Factor VIII polypeptide preparation

Abstract

To provide a Factor VIII polypeptide preparation. [Solution] The present invention provides formulations of factor VIII polypeptides, such as FVIII-Fc, and methods of using the same. The FVIII polypeptide can be a recombinant FVIII protein, a short-acting FVIII protein, or a long-acting FVIII protein. Pharmaceutical formulations containing the FVIII polypeptide can be used for individual prophylaxis, weekly prophylaxis, episodic (on-demand) treatment, or perioperative management of hemophilia. The pharmaceutical composition comprises: (a) a FVIII polypeptide; (b) one or more stabilizers selected from the group consisting of sucrose, trehalose, raffinose, arginine, and mixtures thereof; (c) sodium chloride (NaCl); (d) L-histidine; (e) calcium chloride; and (f) polysorbate 20 or polysorbate 80.

Description

Technical Field

[0001] Background of the Invention The present invention generally relates to the field of treating hemostatic disorders.

Background Art

[0002] Hemophilia A is an X-linked bleeding disorder caused by mutations and / or deletions in the factor VIII (FVIII) gene that result in a deficiency of FVIII activity (Peyvandi, F. et al. Haemophilia 12:82-89 (2006)). This disease is characterized by spontaneous bleeding and excessive bleeding after trauma. Over a long period, repeated bleeding into muscles and joints (often starting in childhood) leads to hemophilic arthropathy and irreversible joint damage. This damage is progressive and can cause very limited joint mobility, muscle atrophy, and chronic pain (Rodriguez-Merchan, E.C., Semin. Thromb. Hemost. 29:87-96 (2003), which is incorporated herein by reference in its entirety).

[0003] The A2 domain is required for the procoagulant activity of the factor VIII molecule. Studies have shown that porcine factor VIII has a procoagulant activity six times higher than human factor VIII (Lollar, P., and E.T. Parker, J. Biol. Chem. 266:12481-12486 (1991)), and that the difference in aggregation activity between human and porcine factor VIII appears to be based on differences in the amino acid sequence between one or more residues in the human A2 domain and the porcine A2 domain (Lollar, P., et al., J. Biol. Chem. 267:23652-23657 (1992)), which are incorporated herein by reference in their entirety.

[0004] The treatment of hemophilia A is by replacement therapy aimed at restoring FVIII activity to 1-5% of normal levels to prevent spontaneous bleeding (Mannucci, P.M., et al., N. Engl. J. Med. 344: 1773-1779 (2001), which is incorporated herein by reference in its entirety). There are plasma-derived recombinant FVIII preparations available for treating bleeding symptoms on demand or for preventing bleeding symptoms that occur by prophylactic treatment. Reduction in mortality, prevention of joint damage, and improvement in quality of life are important outcomes resulting from the development of plasma-derived recombinant FVIII. Long-term protection from bleeding represents another important advance in the treatment of patients with hemophilia A.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Summary of the Invention

Means for Solving the Problems

[0006] The present invention includes a pharmaceutical composition comprising: (a) an FVIII polypeptide; (b) one or more stabilizers selected from sucrose, trehalose, raffinose, arginine, and mixtures thereof; (c) sodium chloride (NaCl); (d) L-histidine; (e) calcium chloride; and (f) polysorbate 20 or polysorbate 80. In one embodiment, mannitol, glycine, alanine, or hydroxyethyl starch is not included in the pharmaceutical composition. In another embodiment, NaCl is the only filler. In other embodiments, the FVIII polypeptide is a long-acting polypeptide or a short-acting polypeptide.

[0007] In other embodiments, the pharmaceutical composition comprises from about 1% (w / v) to about 2.5% (w / v) sucrose, from about 1.3% (w / v) sucrose to about 2.0% (w / v) sucrose, about 1.33% (w / v) sucrose, or about 2.0% (w / v) sucrose, from about 10 mg / ml to about 25 mg / ml sucrose, from about 13 mg / ml to about 20 mg / ml sucrose, about 13.3 mg / ml sucrose, or about 20.0 mg / ml sucrose. Still in other embodiments, the pharmaceutical composition comprises from about 150 mM to about 250 mM NaCl, from about 175 mM to about 225 mM NaCl, from about 200 mM to about 210 mM NaCl, about 205 mM NaCl, from about 8.8 mg / ml to about 14.6 mg / ml NaCl, from about 10 mg / ml to about 13 mg / ml NaCl, about 12.0 mg / ml NaCl, from about 250 mM to about 350 mM NaCl, from about 275 mM to about 325 mM NaCl, about 308 mM NaCl, from about 14.6 mg / ml to about 20.5 mg / ml NaCl, from about 16 mg / ml to about 19 mg / ml NaCl, or about 18.0 mg / ml NaCl.

[0008] In still other embodiments, the pharmaceutical composition comprises L-histidine at about 5 mM to about 15 mM, L-histidine at about 6.64 mM to about 9.8 mM, L-histidine at about 0.75 mg / ml to about 2.25 mg / ml, or L-histidine at about 1.03 mg / ml to about 1.55 mg / ml. In certain embodiments, the pharmaceutical composition comprises calcium chloride at about 5 mM to about 10 mM, calcium chloride at about 5.4 mM to about 8 mM, calcium chloride dihydrate at about 0.75 mg / ml to about 1.5 mg / ml, or calcium chloride dihydrate at about 0.8 mg / ml to about 1.18 mg / ml.

[0009] In some embodiments, the pharmaceutical composition comprises polysorbate 20 or polysorbate 80 at about 0.008% (w / v) to about 0.025% (w / v), polysorbate 20 or polysorbate 80 at about 0.013% (w / v), or polysorbate 20 or polysorbate 80 at about 0.02% (w / v), polysorbate 20 or polysorbate 80 at about 0.08 mg / ml to about 0.25 mg / ml, polysorbate 20 or polysorbate 80 at about 0.13% mg / ml, or polysorbate 20 or polysorbate 80 at about 0.20 mg / ml.

[0010] In other embodiments, the pharmaceutical composition comprises an rFVIIIFc polypeptide and includes a first subunit comprising an amino acid sequence that is at least 90% or 95% identical to amino acids 20 to 1684 of SEQ ID NO: 2 or amino acids 20 to 2578 of SEQ ID NO: 6, and a second subunit comprising an amino acid sequence that is at least 90% to 95% identical to amino acids 21 to 247 of SEQ ID NO: 4. In certain embodiments, the rFVIIIFc polypeptide includes a first subunit comprising amino acids 20 to 1684 of SEQ ID NO: 2 or amino acids 20 to 2578 of SEQ ID NO: 6, and a second subunit comprising amino acids 21 to 247 of SEQ ID NO: 4.

[0011] In certain embodiments, the FVIII polypeptide is present in the pharmaceutical composition at a concentration of about 50 IU / ml to about 2500 IU / ml of the FVIII polypeptide, for example, 83 IU / ml, 167 IU / ml, 250 IU / ml, 333 IU / ml, 500 IU / ml, 667 IU / ml, 1000 IU / ml, 1333 IU / ml, 1667 IU / ml, or 2000 IU / ml. In some embodiments, the FVIII polypeptide is present in the pharmaceutical composition at a concentration of about 100 IU / ml to about 4000 IU / ml of the FVIII polypeptide, for example, 150 IU / ml, 287.5 IU / ml, 431.25 IU / ml, 575 IU / ml, 862.5 IU / ml, 1150 IU / ml, 1725 IU / ml, 2300 IU / ml, 2875 IU / ml, or 3450 IU / ml.

[0012] In other embodiments, the pharmaceutical composition comprises: (a) a long-acting FVIII polypeptide of about 50 IU / ml to about 2500 IU / ml; (b) about 1% (w / v) to about 2.5% (w / v) sucrose; (c) about 150 mM to about 250 mM NaCl; (d) about 5 mM to about 15 mM L-histidine; (e) about 5 mM to about 10 mM calcium chloride; and (f) about 0.008% (w / v) to about 0.025% polysorbate 20 or polysorbate 80. In other embodiments, the pharmaceutical composition comprises about 175 mM to about 225 mM NaCl or about 200 mM to about 210 mM NaCl.

[0013] In yet other embodiments, the pharmaceutical composition comprises: (a) an FVIII polypeptide of about 83 IU / ml, about 167 IU / ml, about 250 IU / ml, about 333 IU / ml, about 500 IU / ml, about 667 IU / ml, about 1000 IU / ml, about 1333 IU / ml, about 1667 IU / ml, or about 2000 IU / ml; (b) about 1.33% (w / v) sucrose; (c) about 205 mM NaCl; (d) about 6.64 mM L-histidine; (e) about 5.4 mM calcium chloride; and (f) about 0.013% (w / v) polysorbate 20 or polysorbate 80.

[0014] In some embodiments, the pharmaceutical composition comprises: (a) an FVIII polypeptide at about 100 IU / ml to about 4000 IU / ml, (b) sucrose at about 1% (w / v) to about 2.5% (w / v), (c) NaCl at about 250 mM to about 350 mM, (d) L-histidine at about 5 mM to about 15 mM, (e) calcium chloride at about 5 mM to about 10 mM, and (f) polysorbate 20 or polysorbate 80 at about 0.008% (w / v) to about 0.025%. In other embodiments, the pharmaceutical composition comprises NaCl at about 275 mM to about 325 mM.

[0015] In certain embodiments, the pharmaceutical composition comprises: (a) an FVIII polypeptide at about 150 IU / ml, about 287.5 IU / ml, about 431.25 IU / ml, about 575 IU / ml, about 862.5 IU / ml, about 1150 IU / ml, about 1725 IU / ml, about 2300 IU / ml, about 2875 IU / ml, or about 3450 IU / ml; (b) about 2.0% (w / v) sucrose; (c) about 308 mM NaCl; (d) about 9.8 mM L-histidine; (e) about 8 mM calcium chloride; and (f) about 0.020% (w / v) polysorbate 20 or polysorbate 80. In other embodiments, the pharmaceutical composition comprises: (a) an FVIII polypeptide at about 50 IU / ml to about 2500 IU / ml; (b) about 10 mg / ml to about 25 mg / ml sucrose; (c) about 8.8 mg / ml to about 14.6 mg / ml NaCl; (d) about 0.75 mg / ml to about 2.25 mg / ml L-histidine; (e) about 0.75 mg / ml to about 1.5 mg / ml calcium chloride dihydrate; and (f) about 0.08 mg / ml to about 0.25 mg / ml polysorbate 20 or polysorbate 80. In some embodiments, the pharmaceutical composition comprises: (a) an FVIII polypeptide at about 83 IU / ml, about 167 IU / ml, about 250 IU / ml, about 333 IU / ml, about 500 IU / ml, about 667 IU / ml, about 1000 IU / ml, about 1333 IU / ml, about 1667 IU / ml, or about 2000 IU / ml; (b) about 13.3 mg / ml sucrose; (c) about 12.0 mg / ml NaCl; (d) about 1.03 mg / ml L-histidine; (e) about 0.8 mg / ml calcium chloride dihydrate; and (f) about 0.13 mg / ml polysorbate 20 or polysorbate 80.

[0016] In other embodiments, the pharmaceutical composition comprises: (a) about 100 IU / ml to about 4000 IU / ml of FVIII polypeptide; (b) about 10 mg / ml to about 25 mg / ml of sucrose; (c) about 14.6 mg / ml to about 20.5 mg / ml of NaCl; (d) about 0.75 mg / ml to about 2.25 mg / ml of L-histidine; (e) about 0.75 mg / ml to about 1.5 mg / ml of calcium chloride dihydrate; and (f) about 0.08 mg / ml to about 0.25 mg / ml of polysorbate 20 or polysorbate 80. In certain embodiments, the pharmaceutical composition comprises: (a) about 150 IU / ml, about 287.5 IU / ml, about 431.25 IU / ml, about 575 IU / ml, about 862.5 IU / ml, about 1150 IU / ml, about 1725 IU / ml, about 2300 IU / ml, about 2875 IU / ml, or about 3450 IU / ml of FVIII polypeptide; (b) about 20.0 mg / ml of sucrose; (c) about 18.0 mg / ml of NaCl; (d) about 1.55 mg / ml of L-histidine; (e) about 1.18 mg / ml of calcium chloride dihydrate; and (f) about 0.20 mg / ml of polysorbate 20 or polysorbate 80.

[0017] In some embodiments, the present invention includes a pharmaceutical kit comprising: (a) a first container containing a lyophilized powder, the powder comprising: (i) an FVIII polypeptide; (ii) one or more stabilizers selected from sucrose, trehalose, raffinose, arginine, and mixtures thereof; (iii) sodium chloride (NaCl); (iv) L-histidine; (v) calcium chloride; (vi) polysorbate 20 or polysorbate 80; and (b) a second container containing sterile water for injection to be combined with the lyophilized powder of the first container. In other embodiments, the pharmaceutical kit comprises: (a) a first container containing a lyophilized powder, the powder comprising: (i) an FVIII polypeptide of about 250 IU, about 500 IU, about 750 IU, about 1000 IU, about 1500 IU, about 2000 IU, about 3000 IU, about 4000 IU, about 5000 IU, or about 6000 IU; (ii) about 40 mg of sucrose; (iii) about 36 mg of sodium chloride; (iv) about 3.1 mg of L-histidine; (v) about 2.40 mg of calcium chloride dihydrate; (v) about 0.40 mg of polysorbate 20 or polysorbate 80; and (b) a second container containing sterile water for injection, the second container containing a volume of sterile water for injection sufficient to produce a solution comprising: (i) an FVIII polypeptide of about 83 IU / ml, about 167 IU / ml, about 250 IU / ml, about 333 IU / ml, about 500 IU / ml, about 667 IU / ml, about 1000 IU / ml, about 1333 IU / ml, about 1667 IU / ml, or about 2000 IU / ml; (ii) about 1.33% (w / v) of sucrose; (iii) about 205 mM of NaCl; (iv) about 6.64 mM of L-histidine; (v) about 5.4 mM of calcium chloride; (vi) about 0.013% (w / v) of polysorbate 20 or polysorbate 80 when combined with the lyophilized powder of the first container.

[0018] In other embodiments, the pharmaceutical kit comprises: (a) a first container containing a lyophilized powder, the powder comprising: (i) an FVIII polypeptide of about 250 IU, about 500 IU, about 750 IU, about 1000 IU, about 1500 IU, about 2000 IU, about 3000 IU, about 4000 IU, about 5000 IU, or about 6000 IU; (ii) about 40 mg of sucrose; (iii) about 36 mg of sodium chloride; (iv) about 3.1 mg of L-histidine; (v) about 2.40 mg of calcium chloride dihydrate; (v) about 0.40 mg of polysorbate 20 or polysorbate 80; and (b) a second container containing sterile water for injection, the second container containing sterile water for injection in a volume sufficient to produce a solution that, when combined with the lyophilized powder of the first container, comprises: (i) an FVIII polypeptide of about 83 IU / ml, about 167 IU / ml, about 250 IU / ml, about 333 IU / ml, about 500 IU / ml, about 667 IU / ml, about 1000 IU / ml, about 1333 IU / ml, about 1667 IU / ml, or about 2000 IU / ml; (ii) about 13.3 mg / ml of sucrose; (iii) about 12.0 mg / ml of NaCl; (iv) about 1.03 mg / ml of L-histidine; (v) about 0.80 mg / ml of calcium chloride; (vi) about 0.13 mg / ml of polysorbate 20 or polysorbate 80.

