation over the tested time period.

[0497] A summary of the aggregation test results is presented in Table 18. An increase of 1.0% or more of HMWS from the initial value set as the corresponding value for 5% sucrose was considered unacceptable. The content of 0% sucrose was found to be unacceptable. Table 18. Stability screening study results according to sucrose content (% HMWS) Sucrose concentration, % Storage in frozen state, % (-80°C) (1 mg / ml) Liquid DS, level in % / h (1 mg / ml) Liquid BDP, level in % / h (4000 IU) Cumulate. % / h (4000 IU) Lyo DP (Δ6M) (4000 IU) Lyo DP (Δ6M) (250 IU) Δ6M Δ5XFT RT 5°C RT 5°C / RT 5°C 30°C 40°C 5°C 30°C 40°C 5 0,3 1,0 0,03 0,01 0,02 0,01 0,3 0,2 0,4 0,0 -0,1 0,0 2 0,3 1,2 0,02 0,02 0,02 0,02 0,5 0,7 0,9 0,0 0,2 0,0 1 0,3 1,3 0,08 0,03 0,04 0,03 0,7 0,9 0,3 0,1 0,0 0,0 0 0,6 2,2 ND ND ND ND 0,2 0,0 -0,2 0,2 0,1 0,1 ND=not determined

[0498] As shown in Table 18, the stability study results indicate that freeze / thaw stability and frozen storage data were favorable for sucrose with contents ranging from 1% to 5% w / v at -80°C. Freeze / thaw stability and frozen storage were unfavorable for all concentrations at -30°C (data not shown). DP samples showed a slight but acceptable increase in aggregation over 6 months at 40°C for the 1% and 2% formulations.

[0499] The glass transition temperature (Tg) of the compositions containing 250 IU or 4000 IU of ephanesoctocog-alpha-based DP was tested at 5°C, 30°C, and 40°C. Tg was measured by modulated differential scanning calorimetry (DSC). The DP-based compositions containing 0, 1, 2, and 5% w / v sucrose were tested. The samples were tested at T0, 1 month, 2 months, 3 months, and 6 months. The results are shown in FIG. 20 (5°C), FIG. 21 (30°C), and FIG. 22 (40°C). In general, it was observed that Tg correlated with the sucrose concentration, with a lower Tg observed at a higher sucrose concentration. No significant changes in Tg were observed at 5°C, 30°C, and 40°C over 6 months for any composition. No significant changes in Tg were observed at different DP concentrations.

[0500] The glass transition temperature (Tg) of the ephanesoctocog-alpha-based drug product (DP) containing 0, 1, 2, and 5% w / v sucrose was measured using differential scanning calorimetry (DSC). The DP samples were tested at two different DP concentrations (4000 IU and 250 IU). The results are shown in FIG. 23.

[0501] The DSC protocol for determining Tg includes: equilibration at 15°C, (ii) temperature modulation by 1°C every 60 seconds, (iii) constant temperature for 5 minutes, (iv) linear temperature increase at 3°C / min to 130°C.

[0502] The residual moisture content of formulations containing lyophilized drug products (Lyo DP) based on ephanesoctocog alfa in an amount of 250 IU or 4000 IU was tested at 5°C, 30°C and 40°C. Lyo DP-based formulations containing 0, 1, 2 and 5% w / v sucrose were tested. The samples were tested at time T0, 1 month, 2 months, 3 months and 6 months. The results are shown in FIG. 24 (5°C), FIG. 25 (30°C) and FIG. 26 (40°C). No significant changes in residual moisture content were observed at 5°C, 30°C and 40°C over 6 months for all formulations. All residual moisture values ​​fell below the specified value of 3%.

[0503] When visually inspected, sucrose concentration did not have any effect on the mass appearance for more than 6 months at 2-8°C, 30°C and 40°C (data not shown).

[0504] Based on the results of these experiments, sucrose concentrations of 1% to 2% (w / v) are acceptable concentrations for ephanesoctocoga alfa-based pharmaceutical compositions.

[0505] The foregoing description of specific embodiments so fully reveals the general nature of the present invention that others, using knowledge within the skill in the art, can easily modify and / or adapt such specific embodiments for various applications without further experimentation, without departing from the general concept of the present invention. Accordingly, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments based on the idea and principle presented herein. It should be understood that the language or terminology in this document is for the purpose of description and not limitation, and therefore, the terminology or language in this description should be interpreted by a skilled person in light of these ideas and principles.

[0506] Other embodiments of the present invention will be apparent to those skilled in the art upon consideration of the description and practice of the present invention disclosed herein. It is intended that the description and examples be considered as illustrative only, and the true scope and spirit of the present invention are indicated in the following claims.

[0507] All patents and publications cited in this document are incorporated herein by reference in their entirety.SEQUENCES Amino acid sequence of protein FVIII (312A) - contains signal peptide: SEQ ID NO: 3 1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVDARFP 51 PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY 101 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG 151 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE 201 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARAWPKM 251 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH 301 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE 351 EPQLRMKNNE EADEYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT 401 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY 451 TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT 501 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR 551 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE 601 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL 651 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS 701 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL 751 SKNNAIEPRS FSQNGTSESA TPESGPGSEP ATSGSETPGT SESATPESGP 801 GSEPATSGSE TPGTSESATP ESGPSAPGSPAGSTP GSPTSTEEGT851 SESATPESGP GSEPATSGSE TPGTSESATP ESGPGSPAGS PTSTEEGSPA 901 GSPTSTEEGT STEPSEGSAP GTSESATPES GPGTSESATP ESGPGTSESA 951 TPESGPGSEP ATSGSETPGS EPATSGSETP GSPAGSPTST EEGTSTEPSE 1001 GSAPGTSTEP SEGSAPGSEP ATSGSETPGT SESATPESGP GTSTEPSEGS 1051 APASSEITRT TLQSDQEEID YDDTISVEMK KEDFDIYDED ENQSPRSFQK 1101 KTRHYFIAAV ERLWDYGMSS SPHVLRNRAQ SGSVPQFKKV VFQEFTDGSF 1151 TQPLYRGELN EHLGLLGPYI RAEVEDNIMV TFRNQASRPY SFYSSLISYE 1201 EDQRQGAEPR KNFVKPNETK TYFWKVQHHM APTKDEFDCK AWAYFSDVDL 1251 EKDVHSGLIG PLLVCHTNTL NPAHGRQVTV QEFALFFTIF DETKSWYFTE 1301 NMERNCRAPC NIQMEDPTFK ENYRFHAING YIMDTLPGLV MAQDQRIRWY 1351 LLSMGSNENI HSIHFSGHVF TVRKKEEYKM ALYNLYPGVF ETVEMLPSKA 1401 GIWRVECLIG EHLHAGMSTL FLVYSNKCQT PLGMASGHIR DFQITASGQY 1451 GQWAPKLARL HYSGSINAWS TKEPFSWIKV DLLAPMIIHG IKTQGARQKF 1501 SSLYISQFII MYSLDGKKWQ TYRGNSTGTL MVFFGNVDSS GIKHNIFNPP 1551 IIARYIRLHP THYSIRSTLR MELMGCDLNS CSMPLGMESK AISDAQITAS 1601 SYFTNMFATW SPSKARLHLQ GRSNAWRPQV NNPKEWLQVD FQKTMKVTGV 1651 TTQGVKSLLT SMYVKEFLIS SSQDGHQWTLFFQNGKVKVF QGNQDSFTPV 1701 VNSLDPPLLT RYLRIHPQSW VHQIALRMEV LGCEAQDLYD KTHTCPPCPA 1751 PELLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG 1801 VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP 1851 IEKTISKAKG QPREPQVYTL PPSRDELTKN QVSLTCLVKG FYPSDIAVEW 1901 ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA 1951 LHNHYTQKSL SLSPG* Nucleotide sequence encoding FVIII 312A: SEQ ID NO: 4 1 ATGCAAATAG AGCTCTCCAC CTGCTTCTTT CTGTGCCTTT TGCGATTCTG 51 CTTTAGTGCC ACCAGAAGAT ACTACCTGGG TGCAGTGGAA CTGTCATGGG 101 ACTATATGCA AAGTGATCTC GGTGAGCTGC CTGTGGATTGCCT AGCGATCTA15 CAAAATCTTT TCCATTCAAC ACCTCAGTCG TGTACAAAAA 201 GACTCTGTTT GTAGAATTCA CGGATCACCT TTTCAACATC GCTAAGCCAA 251 GGCCACCCTG GATGGGTCTG CTAGGTCCTA CCATCCAGGC TGAGGTTTAT 301 GATACAGTCAGTCATTGATTCATT GCTTCCCATC CTGTCAGTCT 351 TCATGCTGTT GGTGTATCCT ACTGGAAAGC TTCTGAGGGA GCTGAATATG 401 ATGATCAGAC CAGTCAAAGG GAGAAAGAAG ATGATAAAGT CTTCCCTGGT 451 GGAAGCCATA CATATGTCTG GGAAGCTG GTCGAGATG 501 CTCTGACCCA CTGTGCCTTA CCTACTCATA TCTTTCTCAT GTGGACCTGG 551 TAAAAGACTT GAATTCAGGC CTCATTGGAG CCCTACTAGT ATGTAGAGAA 601 GGGAGTCTGG CCAAGGAAAA GACACAGACC TTACACAACTTAGTTATGTTAT15 TTTGATGAAG GGAAAAGTTG GCACTCAGAA ACAAAGAACT 701 CCTTGATGCA GGATAGGGAT GCTGCATCTG CTCGGGCCTG GCCTAAAATG 751 CACACAGTCA ATGGTTATGT AAACAGGTCT CTGCCAGGTC TGATTGGATCCAG AGTCAGTAGATT 801 GGCATGTGAT TGGAATGGGC ACCACTCCTG851 AAGTGCACTC AATATTCCTC GAAGGTCACA CATTTCTTGT GAAGGAACCAT 901 CGCCAGGCGT CCTTGGAAAT CTCGCCAATA ACTTTCCTTA CTGCTCAAAC 951 ACTCTTGATG GACCTTGGAC AGTTTCTACT GTTTTGTCAT ATCTCTTCCC 1001 ACCAACATGA TGGCATGGAA GCTTATGTCA AAGTAGACAG CTGTCCAGAG 1051 GAACCCCAAC TACGAATGAA AAATAATGAA GAAGCGGAAG ACTATGATGA 1101 TGATCTTACT GATTCTGAAA TGGATGTGGT CAGGTTTGAT GATGACAACT 1151 CTCCTTCCTT TATCCAAATT CGCTCAGTTG CCAAGAAGCA TCCTAAAACT 1201 TGGGTACATT ACATTGCTGC TGAAGAGGAG GACTGGGACT ATGCTCCCTT 1251 AGTCCTCGCC CCCGATGACA GAAGTTATAA AAGTCAATAT TTGAACAATG 1301 GCCCTCAGCG GATTGGTAGG AAGTACAAAA AAGTCCGATT TATGGCATAC 1351 ACAGATGAAA CCTTTAAGAC TCGTGAAGCT ATTCAGCATG AATCAGGAAT 1401 CTTGGGACCT TTACTTTATG GGGAAGTTGG AGACACACTG TTGATTATAT 1451 TTAAGAATCA AGCAAGCAGA CCATATAACA TCTACCCTCA CGGAATCACT 1501 GATGTCCGTC CTTTGTATTC AAGGATTTA CCAAAAGGTG TAAAACATTT 1551 GATGATTTT CCAATTCTGC CAGGAAT ATTCAAATAT AAATGGACAG 1601 TGACTGTAGA AGATGGGCCA ACTAAATCAG ATCCTCGGTG CCTGACCGCC 1651 TATTACTCTTA GTTTCGTTAA TATGGAGAGAGATCTAGCTT CAGGACTCAT 1701 TGGCCCTCTC CTCATCTGCT ACAAAGAATC TGTAGATCAA AGAGGAAACC 1751 AGATAATGTC AGACAAGAGG AATGTCATCC TGTTTTCTGT ATTTGATGAG 1801 AACCTACCAGTCACCT AGCAGCTA 1851 AGCTGGAGTG CAGCTTGAGG ATCCAGAGTT CCAAGCCTCC