[0019] The invention also includes methods for reducing or decreasing bleeding symptoms, or treating or preventing bleeding disorders, comprising administering a pharmaceutical composition comprising an FVIII polypeptide. In certain embodiments, for example, the following are provided: (Item 1) A pharmaceutical composition comprising: (a) an FVIII polypeptide; (b) one or more stabilizers selected from the group consisting of sucrose, trehalose, raffinose, arginine, and mixtures thereof; (c) sodium chloride (NaCl); (d) L-histidine; (e) calcium chloride; (f) The pharmaceutical composition comprising polysorbate 20 or polysorbate 80. (Item 2) The pharmaceutical composition according to Item 1, which does not contain mannitol, glycine, alanine, or hydroxyethyl starch. (Item 3) The pharmaceutical composition according to Item 1 or 2, wherein NaCl is the only filler. (Item 4) The pharmaceutical composition according to any one of Items 1 to 3, which contains sucrose. (Item 5) The pharmaceutical composition according to Item 4, which contains sucrose at about 1% (w / v) to about 2.5% (w / v). (Item 6) The pharmaceutical composition according to Item 5, which contains sucrose at about 1.3% (w / v) to about 2.0% (w / v). (Item 7) The pharmaceutical composition according to Item 6, which contains sucrose at about 1.33% (w / v) to about 2.0% (w / v). (Item 8) The pharmaceutical composition according to Item 4, which contains sucrose at about 10 mg / ml to about 25 mg / ml. (Item 9) The pharmaceutical composition according to Item 8, which contains sucrose at about 13 mg / ml to about 20 mg / ml. (Item 10) The pharmaceutical composition according to Item 9, which contains sucrose at about 13.3 mg / ml or about 20.0 mg / ml. (Item 11) The pharmaceutical composition according to any one of Items 1 to 10, which contains NaCl at about 150 mM to about 250 mM. (Item 12) The pharmaceutical composition according to Item 11, which contains NaCl at about 175 mM to about 225 mM. (Item 13) The pharmaceutical composition according to Item 12, which contains NaCl at about 200 mM to about 210 mM. (Item 14) The pharmaceutical composition according to Item 13, which contains NaCl at about 205 mM. (Item 15) The pharmaceutical composition according to any one of Items 1 to 10, comprising about 8.8 mg / ml to about 14.6 mg / ml of NaCl. (Item 16) The pharmaceutical composition according to Item 15, comprising about 10 mg / ml to about 13 mg / ml of NaCl. (Item 17) The pharmaceutical composition according to Item 16, comprising about 12.0 mg / ml of NaCl. (Item 18) The pharmaceutical composition according to any one of Items 1 to 10, comprising about 250 mM to about 350 mM of NaCl. (Item 19) The pharmaceutical composition according to Item 18, comprising about 275 mM to about 325 mM of NaCl. (Item 20) The pharmaceutical composition according to Item 19, comprising about 308 mM of NaCl. (Item 21) The pharmaceutical composition according to any one of Items 1 to 10, comprising about 14.6 mg / ml to about 20.5 mg / ml of NaCl. (Item 22) The pharmaceutical composition according to Item 21, comprising about 16 mg / ml to about 19 mg / ml of NaCl. (Item 23) The pharmaceutical composition according to Item 22, comprising about 18.0 mg / ml of NaCl. (Item 24) The pharmaceutical composition according to any one of Items 1 to 23, comprising about 5 mM to about 15 mM of L-histidine. (Item 25) The pharmaceutical composition according to Item 24, comprising about 6.64 mM of L-histidine or about 9.8 mM of L-histidine. (Item 26) The pharmaceutical composition according to any one of Items 1 to 23, comprising about 0.75 mg / ml to about 2.25 mg / ml of L-histidine. (Item 27) The pharmaceutical composition according to Item 26, comprising about 1.03 mg / ml of L-histidine or about 1.55 mg / ml of L-histidine. (Item 28) The pharmaceutical composition according to any one of Items 1 to 27, comprising calcium chloride at about 5 mM to about 10 mM. (Item 29) The pharmaceutical composition according to Item 28, comprising calcium chloride at about 5.4 mM or calcium chloride at about 8 mM. (Item 30) The pharmaceutical composition according to any one of Items 1 to 27, comprising calcium chloride dihydrate at about 0.75 mg / ml to about 1.5 mg / ml. (Item 31) The pharmaceutical composition according to Item 30, comprising calcium chloride dihydrate at about 0.8 mg / ml or calcium chloride dihydrate at about 1.18 mg / ml. (Item 32) The pharmaceutical composition according to any one of Items 1 to 31, comprising polysorbate 20 or polysorbate 80 at about 0.008% (w / v) to about 0.025% (w / v). (Item 33) The pharmaceutical composition according to Item 32, comprising polysorbate 20 or polysorbate 80 at about 0.013% (w / v) or polysorbate 20 or polysorbate 80 at about 0.02% (w / v). (Item 34) The pharmaceutical composition according to any one of Items 1 to 31, comprising polysorbate 20 or polysorbate 80 at about 0.08 mg / ml to about 0.25 mg / ml. (Item 35) The pharmaceutical composition according to Item 34, comprising polysorbate 20 or polysorbate 80 at about 0.13% mg / ml or polysorbate 20 or polysorbate 80 at about 0.20 mg / ml. (Item 36) The pharmaceutical composition according to any one of Items 1 to 35, wherein the FVIII polypeptide comprises a first subunit comprising an amino acid sequence that is at least 90% or 95% identical to amino acids 20 to 1684 of SEQ ID NO: 2 or amino acids 20 to 2578 of SEQ ID NO: 6, and a second subunit comprising an amino acid sequence that is at least 90% to 95% identical to amino acids 21 to 247 of SEQ ID NO: 4. (Item 37) The pharmaceutical composition according to item 36, wherein the FVIII polypeptide comprises a first subunit comprising amino acids 20 to 1684 of SEQ ID NO: 2 or amino acids 20 to 2578 of SEQ ID NO: 6, and a second subunit comprising amino acids 21 to 247 of SEQ ID NO: 4. (Item 38) The pharmaceutical composition according to any one of items 1 to 37, wherein the FVIII polypeptide is present at a concentration of about 50 IU / ml to about 2500 IU / ml. (Item 39) The pharmaceutical composition according to item 38, comprising the FVIII polypeptide at 83 IU / ml, 167 IU / ml, 250 IU / ml, 333 IU / ml, 500 IU / ml, 667 IU / ml, 1000 IU / ml, 1333 IU / ml, 1667 IU / ml, or 2000 IU / ml. (Item 40) The pharmaceutical composition according to any one of items 1 to 37, wherein the FVIII polypeptide is present at a concentration of about 100 IU / ml to about 4000 IU / ml. (Item 41) The pharmaceutical composition according to item 40, comprising the FVIII polypeptide at 150 IU / ml, 287.5 IU / ml, 431.25 IU / ml, 575 IU / ml, 862.5 IU / ml, 1150 IU / ml, 1725 IU / ml, 2300 IU / ml, 2875 IU / ml, or 3450 IU / ml. (Item 42) (a) the FVIII polypeptide at about 50 IU / ml to about 2500 IU / ml; (b) sucrose at about 1% (w / v) to about 2.5% (w / v); (c) NaCl at about 150 mM to about 250 mM; (d) L-histidine at about 5 mM to about 15 mM; (e) calcium chloride at about 5 mM to about 10 mM; (f) polysorbate 20 or polysorbate 80 at about 0.008% (w / v) to about 0.025%, and the pharmaceutical composition according to any one of items 1 to 4. (Item 43) The pharmaceutical composition according to item 42, comprising about 175 mM to about 225 mM of NaCl. (Item 44) The pharmaceutical composition according to item 43, comprising about 200 mM to about 210 mM of NaCl. (Item 45) (a) said FVIII polypeptide at about 83 IU / ml, about 167 IU / ml, about 250 IU / ml, about 333 IU / ml, about 500 IU / ml, about 667 IU / ml, about 1000 IU / ml, 1333 IU / ml, 1667 IU / ml, or 2000 IU / ml, and (b) about 1.33% (w / v) of sucrose, and (c) about 205 mM of NaCl, and (d) about 6.64 mM of L-histidine, and (e) about 5.4 mM of calcium chloride, and (f) about 0.013% (w / v) of polysorbate 20 or polysorbate 80, the pharmaceutical composition according to item 44. (Item 46) (a) said FVIII polypeptide at about 100 IU / ml to about 4000 IU / ml, and (b) about 1% (w / v) to about 2.5% (w / v) of sucrose, and (c) about 250 mM to about 350 mM of NaCl, and (d) about 5 mM to about 15 mM of L-histidine, and (e) about 5 mM to about 10 mM of calcium chloride, and (f) about 0.008% (w / v) to about 0.025% of polysorbate 20 or polysorbate 80, the pharmaceutical composition according to any one of items 1 to 4. (Item 47) The pharmaceutical composition according to item 46, comprising about 275 mM to about 325 mM of NaCl. (Item 48) (a) said FVIII polypeptide at about 150 IU / ml, about 287.5 IU / ml, about 431.25 IU / ml, about 575 IU / ml, about 862.5 IU / ml, about 1150 IU / ml, about 1725 IU / ml, about 2300 IU / ml, about 2875 IU / ml, or about 3450 IU / ml, and (b) about 2.0% (w / v) sucrose, and (c) about 308 mM NaCl, and (d) about 9.8 mM L - histidine, and (e) about 8 mM calcium chloride, and (f) about 0.020% (w / v) polysorbate 20 or polysorbate 80, the pharmaceutical composition according to item 47. (Item 49) (a) said FVIII polypeptide at about 50 IU / ml to about 2500 IU / ml, and (b) sucrose at about 10 mg / ml to about 25 mg / ml, and (c) NaCl at about 8.8 mg / ml to about 14.6 mg / ml, and (d) L - histidine at about 0.75 mg / ml to about 2.25 mg / ml, and (e) calcium chloride dihydrate at about 0.75 mg / ml to about 1.5 mg / ml, and (f) polysorbate 20 or polysorbate 80 at about 0.08 mg / ml to about 0.25 mg / ml, the pharmaceutical composition according to any one of items 1 to 4. (Item 50) The pharmaceutical composition according to item 49, containing about 10 mg / ml to 13 mg / ml of NaCl. (Item 51) (a) said FVIII polypeptide at about 83 IU / ml, about 167 IU / ml, about 250 IU / ml, about 333 IU / ml, about 500 IU / ml, about 667 IU / ml, about 1000 IU / ml, about 1333 IU / ml, about 1667 IU / ml, or about 2000 IU / ml, and (b) about 13.3 mg / ml of sucrose, and (c) about 12.0 mg / ml of NaCl, and (d) about 1.03 mg / ml of L - histidine, (e) About 0.8 mg / ml of calcium chloride dihydrate, and (f) About 0.13 mg / ml of polysorbate 20 or polysorbate 80, the pharmaceutical composition according to item 50. (Item 52) (a) About 100 IU / ml to about 4000 IU / ml of the FVIII polypeptide, and (b) About 10 mg / ml to about 25 mg / ml of sucrose, and (c) About 14.6 mg / ml to about 20.5 mg / ml of NaCl, and (d) About 0.75 mg / ml to about 2.25 mg / ml of L-histidine, and (e) About 0.75 mg / ml to about 1.5 mg / ml of calcium chloride dihydrate, and (f) About 0.08 mg / ml to about 0.25 mg / ml of polysorbate 20 or polysorbate 80, the pharmaceutical composition according to any one of items 1 to 4. (Item 53) The pharmaceutical composition according to item 52, containing about 16 mg / ml to about 19 mg / ml of NaCl. (Item 54) (a) About 150 IU / ml, about 287.5 IU / ml, about 431.25 IU / ml, about 575 IU / ml, about 862.5 IU / ml, about 1150 IU / ml, about 1725 IU / ml, about 2300 IU / ml, about 2875 IU / ml, or about 3450 IU / ml of the FVIII polypeptide, and (b) About 20.0 mg / ml of sucrose, and (c) About 18.0 mg / ml of NaCl, and (d) About 1.55 mg / ml of L-histidine, and (e) About 1.18 mg / ml of calcium chloride dihydrate, and (f) About 0.20 mg / ml of polysorbate 20 or polysorbate 80, the pharmaceutical composition according to item 53. (Item 55) A pharmaceutical kit, (a) A first container containing a lyophilized powder, the powder being (i) the FVIII polypeptide, (ii) one or more stabilizers selected from the group consisting of sucrose, trehalose, raffinose, arginine, and mixtures thereof, (iii) sodium chloride (NaCl), (iv) L-histidine, (v) calcium chloride, (vi) polysorbate 20 or polysorbate 80, and the first container comprising the same; (b) a second container containing sterile water for injection to be combined with the lyophilized powder of the first container, and the pharmaceutical kit comprising the same. (Item 56) The pharmaceutical kit according to Item 55, which does not contain mannitol, glycine, alanine, or hydroxyethyl starch. (Item 57) The pharmaceutical kit according to Item 55 or 56, wherein NaCl is the only filler. (Item 58) (a) A first container containing a lyophilized powder, wherein the powder comprises: (i) about 250 IU, about 500 IU, about 750 IU, about 1000 IU, about 1500 IU, about 2000 IU, about 3000 IU, about 4000 IU, about 5000 IU, or about 6000 IU of the FVIII polypeptide; (ii) about 40 mg of sucrose; (iii) about 36 mg of sodium chloride; (iv) about 3.1 mg of L-histidine; (v) about 2.40 mg of calcium chloride dihydrate; (vi) about 0.40 mg of polysorbate 20 or polysorbate 80, and the first container comprising the same; (b) A second container containing sterile water for injection, which when combined with the lyophilized powder of the first container, (i) respectively, about 83 IU / ml, about 167 IU / ml, about 250 IU / ml, about 333 IU / ml, about 500 IU / ml, about 667 IU / ml, about 1000 IU / ml, about 1333 IU / ml, about 1667 IU / ml, or about 2000 IU / ml of said FVIII polypeptide, and (ii) about 1.33% (w / v) sucrose, and (iii) about 205 mM NaCl, and (iv) about 6.64 mM L-histidine, and (v) about 5.4 mM calcium chloride, and (vi) about 0.013% (w / v) polysorbate 20 or polysorbate 80, in a volume sufficient to produce a solution, a second container containing said sterile water for injection, a pharmaceutical kit according to any one of items 55 to 57. (Item 59) (a) A first container containing a lyophilized powder, said powder being (i) about 250 IU, about 500 IU, about 750 IU, about 1000 IU, about 1500 IU, about 2000 IU, or 3000 IU, about 4000 IU, about 5000 IU, or about 6000 IU of said FVIII polypeptide, and (ii) about 40 mg sucrose, and (iii) about 36 mg sodium chloride, and (iv) about 3.1 mg L-histidine, and (v) about 2.40 mg calcium chloride dihydrate, and (vi) about 0.40 mg polysorbate 20 or polysorbate 80, said first container, and (b) A second container containing sterile water for injection, when combined with said lyophilized powder of said first container, (i) respectively, about 83 IU / ml, about 167 IU / ml, about 250 IU / ml, about 333 IU / ml, about 500 IU / ml, about 667 IU / ml, about 1000 IU / ml, about 1333 IU / ml, about 1667 IU / ml, or about 2000 IU / ml of said FVIII polypeptide, and (ii) about 13.3 mg / ml sucrose, and (iii) about 12.0 mg / ml of NaCl, and (iv) about 1.03 mg / ml of L-histidine, and (v) about 0.80 mg / ml of calcium chloride, and (vi) about 0.13 mg / ml of polysorbate 20 or polysorbate 80, in a volume sufficient to produce a solution, and a second container containing the sterile water for injection, the pharmaceutical kit according to any one of items 55 to 57. (Item 60) The pharmaceutical kit according to any one of items 55 to 59, wherein the first container is a glass vial equipped with a rubber stopper. (Item 61) The pharmaceutical kit according to any one of items 55 to 60, wherein the second container is a syringe body, and the syringe body is associated with a plunger. (Item 62) The pharmaceutical kit according to item 61, further comprising an adapter for connecting the glass vial to the syringe body. (Item 63) The pharmaceutical kit according to item 61 or 62, further comprising an infusion tube for connecting to a needle connected to the syringe suitable for intravenous injection. (Item 64) A method for reducing the annualized bleeding rate in a subject having hemophilia, the method comprising administering to the subject the pharmaceutical composition according to any one of items 1 to 54 at an administration interval of about 3 days or more. The method as described above. (Item 65) The method according to item 64, wherein the administration is prophylactic administration, individualized according to the subject, and results in an annualized bleeding rate of less than about 5.0, less than about 4.9, less than about 4.8, less than about 4.7, less than about 4.6, or less than about 4.5. (Item 66) The method according to item 65, wherein the median of the annualized bleeding rate is about 1.6. (Item 67) The method according to item 64, wherein the administration is prophylactic and results in an annualized bleeding rate of less than about 9.0, less than about 8.9, less than about 8.8, less than about 8.7, less than about 8.6, less than about 8.5, or less than about 8.4 per week. (Item 68) The method according to item 67, wherein the median of the annualized bleeding rate is about 3.6. (Item 69) The method according to item 64, wherein the administration is on-demand or at the time of onset and results in an annualized bleeding rate of less than about 55, less than about 54, less than about 53, less than about 52, less than about 51, less than about 50, less than about 49, less than about 48, or less than about 47. (Item 70) The method according to item 69, wherein the median of the annualized bleeding rate is about 33.6. (Item 71) The method according to any one of items 64 to 70, wherein the pharmaceutical composition comprises an effective amount of the FVIII polypeptide that is from about 20 IU / kg to about 90 IU / kg. (Item 72) The method according to item 71, wherein the effective amount is 20 - 30 IU / kg, 30 - 40 IU / kg, 40 - 50 IU / kg, 50 - 60 IU / kg, 60 - 70 IU / kg, 70 - 80 IU / kg, or 80 - 90 IU / kg. (Item 73) The method according to item 72, wherein the effective amount is 20 IU / kg, 25 IU / kg, 30 IU / kg, 35 IU / kg, 40 IU / kg, 45 IU / kg, 50 IU / kg, 55 IU / kg, 60 IU / kg, 65 IU / kg, 70 IU / kg, 75 IU / kg, 80 IU / kg, 85 IU / kg, or 90 IU / kg. (Item 74) The method according to any one of items 71 to 73, wherein the administration is prophylactic administration and is individualized with an effective amount of from about 25 IU / kg to about 65 IU / kg twice a week or every three days, or from about 50 IU / kg to about 65 IU / kg every four or five days. (Item 75) The method according to item 74, wherein the first dosage of the FVIII polypeptide is about 25 IU / kg and the second dosage of the FVIII polypeptide is about 50 IU / kg. (Item 76) The method according to any one of items 71 to 73, wherein the administration is prophylactic administration and is performed weekly at an effective amount of 65 IU / kg per week. (Item 77) The method according to any one of items 71 to 73, wherein the administration is on-demand or at the time of onset at an effective amount of 10 IU / kg to 75 IU / kg every 12 to 24 hours. (Item 78) The method according to any one of items 64 to 70, wherein the pharmaceutical composition comprises an effective amount that is a standard and fixed dosage across the total body weight. (Item 79) The method according to item 78, wherein the fixed dosage is about 2,000 IU, about 2,500 IU, about 3,000 IU, about 3,500 IU, about 4,000 IU, about 5,000 IU, or about 6,000 IU per dosage. (Item 80) The method according to any one of items 64 to 70, wherein the pharmaceutical composition comprises an effective amount that is a stratified dosage. (Item 81) The method according to any one of items 64 to 70, wherein the trough level of the FVIII polypeptide is more than the normal 1%, more than 2%, or more than 3% after the administration. (Item 82) The FVIII polypeptide has a T 1 / 2ベータ (activity) of about 7 hours to about 48 hours, about 6 hours to about 49 hours, about 5 hours to about 50 hours, and the method according to any one of items 64 to 81. (Item 83) The FVIII polypeptide has a T of at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, or at least about 25 hours 1 / 2ベータThe method according to item 82, having an (activity) average. (Item 84) Said T 1 / 2ベータ The method according to item 83, wherein the (activity) average is about 19 hours. (Item 85) Said T 1 / 2ベータ The method according to item 82 or 83, wherein the (activity) average is at least about 1.5 times higher than the polypeptide consisting of amino acids 20 to 1457 of SEQ ID NO: 2, amino acids 20 to 2351 of SEQ ID NO: 6, or ADVATE®. (Item 86) The method according to any one of items 64 to 85, wherein the plasma trough level of the FVIII polypeptide is maintained at about 1% to about 5%, about 1% to about 6%, about 1% to about 7%, about 1% to about 8%, about 1% to about 9%, about 1% to about 10%, about 1% to about 11%, about 1% to about 12%, about 1% to about 13%, about 1% to about 14%, about 1% to about 15% above the baseline in the subject after said administration. (Item 87) The pharmaceutical composition according to any one of items 1 to 35 or 38 to 54, the pharmaceutical kit according to any one of items 55 to 63, or the method according to any one of items 64 to 86, wherein the FVIII polypeptide is a long-acting FVIII polypeptide comprising an FVIII polypeptide and a heterologous moiety. (Item 88) The pharmaceutical composition, pharmaceutical kit, or method according to item 87, wherein the heterologous moiety is an FcRn binding partner. (Item 89) The pharmaceutical composition, pharmaceutical kit, or method according to item 88, wherein the FcRn binding partner comprises an Fc region. (Item 90) The pharmaceutical composition, pharmaceutical kit, or method according to item 88 or 89, wherein the long-acting FVIII polypeptide further comprises a second FcRn binding partner. (Item 91) The pharmaceutical composition, pharmaceutical kit, or method according to item 90, wherein the second FcRn binding partner comprises a second Fc region. (Item 92) The pharmaceutical composition, pharmaceutical kit, or method according to item 90 or 91, wherein the FcRn binding partner and the second FcRn binding partner are bound to each other. (Item 93) The pharmaceutical composition, pharmaceutical kit, or method according to item 92, wherein the binding is a covalent bond. (Item 94) The pharmaceutical composition, pharmaceutical kit, or method according to item 93, wherein the covalent bond is a disulfide bond. (Item 95) The pharmaceutical composition, pharmaceutical kit, or method according to any one of items 90 to 94, wherein the second FcRn binding partner is not bound to the amino acid sequence by a peptide bond. (Item 96) The pharmaceutical composition, pharmaceutical kit, or method according to any one of items 87 to 95, wherein the long-acting FVIII polypeptide is an FVIII monomer-dimer hybrid. (Item 97) The pharmaceutical composition, pharmaceutical kit, or method according to any one of items 87 to 96, wherein the FVIII polypeptide in the long-acting polypeptide is human FVIII. (Item 98) The pharmaceutical composition, pharmaceutical kit, or method according to any one of items 87 to 97, wherein the FVIII polypeptide in the long-acting polypeptide is full-length FVIII or B-domain-deleted FVIII. (Item 99) The pharmaceutical composition, pharmaceutical kit, or method according to any one of items 87 to 97, wherein the FVIII polypeptide is single-stranded. (Item 100) The pharmaceutical composition, pharmaceutical kit, or method according to any one of items 87 to 97, wherein the FVIII polypeptide is unprocessed. (Item 101) The pharmaceutical composition, pharmaceutical kit, or method according to any one of items 87 to 97, wherein the FVIII polypeptide is within two chains, a first chain comprising the heavy chain of the FVIII polypeptide and a second chain comprising the light chain of the FVIII polypeptide. (Item 102) The pharmaceutical composition, pharmaceutical kit, or method according to any one of items 88 to 101, wherein the FcRn binding partner in the chimeric polypeptide is human Fc. (Item 103) The pharmaceutical composition, pharmaceutical kit, or method according to any one of items 87 to 102, wherein the FVIII polypeptide does not contain a signal sequence and is at least 60%, 70%, 80%, 90%, 95%, 95%, 97%, 98%, 99%, or 100% identical to the FVIII amino acid sequence shown in Table 11A or 11B (amino acids 20 to 1457 of SEQ ID NO: 2 or amino acids 20 to 2351 of SEQ ID NO: 6). (Item 104) The pharmaceutical composition, pharmaceutical kit, or method according to any one of items 88 to 103, wherein the FcRn binding partner does not contain a signal sequence and is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the Fc amino acid sequence shown in Table 11B (amino acids 21 to 247 of SEQ ID NO: 4). (Item 105) The pharmaceutical composition, pharmaceutical kit, or method according to any one of items 90 to 104, wherein the second FcRn binding partner in the chimeric polypeptide is human Fc. (Item 106) The pharmaceutical composition, pharmaceutical kit, or method according to item 105, wherein the second FcRn binding partner does not contain a signal sequence and is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the Fc amino acid sequence shown in Table 11B (amino acids 21 to 247 of SEQ ID NO: 4). (Item 107) The pharmaceutical composition according to any one of items 1 to 54, the pharmaceutical kit according to any one of items 55 to 63, or the method according to any one of items 64 to 106, wherein the pharmaceutical composition is a pharmaceutical composition for perioperative management of bleeding symptoms. (Item 108) The method according to any one of items 64 to 107, wherein the subject requires management or prevention of bleeding or bleeding symptoms. (Item 109) The method according to item 108, wherein the subject requires management or prevention of bleeding in mild bleeding, hemarthrosis, superficial muscle bleeding, soft tissue bleeding, moderate bleeding, intramuscular or soft tissue bleeding due to incision, mucosal bleeding, hematuria, massive bleeding, pharyngeal bleeding, posterior pharyngeal bleeding, retroperitoneal bleeding, central nervous system bleeding, contusion, laceration, abrasion, joint bleeding, epistaxis, oral bleeding, bleeding from the gums, intracranial bleeding, intraperitoneal bleeding, mild spontaneous bleeding, bleeding after major trauma, moderate skin injury, or spontaneous bleeding in joints, muscles, internal organs, or the brain. (Item 110) The method according to item 109, wherein the subject requires management of bleeding associated with surgery or tooth extraction. (Item 111) The method according to item 110, wherein the subject will perform, is performing, or has performed major surgery. (Item 112) The method according to item 111, wherein the major surgery is orthopedic surgery, extensive oral surgery, urological surgery, or hernia surgery. (Item 113) The method according to item 112, wherein the orthopedic surgery is replacement of the knee, hip, or other major joint. (Item 114) The method according to any one of items 64 to 113, wherein the subject requires long-term treatment. (Item 115) The method according to any one of items 64 to 114, wherein the FVIII polypeptide is administered intravenously or subcutaneously. (Item 116) The pharmaceutical composition according to any one of items 18 to 23, 40, 41, 46 to 48, and 52 to 54, wherein the pharmaceutical composition is a composition before lyophilization.

Brief Description of the Drawings

[0020]

Figure 1

Figure 2

Modes for Carrying Out the Invention

[0021] The present invention provides a formulation of factor VIII polypeptide, such as FVIII-Fc, and a method of using the same.

[0022] I. Definitions As used within the specification and the appended claims, it is to be noted that the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. The terms "a" (or "an") and "one or more" and "at least one" may be used interchangeably herein.

[0023] The term "about" is used herein to mean approximately, roughly, around, or within the range of. When the term "about" is used in connection with a numerical range, the range is modified by extending the boundaries above and below the recited numerical values. Generally, the term "about" is used herein to modify a numerical value by a difference of up to 10 percent above or below (higher or lower) the recited value.

[0024] Furthermore, as used herein, "and / or" is considered to be a specific disclosure of each of two particular features or components, including or not including the other. Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0025] To the extent that embodiments are described herein in terms of "comprising", it is understood that similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided, if not otherwise stated.

[0026] Unless defined otherwise, 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 disclosure pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press, The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press, and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide many common dictionaries of terms used in this disclosure to those of ordinary skill in the art.

[0027] Units, prefixes, and symbols are shown in forms acceptable in the International System of Units (SI). Numerical ranges include the numbers defining the range. Unless otherwise specified, amino acid sequences are described with the amino-to-carboxy orientation from left to right. The headings provided herein are not limitations of the various aspects or embodiments of the present disclosure, which can be done by referring to the present specification as a whole. Accordingly, the terms defined immediately below are more fully defined by referring to the entire present specification. Amino acids are referred to herein by either the generally known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides are likewise referred to by their generally accepted one-letter codes.

[0028] The terms "polypeptide", "peptide", and "protein" are used interchangeably and refer to a polymeric compound consisting of amino acid residues linked by covalent bonds.

[0029] The terms "polynucleotide" and "nucleic acid" are used interchangeably and refer to a polymeric compound consisting of nucleotide residues linked by covalent bonds. A polynucleotide can be DNA, cDNA, RNA, single-stranded or double-stranded, a vector, plasmid, phage, or virus. Polynucleotides include those in Table 10 encoding the polypeptides of Table 11 (see Table 10). Polynucleotides also include fragments of the polynucleotides of Table 10, for example, those encoding fragments of the polypeptides of Table 11, for example, Factor VIII, Fc, signal sequences, propeptides, and other fragments of the polypeptides of Table 11.

[0030] As used herein, "administer" means to prohibit or give a pharmaceutical composition comprising a Factor VIII polypeptide of the invention to a subject by a pharmaceutically acceptable route. Examples of administration routes include, but are not limited to, intravenous, e.g., intravenous injection and intravenous infusion, via a central venous access. Further administration routes include subcutaneous, intramuscular, oral, nasal, and pulmonary administration. Advantages of the invention include improved regimen compliance, reduced destruction by bleeding, increased protection of joints from bleeding, prevention of joint damage, reduced morbidity, reduced mortality, long-term protection from bleeding, reduced thromboembolic events, and improved quality of life.

[0031] The terms "long-acting" and "long-lasting" are used interchangeably herein. In one embodiment, the term "long-acting" or "long-lasting" indicates that the FVIII activity as a result of administration of a "long-acting" FVIII polypeptide is longer than the FVIII activity of wild-type FVIII (e.g., ADVATE® or plasma-derived FVIII ("pdFVIII")). The "longer" FVIII activity can be measured by methods optionally known in the art, such as, for example, an aPTT assay, a chromogenic assay, ROTEM®, TGA, etc. In one embodiment, the "longer" FVIII activity can be indicated by T 1 / 2ベータ (activity). In another embodiment, the "longer" FVIII activity can be indicated by the level of FVIII antigen present in plasma, e.g., T 1 / 2ベータ (antigen). In other embodiments, a long-acting or long-lasting FVIII polypeptide acts longer in the coagulation cascade, e.g., is active for a longer period compared to a wild-type FVIII polypeptide, i.e., a polypeptide consisting of amino acids 20-1457 of SEQ ID NO: 2, i.e., SQ BDD FVIII (REFACTO®) or a polypeptide consisting of amino acids 20-2351 of SEQ ID NO: 6 (ADVATE®).

[0032] The terms "short-acting" and "short-lasting" are used interchangeably herein. The terms "short-acting" and "short-lasting" indicate that the FVIII activity as a result of administration of a "short-acting" FVIII polypeptide is similar to, or the same as, or shorter than the FVIII activity of wild-type FVIII (e.g., ADVATE® (amino acids 20-2351 of SEQ ID NO: 6), REFACTO® (amino acids 20-1457 of SEQ ID NO: 2), or plasma-derived FVIII ("pdFVIII")). "Shorter" FVIII activity can be measured, for example, by methods arbitrarily known in the art such as aPTT assay, chromogenic assay, ROTEM®, TGA, etc. Exemplary short-acting FVIII polypeptides include, but are not limited to, wild-type mature FVIII polypeptides (ADVATE®, RECOMBINATE®, or HELIXATE®), or B-domain deleted FVIII polypeptides such as SQ BDD FVIII (REFACTO® and XYNTHA®), or FVIII polypeptides containing 21 amino acids from the B-domain (i.e., SFSQNSRHPSQNPPVLKRHQR, SEQ ID NO: 17) (e.g., NOVOEIGHT®).

[0033] As used herein, the term "chimeric polypeptide" means a polypeptide that contains therein at least two polypeptides (or a part thereof such as a subsequence or peptide, for example) from different sources. A chimeric polypeptide can contain two, three, four, five, six, seven, or more polypeptides or a part thereof from different sources such as different genes, different cDNAs, or different animals or other species. A chimeric polypeptide can contain one or more linkers that link different polypeptides or a part thereof. Thus, the polypeptides or a part thereof can be directly linked within a single chimeric polypeptide, or indirectly linked via a linker, or both. A chimeric polypeptide can further contain additional peptides, such as a signal sequence, and sequences such as 6His and FLAG that are useful for protein purification or detection. In addition, a chimeric polypeptide can have amino acid or peptide additions at the N-terminus and / or C-terminus. In some embodiments, the chimeric polypeptide is a long-acting FVIII polypeptide. Exemplary chimeric polypeptides of the invention are factor VIII-FcRn BP chimeric polypeptides, such as factor VIII-Fc polypeptides, such as FVIIIFc (Table 11) of SEQ ID NO: 2 or 6, with or without its signal sequence and propeptide.

[0034] As used herein, "hybrid" polypeptides and proteins refer to a combination of a chimeric polypeptide and a second polypeptide. The chimeric polypeptide and the second polypeptide in the hybrid can bind to each other via protein-protein interactions such as charge-charge or hydrophobic interactions. The chimeric polypeptide and the second polypeptide in the hybrid can bind to each other via disulfide bonds or other covalent bonds (including plural). Hybrids are described in WO2004 / 101740 and WO2006 / 074199, each of which is hereby incorporated by reference in its entirety. Reference is also made to U.S. Patent Nos. 7,404,956 and 7,348,004, each of which is hereby incorporated by reference in its entirety. The second polypeptide can be a second copy of the same chimeric polypeptide or a different chimeric polypeptide. See Table 11. In one embodiment, the second polypeptide is a polypeptide comprising an FcRn binding partner. The FcRn binding partner binds to FcRn and protects the FcRn binding partner containing molecules from catabolism, thus extending the half-life in plasma. In another embodiment, the chimeric polypeptide is a chimeric Factor VIII-Fc polypeptide and the second polypeptide consists essentially of a single molecule of recombinant B domain deleted human FVIII (BDD-rFVIII) fused to an Fc, e.g., a dimeric Fc domain of human IgG1 without an intervening linker sequence. This hybrid polypeptide is referred to herein as an FVIIIFc monomeric Fc fusion protein, an FVIIIFc monomeric hybrid, a monomeric FVIIIFc hybrid, and an FVIIIFc monomer-dimer. See Table 11A. The examples provide preclinical and clinical data for this hybrid polypeptide.

[0035] The second polypeptide in the hybrid may contain or consist essentially of a sequence that is at least 90% or 95% identical to the amino acid sequence shown in Table 11A(ii) (amino acids 21 - 247 of SEQ ID NO: 4) without a signal sequence, or that is at least 90% or 95% identical to the amino acid sequence shown in Table 11A(ii) (amino acids 1 - 247 of SEQ ID NO: 4) with a signal sequence. The second polypeptide may contain or consist essentially of a sequence that is identical to the amino acid sequence shown in Table 11A(ii) (amino acids 21 - 247 of SEQ ID NO: 4) without a signal sequence, or that is identical to the amino acid sequence shown in Table 11A(ii) (amino acids 1 - 247 of SEQ ID NO: 4) with a signal sequence.

[0036] Factor VIII coagulant activity is expressed as international units (IU). 1 IU of Factor VIII activity approximately corresponds to the amount of Factor VIII in 1 milliliter of normal human plasma. Several assays are available for measuring Factor VIII activity, including one - stage clotting assays (activated partial thromboplastin time; aPTT), thrombin generation assays (TGA), and rotational thromboelastometry (ROTEM®).

[0037] As used herein in the context of formulations according to the present invention, the term "lyophilized" refers to a formulation produced by the freeze - drying method known per se in the art. The solvent (e.g., water) is removed by freezing, followed by sublimation under vacuum and desorption of residual water at elevated temperature. In the pharmaceutical field, lyophilized products typically have a residual moisture content of about 0.1 - 5% (w / w) and exist as a powder or physically stable cake. Lyophilized products are characterized by readily dissolving after addition of a reconstitution medium.

[0038] As used herein, the term "reconstituted formulation" refers to a formulation that has been lyophilized and is redissolved by the addition of a diluent. The diluent may include, but is not limited to, water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), surfactant-containing solution (e.g., 0.01% polysorbate 20 or polysorbate 80), pH buffer solution (e.g., phosphate buffer solution), and combinations thereof.

[0039] As used herein, "dose interval" means the amount of time that elapses between multiple administrations to a subject. Thus, the dose interval can be expressed as a range. The dose interval in the methods of the invention using a chimeric FVIII-FcRn BP, e.g., chimeric FVIII-Fc, can be at least about 1.5 to 8 times longer than the dose interval required for an equivalent amount (IU / kg) of FcRn BP, e.g., factor VIII without an Fc portion (i.e., the polypeptide consisting of the aforementioned FVIII). For example, the dose interval when administering the factor VIII-Fc chimeric polypeptide (or hybrid) of the invention can be at least about 1.5 times longer than the dose interval required for an equivalent amount of factor VIII without an Fc portion (i.e., the polypeptide consisting of factor VIII). The dose interval can be at least about 1.5 to 8 times longer than, for example, the dose interval required for an equivalent amount of factor VIII without an Fc portion (or the polypeptide consisting of factor VIII).

[0040] As used herein, the term "dose frequency" refers to the frequency of administering the FVIII polypeptide within a given time period. The dose frequency can be expressed as the number of administrations per given time period, e.g., once a week or once every two weeks.

[0041] As used herein, the terms "prevention of one or more bleeding episodes" or "prophylactic treatment" mean administering an FVIII polypeptide in multiple administrations to a subject over time to increase the FVIII activity level in the subject's plasma. In one embodiment, "prevention of one or more bleeding episodes" refers to the use of an FVIII polypeptide to prevent or inhibit the occurrence of one or more spontaneous or uncontrollable bleedings or bleeding episodes, or to reduce the frequency of one or more spontaneous or uncontrollable bleedings or bleeding episodes. In another embodiment, the increased FVIII activity level is sufficient to reduce the occurrence of spontaneous bleeding or to prevent bleeding in the event of accidental injury. Prophylactic treatment reduces or prevents bleeding episodes, such as those described under on-demand treatment. Prophylactic treatment can be individualized, for example, as discussed under "dosing interval", to account for variability between patients.

[0042] As used herein, the term "about twice a week" means an approximate number, and "about twice a week" can include twice a week, for example, a first administration over 3 days, a second administration over 3 days, a first administration over 3 days, a second administration over 4 days, a first administration over 4 days, a second administration over 3 days, a first administration over 4 days, a second administration over 4 days. The term "about twice a week" can also include every 3 days, every 4 days, or every 5 days.

[0043] As used herein, the term "about once a week" means an approximate number, and "about once a week" can include every 7 days ± 2 days, that is, every 5 days to every 9 days. Thus, the dosing frequency of "once a week" can be every 5 days, every 6 days, every 7 days, every 8 days, or every 9 days.

[0044] As used herein, the terms "individualized prophylaxis" or "prophylactic and individualized" mean the use of an FVIII polypeptide at individualized dosages and / or dosing intervals or frequencies to prevent or inhibit the occurrence of one or more spontaneous and / or uncontrollable bleeding or bleeding symptoms, or to reduce the frequency of one or more spontaneous and / or uncontrollable bleeding or bleeding symptoms. "Individualized" within the context of prophylaxis is used synonymously throughout this application with "tailored to the individual." For example, "individualized prophylaxis" also means "prophylaxis tailored to the individual," and "prophylactic and individualized" also means "prophylactic and tailored to the individual." In one embodiment, "individualized interval" includes every three days plus or minus two days, i.e., daily to every five days. Thus, the dosing frequency of "prophylaxis at individualized intervals" can be daily, every two days, every three days, every four days, or every five days.

[0045] As used herein, the term "on-demand therapy" is intended to be carried out over a short period of time and means a treatment in response to the current situation such as bleeding symptoms or in response to a predicted short-term need such as a scheduled surgery. "On-demand therapy" is used interchangeably with "at onset" therapy. Conditions that may require on-demand therapy include bleeding symptoms, hemarthrosis, muscle bleeding, oral bleeding, hemorrhage, bleeding into muscle, oral cavity bleeding, trauma, head trauma (trauma capitis), gastrointestinal bleeding, intracranial bleeding, intra-abdominal bleeding, intrathoracic bleeding, fractures, central nervous system bleeding, retropharyngeal bleeding, retroperitoneal bleeding, or bleeding within the iliopsoas sheath. Other bleeding symptoms are also included. The subject may require treatment for surgical prophylaxis, perioperative management, or a surgical procedure. Such surgeries include minor surgeries, major surgeries, tooth extractions, tonsillectomies, other dental / head and neck surgeries, inguinal hernia incisions, synovectomies, total knee arthroplasties, other joint replacement surgeries, craniotomies, bone fixation surgeries, trauma surgeries, intracranial surgeries, intra-abdominal surgeries, or intrathoracic surgeries. Other surgeries are also included.

[0046] Additional conditions that may require on-demand treatment include mild bleeding, hemarthrosis, superficial muscle bleeding, soft tissue bleeding, moderate bleeding, intramuscular or soft tissue bleeding due to incision, mucosal bleeding, hematuria, major bleeding, pharyngeal bleeding, posterior pharyngeal bleeding, retroperitoneal bleeding, central nervous system bleeding, contusion, laceration, abrasion, joint bleeding, epistaxis, oral bleeding, bleeding from the gums, intracranial bleeding, intra-abdominal bleeding, mild spontaneous bleeding, bleeding after major trauma, moderate skin injury, or spontaneous bleeding into joints, muscles, internal organs, or the brain. Additional reasons for on-demand treatment include perioperative management for surgery or tooth extraction, major surgery, extensive oral surgery, urological surgery, hernia surgery, and the need in orthopedic surgery such as knee, hip, or other major joint replacement.

[0047] As used herein, the terms "treatment" or "treating" mean the amelioration or reduction of one or more symptoms of a bleeding disorder or condition, including but not limited to hemophilia B. In one embodiment, "treatment" or "treating" a bleeding disorder or condition includes the prevention of one or more symptoms of the bleeding disorder or condition. In a bleeding disorder or condition caused by a deficiency of FVIII (e.g., low baseline FVIII activity), the terms "treatment" or "treating" mean FVIII replacement therapy. By administering to a subject a pharmaceutical composition comprising an FVIII polypeptide, the subject can achieve and / or maintain a plasma trough level of FVIII activity of about 1 IU / dl or greater than 1 IU / dl. In other embodiments, "treatment" or "treating" means a decrease in the frequency of one or more symptoms of a bleeding disorder or condition, such as spontaneous or uncontrollable bleeding symptoms. However, "treatment" does not necessarily have to result in a cure.

[0048] As used herein, the term "perioperative management" means the use of a pharmaceutical composition comprising an FVIII polypeptide in a surgical procedure, e.g., before, at the same time as, or after surgery. Use for "perioperative management" of one or more bleeding symptoms includes preoperative (i.e., pre-operative), intraoperative (i.e., intra-operative), or postoperative (i.e., post-operative) surgical prophylaxis to prevent one or more bleeding or bleeding symptoms before, during, or after surgery, or to reduce or inhibit idiopathic and / or uncontrollable bleeding symptoms.

[0049] As used herein, "baseline" is the lowest measured plasma factor VIII level in a subject before dosing. Plasma levels of FVIII can be measured at two time points, at screening visit and immediately prior to dosing. Alternatively, (a) for patients with pre-treatment FVIII activity less than 1%, no detectable FVIII antigen, and a nonsense genotype, the baseline can be defined as 0%, (b) for patients with pre-treatment FVIII activity less than 1% and detectable FVIII antigen, the baseline can be set at 0.5%, (c) for patients with pre-treatment FVIII activity between 1 - 2%, the baseline is Cmin (the lowest activity through PK studies), and (d) for patients with pre-treatment FVIII activity 2% or greater, the baseline can be set at 2%. Activity above baseline prior to dosing can be considered residual drug from prior treatment, decayed to baseline, and subtracted from PK data after rFVIIIFc administration.

[0050] As used herein, "T 1 / 2β " or "T 1 / 2ベータ " or "beta HL" is the half-life associated with the elimination phase, t1 / 2β = (ln2) / elimination rate constant associated with the terminal phase. T 1 / 2ベータ can be measured by FVIII activity or by FVIII antigen level in plasma. T 1 / 2ベータ based on activity is denoted as T 1 / 2ベータ (activity), and T 1 / 2ベータ based on FVIII antigen level is T1 / 2ベータ can be expressed as (antigen). T 1 / 2ベータ (activity) and T 1 / 2ベータ Both (antigen) can be expressed as a range or a geometric mean.

[0051] As used herein, "trough" is the lowest plasma FVIII activity level reached after administration of a dose of the chimeric polypeptide of the invention or another FVIII molecule and, if any, before the next dose is administered. Trough is used interchangeably herein with "threshold". The baseline FVIII level is subtracted from the measured FVIII level to calculate the trough level.

[0052] As used herein, the term "annualized bleeding rate" ("ABR") refers to the number of bleeding episodes (including spontaneous and traumatic bleeding) experienced by a subject during a given period extrapolated to 1 year. For example, 2 bleeds in 6 months can represent an ABR of 4. The median ABR provides the median among all observed ABRs, with half of the subjects having individual ABRs below the median and the other half having individual ABRs above the median.

[0053] As used herein, the term "interquartile range" ("IQR") refers to a measure of statistical dispersion and is equal to the difference between the upper and lower quartiles. Unlike the (total) range, the interquartile range is a robust statistic and has a 25% breakdown point and is therefore often preferred over the total range. For a symmetric distribution (where the median is equal to the midline and the mean of the first and third quartiles), half of the IQR is equal to the median absolute deviation (MAD). The median is the corresponding measure of central tendency.

[0054] As used herein, "subject" means a human. The subjects used herein include an individual known to have at least one occurrence of an uncontrollable bleeding symptom, a disease or disorder associated with an uncontrollable bleeding symptom, such as a bleeding disease or disorder, such as an individual diagnosed with hemophilia A, an uncontrollable bleeding symptom, such as a person prone to hemophilia, or any combination thereof. The subject can also include an individual at risk of one or more uncontrollable bleeding symptoms prior to a particular activity, such as surgery, sports activity, or any strenuous exercise. The subject can have a baseline FVIII activity of less than 1%, less than 0.5%, less than 2%, less than 2.5%, less than 3%, or less than 4%. The subject also includes pediatric humans. Pediatric human subjects are from birth to 20 years old, preferably from birth to 18 years old, from birth to 16 years old, from birth to 15 years old, from birth to 12 years old, from birth to 11 years old, from birth to 6 years old, from birth to 5 years old, from birth to 2 years old, or from 2 to 11 years old.

[0055] As used herein, "therapeutic dosage", "dosage", "effective amount", or "dose" means a dosage that achieves a plasma trough level of FVIII activity of at least about 1 IU / dl or greater than 1 IU / dl in a subject to whom a pharmaceutical composition comprising an FVIII polypeptide is administered. For purposes of the present invention, in one embodiment, the "dosage" is at least about 1 IU / dl or greater than 1 IU / dl, at least about 2 IU / dl or greater than 2 IU / dl, at least about 3 IU / dl or greater than 3 IU / dl, at least about 4 IU / dl or greater than 4 IU / dl, or at least about 5 IU / dl or greater than 5 IU / dl of FVIII activity through administration of a pharmaceutical composition comprising an FVIII polypeptide. In another embodiment, the "dosage" reduces or decreases the frequency of bleeding or bleeding disorders. In other embodiments, the "dosage" stops persistent, uncontrolled bleeding or bleeding symptoms. In still other embodiments, the "dosage" prevents spontaneous bleeding or bleeding symptoms in subjects prone to such spontaneous bleeding or bleeding symptoms. The "dosage" or "therapeutic dosage" does not necessarily have to cure hemophilia.

[0056] The term "target joint" is defined as a large joint (e.g., hip, elbow, wrist, shoulder, knee, and ankle joint) in which recurrent bleeding occurs (frequency of bleeding symptoms three or more times into the same joint over a six-month consecutive period).

[0057] As used herein, the term "bleeding episode" adopts the standardized definition of bleeding episodes. A bleeding episode begins with the first sign of bleeding and ends 72 hours after the final treatment for the bleeding, during which any symptoms of bleeding at the same site, or an injection given within 72 hours or less, were considered to be the same bleeding episode. Any injection given to treat a bleeding episode that occurred more than 72 hours after a previous injection was considered to be the first injection to treat a new bleeding episode at the same site. Any bleeding at a different site was considered to be a separate bleeding episode, regardless of the time since the last injection. This definition was proposed by the Subcommittee on FVIII / FIX, which is the Subcommittee on Standards and Criteria of the International Society of Thrombosis and Hemostasis, and has been used in the PedNet multicenter study in hemophilia.

[0058] As used herein, the term "annualized bleeding rate (ABR)" refers to the number of bleeding episodes annualized for each patient using the following formula.

Number

[0059] As used herein, the term "variant" refers to a polynucleotide or polypeptide that is different from the original polynucleotide or polypeptide but maintains its basic properties, such as the coagulation activity of Factor VIII or the Fc (FcRn binding) activity. Generally, variants are generally substantially similar and are identical to the original polynucleotide or polypeptide in many regions. Variants include fragments of polypeptides and polynucleotides, deletions, insertions, and modified forms of the original polypeptide.

[0060] II. Pharmaceutical Compositions The present invention relates to an FVIII polypeptide formulated as a pharmaceutical composition. The pharmaceutical composition can be formulated for administration to humans. The FVIII polypeptide can be a short-acting FVIII polypeptide or a long-acting FVIII polypeptide. The pharmaceutical composition includes a pharmaceutically acceptable carrier, for example, an ion exchanger, alumina, aluminum stearate, lecithin, serum proteins such as human serum proteins, buffering substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of vegetable saturated fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylate, wax, polyethylene-polyoxypropylene-block polymer, polyethylene glycol, and lanolin. Various methods for formulating the present invention are well known in the art.

[0061] In certain formulations provided herein, the FVIII polypeptide is formulated as a cake from a sterile, pyrogen-free, lyophilized white to off-white powder for intravenous (IV) administration. The formulation can be provided in a disposable vial.

[0062] In certain embodiments, FVIII polypeptide (e.g., long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc) formulations are provided in disposable vials manufactured to contain about 83 IU / ml, 167 IU / ml, 250 IU / ml, 333 IU / ml, 500 IU / ml, 667 IU / ml, 1000 IU / ml, 1333 IU / ml, 1667 IU / ml, or 2000 IU / ml of long-acting FVIII polypeptide after reconstitution with an appropriate amount of diluent. In certain embodiments where diluent is added until a final volume of about 3 ml is reached, the disposable vial can nominally contain about 250, about 500, about 750, about 1000, about 1500, about 2000, about 3000, about 4000, about 5000, or about 6000 international units (IU) of FVIII polypeptide (e.g., long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc).

[0063] In certain embodiments, the formulation includes, in addition to the active FVIII polypeptide (e.g., long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), sucrose (which can act as a stabilizer), sodium chloride (which can act as a filler), L-histidine (which can act as a buffer), calcium chloride, and polysorbate 20 or polysorbate 80 (which can act as a stabilizer). The formulation is provided with a diluent that includes a sterile sodium chloride solution. In certain embodiments, the diluent is provided in a pre-filled syringe.

[0064] Accordingly, provided herein are pharmaceutical compositions comprising a specified amount of FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc) (international units) together with the excipients sucrose, NaCl, L-histidine, calcium chloride, and polysorbate 20 or polysorbate 80. The compositions provided herein include various excipients at various concentrations, which 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), weight / volume percent (e.g., grams per 100 ml of diluent), or milligrams per milliliter (mg / ml). The formulations provided herein can contain various excipients in specified amounts at levels of precision ranging from approximately, e.g., simply expressed to one significant figure (e.g., about 0.1% (w / v)), or with further precision, e.g., up to 2, 3, 4, 5, or 6 significant figures (e.g., about 3.88 mg / ml with a precision up to three significant figures). The level of precision required can vary, e.g., depending on the requirements of a given regulatory agency or the manufacturing process. In certain embodiments, the pharmaceutical composition includes a reconstituted formulation that can be provided as a lyophilized product, optionally with a diluent.

[0065] In certain embodiments, the pharmaceutical composition comprises from about 50 IU / ml to about 2500 IU / ml of rFVIIIFc, such as 83 IU / ml, 167 IU / ml, 250 IU / ml, 333 IU / ml, 500 IU / ml, 667 IU / ml, 1000 IU / ml, 1333 IU / ml, 1667 IU / ml, or 2000 IU / ml of FVIII polypeptide (e.g., long-acting or short-acting FVIII polypeptide, such as rFVIIIFc). In certain embodiments, the pharmaceutical composition comprises 83 IU / ml, 167 IU / ml, 250 IU / ml, 333 IU / ml, 500 IU / ml, 667 IU / ml, 1000 IU / ml, 1333 IU / ml, 1667 IU / ml, or 2000 IU / ml of FVIII polypeptide (e.g., long-acting or short-acting FVIII polypeptide, such as rFVIIIFc) in a formulation comprising about 13.3 mg / ml or about 1.33% (w / v) sucrose, about 12.0 mg / ml or about 205 mM NaCl, about 1.03 mg / ml or about 6.64 mM L-histidine, about 0.80 mg / ml or about 5.4 mM calcium chloride dihydrate, and about 0.13 mg / ml or about 0.013% (w / v) polysorbate 20 or polysorbate 80.

[0066] In certain embodiments, the pharmaceutical composition prior to lyophilization comprises from about 100 IU / ml to about 4000 IU / ml of rFVIIIFc, for example, 150 IU / ml, 287.5 IU / ml, 431.25 IU / ml, 575 IU / ml, 862.5 IU / ml, 1150 IU / ml, 1725 IU / ml, 2300 IU / ml, 2875 IU / ml, or 3450 IU / ml of FVIII polypeptide (e.g., long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc). In certain embodiments, the pharmaceutical composition comprises 150 IU / ml, 287.5 IU / ml, 431.25 IU / ml, 575 IU / ml, 862.5 IU / ml, 1150 IU / ml, 1725 IU / ml, 2300 IU / ml, 2875 IU / ml, or 3450 IU / ml of FVIII polypeptide (e.g., long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc) in a formulation comprising about 20.0 mg / ml or about 2.0% (w / v) sucrose, about 18.0 mg / ml or about 308 mM NaCl, about 1.55 mg / ml or about 9.8 mM L-histidine, about 1.18 mg / ml or about 8.0 mM calcium chloride dihydrate, and about 0.2 mg / ml or about 0.02% (w / v) polysorbate 20 or polysorbate 80.

[0067] In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable amount of sucrose. In certain embodiments, the pharmaceutical composition comprises from about 1% (w / v) to about 2.5% (w / v) sucrose, preferably from about 1.3% (w / v) to about 2% (w / v) sucrose, e.g., about 1.33% (w / v) sucrose or about 2.0% (w / v) sucrose. In certain related embodiments, the pharmaceutical composition comprises from about 10 mg / ml to about 25 mg / ml sucrose, preferably from about 13 mg / ml to about 20 mg / ml sucrose, e.g., about 13.3 mg / ml sucrose or about 20.0 mg / ml sucrose. In still other embodiments, stabilizers such as trehalose, raffinose and / or arginine can be used at these concentrations instead of or in combination with sucrose.

[0068] In certain embodiments, the pharmaceutical composition comprises from about 150 mM to about 250 mM NaCl, from about 175 mM to about 225 mM NaCl, and from about 200 mM to about 210 mM NaCl, such as about 205 mM NaCl. In certain related embodiments, the pharmaceutical composition comprises from about 8.8 mg / ml to about 14.6 mg / ml NaCl, from about 10 mg / ml to about 13 mg / ml NaCl, such as about 12.0 mg / ml NaCl. In certain embodiments, NaCl is provided at a desired concentration in a diluent in which a lyophilized product comprising an FVIII polypeptide (such as a long-acting or short-acting FVIII polypeptide, such as rFVIIIFc) is reconstituted.