AACATCATGC 1901 ACAGCATCAA TGGCTATGTT TTTGATAGTT TGCAGTTGTC AGTTTGTTTG 1951 CATGAGGTG0 CATACTGGACT CATTGACTGCTAACTT1 TTCTTCTCTG GATATACCTT CAAACACAAA ATGGTCTATG 2051 AAGACACACT CACCCTATTC CCATTCTCAG GAGAAACTGT CTTCATGTCG 2101 ATGGAAAACC CAGGTCTATG GATTCTGGGG TGCCACACTGAAGAG CAGAGCCTTAAC2 TTCTAGTTGT GACAAGAACA 2201 CTGGTGATTA TTACGAGGAC AGTTATGAAG ATATTTCAGC ATACTTGCTG 2251 AGTAAAACA ATGCCATTGA ACCAAGAAGC TTCTCTCAAA ACGGTACCTC 2301 AGACCGAGTGACTGCT CCCCAGCAG 2351 GGTCTGAGAC ACCCGGGACT TCCGAGAGTG CCACCCCTGA GTCCGGACCC 2401 GGGTCCGAGC CCGCCACTTC CGGCTCCGAA ACTCCCGGCA CAAGCGAGAG 2451 CGCTACCCCA GAGTCAGGAC CAGGAGACCT2 CCCGACCCT2 TACACAGTCCCCAGCC GGCAGTCCCA CTAGCACCGA GGAGGGAACC 2551 TCTGAAAGCG CCACACCCGA ATCAGGGCCA GGGTCTGAGC CTGCTACCAG 2601 CGGCAGCGAG ACACCAGGCA CCTCTGAGTC CGCCACACCA GAGTCCGGAC 2651 CCGGATCTCC CGCTGGGAGC CCCACCTCCA CTGAGGAGGG ATCTCCTGCT 2701 GGCTCTCCAA CATCTACTGA GGAAGGAACC TCAACCGAGC CATCCGAGGG 2751 ATCAGCTCCC GGCACCTCAG AGTCGGCAAC CCCGGAGTCT GGACCCGGAA 2801 CTTCCGAAAG TGCCACACCA GAGTCCGGTC CCGGGACTTC AGAATCAGCA 2851 ACACCCGAGT CCGGCCCTGG GTCTGAACCC GCCACAAGTG GTAGTGAGAC 2901 ACCAGGATCA GAACCTGCTA CCTCAGGGTC AGAGACACCC GGATCTCCGG 2951 CAGGCTCACC AACCTCCACT GAGGAGGGCA CCAGCACAGA ACCAAGCGAG 3001 GGCTCCGCAC CCGGAACAAG CACTGAACCC AGTGAGGGTT CAGCACCCGG 3051 CTCTGAGCCG GCCACAAGTG GCAGTGAGAC ACCCGGCACT TCAGAGAGTG 3101 CCACCCCCGA GAGTGCCCA GGCACTAGTA CCGAGCCCTC TGAAGGCAGT 3151 GCGCCAGCCT CGAGCGAAAT AACTCGTACT ACTCTTCAGT CAGATCAAGA 3201 GGAAATTGAC TATGATGATA CCATATCAGT TGAAATGAAG AAGGAAGATT 3251 TTGACATTTA TGATGAGGAT GAAAATCAGA GCCCCCGCAG CTTTCAAAAG 3301 AAAACACGAC ACTATTTTAT TGCTGCAGTG GAGAGGCTCTGGGATTATGG 3351 GATGAGTAGC TCCCCACATG TTCTAAGAAA CAGGGCTCAG AGTGGCAGTG 3401 TCCCTCAGTT CAAGAAAGTT GTTTTCCAGG AATTTACTGA TGGCTCCTTT 3451 ACTCAGCCCT TAGAATCTGTGATG AGAACACCTG 3501 GCCATATATA AGAGATCAAG TTGAAGATAA TATCATGGTA ACTTTCGAA 3551 ATCAGGCCTC TCGTCCCTAT TCCTTCTATT CTAGCCTTAT TTCTTATGAG 3601 GAAGATCAGA GGCAAGGAGC AGAACCTTGAG3 TCAAACCTAG3ACAAA5 ACTTACTTTT GGAAAGTGCA ACATCATATG GCACCCACTA 3701 AAGATGAGTT TGACTGCAAA GCCTGGGCTT ATTTCTCTGA TGTTGACCTG 3751 GAAAAAGATG TGCACTCAGG CCTGATTGGA CCCCCTCTGAACC TCACT80TACCACAC ATGGGAGACA AGTGAGAGTA CAGGAATTTG 3851 CTCTGTTTTT CACCATCTTT GATGACCA AAAGCTGGTA CTTCACTGAA 3901 AATGGAAA GAAACTGCAG GGCTCCCTGC AATTCCAGA TGGCATCCGATTGATT ATCGAATCA51 CACTCA TACATAATGG 4001 ATACACTACC TGGCTTAGTA ATGGCTCAGG ATCAAAGGAT TCGATGGTAT 4051 CTGCTCAGCA TGGGCAGCAA TGAAAACATC CATTCTATTC ATTTCAGTGG 4101 ACATGTGTTC ACTGAAGTACCAA ATCTCTATCC AGGTGTTTTTGAGACAGTGG AAATGTTACC ATCCAAAGCT 4201 GGAATTTGGC GGGTGGAATG CCTTATTGGC GAGCATCTAC ATGCTGGGAT 4251 GAGCACACTT TTTCTGGTGT ACAGCAATAA GTGTCAGACT CCCCTGGTGT ACAGCAATAA GTGTCAGACT CCCCTGGGAA 43001GACTTCAGAG TGACTTCAGA TTACAGCTTC AGGACAATAT 4351 GGACAGTGGG CCCCAAAGCT GGCCAGACTT CATTATTCCG GATCAATCAA 4401 TGCCTGGAGC ACCAAGGAGC CCTTTTCTTG GATCAAGGTG GATCTGTTGG 4451 CACCAATGAT TATTCAGTGGCCG GCCAAGCCG TCAGAAGTTC 4501 TCCAGCCTCT ACATCTCTCA GTTTATCATC ATGTATAGTC TTGATGGGAA 4551 GAAGTGGCAG ACTTATCGAG GAAATTCCAC TGGAACCTTA ATGGTCTTCT 4601 TTGGCAATGT GGATTCATACCT GCACCATAGATTCATTTA 4651 ATTATTGCTC GATACATCCG TTTGCACCCA ACTCATTATA GCATTCGCAG 4701 CACTCTTCGC ATGGAGTTGA TGGGCTGTGA TTTAAATAGT TGCAGCATGC 4751 CATTGGGAAT GGAGAGTAAA GCAATATCAG ATGCACAGAT TAACCTTCTATC0101 CCAATATGTT TGCCACCTGG TCTCCTTCAA AAGCTCGACT 4851 TCACCTCCAA GGGAGGAGTA ATGCCTGGAG ACCTCAGGTG AATAATCCAA 4901 AAGAGTGGCT GCAAGTGGAC TTCCAGAAGA CAATGAAAGT CACAGGAGTA ACTCAAGTCAA ACTCAAGTCGACT4951 TCTGCTTACC AGCATGTATG TGAAGGAGTT 5001CCTCATCTCC AGCAGTCAAG ATGGCCATCA GTGGACTCTC TTTTTTCAGA 5051 ATGGCAAAGT AAAGGTTTTT CAGGGAAATC AAGACTCCTT CACACCTGTG 5101 GTGAACTCTC TAGACCCACC GTTACTGACT CGCTACCTTC GAATTCACCC 5151 CCAGAGTTGG GTGCACCAGA TTGCCCTGAG GATGGAGGTT CTGGGCTGCG 5201 AGGCACAGGA CCTCTACGAC AAAACTCACA CATGCCCACC GTGCCCAGCT 5251 CCAGAACTCC TGGGCGGACC GTCAGTCTTC CTCTTCCCCC CAAAACCCAA 5301 GGACACCCTC ATGATCTCCC GGACCCCTGA GGTCACATGC GTGGTGGTGG 5351 ACGTGAGCCA CGAAGACCCT GAGGTCAAGT TCAACTGGTA TGTGGACGGC 5401 GTGGAAGTGC ATAATGCCAA GACAAAGCCG CGGGAGGAGC AGTACAACAG 5451 CACGTACCGT GTGGTCAGCG TCCTCACCGT CCTGCACCAA GACTGGCTGA 5501 ATGGCAAGGA GTACAAGTGC AAGGTCTCCA ACAAAGCCCT CCCAGCCCCC 5551 ATCGAGAAAA CCATCTCCAA AGCCAAAGGG CAGCCCCGAG AACCACAGGT 5601 GTACACCCTG CCCCCATCCC GGGATGAGCT GACCAAGAAC CAAGTTAGCC 5651 TGACCTGCCT GGTCAAAGGC TTCTATCCCA GCGACATCGC CGTGGAGTGG 5701 GAGAGCAATG GGCAGCCGGA GAACAACTAC AAGACCACGC CTCCCGTGTT 5751 GGACTCCGAC GGCTCCTTCT TCCTCTACTC CAAGCTCACC GTGGACAAGA 5801 GCAGGTGGCA GCAGGGGAAC GTCTTCTCATGCTCCGTGAT GCATGAGGCT 5851 CTGCACAACC ACTACACGCA GAAGAGCCTC TCCCTGTCTC CGGGTTGA Amino acid sequence of protein VWF059A - contains signal peptide and D1D2 region: SEQ ID NO: 5 1 MIPARFAGVL LALALILPGT LCAEGTRGRS STARCSLFGS DFVNTFDGSM 51 YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG 101 TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI DGSGNFQVLL 151 SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS WALSSGEQWC 201 ERASPPSSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL VDPEPFVALC 251 EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME 301 YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC 351 VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD 401 NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC TRSVTVRLPG 451 LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV RLSYGEDLQM 501 DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG LAEPRVEDFG 551 NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS 601 PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL 651 NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP ​​PGLYMDERGD 701 CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM SGVPGSLLPD 751 AVLSSPLSHR SKRSLSCRPP MVKLVCPADN LRAEGLECTK TCQNYDLECM 801 SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE TVKIGCNTCV851 CRDRKWNCTD HVCDATCSTI GMAHYLTFDG LKYLFPGECQ YVLVQDYCGS 901 NPGTFRILVG NKGCSHPSVK CKKRVTILVE GGEIELFDGE VNVKRPMKDE 951 THFEVVESGR YIILLLGKAL SVVWDRHLSI SVVLKQTYQE KVCGLCGNFD 1001 GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQ CADTRKVPLD SSPATCHNNI 1051 MKQTMVDSSC RILTSDVFQD CNKLVDPEPY LDVCIYDTCS CESIGDCAAF 1101 CDTIAAYAHV CAQHGKVVTW RTATLCPQSC EERNLRENGY EAEWRYNSCA 1151 PACQVTCQHP EPLACPVQCV EGCHAHCPPG KILDELLQTC VDPEDCPVCE 1201 VAGRRFASGK KVTLNPSDPE HCQICHCDVV NLTCEACQEP GTSESATPES 1251 GPGSEPATSG SETPGTSESA TPESGPGSEP ATSGSETPGT SESATPESGP 1301 GTSTEPSEGS APGSPAGSPT STEEGTSESA TPESGPGSEP ATSGSETPGT 1351 SESATPESGP GSPAGSPTST EEGSPAGSPT STEEGASSDK NTGDYYEDSY 1401 EDISAYLLSK NNAIEPRSFS DKTHTCPPCP APELLGGPSV FLFPPKPKDT 1451 LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY 1501 RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT 1551 LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS 1601 DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG* Nucleotide sequence encoding protein VWF059A: SEQ ID NO: 6 1 ATGATTCCTG CCAGATTTGC CGGGGTGCTG CTTGCTCTGG CCCTCATTTT 51 GCCAGGGACC CTTTGTGCAG AAGGAACTCG CGGCAGGTCA TCCACGGCCC 101 GATGCAGCCT TTTCGGAAGT GACTTCGTCA ACACCTTTGA TGGGAGCATG 151 TACAGCTTTG CGGGATACTG CAGTTACCTC CTGGCAGGGG GCTGCCAGAA 201 ACGCTCCTCTTC TCGATTATTG GGGACTTCCA GAATGGCAAG AGAGTGAGCC 251 TCTCCGTGTA TCTTGGGGAA TTTTTTGACA TCCATTTGTT TGTCAATGGT 301 ACCGTGACAC AGGGGGACCA AAGAGTCTCC ATGCCCTATG CCTCCAAAGG 351 GCTGTATCTA GAAACTGAGG CTGGGTACTA CAAGCTGTCC GGTGAGGCCT 401 ATGGCTTTGT GGCCAGGATC GATGGCAGCG GCAACTTTCA AGTCCTGCTG 451 TCAGACACAGAT ACTTCAACAA GACCTGCGGG CTGTGTGGCA ACTTTAACAT 501 CTTTGCTGAA GATGACTTTA TGACCCAAGA AGGGACCTTG ACCTCGGACC 551 CTTATGACTT TGCCAACTCA TGGGCTCTGA GCAGTGGAGA ACAGTGGTGT 601 GAACGGGCAT CTCCTCCCAG CAGCTCATGC AACATCTCCT CTGGGGAAAT 651 GCAGAAGGGC CTTGGGGAGC AGTGCCAGCT TCTGAAGGC ACCTCGGTGT 701 TTGCCCGCTG CCACCCTCTG GTGGACCCCG AGCCTTTTGT GGCCCTGTGT 751 GAGAAGACTT TGTGTGAGTG TGCTGGGGGG CTGGAGTGCG CCTGCCCTGC 801 CCTCCTGGAG TACGCCCGGA CCTGTGCCCA GGAGGGAATG GTGCTGTACG851 GCTGGACCGA CCACAGCGCG TGCAGCCCAG TGTGCCCTGC TGGTATGGAG 901 TATAGGCAGT GTGTGTCCCC TTGCGCCAGG ACCTGCCAGA GCCTGCACAT 951 CAATGAAATG TGTCAGGAGC GATGCGTGGA TGGCTGCAGC TGCCCTGAGG 1001 GACAGCTCCT GGATGAAGGC CTCTGCGTGG AGAGCACCGA GTGTCCCTGC 1051 GTGCATTCCG GAAAGCGCTA CCCTCCCGGC ACCTCCCTCT CTCGAGACTG 1101 CAACACCTGC ATTTGCCGAA ACAGCCAGTG GATCTGCAGC AATGAAGAAT 1151 GTCCAGGGGA GTGCCTTGTC ACTGGTCAAT CCCACTTCAA GAGCTTTGAC 1201 AACAGATACT TCACCTTCAG TGGGATCTGC CAGTACCTGC TGGCCCGGGA 1251 TTGCCAGGAC CACTCCTTCT CCATTGTCAT TGAGACTGTC CAGTGTGCTG 1301 ATGACCGCGA CGCTGTGTGC ACCCGCTCCG TCACCGTCCG GCTGCCTGGC 1351 CTGCACAACA GCCTTGTGAA ACTGAAGCAT GGGGCAGGAG TTGCCATGGA 1401 TGGCCAGGAC ATCCAGCTCC CCCTCCTGAA AGGTGACCTC CGCATCCAGC 1451 ATACAGTGAC GGCCTCCGTG CGCCTCAGCT ACGGGGAGGA CCTGCAGATG 1501 GACTGGGATG GCCGCGGGAG GCTGCTGGTG AAGCTGTCCC CCGTCTATGC 1551 CGGGAAGACC TGCGGCCTGT GTGGGAATTA CAATGGCAAC CAGGGCGACG 1601 ACTTCCTTAC CCCCTCTGGG CTGGCGGAGC CCCGGGTGGA GGACTTCGGG 1651 AACGCCTGGA AGCTGCACGG GGACTGCCAGGACCTGCAGA AGCAGCACAG 1701 CGATCCCTGC GCCCTCAACC CGCGCATGAC CAGGTTCTCC GAGGAGGCGT 1751 GCGCGGTCCT GACGTCCCCC ACATTCGAGG CCTGCCATCG TGCCGTCAGC 1801 CCGCTGCCCT ACCTGCGGAA CTGCCGCTAC GACGTGTGCT CCTGCTCGGA 1851 CGGCCGCGAG TGCCTGTGCG GCGCCCTGGC CAGCTATGCC GCGGCCTGCG 1901 CGGGGAGAGG CGTGCGCGTC GCGTGGCGCG AGCCAGGCCG CTGTGAGCTG 1951 AACTGCCCGA AAGGCCAGGT GTACCTGCAG TGCGGGACCC CCTGCAACCT 2001 GACCTGCCGC TCTCTCTCTT ACCCGGATGA GGAATGCAAT GAGGCCTGCC 2051 TGGAGGGCTG CTTCTGCCCC CCAGGGCTCT ACATGGATGA GAGGGGGGAC 2101 TGCGTGCCCA AGGCCCAGTG CCCCTGTTAC TATGACGGTG AGATCTTCCA 2151 GCCAGAAGAC ATCTTCTCAG ACCATCACAC CATGTGCTAC TGTGAGGATG 2201 GCTTCATGCA CTGTACCATG AGTGGAGTCC CCGGAAGCTT GCTGCCTGAC 2251 GCTGTCCTCA GCAGTCCCCT GTCTCATCGC AGCAAAAGGA GCCTATCCTG 