[0069] In another embodiment, the composition of the present invention does not contain fillers such as mannitol, glycine, alanine, or hydroxyethyl starch. In other embodiments, NaCl is the only filler.

[0070] In certain embodiments, the pharmaceutical composition prior to lyophilization comprises from about 250 mM to about 350 mM NaCl, from about 275 mM to about 325 mM NaCl, such as about 308 mM NaCl. In certain related embodiments, the pharmaceutical composition comprises from about 14.6 mg / ml to about 20.5 mg / ml NaCl, from about 16 mg / ml to about 19 mg / ml NaCl, such as about 18.0 mg / ml NaCl.

[0071] In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable amount of L-histidine. In certain embodiments, the pharmaceutical composition comprises from about 5 mM to about 15 mM of L-histidine, such as, for example, about 6.64 mM of L-histidine or about 9.8 mM of L-histidine. In certain related embodiments, the pharmaceutical composition comprises from about 0.75 mg / ml to about 2.25 mg / ml of L-histidine, such as, for example, about 1.03 mg / ml of L-histidine or about 1.55 mg / ml of L-histidine. In certain embodiments, L-histidine is provided as part of a lyophilized product that provides L-histidine at the desired concentration upon reconstitution with an appropriate amount of diluent.

[0072] In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable amount of calcium chloride. In certain embodiments, the pharmaceutical composition comprises from about 5 mM to about 10 mM of calcium chloride, such as, for example, about 5.4 mM of calcium chloride or about 8 mM of calcium chloride. In certain related embodiments, the pharmaceutical composition comprises from about 0.75 mg / ml to about 1.5 mg / ml of calcium chloride dihydrate, such as, for example, about 0.8 mg / ml of calcium chloride dihydrate or about 1.18 mg / ml of calcium chloride dihydrate. In certain embodiments, calcium chloride is provided as part of a lyophilized product that provides calcium chloride at the desired concentration upon reconstitution with an appropriate amount of diluent.

[0073] In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable amount of polysorbate 20 or polysorbate 80. In certain related embodiments, the pharmaceutical composition comprises from about 0.008% (w / v) to about 0.025% (w / v) of polysorbate 20 or polysorbate 80, such as about 0.013% (w / v) of polysorbate 20 or polysorbate 80 or about 0.02% (w / v) of polysorbate 20 or polysorbate 80. In certain related embodiments, the pharmaceutical composition comprises from about 0.08 mg / ml to about 0.25 mg / ml of polysorbate 20 or polysorbate 80, such as about 0.13% mg / ml of polysorbate 20 or polysorbate 80 or about 0.20 mg / ml of polysorbate 20 or polysorbate 80. In certain embodiments, the polysorbate 20 or polysorbate 80 is provided as part of a lyophilizate that provides the desired concentration of polysorbate 20 or polysorbate 80 upon reconstitution with a suitable diluent.

[0074] In certain embodiments, the pharmaceutical composition comprises from about 50 IU / ml to about 2500 IU / ml of FVIII polypeptide (such as a long-acting or short-acting FVIII polypeptide, such as rFVIIIFc), from about 1% (w / v) to about 2.5% (w / v) of sucrose, from about 150 mM to about 250 mM of NaCl, from about 5 mM to about 15 mM of L-histidine, from about 5 mM to about 10 mM of calcium chloride, and from about 0.008% (w / v) to about 0.025% of polysorbate 20 or polysorbate 80. In certain embodiments, the pharmaceutical composition is provided as a lyophilizate and a diluent. In certain embodiments, the amount of the lyophilizate provides about 3 ml of the pharmaceutical composition having the desired components at the desired concentration.

[0075] In certain embodiments, the pharmaceutical composition comprises from about 50 IU / ml to about 2500 IU / ml of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), from about 10 mg / ml to about 25 mg / ml of sucrose, from about 8.8 mg / ml to about 14.6 mg / ml of NaCl, from about 0.75 mg / ml to about 2.25 mg / ml of L-histidine, from about 0.75 mg / ml to about 1.5 mg / ml of calcium chloride dihydrate, and from about 0.08 mg / ml to about 0.25 mg / ml of polysorbate 20 or polysorbate 80. In certain embodiments, the pharmaceutical composition is provided as a lyophilizate and a diluent. In certain embodiments, the amount of the lyophilizate provides about 3 ml of the pharmaceutical composition having the desired components at the desired concentration.

[0076] Exemplary compositions are provided in Tables 1 and 2 of the Examples.

[0077] For example, the present disclosure provides a pharmaceutical composition comprising an FVIII polypeptide of about 83 IU / ml (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), about 1.33% (w / v) sucrose, about 205 mM NaCl, about 6.64 mM L-histidine, about 5.4 mM calcium chloride dihydrate, and about 0.013% (w / v) polysorbate 20 or polysorbate 80. The present disclosure further provides a pharmaceutical composition comprising an FVIII polypeptide of about 167 IU / ml (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), about 1.33% (w / v) sucrose, about 205 mM NaCl, about 6.64 mM L-histidine, about 5.4 mM calcium chloride dihydrate, and about 0.013% (w / v) polysorbate 20 or polysorbate 80. The present disclosure further provides a pharmaceutical composition comprising an FVIII polypeptide of about 250 IU / ml (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), about 1.33% (w / v) sucrose, about 205 mM NaCl, about 6.64 mM L-histidine, about 5.4 mM calcium chloride dihydrate, and about 0.013% (w / v) polysorbate 20 or polysorbate 80. The present disclosure further provides a pharmaceutical composition comprising an FVIII polypeptide of about 333 IU / ml (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), about 1.33% (w / v) sucrose, about 205 mM NaCl, about 6.64 mM L-histidine, about 5.4 mM calcium chloride dihydrate, and about 0.013% (w / v) polysorbate 20 or polysorbate 80. The present disclosure further provides a pharmaceutical composition comprising an FVIII polypeptide of about 500 IU / ml (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), about 1.33% (w / v) sucrose, about 205 mM NaCl, about 6.64 mM L-histidine, about 5.4 mM calcium chloride dihydrate, and about 0.013% (w / v) polysorbate 20 or polysorbate 80.The present disclosure further provides a pharmaceutical composition comprising an FVIII polypeptide of about 667 IU / ml (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), about 1.33% (w / v) sucrose, about 205 mM NaCl, about 6.64 mM L-histidine, about 5.4 mM calcium chloride dihydrate, and about 0.013% (w / v) polysorbate 20 or polysorbate 80. The present disclosure further provides a pharmaceutical composition comprising an FVIII polypeptide of about 1000 IU / ml (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), about 1.33% (w / v) sucrose, about 205 mM NaCl, about 6.64 mM L-histidine, about 5.4 mM calcium chloride, and about 0.013% (w / v) polysorbate 20 or polysorbate 80. The present disclosure also provides a pharmaceutical composition comprising an FVIII polypeptide of about 1333 IU / ml (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), about 1.33% (w / v) sucrose, about 205 mM NaCl, about 6.64 mM L-histidine, about 5.4 mM calcium chloride, and about 0.013% (w / v) polysorbate 20 or polysorbate 80. In some embodiments, the present disclosure provides a pharmaceutical composition comprising an FVIII polypeptide of about 1667 IU / ml (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), about 1.33% (w / v) sucrose, about 205 mM NaCl, about 6.64 mM L-histidine, about 5.4 mM calcium chloride, and about 0.013% (w / v) polysorbate 20 or polysorbate 80. In other embodiments, the present disclosure provides a pharmaceutical composition comprising an FVIII polypeptide of about 2000 IU / ml (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), about 1.33% (w / v) sucrose, about 205 mM NaCl, about 6.64 mM L-histidine, about 5.4 mM calcium chloride, and about 0.013% (w / v) polysorbate 20 or polysorbate 80.

[0078] The present disclosure further provides a pharmaceutical composition comprising an FVIII polypeptide of about 83 IU / ml (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), about 13.3 mg / ml of sucrose, about 12.0 mg / ml of NaCl, about 1.03 mg / ml of L-histidine, about 0.80 mg / ml of calcium chloride dihydrate, and about 0.10 mg / ml of polysorbate 20 or polysorbate 80. The present disclosure further provides a pharmaceutical composition comprising an FVIII polypeptide of about 1673 IU / ml (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), about 13.3 mg / ml of sucrose, about 12.0 mg / ml of NaCl, about 1.03 mg / ml of L-histidine, about 0.80 mg / ml of calcium chloride dihydrate, and about 0.10 mg / ml of polysorbate 20 or polysorbate 80. The present disclosure further provides a pharmaceutical composition comprising an FVIII polypeptide of about 250 IU / ml (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), about 13.3 mg / ml of sucrose, about 12.0 mg / ml of NaCl, about 1.03 mg / ml of L-histidine, about 0.80 mg / ml of calcium chloride dihydrate, and about 0.10 mg / ml of polysorbate 20 or polysorbate 80. The present disclosure further provides a pharmaceutical composition comprising an FVIII polypeptide of about 333 IU / ml (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), about 13.3 mg / ml of sucrose, about 12.0 mg / ml of NaCl, about 1.03 mg / ml of L-histidine, about 0.80 mg / ml of calcium chloride dihydrate, and about 0.10 mg / ml of polysorbate 20 or polysorbate 80.The present disclosure further provides a pharmaceutical composition comprising about 500 IU / ml of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), about 13.3 mg / ml of sucrose, about 12.0 mg / ml of NaCl, about 1.03 mg / ml of L-histidine, about 0.80 mg / ml of calcium chloride dihydrate, and about 0.10 mg / ml of polysorbate 20 or polysorbate 80. The present disclosure further provides a pharmaceutical composition comprising about 667 IU / ml of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), about 13.3 mg / ml of sucrose, about 12.0 mg / ml of NaCl, about 1.03 mg / ml of L-histidine, about 0.80 mg / ml of calcium chloride dihydrate, and about 0.10 mg / ml of polysorbate 20 or polysorbate 80. The present disclosure further provides a pharmaceutical composition comprising about 1000 IU / ml of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), about 13.3 mg / ml of sucrose, about 12.0 mg / ml of NaCl, about 1.03 mg / ml of L-histidine, about 0.80 mg / ml of calcium chloride dihydrate, and about 0.10 mg / ml of polysorbate 20 or polysorbate 80. The present disclosure also provides a pharmaceutical composition comprising about 1333 IU / ml of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), about 13.3 mg / ml of sucrose, about 12.0 mg / ml of NaCl, about 1.03 mg / ml of L-histidine, about 0.80 mg / ml of calcium chloride dihydrate, and about 0.10 mg / ml of polysorbate 20 or polysorbate 80.The present disclosure provides a pharmaceutical composition comprising an FVIII polypeptide at about 1667 IU / ml (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), about 13.3 mg / ml of sucrose, about 12.0 mg / ml of NaCl, about 1.03 mg / ml of L-histidine, about 0.80 mg / ml of calcium chloride dihydrate, and about 0.10 mg / ml of polysorbate 20 or polysorbate 80. In other embodiments, the present disclosure provides a pharmaceutical composition comprising an FVIII polypeptide at about 2000 IU / ml (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), about 13.3 mg / ml of sucrose, about 12.0 mg / ml of NaCl, about 1.03 mg / ml of L-histidine, about 0.80 mg / ml of calcium chloride dihydrate, and about 0.10 mg / ml of polysorbate 20 or polysorbate 80.

[0079] The present disclosure also provides components of a pharmaceutical kit. Such a kit includes one or more containers and any accessories. The kits provided herein facilitate the administration of an effective amount of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc) to a subject in need thereof. In certain embodiments, the kit facilitates the administration of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc) via intravenous infusion. In certain embodiments, the kit facilitates the self-administration of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc) via intravenous infusion.

[0080] In certain embodiments, the present disclosure provides a pharmaceutical kit comprising a first container containing a lyophilized powder or cake, wherein the powder or cake comprises (i) an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), (ii) sucrose (and / or trehalose, raffinose, arginine), (iii) NaCl, (iv) L-histidine, (v) calcium chloride dihydrate, and (vi) polysorbate 20 or polysorbate 80; and a second container containing sterile water for injection to be combined with the lyophilized powder of the first container. In certain embodiments, a diluent sufficient to produce a formulation of about 3 ml of FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc) having the desired properties disclosed herein is provided. In certain embodiments, the second container is a filled syringe associated with a plunger to allow addition of the diluent to the first container, reconstitution of the contents of the first container, and transfer back into a syringe. In certain embodiments, the kit further provides an adapter for attaching a syringe to the first container. In certain embodiments, the kit further provides a needle and an infusion tube attached to the syringe containing the reconstituted FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc) formulation to allow IV infusion of the formulation.

[0081] In certain embodiments, the FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc) is provided in a total amount of about 200 IU to about 6000 IU, such as about 250 IU, about 500 IU, about 750 IU, about 1000 IU, about 1500 IU, about 2000 IU, about 3000 IU, about 4000 IU, about 5000 IU, or about 6000 IU.

[0082] In one embodiment, there is provided a first container containing a lyophilized powder, the powder comprising: (i) about 250 IU of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); (ii) about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg of L-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi) about 0.40 mg of polysorbate 20 or polysorbate 80; and a second container containing sterile water for injection, the second container containing sterile water for injection in a volume sufficient to produce a solution comprising: (i) about 83 IU / ml of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); (ii) about 1.33% (w / v) of sucrose; (iii) about 205 mM of NaCl; (iv) about 6.64 mM of L-histidine; (v) about 5.4 mM of calcium chloride; and (vi) about 0.013% (w / v) of polysorbate 20 or polysorbate 80 when combined with the lyophilized powder of the first container. A pharmaceutical kit is provided comprising the first container and the second container.

[0083] In a further embodiment, there is provided a first container containing a lyophilized powder, the powder comprising: (i) about 500 IU of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); (ii) about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg of L-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi) about 0.40 mg of polysorbate 20 or polysorbate 80; and a second container containing sterile water for injection, the second container containing sterile water for injection in a volume sufficient to produce a solution comprising: (i) about 167 IU / ml of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); (ii) about 1.33% (w / v) of sucrose; (iii) about 205 mM of NaCl; (iv) about 6.64 mM of L-histidine; (v) about 5.4 mM of calcium chloride; and (vi) about 0.013% (w / v) of polysorbate 20 or polysorbate 80 when combined with the lyophilized powder of the first container. A pharmaceutical kit is provided comprising the first container and the second container.

[0084] In a further embodiment, a first container containing a lyophilized powder, the powder comprising (i) about 750 IU of FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), (ii) about 40 mg of sucrose, (iii) about 36 mg of NaCl, (iv) about 3.1 mg of L-histidine, (v) about 2.4 mg of calcium chloride dihydrate, and (vi) about 0.40 mg of polysorbate 20 or polysorbate 80; and a second container containing sterile water for injection, the second container containing sterile water for injection in a volume sufficient to produce a solution comprising (i) about 250 IU / ml of FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), (ii) about 1.33% (w / v) of sucrose, (iii) about 205 mM of NaCl, (iv) about 6.64 mM of L-histidine, (v) about 5.4 mM of calcium chloride, and (vi) about 0.013% (w / v) of polysorbate 20 or polysorbate 80 when combined with the lyophilized powder of the first container, are provided in a pharmaceutical kit.

[0085] In a further embodiment, there is provided a first container containing a lyophilized powder, the powder comprising: (i) about 1000 IU of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); (ii) about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg of L-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi) about 0.40 mg of polysorbate 20 or polysorbate 80; and a second container containing sterile water for injection, the second container containing sterile water for injection in a volume sufficient to produce a solution comprising: (i) about 333 IU / ml of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); (ii) about 1.33% (w / v) of sucrose; (iii) about 205 mM of NaCl; (iv) about 6.64 mM of L-histidine; (v) about 5.4 mM of calcium chloride; and (vi) about 0.013% (w / v) of polysorbate 20 or polysorbate 80 when combined with the lyophilized powder of the first container. A pharmaceutical kit is provided comprising the first container and the second container.

[0086] In a further embodiment, there is provided a first container containing a lyophilized powder, the powder comprising: (i) about 1500 IU of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); (ii) about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg of L-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi) about 0.40 mg of polysorbate 20 or polysorbate 80; and a second container containing sterile water for injection, the second container containing sterile water for injection in a volume sufficient to produce a solution comprising: (i) about 500 IU / ml of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); (ii) about 1.33% (w / v) of sucrose; (iii) about 205 mM of NaCl; (iv) about 6.64 mM of L-histidine; (v) about 5.4 mM of calcium chloride; and (vi) about 0.013% (w / v) of polysorbate 20 or polysorbate 80 when combined with the lyophilized powder of the first container. A pharmaceutical kit is provided comprising the first container and the second container.

[0087] In a further embodiment, a first container containing a lyophilized powder, the powder comprising: (i) about 2000 IU of FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), (ii) about 40 mg of sucrose, (iii) about 36 mg of NaCl, (iv) about 3.1 mg of L-histidine, (v) about 2.4 mg of calcium chloride dihydrate, and (vi) about 0.40 mg of polysorbate 20 or polysorbate 80; and a second container containing sterile water for injection, the second container containing sterile water for injection in a volume sufficient to produce a solution comprising: (i) about 667 IU / ml of FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), (ii) about 1.33% (w / v) of sucrose, (iii) about 205 mM of NaCl, (iv) about 6.64 mM of L-histidine, (v) about 5.4 mM of calcium chloride, and (vi) about 0.013% (w / v) of polysorbate 20 or polysorbate 80 when combined with the lyophilized powder of the first container. A pharmaceutical kit is provided comprising the first container and the second container.

[0088] In a further embodiment, there is provided a first container containing a lyophilized powder, the powder comprising: (i) about 3000 IU of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); (ii) about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg of L-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi) about 0.40 mg of polysorbate 20 or polysorbate 80; and a second container containing sterile water for injection, the second container containing sterile water for injection in a volume sufficient to produce a solution comprising: (i) about 1000 IU / ml of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); (ii) about 1.33% (w / v) of sucrose; (iii) about 205 mM of NaCl; (iv) about 6.64 mM of L-histidine; (v) about 5.4 mM of calcium chloride; and (vi) about 0.013% (w / v) of polysorbate 20 or polysorbate 80 when combined with the lyophilized powder of the first container. A pharmaceutical kit is provided comprising the first container and the second container.

[0089] In a further embodiment, there is provided a first container containing a lyophilized powder, the powder comprising: (i) about 4000 IU of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); (ii) about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg of L-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi) about 0.40 mg of polysorbate 20 or polysorbate 80; and a second container containing sterile water for injection, the second container containing sterile water for injection in a volume sufficient to produce a solution comprising: (i) about 1333 IU / ml of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); (ii) about 1.33% (w / v) of sucrose; (iii) about 205 mM of NaCl; (iv) about 6.64 mM of L-histidine; (v) about 5.4 mM of calcium chloride; and (vi) about 0.013% (w / v) of polysorbate 20 or polysorbate 80 when combined with the lyophilized powder of the first container. A pharmaceutical kit is provided comprising the first container and the second container.

[0090] In a further embodiment, a first container containing a lyophilized powder, the powder comprising: (i) about 5000 IU of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); (ii) about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg of L-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi) about 0.40 mg of polysorbate 20 or polysorbate 80; a first container; and a second container containing sterile water for injection, the second container containing sterile water for injection in a volume sufficient to produce a solution comprising: (i) about 1667 IU / ml of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); (ii) about 1.33% (w / v) of sucrose; (iii) about 205 mM of NaCl; (iv) about 6.64 mM of L-histidine; (v) about 5.4 mM of calcium chloride; and (vi) about 0.013% (w / v) of polysorbate 20 or polysorbate 80 when combined with the lyophilized powder of the first container. A pharmaceutical kit is provided.

[0091] In a further embodiment, a first container containing a lyophilized powder, the powder comprising: (i) about 6000 IU of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); (ii) about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg of L-histidine; (v) about 2.4 mg of calcium chloride dihydrate; (vi) about 0.40 mg of polysorbate 20 or polysorbate 80; and a second container containing sterile water for injection, the second container containing sterile water for injection in a volume sufficient to produce a solution comprising: (i) about 2000 IU / ml of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); (ii) about 1.33% (w / v) of sucrose; (iii) about 205 mM of NaCl; (iv) about 6.64 mM of L-histidine; (v) about 5.4 mM of calcium chloride; (vi) about 0.013% (w / v) of polysorbate 20 or polysorbate 80 when combined with the lyophilized powder of the first container. A pharmaceutical kit is provided comprising the first container and the second container.

[0092] In a further embodiment, there is provided a first container containing a lyophilized powder, the powder comprising: (i) about 250 IU of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); (ii) about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg of L-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi) about 0.40 mg of polysorbate 20 or polysorbate 80; and a second container containing sterile water for injection, the second container containing sterile water for injection in a volume sufficient to produce a solution comprising: (i) about 83 IU / ml of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); (ii) about 13.3 mg / ml of sucrose; (iii) about 12.0 mg / ml of NaCl; (iv) about 1.03 mg / ml of L-histidine; (v) about 0.80 mg / ml of calcium chloride dihydrate; and (vi) about 0.13 mg / ml of polysorbate 20 or polysorbate 80 when combined with the lyophilized powder of the first container. A pharmaceutical kit is provided comprising the first container and the second container.

[0093] In a further embodiment, there is provided a first container containing a lyophilized powder, the powder comprising: (i) about 500 IU of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); (ii) about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg of L-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi) about 0.40 mg of polysorbate 20 or polysorbate 80; and a second container containing sterile water for injection, the second container containing sterile water for injection in a volume sufficient to produce a solution comprising: (i) about 167 IU / ml of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); (ii) about 13.3 mg / ml of sucrose; (iii) about 12.0 mg / ml of NaCl; (iv) about 1.03 mg / ml of L-histidine; (v) about 0.80 mg / ml of calcium chloride dihydrate; and (vi) about 0.13 mg / ml of polysorbate 20 or polysorbate 80 when combined with the lyophilized powder of the first container. A pharmaceutical kit is provided comprising the first container and the second container.

[0094] In a further embodiment, a first container containing a lyophilized powder, the powder comprising: (i) about 750 IU of FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); (ii) about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg of L-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi) about 0.40 mg of polysorbate 20 or polysorbate 80; and a second container containing sterile water for injection, the second container containing sterile water for injection in a volume sufficient to produce a solution comprising: (i) about 250 IU / ml of FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); (ii) about 13.3 mg / ml of sucrose; (iii) about 12.0 mg / ml of NaCl; (iv) about 1.03 mg / ml of L-histidine; (v) about 0.80 mg / ml of calcium chloride dihydrate; and (vi) about 0.13 mg / ml of polysorbate 20 or polysorbate 80 when combined with the lyophilized powder of the first container. A pharmaceutical kit is provided.

[0095] In a further embodiment, there is provided a first container containing a lyophilized powder, the powder comprising: (i) about 1000 IU of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); (ii) about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg of L-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi) about 0.40 mg of polysorbate 20 or polysorbate 80; and a second container containing sterile water for injection, the second container containing sterile water for injection in a volume sufficient to produce a solution comprising: (i) about 333 IU / ml of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); (ii) about 13.3 mg / ml of sucrose; (iii) about 12.0 mg / ml of NaCl; (iv) about 1.03 mg / ml of L-histidine; (v) about 0.80 mg / ml of calcium chloride dihydrate; and (vi) about 0.13 mg / ml of polysorbate 20 or polysorbate 80 when combined with the lyophilized powder of the first container. A pharmaceutical kit is provided comprising the first container and the second container.