2301 TCGGCCCCCC ATGGTCAAGC TGGTGTGTCC CGCTGACAAC CTGCGGGCTG 2351 AAGGGCTCGA GTGTACCAAA ACGTGCCAGA ACTATGACCT GGAGTGCATG 2401 AGCATGGGCT GTGTCTCTGG CTGCCTCTGC CCCCCGGGCA TGGTCCGGCA 2451 TGAGAACAGA TGTGTGGCCC TGGAAAGGTG TCCCTGCTTC CATCAGGGCA 2501 AGGAGTATGCCCCTGGAGAA ACAGTGAAGA TTGGCTGCAA CACTTGTGTC 2551 TGTCGGGACC GGAAGTGGAA CTGCACAGAC CATGTGTGTG ATGCCACGTG 2601 CTCCACGATC GGCATGGCCC ACTACCTCAC CTTCGACGGG CTCAAATACC 2651 TGTTCCCCGG GGAGTGCCAG TACGTTCTGG TGCAGGATTA CTGCGGCAGT 2701 AACCCTGGGA CCTTTCGGAT CCTAGTGGGG AATAAGGGAT GCAGCCACCC 2751 CTCAGTGAAA TGCAAGAAAC GGGTCACCAT CCTGGTGGAG GGAGGAGAGA 2801 TTGAGCTGTT TGACGGGGAG GTGAATGTGA AGAGGCCCAT GAAGGATGAG 2851 ACTCACTTTG AGGTGGTGGA GTCTGGCCGG TACATCATTC TGCTGCTGGG 2901 CAAAGCCCTC TCCGTGGTCT GGGACCGCCA CCTGAGCATC TCCGTGGTCC 2951 TGAAGCAGAC ATACCAGGAG AAAGTGTGTG GCCTGTGTGG GAATTTTGAT 3001 GGCATCCAGA ACAATGACCT CACCAGCAGC AACCTCCAAG TGGAGGAAGA 3051 CCCTGTGGAC TTTGGGAACT CCTGGAAAGT GAGCTCGCAG TGTGCTGACA 3101 CCAGAAAAGT GCCTCTGGAC TCATCCCCTG CCACCTGCCA TAACAACATC 3151 ATGAAGCAGA CGATGGTGGA TTCCTCCTGT AGAATCCTTA CCAGTGACGT 3201 CTTCCAGGAC TGCAACAAGC TGGTGGACCC CGAGCCATAT CTGGATGTCT 3251 GCATTTACGA CACCTGCTCC TGTGAGTCCA TTGGGGACTG CGCCGCATTC 3301 TGCGACACCA TTGCTGCCTA TGCCCACGTG TGTGCCCAGCATGGCAAGGT 3351 GGTGACCTGG AGGACGGCCA CATTGTGCCC CCAGAGCTGC GAGGAGAGGA 3401 ATCTCCGGGA GAACGGGTAT GAGGCTGAGT GGCGCTATAA CAGCTGTGCA 3451 CCTGCCTGTC AAGTCACGTG TCAGCACCCT GAGCCACTGG CCTGCCCTGT 3501 GCAGTGTGTG GAGGGCTGCC ATGCCCACTG CCCTCCAGGG AAAATCCTGG 3551 ATGAGCTTTT GCAGACCTGC GTTGACCCTG AAGACTGTCC AGTGTGTGAG 3601 GTGGCTGGCC GGCGTTTTGC CTCAGGAAAG AAAGTCACCT TGAATCCCAG 3651 TGACCCTGAG CACTGCCAGA TTTGCCACTG TGATGTTGTC AACCTCACCT 3701 GTGAAGCCTG CCAGGAGCCG GGTACATCAG AGAGCGCCAC CCCTGAAAGT 3751 GGTCCCGGGA GCGAGCCAGC CACATCTGGG TCGGAAACGC CAGGCACATC 3801 CGAGTCTGCA ACTCCCGAGT CCGGACCTGG CTCCGAGCCT GCCACTAGCG 3851 GCTCCGAGAC TCCGGGAACT TCCGAGAGCG CTACACCAGA AAGCGGACCC 3901 GGAACCAGTA CCGAACCTAG CGAGGGCTCT GCTCCGGGCA GCCCAGCCGG 3951 CTCTCCTACA TCCACGGAGG AGGGCACTTC CGAATCCGCC ACCCCGGAGT 4001 CAGGGCCAGG ATCTGAACCC GCTACCTCAG GCAGTGAGAC GCCAGGAACG 4051 AGCGAGTCCG CTACACCGGA GAGTGGGCCA GGGAGCCCTG CTGGATCTCC 4101 TACGTCCACT GAGGAAGGGT CACCAGCGGG CTCGCCCACC AGCACTGAAG 4151 AAGGTGCCTC GTCTGACAAGAACACTGGTG ATTATTACGA GGACAGTTAT 4201 GAAGATATTT CAGCATACTT GCTGAGTAAA AACAATGCCA TTGAACCAAG 4251 AAGCTTCTCT GACAAAACTC ACACATGCCC ACCGTGCCCA GCTCCCAGTCG ACCTCAAAGTCG TCCCGCTGCCA CAAGGACACC 4351 CTCATGATCT CCCGGACCCC TGAGGTCACA TGCGTGGTGG TGGACGTGAG 4401 CCACGAAGAC CCTGAGGTCA AGTTCAACTG GTATGTGGAC GGCGTGGAAG 4451 TGCATAAT5CAAGGTACACAAGAG4 CCGGCAT CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC CAAGACTGGC TGAATGGCAA 4551 GGAGTACAAG TGCAAGGTCT CCAACAAAGC CCTCCCAGCC CCCATCGAGA 4601 AAACCATCTC CAAAGCCAAA GGGCAGCCCTCC GAGAATGACCCAGC5 GGTCG6 GCTGACCAAG AACCAGTTA GCCTGACCTG 4701 CCTGGTCAAA GGCTTCTATC CCAGCGACAT CGCCGTGGAG TGGGAGAGCA 4751 ATGGGCAGCC GGAGAACAAC TACAGACCA CGCCTCCCGT GTTGGACTGATTGCTCTCTAG 4701 ACCGTGGACA AGAGCAGGTG 4851 GCAGCAGGGG AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA 4901 ACCACTACAC GCAGAAGAGC CTCTCCCTGT CTCCGGGTTG A --->SEQUENCE LIST <110> Bioverativ Therapeutics, Inc. <120> COMPOSITIONS BASED ON CHIMERIC PROTEINS CONTAINING FACTOR VIII AND OPTIONS FOR APPLICATION <130> 730463: SA9-480PC <150> 63 / 214,245 <151> 2021-06-23 <150> 63 / 214,246 <151> 2021-06-23 <150> 63 / 214,752 <151> 2021-06-24 <150> 63 / 231,909 <151> 2021-08-11 <160> 24 <170> PatentIn version 3.5 <210> 1 <211> 1946 <212> PROTEIN <213> Artificial sequence <220> <223> Synthesized <400> 1Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr1 5 10 15Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro20 25 30Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys35 40 45Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile Ala Lys Pro50 55 60Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val65 70 75 80Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val85 90 95Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala100 105 110Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp AspLys Val115 120 125Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn130 135 140Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser145 150 155 160His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu165 170 175Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu180 185 190His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp195 200 205His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser210 215 220Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg225 230 235 240Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His245 250 255Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu260 265 270Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile275 280 285Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly290 295 300Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly Met305 310 315 320Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg325 330 335Met LysAsn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp340 345 350Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe355 360 365Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His370 375 380Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu385 390 395 400Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro405 410 415Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr420 425 430Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile435 440 445Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile450 455 460Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile465 470 475 480Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys485 490 495His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys500 505 510Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys515 520 525Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala530 535 540Ser Gly Leu Ile Gly Pro Leu Leu IleCys Tyr Lys Glu Ser Val Asp545 550 555 560Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe565 570 575Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln580 585 590Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe595 600 605Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser610 615 620Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu625 630 635 640Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr645 650 655Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro660 665 670Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp675 680 685Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala690 695 700Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu705 710 715 720Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala725 730 735Ile Glu Pro Arg Ser Phe Ser Gln Asn Gly Thr Ser Glu Ser Ala Thr740 745 750Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser GluThr755 760 765Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu770 775 780Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr785 790 795 800Pro Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala805 810 815Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser820 825 830Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser835 840 845Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly850 855 860Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Ser Pro865 870 875 880Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Thr Glu Pro Ser885 890 895Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly900 905 910Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser915 920 925Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser930 935 940Gly Ser Glu Thr Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr945 950 955 960Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser965 970 975Thr Glu ProSer Glu Gly Ser Ala Pro Gly Thr Ser Thr Glu Pro Ser980 985 990Glu Gly Ser Ala Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr995 1000 1005Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr1010 1015 1020Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Ala Ser Ser Glu Ile1025 1030 1035Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp1040 1045 1050Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr1055 1060 1065Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr1070 1075 1080Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly1085 1090 1095Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly1100 1105 1110Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp1115 1120 1125Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His1130 1135 1140Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn1145 1150 1155Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe1160 1165 1170Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln GlyAla1175 1180 1185Glu Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr1190 1195 1200Phe Trp Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu Phe1205 1210 1215Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys1220 1225 1230Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr1235 1240 1245Asn Thr Leu Asn Pro Ala His Gly Arg Gln Val Thr Val Gln Glu1250 1255 1260Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr1265 1270 1275Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn Ile1280 1285 1290Gln Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala1295 1300 1305Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met Ala1310 1315 1320Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn1325 1330 1335Glu Asn Ile His Ser Ile His Phe Ser Gly His Val Phe Thr Val1340 1345 1350Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro1355 