[0096] In a further embodiment, there is provided a first container containing a lyophilized powder, the powder comprising: (i) about 1500 IU of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); (ii) about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg of L-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi) about 0.40 mg of polysorbate 20 or polysorbate 80; and a second container containing sterile water for injection, the second container containing sterile water for injection in a volume sufficient to produce a solution comprising: (i) about 500 IU / ml of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); (ii) about 13.3 mg / ml of sucrose; (iii) about 12.0 mg / ml of NaCl; (iv) about 1.03 mg / ml of L-histidine; (v) about 0.80 mg / ml of calcium chloride dihydrate; and (vi) about 0.13 mg / ml of polysorbate 20 or polysorbate 80 when combined with the lyophilized powder of the first container. A pharmaceutical kit is provided comprising the first container and the second container.

[0097] In a further embodiment, there is provided a first container containing a lyophilized powder, the powder comprising: (i) about 2000 IU of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); (ii) about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg of L-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi) about 0.40 mg of polysorbate 20 or polysorbate 80; and a second container containing sterile water for injection, the second container containing sterile water for injection in a volume sufficient to produce a solution comprising: (i) about 667 IU / ml of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); (ii) about 13.3 mg / ml of sucrose; (iii) about 12.0 mg / ml of NaCl; (iv) about 1.03 mg / ml of L-histidine; (v) about 0.80 mg / ml of calcium chloride dihydrate; and (vi) about 0.13 mg / ml of polysorbate 20 or polysorbate 80 when combined with the lyophilized powder of the first container. A pharmaceutical kit is provided comprising the first container and the second container.

[0098] In a further embodiment, there is provided a first container containing a lyophilized powder, the powder comprising: (i) about 3000 IU of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); (ii) about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg of L-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi) about 0.40 mg of polysorbate 20 or polysorbate 80; and a second container containing sterile water for injection, the second container containing sterile water for injection in a volume sufficient to produce a solution comprising: (i) about 1000 IU / ml of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); (ii) about 13.3 mg / ml of sucrose; (iii) about 12.0 mg / ml of NaCl; (iv) about 1.03 mg / ml of L-histidine; (v) about 0.80 mg / ml of calcium chloride dihydrate; and (vi) about 0.13 mg / ml of polysorbate 20 or polysorbate 80 when combined with the lyophilized powder of the first container. A pharmaceutical kit is provided comprising the first container and the second container.

[0099] In a further embodiment, there is provided a first container containing a lyophilized powder, the powder comprising: (i) about 4000 IU of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); (ii) about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg of L-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi) about 0.40 mg of polysorbate 20 or polysorbate 80; and a second container containing sterile water for injection, the second container containing sterile water for injection in a volume sufficient to produce a solution comprising: (i) about 1333 IU / ml of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); (ii) about 13.3 mg / ml of sucrose; (iii) about 12.0 mg / ml of NaCl; (iv) about 1.03 mg / ml of L-histidine; (v) about 0.80 mg / ml of calcium chloride dihydrate; and (vi) about 0.13 mg / ml of polysorbate 20 or polysorbate 80 when combined with the lyophilized powder of the first container. A pharmaceutical kit is provided comprising the first container and the second container.

[0100] In a further embodiment, a first container containing a lyophilized powder, the powder comprising: (i) about 5000 IU of FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); (ii) about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg of L-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi) about 0.40 mg of polysorbate 20 or polysorbate 80; and a second container containing sterile water for injection, the second container containing sterile water for injection in a volume sufficient to produce a solution comprising: (i) about 1667 IU / ml of FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); (ii) about 13.3 mg / ml of sucrose; (iii) about 12.0 mg / ml of NaCl; (iv) about 1.03 mg / ml of L-histidine; (v) about 0.80 mg / ml of calcium chloride dihydrate; and (vi) about 0.13 mg / ml of polysorbate 20 or polysorbate 80 when combined with the lyophilized powder of the first container. A pharmaceutical kit is provided comprising the first container and the second container.

[0101] In a further embodiment, a first container containing a lyophilized powder, the powder comprising: (i) about 6000 IU of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), (ii) about 40 mg of sucrose, (iii) about 36 mg of NaCl, (iv) about 3.1 mg of L-histidine, (v) about 2.4 mg of calcium chloride dihydrate, and (vi) about 0.40 mg of polysorbate 20 or polysorbate 80; and a second container containing sterile water for injection, the second container containing an amount of sterile water for injection sufficient to produce a solution comprising: (i) about 2000 IU / ml of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), (ii) about 13.3 mg / ml of sucrose, (iii) about 12.0 mg / ml of NaCl, (iv) about 1.03 mg / ml of L-histidine, (v) about 0.80 mg / ml of calcium chloride dihydrate, and (vi) about 0.13 mg / ml of polysorbate 20 or polysorbate 80 when combined with the lyophilized powder of the first container. A pharmaceutical kit is provided comprising the first container and the second container.

[0102] In certain embodiments, the first container of the pharmaceutical kit provided herein is a glass vial with a rubber stopper. In certain embodiments, the second container of the pharmaceutical kit provided herein is a syringe body associated with a plunger. In certain embodiments, the syringe is a pre-filled syringe containing a diluent. In certain embodiments, the pharmaceutical kit provided herein further comprises an adapter for connecting the glass vial to the syringe body. In certain embodiments, the pharmaceutical kit provided herein further comprises an infusion tube that connects to a needle that attaches to a syringe suitable for intravenous infusion.

[0103] In certain embodiments, the desired administration of an FVIII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide, such as rFVIIIFc) can be achieved by use of one of the pharmaceutical kits provided herein. In certain embodiments, two or more pharmaceutical kits can be used to achieve the desired administration. Methods are provided herein for combining or pooling the formulations contained in two or more pharmaceutical kits, as provided herein, to achieve the desired administration.

[0104] In some embodiments, the FVIII polypeptide for the pharmaceutical composition is a short-acting FVIII polypeptide. In other embodiments, the FVIII polypeptide for the pharmaceutical composition is a long-acting FVIII polypeptide.

[0105] In some embodiments, the long-acting FVIII polypeptide in the pharmaceutical composition comprises a Factor VIII moiety and a non-Factor VIII moiety, such as a heterologous moiety. In one embodiment, the heterologous moiety is capable of extending the in vivo or in vitro half-life of the FVIII polypeptide. Exemplary non-Factor VIII moieties include, but are not limited to, Fc, albumin, a PAS sequence, transferrin, CTP (the 28 amino acid C-terminal peptide (CTP) of human chorionic gonadotropin (hCG) having four O-glycans), polyethylene glycol (PEG), hydroxyethyl starch (HES), an albumin-binding polypeptide, an albumin-binding small molecule, or any combination thereof. Exemplary long-acting polypeptides of the invention include, for example, Factor VIII Fc polypeptide, Factor VIII albumin polypeptide, Factor VIII PAS polypeptide, Factor VIII transferrin polypeptide, Factor VIII CTP polypeptide, Factor VIII PEG polypeptide, Factor VIII HES polypeptide, Factor VIII albumin-binding polypeptide polypeptide, or Factor VIII albumin-binding small molecule polypeptide.

[0106] Factor VIII (the Factor VIII portion of the long-acting FVIII polypeptide or the FVIII of the short-acting FVIII polypeptide) may be at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the Factor VIII amino acid sequences shown in Table 11 (amino acids 20 to 1457 of SEQ ID NO: 2, amino acids 20 to 2351 of SEQ ID NO: 6) without a signal sequence, and the Factor VIII portion has Factor VIII activity. Factor VIII (the Factor VIII portion of the chimeric polypeptide) may be identical to the Factor VIII amino acid sequences shown in Table 11 (amino acids 20 to 1457 of SEQ ID NO: 2, amino acids 20 to 2351 of SEQ ID NO: 6) without a signal sequence.

[0107] Factor VIII (the Factor VIII portion of the long-acting FVIII polypeptide or the FVIII of the short-acting FVIII polypeptide) may be at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the Factor VIII amino acid sequences shown in Table 11 (amino acids 1 to 1457 of SEQ ID NO: 2, amino acids 1 to 2351 of SEQ ID NO: 6) with a signal sequence, and the Factor VIII portion has Factor VIII activity. Factor VIII (the Factor VIII portion of the long-acting FVIII polypeptide or the short-acting FVIII polypeptide) may be identical to the Factor VIII amino acid sequences shown in Table 11 (amino acids 1 to 1457 of SEQ ID NO: 2 and amino acids 1 to 2351 of SEQ ID NO: 6) with a signal sequence.

[0108] The long-acting polypeptide may contain a sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the Factor VIII and Fc amino acid sequences shown in Table 11A(i) (amino acids 20-1684 of SEQ ID NO: 2) without a signal sequence, or at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the Factor VIII and Fc amino acid sequences shown in Table 11A(i) (amino acids 1-1684 of SEQ ID NO: 2) with a signal sequence, and the said sequence has Factor VIII activity. The Factor VIII activity can be measured by an activated partial thromboplastin time (aPTT) assay, a chromogenic assay, or other known methods. The long-acting FVIII polypeptide may contain a sequence that is identical to the Factor VIII and Fc amino acid sequences shown in Table 11A(i) (amino acids 20-1684 of SEQ ID NO: 2) without a signal sequence, or identical to the Factor VIII and Fc amino acid sequences shown in Table 11A(i) (amino acids 1-1684 of SEQ ID NO: 2) with a signal sequence.

[0109] As discussed above, exemplary long-acting polypeptides also include Factor VIII fused to one or more albumin polypeptides, albumin-binding polypeptides, or albumin-binding small molecules. In one embodiment, the albumin is human albumin. The albumin or albumin-binding protein can be fused to either the N-terminus or the C-terminus of FVIII, or inserted between two amino acids in FVIII. Examples of albumin that can be used in the present invention, such as fragments thereof, are known. For example, U.S. Patent No. 7,592,010, U.S. Patent No. 6,686,179, and Schulte, Thrombosis Res. 124 Suppl. 2: S6-S8 (2009) (each of which is hereby incorporated by reference in its entirety).

[0110] Albumin-binding polypeptides can impair, but are not limited to, bacterial albumin-binding domains, albumin-binding peptides, or albumin-binding antibody fragments that can bind to albumin. Domain 3 of streptococcal protein G, disclosed by Kraulis et al., FEBS Lett. 378:190-194 (1996) and Linhult et al., Protein Sci. 11:206-213 (2002), is an example of a bacterial albumin-binding domain. Examples of albumin-binding peptides include a series of peptides having the core sequence DICLPRWGCLW (SEQ ID NO:7). See, for example, Dennis et al., J. Biol. Chem. 2002, 277:35035-35043 (2002). Examples of albumin-binding antibody fragments are disclosed in Muller and Kontermann, Curr. Opin. Mol. Ther. 9:319-326 (2007); Roovers et al., Cancer Immunol. Immunother. 56:303-317 (2007) and Holt et al., Prot. Eng. Design Sci., 21:283-288 (2008) (which are incorporated herein by reference in their entirety).

[0111] In certain embodiments, the long-acting FVIII polypeptide of the invention comprises at least one binding site for a non-polypeptide small molecule, variant, or derivative that can bind to albumin. An example of such an albumin-binding moiety is 2-(3-maleimidopropanamido)-6-(4-(4-iodophenyl)butanamido)hexanoic acid (the "Albu" tag) disclosed by Trusselet et al., Bioconjugate Chem. 20:2286-2292 (2009).

[0112] As discussed above, exemplary long-acting polypeptides also include Factor VIII fused to at least one C-terminal peptide (CTP) of the β subunit of human chorionic gonadotropin, or fragments, variants, or derivatives thereof. The CTP can be fused to either the N-terminus or the C-terminus of Factor VIII, FVIII, or inserted between two amino acids in FVIII. One or more CTP peptides fused or inserted into a recombinant protein are known to increase the in vivo half-life of the protein. See, for example, U.S. Patent No. 5,712,122, which is incorporated herein by reference in its entirety. Exemplary CTP peptides include DPRFQDSSSSKAPPPSLPSPSRLPGPSDTPIL (SEQ ID NO: 8) or SSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 9). See, for example, U.S. Patent Application Publication No. 2009 / 0087411 A1, which is incorporated by reference.

[0113] As discussed above, exemplary long-acting FVIII polypeptides also include Factor VIII fused to at least one PAS sequence, or fragments, variants, or derivatives thereof. The PAS sequence can be fused to either the N-terminus or the C-terminus of FVIII, or inserted between two amino acids in FVIII. As used herein, a PAS peptide or PAS sequence means an amino acid sequence that consists mainly of alanine and serine residues, or consists mainly of alanine, serine, and proline residues, and that forms a random coil conformation under physiological conditions. Thus, a PAS sequence is a building block, amino acid polymer, or sequence cassette that contains, consists essentially of, or consists of alanine, serine, and proline and can be used as part of a heterologous moiety in a chimeric protein. The amino acid polymer can also form a random coil conformation when residues other than alanine, serine, and proline are added as minor components to the PAS sequence. By "minor component" is meant that an amino acid other than alanine, serine, and proline can be added to the PAS sequence to some extent, for example, up to about 12% of the amino acids, i.e., about 12 out of 100 amino acids of the PAS sequence, up to about 10%, up to about 9%, up to about 8%, about 6%, about 5%, about 4%, about 3%, i.e., about 2%, or about 1%. Amino acids different from alanine, serine, and proline can be selected from the group consisting of Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr, and Val. Under physiological conditions, the PAS peptide forms a random coil conformation, thereby mediating increased in vivo and / or in vitro stability for the recombinant proteins of the invention and having procoagulant activity.

[0114] Non-limiting examples of PAS peptides include ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 10), AAPASPAPAAPSAPAPAAPS (SEQ ID NO: 11), APSSPSPSAPSSPSPASPSS (SEQ ID NO: 12), APSSPSPSAPSSPSPASPS (SEQ ID NO: 13), SSPSAPSPSSPASPSPSSPA (SEQ ID NO: 14), AASPAAPSAPPAAASPAAPSAPPA (SEQ ID NO: 15), ASAAAPAAASAAASAPSAAA (SEQ ID NO: 16), or any variants, derivatives, fragments, or combinations thereof. Further examples of PAS sequences are known, for example, from US Patent Publication No. 2010 / 0292130 A1 and PCT Application Publication No. WO2008 / 155134 A1, and European Patent EP2173890.

[0115] As discussed above, exemplary long-acting FVIII polypeptides also include Factor VIII fused to at least one transferrin peptide, or fragments, variants, or derivatives thereof. The at least one transferrin peptide can be fused either to the N-terminus of FVIII or the C-terminus of FVIII, or inserted between two amino acids in FVIII. Any transferrin can be fused or inserted into the recombinant FVIII protein of the invention. As an example, wild-type human Tf (Tf) is a protein of approximately 679 amino acids of approximately 75 kDa (without considering glycosylation), having two major domains N (about 330 amino acids) and C (about 340 amino acids), which appears to be derived from gene duplication. See GenBank accession numbers NM001063, XM002793, M12530, XM039845, XM039847, and S95936 (www.ncbi.nlm.nih.gov) (all of which are hereby incorporated by reference in their entirety).

[0116] Transferrin transports iron through transferrin receptor (TfR)-mediated endocytosis. After the iron is released into the endosomal compartment and the Tf-TfR complex is recycled to the cell surface, Tf is released back into the extracellular space for the next iron transport cycle. Tf has a long half-life exceeding 14 to 17 days (Li et al., Trends Pharmacol. Sci. 23:206-209 (2002)). Transferrin fusion proteins have been studied for half-life extension, targeted delivery for cancer therapy, oral delivery, and sustained activation of proinsulin (Brandsma et al., Biotechnol. Adv., 29:230-238 (2011), Bai et al., Proc. Natl. Acad. Sci. USA 102:7292-729 6 (2005), Kim et al., J. Pharmacol. Exp. Ther., 334:682-692 (2010), Wang et al., J. Controlled Release 155:386-392 (2011)).

[0117] As discussed above, an exemplary long-acting FVIII polypeptide also comprises Factor VIII fused to at least one polyethylene glycol (PEG) moiety.

[0118] PEGylated FVIII can refer to a complex formed between FVIII and at least one polyethylene glycol (PEG) molecule. PEG is commercially available in a wide range of molecular weights and average molecular weights. Typical examples of the range of average molecular weights of PEG include, but are not limited to, about 200, about 300, about 400, about 600, about 1000, about 1300 - 1600, about 1450, about 2000, about 3000, about 3000 - 3750, about 3350, about 3000 - 7000, about 3500 - 4500, about 5000 - 7000, about 7000 - 9000, about 8000, about 10000, about 8500 - 11500, about 16000 - 24000, about 35000, about 40000, about 60000, and about 80000 daltons. These average molecular weights are provided merely as examples and are in no way intended to be limiting.

[0119] The long - acting FVIII polypeptides useful in the present invention can be PEGylated to include mono - or poly - (e.g., 2 - 4) PEG moieties. PEGylation can be carried out by any of the PEGylation reactions known in the art. Methods for preparing PEGylated protein products generally involve (i) reacting a polypeptide with a polyethylene glycol (such as a reactive ester or aldehyde derivative of PEG) under conditions such that the peptide of the present invention binds to one or more PEG groups, and (ii) obtaining the reaction product(s). Generally, the optimal reaction conditions for the reaction are determined individually based on known parameters and the desired results.

[0120] For example, Malik F et al., Exp. Hematol. 20:1028 - 35 (1992), Francis, Focus on Growth Factors There are several PEG conjugation methods available to those skilled in the art, such as those described in 3(2):4-10(1992), European Patent Publication Nos. EP0401384, EP0154316, and EP0401384, as well as International Patent Application Publication Nos. WO92 / 16221 and WO95 / 34326. As a non-limiting example, an FVIII variant can contain cysteine substituents in one or more insertion sites in FVIII, and the cysteine can be further complexed to a PEG polymer. See Mei et al., Blood 116:270-279(2010) and U.S. Patent No. 7,632,921, which are hereby incorporated by reference in their entirety.

[0121] As discussed above, exemplary long-acting FVIII polypeptides also include Factor VIII fused to at least one hydroxyethyl starch (HES) polymer. HES is a derivative of naturally occurring amylopectin and is degraded by α-amylase in the body. HES exhibits advantageous biological properties and is used in clinics as a blood volume expander and in hemodilution therapy. See, for example, Sommermeyer et al., Krankenhauspharmazie 8:271-278(1987), and Weidler et al., Arzneim.-Forschung / Drug Res. 41:494-498(1991).

[0122] HES is mainly characterized by its molecular weight distribution and degree of substitution. HES has an average molecular weight (weight average) of 1 to 300 kD, 2 to 200 kD, 3 to 100 kD, or 4 to 70 kD. Hydroxyethyl starch may further exhibit a molar degree of substitution of 0.1 to 3, 0.1 to 2, 0.1 to 0.9, or 0.1 to 0.8, and a C2:C6 substitution ratio in the range of 2 to 20 with respect to the hydroxyethyl group. HES having an average molecular weight of about 130 kD is Fresenius' VOLUVEN®. VOLUVEN® is an artificial colloid used, for example, for volume replacement in therapeutic indications for the treatment and prevention of reduced blood volume. There are several HES conjugation methods available to those skilled in the art, such as the same PEG conjugation method described above.

[0123] In some embodiments, the long-acting FVIII polypeptide comprising the FVIII moiety has an increased half-life (t 1 / 2 ) compared to a polypeptide consisting of the same FVIII moiety without an FVIII-unrelated moiety. The long-acting FVIII polypeptide with an increased t 1 / 2 may be referred to herein as long-lasting FVIII. The long-acting chimeric factor VIII polypeptide includes, for example, factor VIII fused to Fc (including chimeric factor VIII polypeptides in hybrid forms such as FVIIIFc monomer-dimer hybrids; see Examples, Table 11A, and U.S. Patent Nos. 7,404,956 and 7,348,004) and factor VIII fused to albumin.

[0124] As used herein, the Factor VIII polypeptide is a functional Factor VIII polypeptide whose normal role is blood coagulation, unless otherwise specified. Thus, the term Factor VIII includes variant polypeptides that are functional. The Factor VIII protein can be a human, porcine, canine, and murine Factor VIII protein. Full-length polypeptide and polynucleotide sequences are known in many functional fragments, variants, and modified forms. Examples of the human Factor VIII sequence are shown as subsequences in SEQ ID NO: 2 or 6 (Table 11). Factor VIII polypeptides include, for example, full-length Factor VIII, full-length Factor VIII lacking Met at the N-terminus, mature Factor VIII (lacking the signal sequence), mature Factor VIII having an additional Met at the N-terminus, and / or Factor VIII with a complete or partial deletion of the B domain. Factor VIII variants include B domain deletions, whether partial or complete deletions.

[0125] A very large number of functional Factor VIII variants are known as discussed above and below. In addition, hundreds of non-functional mutations in Factor VIII have been identified in hemophilia patients, and it has been determined that the effect of these mutations on Factor VIII function is due largely to the location within the three-dimensional structure of Factor VIII rather than the nature of the substitution (Cutler et al., Hum. Mutat. 19:274-8 (2002), which is incorporated herein by reference in its entirety). In addition, comparison between Factor VIII from humans and other species has identified conserved residues that are likely required for function (Cameron et al., Thromb. Haemost. 79:317-22 (1998), US 6,251,632, which are incorporated herein by reference in their entirety).

[0126] The human Factor VIII gene has been isolated or expressed in mammalian cells (Toole, J.J., et al., Nature 312:342-347 (1984), Gitschier, J., et al., Nature 312:326-330 (1984), Wood, W.I., et al., Nature 312:330-337 (1984), Vehar, G.A., et al., Nature 312:337-342 (1984), WO87 / 04187, WO88 / 08035, WO88 / 03558, U.S. Patent No. 4,757,006, each of which is incorporated herein by reference in its entirety), and the amino acid sequence was deduced from cDNA. U.S. Patent No. 4,965,199 (Capon et al.) (incorporated herein by reference in its entirety) discloses a DNA recombination method for the production of Factor VIII in mammalian host cells and the purification of human Factor VIII. Human Factor VIII expression has been reported in CHO (Chinese hamster ovary) cells and BHK (baby hamster kidney cells). Human Factor VIII has been modified to delete some or all of the B domain (U.S. Patent Nos. 4,994,371 and 4,868,112, each of which is incorporated herein by reference in its entirety), and replacement of the human Factor VIII B domain with the human Factor V B domain has been carried out (U.S. Patent No. 5,004,803, incorporated herein by reference in its entirety). The cDNA sequence encoding human Factor VIII and the predicted amino acid sequence are shown in SEQ ID NOs: 1 and 2 of U.S. Patent Application Publication No. 2005 / 0100990 (incorporated herein by reference in its entirety).

[0127] U.S. Patent No. 5,859,204 (Lollar, J.S.), which is hereby incorporated by reference in its entirety, reports on functional variants of Factor VIII having reduced antigenicity and reduced immune response. U.S. Patent No. 6,376,463 (Lollar, J.S.), which is hereby incorporated by reference in its entirety, also reports on variants of Factor VIII having reduced immune response. U.S. Patent Application Publication No. 2005 / 0100990 (Saenko et al.) (which is hereby incorporated by reference in its entirety) reports on functional mutations in the A2 domain of Factor VIII.

[0128] Several functional Factor VIII molecules containing B domain deletions are disclosed in US6,316,226 and US6,346,513, both assigned to Baxter; US7,041,635 assigned to In2Gen; US5,789,203, US6,060,447, US5,595,886, and US6,228,620 assigned to Chiron; US5,972,885 and US6,048,720 assigned to Biovitrum; US5,543,502 and US5,610,278 assigned to Novo Nordisk; US5,171,844 assigned to Immuno Ag; US5,112,950 assigned to Transgene S.A.; US4,868,112 assigned to Genetics Institute, each of which is hereby incorporated by reference in its entirety.

[0129] The porcine factor VIII sequence has been published (Toole, J.J., et al., Proc. Natl. Acad. Sci. USA 83:5939-5942 (1986), which is hereby incorporated by reference in its entirety), and the complete porcine cDNA sequence obtained from PCR amplification of the factor VIII sequence from a porcine spleen cDNA library has been reported (Healey, J.F. et al., Blood 88:4209-4214 (1996), which is hereby incorporated by reference in its entirety). Hybrid human / porcine factor VIII with all domains, all subunits, and substitutions of specific amino acid sequences are disclosed in U.S. Patent No. 5,364,771 by Lollar and Runge, and in WO93 / 20093, both of which are hereby incorporated by reference in their entirety. More recently, the nucleotide and corresponding amino acid sequences of the A1 and A2 domains of porcine factor VIII and chimeric factor VIII, together with porcine A1 and / or A2 domains substituted with the corresponding human domains, were reported in WO94 / 11503 (which is hereby incorporated by reference in its entirety). U.S. Patent No. 5,859,204 (Lollar, J.S.) also discloses porcine cDNA and the deduced amino acid sequence. U.S. Patent No. 6,458,563, assigned to Emory (which is hereby incorporated by reference in its entirety), discloses B domain-deleted porcine factor VIII.

[0130] As used herein, the Factor VIII "B domain" is the same as the B domain known in the art, defined by internal amino acid sequence identity and proteolytic cleavage sites by thrombin (e.g., residues Ser741-Arg1648 of full-length mature human Factor VIII). Other human Factor VIII domains are defined by the following amino acid residues: A1, residues Ala1-Arg372; A2, residues Ser373-Arg740; A3, residues Ser1690-Ile2032; C1, residues Arg2033-Asn2172; C2, residues Ser2173-Tyr2332. The A3-C1-C2 sequence includes residues Ser1690-Tyr2332. The remaining sequence, residues Glu1649-Arg1689, is commonly referred to as the Factor VIII light chain activation peptide. For porcine, murine, and canine Factor VIII, the positions of the boundaries for all domains including the B domain are also known in the art. In one embodiment, the B domain of Factor VIII is deleted ("B domain deleted Factor VIII" or "BDD FVIII"). An example of BDD FVIII is REFACTO® (recombinant BDD FVIII), which has the sequence as the Factor VIII portion of the sequence in Table 11A(i) (amino acids 1-1457 or 20-1457 of SEQ ID NO: 2). In another embodiment, the B domain deleted Factor VIII contains an intact intracellular processing site, which corresponds to the arginine at residue 754 of the B domain deleted Factor VIII, which corresponds to the arginine residue 773 of SEQ ID NO: 2 or residue 1648 of full-length Factor VIII, which corresponds to the arginine residue 1667 of SEQ ID NO: 6. Sequence residue numbers used herein that do not refer to any SEQ ID NO correspond to the Factor VIII sequence without the signal peptide sequence (19 amino acids), unless otherwise specified. For example, S743 / Q1638 of full-length Factor VIII corresponds to S762 / Q1657 of SEQ ID NO: 6 as a result of the 19-amino acid signal peptide sequence.In other embodiments, the B-domain deleted FVIII comprises a substitution or mutation at the amino acid position corresponding to arginine 1645, a substitution or mutation at the amino acid position corresponding to arginine 1648, or a substitution or mutation at the amino acid positions corresponding to both arginine 1645 and arginine 1648 in the full-length Factor VIII. In some embodiments, the amino acid substituted at the amino acid position corresponding to arginine 1645 is a different amino acid than the amino acid substituted at the amino acid position corresponding to arginine 1648. In certain embodiments, the substitution or mutation is an amino acid other than arginine, for example, alanine.

[0131] The "Factor VIII lacking the B domain" can have complete or partial deletions disclosed in U.S. Patent Nos. 6,316,226, 6,346,513, 7,041,635, 5,789,203, 6,060,447, 5,595,886, 6,228,620, 5,972,885, 6,048,720, 5,543,502, 5,610,278, 5,171,844, 5,112,950, 4,868,112, and 6,458,563 (each of which is hereby incorporated by reference in its entirety). In some embodiments, the Factor VIII lacking the B domain sequence of the present invention includes any one of the deletions disclosed in column 4, row 4 to column 5, row 28 and Examples 1 to 5 of U.S. Patent No. 6,316,226 (similarly, US6,346,513). In some embodiments, the Factor VIII lacking the B domain of the present invention has the deletions disclosed in column 2, rows 26 to 51 and Examples 5 to 8 of U.S. Patent No. 5,789,203 (similarly, US6,060,447, US5,595,886, and US6,228,620). In some embodiments, the Factor VIII lacking the B domain has deletions described in column 1, row 25 to column 2, row 40 of U.S. Patent No. 5,972,885; columns 6, rows 1 to 22 and Example 1 of U.S. Patent No. 6,048,720; column 2, rows 17 to 46 of U.S. Patent No. 5,543,502; column 4, row 22 to column 5, row 36 of U.S. Patent No. 5,171,844; column 2, rows 55 to 68, Figure 2, and Example 1 of U.S. Patent No. 5,112,950; column 2, row 2 to column 19, row 21 and Table 2 of U.S. Patent No. 4,868,112; column 2, row 1 to column 3, row 19, column 3, row 40 to column 4, row 67, column 7, row 43 to column 8, row 26, column 11, row 5 to column 13, row 39 of U.S. Patent No. 7,041,635; or column 4, rows 25 to 53 of U.S. Patent No. 6,458,563.In some embodiments, the B-domain deleted Factor VIII has a deletion of most of the B domain but still contains the amino-terminal sequence of the B domain that is essential for in vivo proteolytic processing into the two polypeptide chains of the primary translation product (i.e., the intracellular processing site), as disclosed in WO91 / 09122, which is hereby incorporated by reference in its entirety. In some embodiments, the B-domain deleted Factor VIII is constructed by deletion of amino acids 747-1638, i.e., substantially complete deletion of the B domain. Hoeben R.C., et al. J. Biol. Chem. 265(13):7318-7323(1990), which is hereby incorporated by reference in its entirety. The B-domain deleted Factor VIII can also contain deletions of amino acids 771-1666 or 868-1562 of Factor VIII. Meulien P., et al. Protein Eng. 2(4):301-6(1988), which is hereby incorporated by reference in its entirety. Further B-domain deletions that are part of the present invention include, for example, deletions of amino acids 982-1562 or 760-1639 (Toole et al., Proc. Natl. Acad. Sci. U.S.A. 83:5939-5942(1986)), 797-1562 (Eaton et al., Biochemistry 25:8343-8347(1986)), 741-1646 (Kaufman (PCT Published Application No. WO87 / 04187)), 747-1560 (Sarver et al., DNA 6:553-564(1987)), 741-1648 (Pasek (PCT Application No. 88 / 00831)), 816-1598 or 741-1689 (Lagner (Behring Inst. Mitt. (1988) No82:16-25, EP295597), each of which is hereby incorporated by reference in its entirety.In some embodiments, the B domain deleted FVIII contains a partial deletion in the B domain, i.e., it has 21 amino acids derived from the B domain (i.e., SFSQNSRHPSQNPPVLKRHQR of SEQ ID NO: 17) as disclosed in U.S. Patent Application Publication No. 20100286067 and U.S. Patent Application Publication No. US2012009384, both of which are incorporated herein by reference in their entirety. Each of the aforementioned deletions can be made in any Factor VIII sequence.

[0132] In one embodiment, the B domain deleted Factor VIII portion in the FVIII polypeptide is processed into two chains connected (or linked) by a metal bond, wherein the first chain contains the heavy chain (A1 - A2 - partial B) and the second chain contains the light chain (A3 - C1 - C2). In another embodiment, the B domain deleted Factor VIII portion is a single - chain Factor VIII. The single - chain Factor VIII can contain an intracellular processing site corresponding to arginine at residue 754 of the B domain - deleted Factor VIII (residue 773 of SEQ ID NO: 2), or at residue 1648 of the full - length Factor VIII (residue 1657 of SEQ ID NO: 6).

[0133] The metal bond between the heavy chain and the light chain can be any metal known in the art. For example, the metal useful in the present invention can be a divalent metal ion. Metals that can be used to link the heavy chain and the light chain include Ca 2+ , Mn 2+ or Cu 2+ and are not limited thereto. Fatouros et al., Intern. J. Pharm. 155(1):121 - 131(1997), Wakabayashi et al., JBC. 279(13):12677 - 12684(2004).

[0134] The FVIII polypeptides used herein can include processed Factor VIII or single-chain Factor VIII or combinations thereof. As used herein, "processed Factor VIII" means Factor VIII cleaved at arginine 1648 (for full-length Factor VIII) or arginine 754 (for B-domain-deleted Factor VIII), i.e., Factor VIII cleaved at an intracellular processing site. Cleavage at the intracellular processing site results in processed Factor VIII comprising two polypeptide chains, a first chain that is the heavy chain and a second chain that is the light chain. For example, a processed Factor VIII Fc fusion protein (i.e., the heavy and light chains fused to Fc) migrates at approximately 90 kDa and 130 kDa, respectively, in non-reducing SDS-PAGE and at 90 kDa and 105 kDa, respectively, in reducing SDS-PAGE. Thus, in one embodiment, in a long-acting polypeptide or in a short-acting FVIII polypeptide, at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the Factor VIII portion is processed Factor VIII. In another embodiment, in a long-acting polypeptide or in a short-acting FVIII polypeptide, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the Factor VIII portion is processed Factor VIII. In certain embodiments, an FVIII polypeptide comprising processed Factor VIII is purified (isolated) from a polypeptide comprising single-chain Factor VIII, and in a long-acting polypeptide or in a short-acting FVIII polypeptide, at least about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the Factor VIII portion is processed Factor VIII. In some embodiments, the FVIII polypeptide comprises about 15% - 25% of a single-chain FVIII polypeptide and about 75% - about 85% of a processed FVIII polypeptide.

[0135] As used herein, "single-chain Factor VIII", "SC Factor VIII", or "SCFVIII" means Factor VIII that is not cleaved at the arginine site (residue 1648 for full-length Factor VIII (i.e., residue 1667 of SEQ ID NO: 6), or residue 754 for B-domain-deleted Factor VIII (i.e., residue 773 of SEQ ID NO: 2)). Thus, the single-chain Factor VIII in the FVIII polypeptide used herein contains a single chain. In one embodiment, the single-chain Factor VIII contains an intact intracellular processing site. In another embodiment, the single-chain Factor VIII of the present invention contains a substitution or mutation at the amino acid position corresponding to arginine 1645 in full-length Factor VIII, a substitution or mutation at the amino acid position corresponding to arginine 1648, or a substitution or mutation at the amino acid positions corresponding to arginine 1645 and arginine 1648. In other embodiments, the amino acid substituted at the amino acid position corresponding to arginine 1645 is a different amino acid from the amino acid substituted at the amino acid position corresponding to arginine 1648. In certain embodiments, the substitution or mutation is an amino acid other than arginine, such as isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, selenocysteine, serine, tyrosine, histidine, ornithine, pyrrolidine, or taurine. The single-chain Factor VIII Fc fusion protein can migrate at approximately 220 kDa in non-reducing SDS-PAGE and at approximately 195 kDa in reducing SDS-PAGE.

[0136] The Factor VIII moiety in the FVIII polypeptide used herein has Factor VIII activity. The Factor VIII activity can be measured by any method known in the art. For example, one of those methods can be a chromogenic assay. The chromogenic assay mechanism is based on the principle of the blood coagulation cascade that activated Factor VIII accelerates the conversion of Factor X to Factor Xa in the presence of activated Factor IX, phospholipids, and calcium ions. Factor Xa activity is evaluated by hydrolysis of a p-nitroanilide (pNA) substrate specific for Factor Xa. The initial rate of release of p-nitroaniline measured at 405 nM is directly proportional to Factor Xa activity and thus directly proportional to the Factor VIII activity in the sample. The chromogenic assay is recommended by the Factor VIII and Factor IX Subcommittee of the Scientific and Standardization Committee (SSC) of the International Society on Thrombosis and Hemostasis (ISTH). Since 1994, the chromogenic assay has also been the reference method for the assignment of the potency of FVIII concentrates in the European Pharmacopoeia. Thus, in one embodiment, an FVIII polypeptide comprising a single-chain Factor VIII has Factor VIII activity equivalent to that of an FVIII polypeptide comprising a processed Factor VIII when the Factor VIII activity is measured in vitro by a chromogenic assay.

[0137] In certain embodiments, the long-acting FVIII polypeptide is an FVIII monomer-dimer hybrid. To obtain the FVIII monomer-dimer hybrid, the coding sequence of human recombinant B domain-deleted FVIII was obtained by reverse transcription polymerase chain reaction (RT-PCR) from human liver poly A RNA (Clontech) using FVIII-specific primers. The FVIII sequence includes the native signal sequence for FVIII. The B domain deletion was from serine 743 (S743; 2287 bp) to glutamine 1638 (Q1638; 4969 bp), for a total deletion of 2682 bp. Next, the coding sequence for human recombinant Fc was obtained by RT-PCR from a human leukocyte cDNA library (Clontech) using Fc-specific primers. The primers were designed such that the B-domain-deleted FVIII sequence was fused directly to the N-terminus of the Fc sequence without an intervening linker. The FVIIIFc cDNA sequence was cloned into the mammalian dual expression vector pBUDCE4.1 (Invitrogen) under the control of the CMV promoter. A second identical Fc sequence containing the mouse Igk signal sequence was obtained by RT-PCR and cloned downstream of the second promoter EF1α in the expression vector pBUDCE4.1.

[0138] In one embodiment, the FcRn binding partner can be the Fc region. The FcRn binding partner can be specifically bound by the FcRn receptor, and as a result, is any molecule that results in active transport of the FcRn binding partner by the FcRn receptor. Thus, the term Fc includes any variant of the functional IgG Fc. The region of the Fc portion of IgG that binds to the FcRn receptor has been described based on X-ray crystallography (Burmeister et al., Nature 372:379 (1994), which is hereby incorporated by reference in its entirety). The main contact region of the Fc with FcRn is near the junction of the CH2 and CH3 domains. All of the Fc-FcRn contacts are within one Ig heavy chain. The FcRn binding partner includes, for example, whole IgG, Fc fragments of IgG, and other fragments of IgG that include the complete binding region for FcRn. The main contact sites include amino acid residues 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 of the CH2 domain, and amino acid residues 385-387, 428, and 433-436 of the CH3 domain. All references made to immunoglobulin or immunoglobulin fragments, or amino acid numbering of regions, are based on Kabat et al. 1991, Sequences of Proteins of Immunological Interest, U.S. Department of Public Health, Bethesda; MD, which is hereby incorporated by reference in its entirety. (The FcRn receptor has been isolated from several mammalian species including humans. The sequences of human FcRn, rat FcRn, and mouse FcRn are known (Story et al., J. Exp. Med. 180:2377 (1994), which is hereby incorporated by reference in its entirety). The Fc can include the CH2 and CH3 domains of the immunoglobulin, with or without the hinge region of the immunoglobulin. Exemplary Fc variants are provided in WO2004 / 101740 and WO2006 / 074199, which are hereby incorporated by reference in their entirety.)

[0139] The Fc (or the Fc portion of a chimeric polypeptide) can comprise one or more mutations and combinations of mutations.

[0140] The Fc (or the Fc portion of a chimeric polypeptide) can comprise mutations that confer an increased half-life, such as M252Y, S254T, T256E, and combinations thereof, disclosed in Oganesyan et al., Mol. Immunol. 46:1750 (2009), which is hereby incorporated by reference in its entirety; H433K, N434F, and combinations thereof, disclosed in Vaccaro et al., Nat. Biotechnol. 23:1283 (2005), which is hereby incorporated by reference in its entirety; mutants disclosed in paragraphs

[0012] on pages 1-2 and Examples 9 and 10 of US2009 / 0264627A1, which is hereby incorporated by reference in its entirety; and mutants disclosed in paragraphs

[0014] -

[0021] on page 2 of US20090163699A1, which is hereby incorporated by reference in its entirety.

[0141] The Fc (or the Fc portion of a chimeric polypeptide) may also include, for example, the following mutations: It is also possible to modify the Fc region of IgG according to well-known techniques such as site-directed mutagenesis to obtain a modified IgG or Fc fragment or a portion thereof that would be bound by FcRn. Such modifications include, for example, modifications that are far from the FcRn contact site, as well as modifications within the contact site that retain or even enhance binding to FcRn. For example, without significantly impairing the Fc binding affinity for FcRn, the following single amino acid residues in human IgG1 Fc (Fcy1): P238A, S239A, K246A, K248A, D249A, M252A, T256A, E258A, T260A, D265A, S267A, H268A, E269A, D270A, E272A, L274A, N276A, Y278A, D280A, V282A, E283A, H285A, N286A, T289A, K290A, R292A, E293A, E294A, Q295A, Y296F, N297A, S298A, Y300F, R301A, V303A, V305A, T307A, L309A, Q311A, D312A, N315A, K317A, E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A330Q, A330S, P331A, P331S, E333A, K334A, T335A, S337A, K338A, K340A, Q342A, R344A, E345A, Q347A, R355A, E356A, M358A, T359A, K360A, N361A, Q362A, Y373A, S375A, D376A, A378Q, E380A, E382A, S383A, N384A, Q386A, E388A, N389A, N390A, Y391F, K392A, L398A, S400A, D401A, D413A, K414A, R416A, Q418A, Q419A, N421A, V422A, S424A, E430A, N434A, T437A, Q438A, K439A, S440A, S444A, and K447A can be substituted, where, for example, P238A indicates that the wild-type proline at position number 238 has been substituted by alanine. In addition to alanine, other amino acids can also substitute the wild-type amino acid at the positions specified above.It is also possible to introduce a single mutation into the Fc to generate over 100 FcRn binding partners that are different from native Fc. Furthermore, it is possible to introduce combinations of two, three, or more of these individual mutations together to generate hundreds more FcRn binding partners. Some of these mutations can also confer new functionality to the FcRn binding partners. For example, one embodiment incorporates N297A, removing a highly conserved N-glycosylation site. The effect of this mutation is to reduce immunogenicity, thereby improving the circulating half-life of the FcRn binding partner and making the FcRn binding partner unable to bind to FcyRI, FcyRIIA, FcyRIIB, and FcyRIIIA without compromising its affinity for FcRn (Routledge et al. 1995, Transplantation 60:847, which is incorporated herein by reference in its entirety; Friend et al. 1999, Transplantation 68:1632, which is incorporated herein by reference in its entirety; Shields et al. 1995, J. Biol. Chem. 276:6591, which is incorporated herein by reference in its entirety). Furthermore, at least three human Fc gamma receptors appear to recognize binding sites on IgG within the lower hinge region, generally amino acids 234-237. Thus, another example of new functionality and potentially reduced immunogenicity can also arise from mutations in this region, for example, by substituting the "ELLG" of amino acids 233-236 of human IgG1 with the corresponding sequence "PVA" from IgG2 (with one amino acid deletion). FcyRI, FcyRII, and FcyRIII, which mediate various effector functions, have been shown not to bind to IgG1 when such mutations are introduced (Ward and Ghetie, Therapeutic Immunology 2:77 (1995), which is incorporated herein by reference in its entirety; and Armour et al., Eur. J. Immunol. 29:2613 (1999), which is incorporated herein by reference in its entirety).As a further example of additional functionality resulting from the mutations described above, in some cases it may also be possible to increase the affinity for FcRn relative to the wild-type. This increase in affinity may reflect an increase in the "on" rate, a decrease in the "off" rate, or both an increase in the "on" rate and a decrease in the "off" rate. Mutations that are thought to increase the affinity for FcRn include, for example, T256A, T307A, E380A, and N434A (Shields et al., J. Biol. Chem. 276:6591 (2001), which is hereby incorporated by reference in its entirety).

[0142] Fc (or the Fc portion of the chimeric polypeptide) can be at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the Fc amino acid sequence shown in Table 11 (amino acids 1458-1684 of SEQ ID NO: 2 or amino acids 2352-2578 of SEQ ID NO: 6). Fc (or the Fc portion of the chimeric polypeptide) can be identical to the Fc amino acid sequence shown in Table 11 (amino acids 1458-1684 of SEQ ID NO: 2 and amino acids 2352-2578 of SEQ ID NO: 6).

[0143] As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence or polypeptide of the invention can usually be determined using known computer programs. In one embodiment, a method for determining the best overall match between a query sequence (reference or original sequence) and a target sequence, also referred to as global sequence alignment, can also be determined using the FASTDB computer program based on the algorithm of Brutlag et al., Comp. App. Biosci. 6:237-245 (1990), the entirety of which is incorporated herein by reference. In sequence alignment, both the query and target sequences are DNA sequences. RNA sequences can be compared by converting U (uridine) to T (thymine). The results of the aforementioned global sequence alignment are expressed as percent identity. In another embodiment, the parameters used for FASTDB alignment of DNA sequences to calculate percent identity are as follows: matrix = single, k-tuple = 4, mismatch penalty = 1, join penalty = 30, randomization group length = 0, cutoff score = 1, gap penalty = 5, gap size penalty = 0.05, window size = 500 or the shorter of the length of the target nucleotide sequence.

[0144] If the target array is shorter than the query array due to a 5' or 3' deletion rather than an internal deletion, manual correction must be performed on the result. This is because the FASTDB program does not handle 5' and 3' shortening of the target array when calculating percent identity. For a target array shortened at the 5' or 3' end, the percent identity is corrected by calculating, as a percentage of the total bases of the query array, the number of bases of the query array that do not match / align at the 5' and 3' of the target array when compared to the query array. Whether a nucleotide matches / aligns is determined by the result of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity and calculated by the above FASTDB program using specific parameters to arrive at the final percent identity score. This corrected score is the one used for the purposes of the present invention. Only the bases outside the 5' and 3' bases of the target array, as indicated by the FASTDB alignment, that do not match / align with the query array are calculated for the purpose of manual adjustment of the percent identity score.

[0145] For example, a target sequence of 90 bases is aligned with a query sequence of 100 bases to determine percent identity. A deletion occurs at the 5' end of the target sequence, and thus the FASTDB alignment does not show a match / alignment for the first 10 bases at the 5' end. Since 10 unpaired bases represent 10% (the number of bases at the non-matching 5' and 3' ends / total number of bases in the query sequence), 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases match perfectly, the final percent identity is 90%. In another example, a target sequence of 90 bases is compared to a query sequence of 100 bases. This time the deletion is an internal deletion, and as a result, there are no bases on the 5' or 3' end of the target sequence that do not match / align with the query. In this case, the percent identity calculated by FASTDB is not corrected manually. Again, only the bases at the 5' and 3' ends of the target sequence that do not match / align with the query sequence are corrected manually. No other manual corrections are made for the purposes of the present invention.

[0146] The amino acid sequence of the target polypeptide is intended to be identical to the query sequence, except that the query amino acid sequence and a polypeptide having an amino acid sequence that is at least, for example, 95% "identical" can include up to 5 amino acid changes per 100 amino acids of the query amino acid sequence. That is, in order to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the query amino acid sequence, up to 5% of the amino acid residues in the target sequence may be inserted, deleted, (lost), or substituted with another amino acid. These changes to the reference sequence can occur at the amino or carboxy terminal positions of the reference amino acid sequence, or at any position between these termini, either scattered among the residues in the reference sequence or as one or more groups of contiguous residues within the reference sequence.

[0147] As a practical matter, whether any particular polypeptide is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, for example, the amino acid sequence of SEQ ID NO: 2 (Factor VIII moiety, Fc moiety, individually or together) or 4, or a known Factor VIII or Fc polypeptide sequence, can typically be determined using known computer programs. In one embodiment, the method for determining the best overall match between a query sequence (reference or original sequence) and a target sequence, also referred to as global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al., Comp. App. Biosci. 6:237-245 (1990), which is hereby incorporated by reference in its entirety. In sequence alignment, the query and target sequences are either both nucleotide sequences or both amino acid sequences. The result of the global sequence alignment is expressed as percent identity. In another embodiment, the parameters used in the FASTDB amino acid alignment are: matrix = PAM 0, k-tuple = 2, mismatch penalty = 1, join penalty = 20, randomization group length = 0, cutoff score = 1, window size = the length of the sequence or 500, whichever is shorter, gap penalty = 5, gap size penalty = 0.05, window size = the length of the target amino acid sequence or 500, whichever is shorter.