1360 1365Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile1370 1375 1380Trp Arg Val Glu Cys Leu Ile GlyGlu His Leu His Ala Gly Met1385 1390 1395Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu1400 1405 1410Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser1415 1420 1425Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr1430 1435 1440Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp1445 1450 1455Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys1460 1465 1470Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln1475 1480 1485Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr1490 1495 1500Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val1505 1510 1515Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Ile1520 1525 1530Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser1535 1540 1545Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser1550 1555 1560Met Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile1565 1570 1575Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro1580 1585 1590Ser LysAla Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg1595 1600 1605Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln1610 1615 1620Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys Ser1625 1630 1635Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser1640 1645 1650Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val1655 1660 1665Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn1670 1675 1680Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro1685 1690 1695Gln Ser Trp Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly1700 1705 1710Cys Glu Ala Gln Asp Leu Tyr Asp Lys Thr His Thr Cys Pro Pro1715 1720 1725Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe1730 1735 1740Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu1745 1750 1755Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val1760 1765 1770Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys1775 1780 1785Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg ValVal1790 1795 1800Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu1805 1810 1815Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Pro Ile Glu Pro Gln Val1835 1840 1845Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val1850 1855 1860Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala1865 1870 1870 1750 Glu Glu Gl As Ser Gln Pro Glu Asn Asn Tyr Lys Thr1880 1885 1890Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser1895 1900 1905Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Valley Phe 1915 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln1925 1930 1935Lys Ser Leu Ser Leu Ser Pro Gly1940 1945 <210> 2 <211> 883 <212> PROTEIN <213> Artificial sequence <220> <223> Synthesized <400> 2Ser Leu Ser Cys Arg Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp1 5 10 15Asn Leu Arg Ala Glu Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr20 25 30Asp Leu Glu Cys Met Ser Met Gly Cys Val SerGly Cys Leu Cys Pro35 40 45Pro Gly Met Val Arg His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys50 55 60Pro Cys Phe His Gln Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys65 70 75 80Ile Gly Cys Asn Thr Cys Val Cys Arg Asp Arg Lys Trp Asn Cys Thr85 90 95Asp His Val Cys Asp Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr100 105 110Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr115 120 125Val Leu Val Gln Asp Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile130 135 140Leu Val Gly Asn Lys Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys145 150 155 160Arg Val Thr Ile Leu Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly165 170 175Glu Val Asn Val Lys Arg Pro Met Lys Asp Glu Thr His Phe Glu Val180 185 190Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser195 200 205Val Val Trp Asp Arg His Leu Ser Ile Ser Val Val Leu Lys Gln Thr210 215 220Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln225 230 235 240Asn Asn Asp Leu Thr Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val245 250 255Asp PheGly Asn Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg260 265 270Lys Val Pro Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met275 280 285Lys Gln Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val290 295 300Phe Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val305 310 315 320Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala Ala325 330 335Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln His Gly340 345 350Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln Ser Cys Glu355 360 365Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Ala Glu Trp Arg Tyr Asn370 375 380Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln His Pro Glu Pro Leu385 390 395 400Ala Cys Pro Val Gln Cys Val Glu Gly Cys His Ala His Cys Pro Pr...

Claims

1. A pharmaceutical composition containing: (a) from about 75 IU / mL to about 2000 IU / mL of a chimeric protein containing a first polypeptide chain that comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1, and a second polypeptide chain that comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2; (b) from about 1% (w / v) to about 7.5% (w / v) sucrose; (c) from about 5 mM to about 15 mM histidine; (d) from about 200 mM to about 300 mM arginine; (e) from about 2.5 mM to about 10 mM calcium chloride; and (f) from about 0.008% (w / v) to about 0.1% (w / v) polysorbate 80, wherein the pharmaceutical composition is characterized by a pH of from about 6.5 to about 7.5, and where "approximately" means ± 10%.

2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is characterized by a pH of approximately 7.

0.

3. The pharmaceutical composition of claim 1 or 2, wherein the pharmaceutical composition is characterized by a pH of approximately 6.

8.

4. The pharmaceutical composition according to any one of claims 1-3, wherein the histidine is L-histidine and wherein the arginine is L-arginine.

5. The pharmaceutical composition according to claim 4, which contains L-arginine HCl.

6. A pharmaceutical composition according to any one of claims 1-5, which contains calcium chloride dihydrate.

7. A pharmaceutical composition according to any one of claims 1-6, wherein the first polypeptide chain comprises an amino acid sequence presented under SEQ ID NO: 1, and the second polypeptide chain comprises an amino acid sequence presented under SEQ ID NO: 2, wherein the first polypeptide chain and the second polypeptide chain are covalently linked by two disulfide bonds between the Fc domains in the first and second polypeptide chains.

8. A pharmaceutical composition according to any one of claims 1-7, wherein the chimeric protein is ephanesoctocog-alpha.

9. A pharmaceutical composition according to any one of claims 1-8, which contains approximately 250 IU, 500 IU, 1000 IU, 2000 IU, 3000 IU or 4000 IU of the chimeric protein.

10. A pharmaceutical composition according to any one of paragraphs 1-9, wherein the pharmaceutical composition comprises: (a) approximately 5% (w / v) sucrose; (b) approximately 10 mM L-histidine; (c) approximately 250 mM L-arginine HCl; (d) approximately 5 mM calcium chloride; and (e) approximately 0.05% (w / v) polysorbate 80.

11. Pharmaceutical kit containing: (i) a first container containing a lyophilized pharmaceutical composition comprising: (a) a chimeric protein containing a first polypeptide chain that comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1, and a second polypeptide chain that comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2, a constant region or a portion thereof; (b) from about 30 mg to about 200 mg sucrose; (c) from about 2.5 mg to about 7.5 mg of histidine; (d) from about 140 mg to about 200 mg of arginine; (e) from about 1.5 mg to about 5 mg of calcium chloride; and (f) from about 1 mg to about 5 mg of polysorbate 80; and (ii) a second container containing sterile water, where "approximately" means ± 10%.

12. The pharmaceutical kit according to claim 11, wherein the histidine is L-histidine.

13. The pharmaceutical kit according to claim 11 or 12, wherein the arginine is L-arginine.

14. A pharmaceutical kit according to any one of claims 11-13, which contains L-arginine HCl.

15. A pharmaceutical kit according to any one of claims 11-14, wherein the lyophilized pharmaceutical composition is characterized by a moisture content of less than 1.6%.

16. A pharmaceutical kit according to any one of claims 11-15, wherein the first container contains 250 IU, 500 IU, 1000 IU, 2000 IU, 3000 IU or 4000 IU of the chimeric protein.

17. A pharmaceutical kit according to any one of claims 11-16, wherein the chimeric protein is ephanesoctocog-alpha.

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