[0148] If the target array is shorter than the query array due to N-terminal or C-terminal deletion rather than internal deletion, manual correction must be made to the results. This is because the FASTDB program does not handle N-terminal and C-terminal shortening of the target array when calculating global percent identity. For the target array shortened at the N-terminal and C-terminal, the percent identity is corrected by calculating, as a percentage of the total bases of the query array, the number of residues of the query array that do not match / align with the corresponding target residues at the N-terminal and C-terminal of the target array when compared with the query array. Whether a residue matches / aligns is determined by the result of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity and calculated by the above FASTDB program using specific parameters to reach the final percent identity score. This final percent identity score is the one used for the purposes of the present invention. Only the residues belonging to the N-terminal and C-terminal of the target array that do not match / align with the query array are considered for the purpose of manually adjusting the percent identity score. That is, only the query residues are aligned outside the outermost N-terminal and C-terminal residues of the target array.

[0149] For example, a target sequence of 90 amino acid residues is aligned with a query sequence of 100 residues to determine percent identity. The deletion occurs at the N-terminus of the target sequence, and thus the FASTDB alignment does not show a match / alignment of the first 10 residues at the N-terminus. Since 10 unpaired residues represent 10% (the number of residues at the non-matching N-terminus and C-terminus / the total number of residues in the query sequence), 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues are completely identical, the final percent identity is 90%. In another example, a target sequence of 90 residues is compared to a query sequence of 100 residues. This time the deletion is an internal deletion, and as a result, there are no residues on the N-terminus and C-terminus of the target sequence that do not match / align with the query. In this case, the percent identity calculated by FASTDB is not manually corrected. Again, only residues that are aligned outside the N-terminus and C-terminus of the target sequence and do not match / align with the query sequence, as shown in the FASTDB alignment, are manually corrected. No other manual corrections are made for the purposes of the present invention.

[0150] Polynucleotide variants can include changes in the coding region, non-coding region, or both. In one embodiment, the polynucleotide variant includes changes that produce silent substitutions, additions, or deletions, but do not change the nature or activity of the encoded polypeptide. In another embodiment, the nucleotide variant is produced by a silent substitution due to the degeneracy of the genetic code. In other embodiments, variants in which 5 - 10, 1 - 5, or 1 - 2 amino acids are substituted, deleted, or added in any combination. Polynucleotide variants can be generated for various reasons, for example, to optimize codon expression for a particular host (such as changing the codons in a human mRNA for a bacterial host such as E. coli).

[0151] Spontaneous mutants are referred to as "allelic mutants" and refer to one of several alternative forms of a gene that occupies a given locus on the chromosome of an organism (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allelic mutants may differ at the polynucleotide and / or polypeptide levels and are included in the present invention. Alternatively, non-spontaneous mutants can be generated by mutagenesis techniques or direct synthesis.

[0152] Using known methods of protein engineering and recombinant DNA techniques, mutants can be made to improve or modify the characteristics of a polypeptide. For example, one or more amino acids can be deleted from the N-terminus or C-terminus of a secreted protein without substantial loss of biological function. Ron et al., J. Biol. Chem. 268:2984-2988 (1993) (incorporated herein by reference in its entirety) reported that mutant KGF protein still had heparin-binding activity even after deletion of 3, 8, or 27 amino-terminal amino acid residues. Similarly, interferon gamma was shown to have up to a 10-fold increase in activity after deletion of 8-10 amino acid residues from the carboxy terminus of this protein (Dobeli et al., J. Biotechnology 7:199-216 (1988), incorporated herein by reference in their entirety).

[0153] Furthermore, there is sufficient evidence demonstrating that variants often maintain a biological activity similar to that of the naturally occurring protein. For example, Gayle and co-workers (J. Biol. Chem 268:22105-22111 (1993), incorporated herein by reference in its entirety) performed a large-scale mutagenesis analysis of human cytokine IL-1α. They utilized random mutagenesis to generate over 3,500 individual IL-1α variants, each having on average 2.5 amino acid changes over the entire length of the molecule. Multiple mutations were examined for each possible amino acid position. The researchers found that "most of the molecule can be modified with little effect on either [binding or biological activity]." (See abstract.) In fact, out of the over 3,500 nucleotide sequences examined, only 23 unique amino acid sequences produced proteins that were significantly different from the wild-type activity.

[0154] As described above, polypeptide variants include, for example, modified polypeptides. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of cross-links by covalent bonding, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation (Mei et al., Blood 116:270-79 (2010), which is hereby incorporated by reference in its entirety), proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, addition of an amino acid to a protein mediated by transfer RNA (e.g., arginylation), and ubiquitination. In some embodiments, Factor VIII is modified, e.g., pegylated, at any convenient position. In some embodiments, Factor VIII is pegylated at surface-exposed amino acids, e.g., engineered cysteines, which can be surface-exposed cysteines. Ibid. In some embodiments, the modified Factor VIII, e.g., pegylated Factor VIII, is a long-acting Factor VIII.

[0155] III. Methods of Administration The treatment of hemophilia A is replacement therapy aimed at restoring FVIII activity to 1-5% of normal levels to prevent spontaneous bleeding (Mannucci, P.M., et al., N. Engl. J. Med. 344:1773-9 (2001), which is hereby incorporated by reference in its entirety).

[0156] The present invention also includes a method of administering the pharmaceutical composition of the present invention. The present invention provides a method of administering a pharmaceutical composition to a human subject (e.g., a human patient) in need thereof, which comprises administering a therapeutically effective amount of a Factor VIII polypeptide, e.g., a long-acting FVIII polypeptide or a short-acting FVIII polypeptide, e.g., a Factor VIII-Fc polypeptide, or a hybrid of such polypeptides, to the subject at dosing intervals.

[0157] In some embodiments, the pharmaceutical composition of the present invention is used to reduce or decrease one or more bleeding symptoms or frequencies (e.g., bleeding disorders) in a subject in need thereof. In other embodiments, the pharmaceutical composition of the present invention is used to treat or prevent a bleeding disorder in a subject in need thereof.

[0158] Bleeding disorders can be caused by blood coagulation disorders. Blood coagulation disorders can also be referred to as coagulation disorders. In one example, a blood coagulation disorder that can be treated with the pharmaceutical composition of the present disclosure is hemophilia. In another example, a blood coagulation disorder that can be treated with the pharmaceutical composition of the present disclosure is hemophilia A.

[0159] In some embodiments, the types of bleeding associated with bleeding disorders are selected from hemarthrosis, muscle bleeding, oral bleed, hemorrhage, bleeding into muscle, oral hemorrhage, trauma, head trauma, gastrointestinal bleeding, intracranial bleeding, intra-abdominal bleeding, intrathoracic bleeding, fractures, central nervous system bleeding, retropharyngeal bleeding, retroperitoneal bleeding, or bleeding within the iliopsoas sheath.

[0160] In other embodiments, a subject suffering from a bleeding disorder requires treatment for surgery, such as including surgical prophylaxis or perioperative management. In one example, the surgery is selected from minor surgery and major surgery. Exemplary surgical procedures include tooth extraction, tonsillectomy, inguinal hernia incision, synovectomy, craniotomy, bone fixation, trauma surgery, intracranial surgery, intraperitoneal surgery, intrathoracic surgery, or joint replacement surgery (e.g., total knee arthroplasty, hip arthroplasty, etc.), cardiac surgery, and cesarean section.

[0161] The present invention also provides a method for reducing or decreasing the annualized bleeding rate (ABR) in a subject with hemophilia, which includes administering an effective amount of an FVIII polypeptide to the subject. In one embodiment, the long-acting FVIII polypeptide is administered at an administration interval of every 3 days or more. In another embodiment, the effective amount is from about 20 IU / kg to about 90 IU / kg. In other embodiments, the effective amount is 20 - 30 IU / kg, 30 - 40 IU / kg, 40 - 50 IU / kg, 50 - 60 IU / kg, 60 - 70 IU / kg, 70 - 80 IU / kg, or 80 - 90 IU / kg. In still other embodiments, the effective amount is 20 IU / kg, 25 IU / kg, 30 IU / kg, 35 IU / kg, 40 IU / kg, 45 IU / kg, 50 IU / kg, 55 IU / kg, 60 IU / kg, 65 IU / kg, 70 IU / kg, 75 IU / kg, 80 IU / kg, 85 IU / kg, or 90 IU / kg.

[0162] In certain embodiments, administration of the FVIII polypeptide is for individualized prophylaxis and results in an ABR of less than about 5.5, less than about 5.4, less than about 5.3, less than about 5.2, less than about 5.1, less than about 5.0, less than about 4.9, less than about 4.8, less than about 4.7, less than about 4.6, or less than about 4.5. In other embodiments, administration results in an ABR of from about 4.7 to 0. In some embodiments, the median of the ABR is about 1.6. In still other embodiments, the mean of the ABR is about 2.9. In one aspect of an individualized prophylaxis regimen, the effective amount is from about 25 IU / kg to about 65 IU / kg administered every 3 or 5 days. For example, the effective amount is from about 25 IU / kg to about 65 IU / kg administered every 3 days. In another aspect, the effective amount is from about 50 IU / kg to about 65 IU / kg every 4 or 5 days. In other aspects, the effective amount is up to 65 IU / kg every 3 days. In still other embodiments, the effective amount of the FVIII polypeptide is subsequently modified based on the patient's pharmacokinetic profile. In one example, the patient is initially administered two doses, a first dose of 25 IU / kg on day 1 and a second dose of 50 IU / kg on day 4. If the pharmacokinetic data shows a trough level of less than 1% of normal FVIII activity after the first two administrations, the patient is administered from about 25 IU / kg to about 65 IU / kg every 3 days. In another example, if the pharmacokinetic data shows a trough level greater than 1% of normal FVIII activity for 5 days following administration, the patient is administered from about 50 IU / kg to about 65 IU / kg every 5 days. If a patient still experiences more than two spontaneous bleedings over an 8-week period after being administered from about 50 IU / kg to about 65 IU / kg every 5 days, the patient is administered one of the following: (1) up to about 65 IU / kg every 3 days (for a normal maximum 5% target trough level), (2) from about 50 IU / kg to about 65 IU / kg every 4 days (for a normal maximum 5% target trough level), or (3) from about 25 IU / kg to about 65 IU / kg every 3 days (for a normal maximum 1% - 3% target trough level). If a patient receiving from about 50 IU / kg to about 65 IU / kg every 4 days still experiences more than two breakthrough bleedings, the maximum dose can be up to about 65 IU / kg every 3 days.In some embodiments, the interquartile range (IQR) of ABR for an individualized prophylaxis regimen is 0 to 4.7.

[0163] In some embodiments, the administration of the FVIII polypeptide is for prophylaxis on a weekly basis and results in an ABR of less than about 9.0, less than about 8.9, less than about 8.8, less than about 8.7, less than about 8.6, less than about 8.5, or less than about 8.4. In one example, the administration results in an ABR of about 8.4 to 0. In another example, the median of the ABR for prophylaxis on a weekly basis is about 3.6. In other examples, the mean of the ABR for prophylaxis on a weekly basis is about 8.8. In other embodiments, the effective amount for prophylaxis on a weekly basis is about 65 IU / kg once a week, for example, every 5 days, every 6 days, every 7 days, every 8 days, or every 9 days. In some embodiments, the IQR of the ABR for a weekly prophylaxis regimen is 1.9 to 8.4.

[0164] In other embodiments, the administration of the FVIII polypeptide is for treatment at the time of onset or on demand and results in an ABR of less than about 55, less than about 54, less than about 53, less than about 52, less than about 51, less than about 50, less than about 49, less than about 48, or less than about 47. In one example, the administration results in an ABR of about 49 to 0, for example, 48.7 to 0. In another example, the median of the ABR for treatment at the time of onset (on demand) is about 33.5. In other examples, the mean of the ABR for treatment at the time of onset (on demand) is about 37.23. In other embodiments, the effective amount for on-demand treatment is about 10 IU / kg to 75 IU / kg every 12 to 24 hours. In some embodiments, the IQR of the ABR for on-demand treatment is 21.1 to 48.7.

[0165] In some embodiments, the effective amount for individualized prophylaxis, weekly prophylaxis, or treatment at onset of disease is a fixed dosage or a stratified dosage. In one aspect, the fixed dosage is about 2,000 IU per dosage, about 2,500 IU per dosage, about 3,000 IU per dosage, about 3,500 IU per dosage, or about 4,000 IU per dosage.

[0166] The dosing interval for the long-acting FVIII polypeptide is at least about 1.5 to 6 times longer, 1.5 to 5 times longer, 1.5 to 4 times longer, 1.5 to 3 times longer, or 1.5 to 2 times longer than the dosing interval required for an equivalent dosing of factor VIII (a polypeptide consisting of the factor VIII portion) without an Fc portion, for example, without a non-factor VIII portion. The dosing interval can be at least about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, or 6 times longer than the dosing interval required for an equivalent dosing of factor VIII (a polypeptide consisting of the factor VIII portion) without an Fc portion, for example, without a non-factor VIII portion. The dosing interval can be every about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days.

[0167] The methods of the invention can be practiced on subjects in need of prophylactic treatment or on-demand treatment.

[0168] For on-demand treatment, the dosing interval of the pharmaceutical composition comprising the FVIII polypeptide is once every about 24 to 36, 24 to 48, 24 to 72, 24 to 96, 24 to 120, 24 to 144, 24 to 168, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 hours, or more.

[0169] In one embodiment, the subject requires on-demand (at onset) treatment. In another embodiment, the on-demand (at onset) treatment resolves more than 80% (more than 80%, more than 81%, more than 82%, more than 83%, more than 84%, more than 85%, more than 86%, more than 87%, more than 88%, more than 89%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or 100%) or 80 - 100%, 80 - 90%, 85 - 90%, 90 - 100%, 90 - 95%, or 95 - 100% of the bleeding (e.g., acute bleeding) in a single administration. In another embodiment, more than 80% (more than 81%, more than 82%, more than 83%, more than 84%, more than 85%, more than 86%, more than 87%, more than 88%, more than 89%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, or 100%) or 80 - 100%, 80 - 90%, 85 - 90%, 90 - 100%, 90 - 95%, or 95 - 100% of the bleeding symptoms are evaluated as very good or good by a physician after on-demand (at onset) treatment. In other embodiments, more than 5% (more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 11%, more than 12%, more than 13%, more than 14%, more than 15%, more than 16%, more than 17%, more than 18%, more than 19%, more than 20%), or 5 - 20%, 5 - 15%, 5 - 10%, 10 - 20%, or 10 - 15% of the bleeding symptoms are evaluated as fair by a physician after on-demand treatment.

[0170] In other embodiments, a pharmaceutical composition comprising an FVIII polypeptide is used for prophylaxis. Prophylaxis can be demonstrated by better Cmax, better Tmax, and / or better mean residence time versus short-acting FVIII. In some embodiments, prophylaxis does not result in breakthrough bleeding symptoms within about 24, 36, 48, 72, or 96 hours (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 96, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours) after injection (e.g., the last injection). In certain embodiments, prophylaxis results in a greater than 30% (e.g., greater than 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 96, 87, 88, 89, or 90%, e.g., greater than 50%) average reduction in annualized bleeding symptoms at a weekly dosage (e.g., at 65 IU / kg).

[0171] In another example, the subject is treated simultaneously with FIX. Since the compounds of the present invention can activate FIXa, they can be used to pre-activate FIXa polypeptide prior to administration of FIXa to the subject.

[0172] IV. Manufacturing Method The FVIII polypeptide can be produced in a host cell containing a vector encoding the FVIII polypeptide. In one embodiment, the host cell is transformed with one or more vectors comprising a first nucleotide sequence encoding an FVIII polypeptide and a first FcRn polypeptide, a second nucleotide sequence encoding a second FcRn polypeptide, and optionally a third nucleotide sequence encoding a protein convertase (e.g., PC5). As used herein, an expression vector refers to any nucleic acid construct that contains the elements necessary for transcription and translation of an inserted coding sequence, or in the case of an RNA viral vector, the elements necessary for replication and translation, when introduced into a suitable host cell. Expression vectors can include plasmids, phagemids, viruses, and their derivatives.

[0173] The gene expression control sequences used herein are any regulatory nucleotide sequences, such as promoter sequences or combinations of promoter enhancers, that facilitate the efficient transcription and translation of the coding nucleic acid to which they are operably linked. The gene expression control sequences can be, for example, mammalian or viral promoters such as constitutive or inducible promoters. Constitutive mammalian promoters include, but are not limited to, promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin promoter, and other constitutive promoters. Exemplary viral promoters that function constitutively in eukaryotic cells include, for example, cytomegalovirus (CMV), simian virus (e.g., SV40), papillomavirus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, promoters from cytomegalovirus, long terminal repeats (LTRs) of Moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus. Other constitutive promoters are known to those skilled in the art. Promoters useful as gene expression sequences of the present invention also include inducible promoters. Inducible promoters are expressed in the presence of an inducer. For example, the metallothionein promoter is induced to promote transcription and translation in the presence of certain metal ions. Other inducible promoters are known to those skilled in the art.

[0174] Examples of vectors include, but are not limited to, viral vectors or plasmid vectors. Plasmid vectors are widely described in the art and are well known to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the past few years, plasmid vectors have been found to be particularly advantageous for delivering genes into cells in vivo because they can replicate in the host genome but cannot integrate into it. However, these plasmids having a promoter compatible with the host cell can express peptides from genes operably encoded within the plasmid. Some commonly used plasmids available from commercial suppliers include pBR322, pUC18, pUC19, various pcDNA plasmids, pRC / CMV, various pCMV plasmids, pSV40, and pBlueScript. Further examples of specific plasmids include pcDNA3.1, catalog number V79020; pcDNA3.1 / hygro, catalog number V87020; pcDNA4 / myc-His, catalog number V86320; and pBudCE4.1, catalog number V53220 (all from Invitrogen (Carlsbad, CA)). Other plasmids are well known to those skilled in the art. Additionally, plasmids can be custom designed using standard molecular biology techniques to remove and / or add specific fragments of DNA.

[0175] The expression vector or vector is then transfected or co-transfected into suitable target cells that will express the polypeptide. Transfection techniques known in the art include, but are not limited to, calcium phosphate precipitation (Wigler et al. (1978) Cell 14:725), electroporation (Neumann et al. (1982) EMBO J 1:841), and liposome-based reagents. A variety of host-expression vector systems can be utilized to express the proteins described herein, including both prokaryotic and eukaryotic cells. These include microorganisms such as bacteria (e.g., E. coli) transformed with recombinant bacteriophage DNA or plasmid DNA expression vectors containing appropriate coding sequences; yeast or filamentous fungi transformed with recombinant yeast or fungal expression vectors containing appropriate coding sequences; insect cell lines infected with recombinant virus expression vectors (e.g., baculovirus) containing appropriate coding sequences; plant cell lines infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus or tobacco mosaic virus) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing appropriate coding sequences; or animal cell lines including mammalian cells (e.g., HEK293, CHO, Cos, HeLa, HKB11, and BHK cells), but are not limited thereto.

[0176] In one embodiment, the host cell is a eukaryotic cell. As used herein, a eukaryotic cell refers to any animal or plant cell having a definitive nucleus. Eukaryotic cells of animals include cells of vertebrates, such as mammalian cells, and invertebrates, such as insect cells. Eukaryotic cells of plants can specifically include, without limitation, yeast cells. Eukaryotic cells are distinct from prokaryotic cells, such as bacteria.

[0177] In certain embodiments, the eukaryotic cell is a mammalian cell. A mammalian cell is any cell derived from a mammal. Mammalian cells specifically include, but are not limited to, mammalian cell lines. In one embodiment, the mammalian cell is a human cell. In another embodiment, the mammalian cell is a HEK293 cell, which is a human embryonic kidney cell line. HEK293 cells are available from the American Type Culture Collection, Manassas, VA, as CRL-1533, and from Invitrogen (Carlsbad, Calif.) as 293-H cells with catalog number 11631-+017 or 293-F cells with catalog number 11625-019. In some embodiments, the mammalian cell is a PER.C6® cell, which is a human cell line derived from the retina. PER.C6® cells are available from Crucell (Leiden, Netherlands). In other embodiments, the mammalian cell is a Chinese hamster ovary (CHO) cell. CHO cells are available from the American Type Culture Collection, Manassas, VA. (e.g., CHO-K1; CCL-61). Still in other embodiments, the mammalian cell is a baby hamster kidney (BHK) cell. BHK cells are available from the American Type Culture Collection, Manassas, Va. (e.g., CRL-1632). In some embodiments, the mammalian cell is an HKB11 cell, which is a hybrid cell line of HEK293 cells and a human B cell line. Mei et al., Mol. Biotechnol. 34(2):165-78(2006).

[0178] The method can further include a purification step. Various known purification steps are well known in the art.

Example

[0179] Example 1. Description of the Product rFVIIIFc is a long-acting fully recombinant fusion protein consisting of human coagulation factor VIII (FVIII) covalently linked to the Fc domain of human immunoglobulin G1 (IgG1). The factor VIII portion of rFVIIIFc has a primary amino acid sequence and post-translational modifications equivalent to the 90 + 80 kDa form of factor VIII (i.e., B domain deleted). The Fc domain of rFVIIIFc contains the hinge, CH2, and CH3 regions of IgG1. rFVIIIFc contains 1882 amino acids with an apparent molecular weight of approximately 220 kilodaltons.

[0180] rFVIIIFc is produced by recombinant DNA techniques in a well-characterized human embryonic kidney (HEK) cell line. The cell line expresses rFVIIIFc in a defined cell culture medium that does not contain any proteins of animal or human origin. rFVIIIFc is purified by a series of chromatography steps that do not require the use of monoclonal antibodies. The process includes a detergent viral inactivation step and multiple virus clearance steps including an affinity chromatography step and a 15 nm virus retention nanofiltration step. No human or animal additives are used in the cell culture, purification, and formulation processes.

[0181] rFVIIIFc falls into the drug therapy group: hemostatic agents, blood coagulation factor VIII. It is provided as a cake from a sterile, preservative-free, non-pyrogenic, lyophilized white to off-white powder for intravenous (IV) administration in a single-use vial, accompanied by a liquid diluent in a filled syringe. In addition to rFVIIIFc, the pharmaceutical composition contains sucrose, sodium chloride, L-histidine, calcium chloride, and polysorbate 20 or polysorbate 80 in the lyophilized product and is contained in sterile water for injection. Each single-use vial nominally contains 250, 500, 750, 1000, 1500, 2000, 3000, 4000, 5000, or 6000 international units (IU) of rFVIIIFc. When reconstituted with the provided diluent, the product contains the following excipients: sucrose, sodium chloride, L-histidine, calcium chloride, and polysorbate 20 or polysorbate 80 at the concentrations shown in Table 1 or Table 2 below. The pharmaceutical composition is formulated for intravenous administration only after reconstitution.

[0182] Each pack contains a powder vial (type 1 glass) with a stopper (butyl) and flip-off seal (aluminum), 3 ml of solvent in a filled syringe (type 1 glass) with a plunger stopper (butyl), a tip cap (butyl), and a sterile vial adapter reconstitution device.

Table 1

Table 2

[0183] Example 2. Method of formulation The rFVIIIFc pharmaceutical product is a sterile, lyophilized powder for injection intended for intravenous administration. It is supplied in sterile, pre-filled, single-use vials nominally containing 250, 500, 750, 1000, 1500, 2000, 3000, 4000, 5000, and 6000 IU per vial. The vials are 10 mL United States Pharmacopeia / European Pharmacopeia Type 1 glass vials sealed with a 20 mm Teflon®-coated butyl rubber lyophilization stopper and an aluminum flip-off crimp seal. The nominal fill volume target for 250 - 6000 IU vials is 3 mL prior to lyophilization. The composition of the formulation excipients prior to lyophilization is the same for all dosage strengths. The injectable powder is reconstituted with 3 mL of diluent containing sterile water for injection provided in a sterile, pre-filled syringe.

[0184] The composition of the drug product solution prior to lyophilization is presented in Table 3. The composition of the drug product following reconstitution is presented in Table 1 or Table 2 (Example 1).

Table 3

[0185] Example 3. Dosage and Method of Administration / Method for Calculating Initial Estimated Dosage rFVIIIFc is a long-acting antihemophilic factor (recombinant) indicated for use in adults and children (12 years of age and older) with hemophilia A (congenital factor VIII deficiency) for, for example, the control and prevention of bleeding episodes, routine prophylaxis to prevent or reduce the frequency of bleeding episodes, and perioperative management (surgical prophylaxis).

[0186] The dosage of rFVIIIFc can be estimated as described in this example, but can also be determined by standard tests such as the FVIII activity assay described elsewhere in this specification.

[0187] One IU of rFVIIIFc per kg of body weight is expected to increase the circulating level of factor VIII by 2 [IU / dL]. rFVIIIFc has been shown to have a long circulating half-life.

[0188] Since patients can vary in their pharmacokinetics (e.g., half-life, in vivo recovery) and clinical response to rFVIIIFc, the expected in vivo peak increase in factor VIII level is expressed as IU / dL (or normal %), or the required dose can be estimated using the following formula: IU / dL (or normal %) = [total dose (IU) / body weight (kg)] × 2 (IU / dL per kg of body weight) Or Dose (IU) = body weight (kg) × desired factor VIII increase (IU / dL or normal %) × 0.5 (IU / kg per IU / dL)

[0189] The following table (Table 4) can be used to guide dosing in bleeding symptoms:

Table 4

[0190] Subsequent doses and duration of treatment depend on the individual clinical response, severity of factor VIII deficiency, and location and extent of bleeding.

[0191] The following table (Table 5) can be used to guide dosing for perioperative management (surgical prophylaxis):

Table 5

[0192] For routine prophylaxis, the recommended regimen is 50 IU / kg every 3 - 5 days. Dosing can be adjusted based on the patient's response in the range of 25 - 65 IU / kg.

[0193] The recommended dose for weekly prophylaxis is 65 IU / kg.

[0194] rFVIIIFc is contraindicated in patients who have demonstrated a severe allergic reaction, including anaphylaxis, to the product or its components. Severe allergic reactions have not been observed in clinical trials; however, these are known to occur with the use of other factor VIII replacement factors.

[0195] Clinical responses to rFVIIIFc vary. If bleeding is not controlled by the recommended dosage, plasma levels of factor VIII are determined and a sufficient dosage of rFVIIIFc may be administered to achieve a satisfactory clinical response. If the patient's plasma factor VIII levels do not rise as expected or if bleeding is not controlled after rFVIIIFc administration, the presence of inhibitors (neutralizing antibodies) should be suspected and appropriate tests should be performed. Patients using rFVIIIFc can be monitored for the development of factor VIII inhibitors by appropriate clinical observations and laboratory tests known to those of skill in the art.

[0196] The patient's plasma can be monitored for factor VIII activity levels (e.g., by one-stage clotting assay to confirm that appropriate factor VIII levels are achieved or maintained when clinically indicated). The patient's plasma can be further monitored for the development of factor VIII inhibitors.

[0197] Example 4. Phase 3 Clinical Trial of Recombinant Fc-Fused Factor VIII with Extended Half-Life (the "A-LONG" Study) Summary of the A-LONG Study In the A-LONG study, 165 male patients 12 years of age and older were enrolled. The A-LONG study had three treatment arms: individualized prophylaxis, prophylaxis on a weekly basis, and on-demand treatment (arms 1, 2, and 3, respectively). In subgroups of patients across the treatment arms, rFVIIIFc was evaluated in the perioperative management of patients who required major surgical procedures during the study.

[0198] Generally, 92.7 percent of the patients completed the study. Recombinant FVIIIFc was generally well tolerated. No inhibitors of rFVIIIFc were detected, and no cases of hypersensitivity were reported in any of the patients (all of them had switched from commercially available factor VIII products). No serious adverse events were evaluated by the researchers as drug-related.

[0199] The most common adverse events (incidence rate of 5 percent or more) occurring outside the perioperative management period were nasopharyngitis, arthralgia, headache, and upper respiratory tract infection.

[0200] The median (mean) annualized bleeding rate (ABR), including spontaneous and traumatic bleeding, was 1.6 (2.9) in the individualized prophylaxis arm, 3.6 (8.8) in the arm for prophylaxis on a weekly basis, and 33.6 (37.2) in the on-demand treatment arm. In the individualized prophylaxis arm, the median dosing interval was 3.5 days. During the last three months of the study, 30 percent of the patients in the individualized prophylaxis arm achieved an average dosing interval of at least 5 days.

[0201] Bleeding control was evaluated in all patients who experienced bleeding symptoms during the study. Generally, 98 percent of the bleeding symptoms were controlled by one or two injections of rFVIIIFc.

[0202] In addition, rFVIIIFc was evaluated in the perioperative management of 9 patients who underwent 9 major surgical procedures. The treating physicians evaluated the hemostatic efficacy of rFVIIIFc as excellent or good in 100% of these surgeries.

[0203] A-LONG included pharmacokinetic (PK) analysis of rFVIIIFc in all patients in the study. In a protocol-defined subset of patients with extensive PK sampling, the approximate final half-life of rFVIIIFc was 19.0 hours compared to 12.4 hours for ADVATE® [Antihemophilic Factor (Recombinant), Method without Plasma / Albumin], consistent with results obtained in the Phase 1 / 2 study of rFVIIIFc.

[0204] For the A-LONG study and rFVIIIFc program, A-LONG was a global, open-label, multicenter Phase 3 study that evaluated the efficacy, safety, and pharmacokinetics of intravenously administered rFVIIIFc. The study was designed to evaluate rFVIIIFc in the control and prevention of bleeding, routine prophylaxis, and perioperative management in patients with hemophilia A. A-LONG included 60 hemophilia treatment centers in 19 countries on 6 continents.

[0205] The A-LONG study had three treatment arms. In Arm 1 (Individualized prophylaxis; n = 117), patients were treated with 25 - 65 IU / kg of rFVIIIFc at individualized intervals of 3 - 5 days to maintain sufficient factor trough levels to prevent bleeding. In Arm 2 (For weekly prophylaxis; n = 24), patients were treated with a weekly dose of 65 IU / kg. In Arm 3 (On-demand treatment; n = 23), patients received on-demand rFVIIIFc treatment for bleeding as needed. In subgroups of patients across the treatment arms, rFVIIIFc was evaluated in a surgical setting.

[0206] The primary efficacy measures were the annualized bleeding rates per patient for Arm 1 vs. Arm 3 and the pharmacokinetics of rFVIIIFc vs. rFVIII (one-stage clotting [activated partial thromboplastin time] assay and chromogenic assay calibrated against normal human reference plasma, with potency attributable to World Health Organization (WHO) standards). Safety endpoints included the occurrence of adverse events and inhibitor development (Nijmegen modified Bethesda assay) in patients studied for up to 54 weeks. Secondary efficacy endpoints included the ABR for Arm 2 vs. Arm 3, the annualized number of spontaneous and joint bleeding episodes per patient, the number of injections and dose per injection to resolve bleeding, the investigators' assessment of the patient response to surgery with rFVIIIFc using a bleeding response scale (surgical subgroup only), treatment of bleeding symptoms, and the patient response to the pharmacokinetics of rFVIIIFc vs. ADVATE®. Non-neutralizing antibodies (NNAs) were evaluated using an electrochemiluminescence-based anti-rFVIIIFc binding antibody assay.

[0207] Ongoing clinical studies of rFVIIIFc include the A-LONG and ASPIRE studies for children. The A-LONG study for children is a Phase 3 open-label study in previously treated pediatric patients less than 12 years of age with hemophilia A, with active patient recruitment. ASPIRE is a long-term open-label study for patients who have completed the A-LONG study or will complete the A-LONG study for children.

[0208] A-LONG Study Design Design: Global open-label multicenter Phase 3 study. The study protocol was approved by the local institutional review board at each participating site, and the study was conducted in accordance with the International Conference on Harmonization guidelines for Good Clinical Practice.

[0209] Objective: To evaluate the efficacy and safety of intravenously administered recombinant factor VIII Fc fusion protein (rFVIIIFc) in the control and prevention of bleeding symptoms, routine prophylaxis, and perioperative management in individuals with severe hemophilia A.

[0210] Important inclusion criteria: Male 12 years of age or older and at least 40 kg Diagnosis of severe hemophilia A defined as < 1% ( < 1 IU / dL) endogenous factor VIII (FVIII) activity Previously treated patients (PTP): History of 150 or more previous documented exposure days (ED) with any currently marketed FVIII product, prophylaxis with FVIII products more than once a week (for Arm 1 only), or 12 or more bleedings experienced in the past 12 months at the time of onset regimen No current / previous FVIII inhibitor with measurable activity. No history of inhibitor, or history of hypersensitivity or anaphylaxis related to any FVIII or intravenous immunoglobulin administration All subjects (or guardians in the case of minors) gave written informed consent prior to study participation.

[0211] Treatment arms: Details of the A-LONG study design are described below. Arm 1 (individualized prophylaxis) was administered 25 - 65 IU / kg every 3 - 5 days (maximum dosing interval of 5 days). Subjects were treated with an initial dose of 25 IU / kg on Day 1 and 50 IU / kg on Day 4, which were then adjusted to maintain a sufficient factor trough level to prevent bleeding. If a subject experienced 2 or more moderate / severe bleedings over an 8-week period, further dose adjustment to a target trough level of 3 - 5 IU / dL was permitted after Week 7; 10 - 20 IU / kg of rFVIIIFc for minor bleeding symptoms (target FVIII of 20 - 40 IU / dL); 15 - 30 IU / kg of rFVIIIFc for moderate to major bleeding symptoms (target FVIII of 30 - 60 IU / dL); 40 - 50 IU / kg of rFVIIIFc for major to life-threatening bleeding symptoms (target FVIII of 80 - 100 IU / dL).

[0212] Arm 2 (for weekly prophylaxis) was administered at a dose of 65 IU / kg. Subjects were treated at 65 IU / kg once weekly without dose or interval adjustment.

[0213] Arm 3 (onset [on-demand] treatment) was administered 10 - 50 IU / kg. Subjects received treatment with rFVIIIFc at the onset of bleeding as needed. A perioperative management subgroup was established. In this subgroup, rFVIIIFc was administered before and following surgery in a subset of patients who required major surgical procedures during the study. Subjects in any treatment arm could be enrolled in the surgical subgroup. Eligibility: required major surgery; had a negative inhibitor titer for rFVIIIFc for 12 or more exposure days within 4 weeks prior to and following this period; had completed at least a brief PK sample collection.

[0214] All subjects (except for the sequential PK subgroup) received PK sample collection from pre-dose with rFVIIIFc post-dose up to 96 hours according to schedule: pre-dose, 30 (±3) minutes from the start of injection, 3 hours (±15 minutes), 72 (±2) hours (day 3), and 96 (±2) hours (day 4).

[0215] Pharmacokinetic (PK) evaluations were performed. All subjects in all arms had an initial PK evaluation after their first dose of rFVIIIFc. A subset of subjects from Arm 1 was assigned to sequential PK subgroups as designated by the protocol to compare the stability of the PK properties of rFVIIIFc over time and the stability of recombinant factor VIII (rFVIII, ADVATE® [Antihemophilic Factor (Recombinant) without Plasma / Albumin], octogam alpha) as follows: Prior to treatment in Arm 1, PK was evaluated after a single dose of ADVATE® 50 IU / kg. PK was then evaluated in these same subjects after a single dose of 50 IU / kg of rFVIIIFc. The PK of rFVIIIFc was repeated at 12 - 24 weeks.

[0216] Details of the design of sequential PK subgroup (arm 1) administration and PK sample collection are described in Figure 1.

[0217] (Included in the first readout) Key efficacy outcome measures: a. Annualized bleeding rate (ABR) in arm 1 vs arm 3 b. Individualized prophylaxis arm compared to treatment arm at onset c. Number of injections required to resolve one bleeding symptom d. Number of injections required to resolve multiple bleeding symptoms e. Median dose required to resolve bleeding symptoms f. Physician assessment of subject response to surgery with rFVIIIFc using a 4-point scale

[0218] PK outcome measures include the following: a. The primary PK assessment was based on FVIII activity levels determined in the central laboratory by one-stage clotting assay and by chromogenic assay against commercially available plasma standards. b. PK of rFVIIIFc and ADVATE® c. The PK properties of rFVIIIFc were compared to rFVIII (ADVATE®; sequential PK subgroups). Generally, the study duration was 75 weeks or less for all subjects. The primary efficacy endpoint was the annualized bleeding rate (ABR; arms 1 and 2 vs arm 3). Prophylactic dosing and intervals, the number of injections required to treat bleeding symptoms, and perioperative hemostasis were evaluated.

[0219] Key safety outcome measures include the following: a. Incidence of inhibitor generation. The study was powered to detect the incidence of inhibitors with two-sided 95% confidence intervals using the exact Clopper-Pearson method if two cases of inhibitor formation were observed. b. Incidence of adverse events (AEs) occurring outside the perioperative management period

[0220] Treatment compliance (compliance measurement) was evaluated using the patient's electronic diary.

[0221] A-LONG results Subjects A total of 165 subjects were enrolled in the study. The median age was 30 years (range 12 - 65), and 8% were under 18 years old. The number of patients in each arm of the study was as follows: Arm 1 (individualized prophylaxis), n = 118; Arm 2 (for weekly prophylaxis), n = 24; Arm 3 (treatment at onset), n = 23; and the perioperative management subgroup, n = 9, 9 surgeries (8 subjects from Arm 1 and 1 subject from Arm 2). Subjects from each treatment arm were eligible to enter the surgery subgroup if they required major surgery, and had ≥12 ED and a negative inhibitor titer against rFVIIIFc within 4 weeks prior to and during this period and before surgery. Patients who had received previous treatment at onset had a higher median bleeding symptoms in the 12 months prior to the study, and a higher proportion of patients in Arms 2 and 3 had target joints. For patients who had previous prophylaxis, 87% reported being injected at least 3 times a week.

[0222] 92.7% of the subjects completed the study. Overall, 153 subjects (approximately 93%) completed the study; 112 / 118 (95%) in Arm 1, 19 / 24 (79%) in Arm 2, and 22 / 23 (96%) in Arm 3. Reasons for early discontinuation: Arm 1 - subject discontinuation (n = 2), physician's decision (n = 2), other (n = 1), death (n = 1); Arm 2 - subject discontinuation (n = 2; potentially contributed by adverse events (AE) related to the study drug), AE (n = 2), other (n = 1); Arm 3 - other (n = 1).

[0223] The age, race, and geography of the subjects represented a global hemophilia A population with access to treatment, as summarized in Table 6.

Table 6-1

Table 6-2

[0224] Subjects received rFVIIFc for a median (minimum–maximum) of 30.5 (less than 1–54) weeks at the median (minimum–maximum) of 32.1 (9, 54), 28.0 (less than 1, 38), and 28.9 (15, 32) weeks in Arms 1, 2, and 3, respectively. Overall, 111 subjects (67.7%) had 50 or more EDs for drug research. A total of 9356 injections were administered during the study to correspond to 9170 EDs (100.2 person-years of exposure). Generally, 93.6% of patients adhered to both the prescribed dosing and intervals in the prophylaxis arm.

[0225] Generally, 93.6% of subjects adhered to both the prescribed dosing and the prescribed dosing intervals in the prophylaxis treatment arm.

[0226] Efficacy Overall, 163 subjects were included in the efficacy analysis. One subject was excluded for receiving only ADVATE® (Arm 1), and another subject discontinued prior to efficacy evaluation (Arm 2). The median ABR with 25th and 75th percentiles (interquartile range [IQR]) was as follows: Arm 1—individualized prophylaxis arm: 1.6 (0.0, 4.7); Arm 2—arm for prophylaxis on a weekly basis: 3.6 (1.9, 8.4); and Arm 3—treatment-at-onset arm: 33.6 (21.1, 48.7). Most bleeding symptoms were sudden. In Arms 1, 2, and 3, 45.3%, 17.4%, and 0% had no bleeding symptoms, and 13.7%, 34.8%, and 0% had one bleeding symptom, respectively.

[0227] Median dosing interval: a. In the individualized prophylaxis arm, the median dosing interval was 3.5 days during the last 3 months of the study. b. Thirty percent of patients in the individualized prophylaxis arm achieved an average dosing interval of at least 5 days. c. Generally, the median (IQR) dosing interval by individualized prophylaxis aiming to achieve an FVIII trough of at least 1 - 3 IU / dL (the maximum 5 - day dosing interval allowed in the protocol) was 3.5 (3.2 - 4.4) days based on a median (IQR) weekly dosing of 78 (72 - 91) IU / kg (Table 7). d. Approximately 30% of the subjects achieved a mean dosing interval of 5 days over the last 3 months of the study. 100% of the subjects had a mean dosing interval of 2 days or more throughout the study.

[0228] The median dose per injection for the Arm 3 onset regimen was 26.5 IU / kg (n = 23), and the median total dose per bleeding episode was 27.4 IU / kg (n = 23).

Table 7

[0229] Bleeding control: Approximately 98% (97.7%) of bleeding episodes were controlled by one or two injections of rFVIIIFc. 87.3% of bleeding episodes were controlled by one injection. 1.7% required three injections.

[0230] Peri - operative management: Generally, nine major surgeries were performed in nine subjects, including knee arthroplasty (n = 5), laparoscopic inguinal hernia repair (n = 2), appendectomy (n = 1), and arthroscopy (n = 1) (8 subjects from Arm 1; 1 subject from Arm 2). Treating physicians evaluated the hemostatic effectiveness of rFVIIIFc as excellent (8 / 9) or good (1 / 9) in 100% of these surgeries. The median (minimum, maximum) estimated blood loss available for 7 / 9 of the surgeries was 15.0 (0, 600) mL during the surgery and 0.0 (0, 1100) mL (post - surgical drainage) after the surgery.

[0231] PK PK data comparing rFVIIIFc and ADVATE® available from 28 subjects in the sequential PK subgroup of Arm 1 are summarized in Table 7. a. rFVIIIFc demonstrated an elimination half-life and mean residence time approximately 50% longer compared to ADVATE® (P<0.001) (Figure 2) due to a 35% decrease in rFVIIIFc clearance with respect to ADVATE®. b. The geometric mean time to an FVIII activity of 1 IU / dL following a 50-IU / kg dose was approximately 5 days for rFVIIIFc versus approximately 3 days for ADVATE® (P<0.001; Table 7, Figure 2). c. The incremental recovery for rFVIIIFc was clinically equivalent to ADVATE®. d. There was no shift in the PK properties of rFVIIIFc, as demonstrated by equivalent PK properties between baseline and repeat rFVIIIFc PK profiles. e. Similar PK results were obtained when the analysis was based on a chromogenic FVIII assay. f. One-stage and chromogenic clotting assays accurately and precisely measured both rFVIIIFc and ADVATE® using commercially available plasma FVIII standards.

[0232] The geometric mean (95% confidence interval) terminal half-life of rFVIIIFc was approximately 19.0 hours (17.0, 21.1), which was 1.53-fold longer than that of ADVATE® (approximately 12.4 (11.1, 13.9) hours).

[0233] The geometric mean time (IQR) to 1% FVIII activity following 50 IU / kg of rFVIIIFc was approximately 5 days (4.92 (4.43, 5.46) days). An equivalent PK profile of rFVIIIFc was observed at week 14. The median dosing interval with individualized prophylaxis was 3.5 days, and the median weekly dose was 78 IU / kg; approximately 30% of subjects achieved a mean dosing interval of more than 5 days over the last 3 months of the study (subjects in the study for more than 6 months), as shown in Table 8. 98% of bleeding episodes were controlled with 1 - 2 injections. [[Table 8]]

[0234] Safety Inhibitors were not detected in rFVIIIFc, and no cases of anaphylaxis, allergy, or serious thromboembolic events were reported.

[0235] rFVIIIFc was generally well - tolerated, and no serious adverse events were evaluated as being related to rFVIIIFc.

[0236] The most common AEs (occurring in more than 5%) occurring outside the peri - operative management period were nasopharyngitis, arthralgia (joint pain), headache, and upper respiratory tract infection, as summarized in Table 9, regardless of causal relationship.

[0237] Twelve subjects (7.3%) experienced at least one serious adverse event (SAE) outside the peri - operative management period.

[0238] None of the SAEs were evaluated by the investigators as being drug - related.

[0239] Minor occurrences of non-neutralizing antibodies (NNAs) were observed in the NNA assay, which is approximately 20-fold more sensitive than the Bethesda assay. Five subjects were found to be positive for NNAs at baseline, but all had at least one negative assessment during the study. Six subjects, four in the Arm 1 study and two in the Arm 2 study, seroconverted during the study. However, in almost all cases, the antibodies were transient and of low titer, and in all cases, the antibodies were directed against FVIII.

[0240] Overall, 14 (8.5%) subjects reported serious adverse events (SAEs). None of the SAEs were determined by the investigator to be related to the drug study, and no SAEs were experienced by more than one subject. One death occurred during the study, which was due to overdose of various medications and was evaluated by the investigator to be unrelated to rFVIIIFc. [Table 9]

[0241] Summary An individualized weekly prophylaxis regimen resulted in a low single-digit median annualized bleeding rate.

[0242] In the individualized prophylaxis arm, the median dosing interval was 3.5 days. During the last three months of the study, 30 percent of the patients in the individualized prophylaxis arm achieved an average dosing interval of at least five days.

[0243] 98% of bleeding episodes were controlled with one or two injections of rFVIIIFc. Overall, 757 bleeding episodes (Arm 1 = 209, Arm 2 = 92, Arm 3 = 456) were treated in 106 of 164 subjects. 87.3% required a single injection of rFVIIIFc for resolution, and 97.8% required two or more injections. A total of 85.6%, 80.4%, and 89.5% of bleeding episodes were resolved with a single injection of rFVIIIFc in Arms 1, 2, and 3, respectively.

[0244] The hemostatic efficacy of rFVIIIFc during surgery was evaluated by the treating physician as excellent or good in 100% of surgeries.

[0245] The half-life of rFVIIIFc was approximately 19.0 hours compared to 12.4 hours for ADVATE®.

[0246] There were no subjects who developed inhibitors or who experienced hypersensitivity reactions to rFVIIIFc.

[0247] Recombinant FVIIIFc was generally well tolerated.

[0248] A-LONG was the largest registered worldwide pivotal Phase 3 study of a long-acting rFVIII in severe hemophilia A conducted to date. The study demonstrated that rFVIIIFc offers the potential for significantly reduced injection frequency, decreased treatment burden, and improved clinical outcomes for bleeding prevention in patients with severe hemophilia A.

[0249] Objectives of the study design: The study was designed to evaluate the efficacy and safety of rFVIIIFc in the control and prevention of bleeding episodes, routine prophylaxis, and perioperative management in subjects with severe hemophilia A. In addition, the study was designed to evaluate the effective dose and interval of rFVIIIFc for prophylaxis and the feasibility of weekly treatment at a dose of 65 IU / kg.

[0250] On-demand treatment is the administration of replacement factor only when needed to treat bleeding symptoms after bleeding has started. Prophylactic treatment is the regular administration of replacement factor to prevent bleeding symptoms.

[0251] The starting regimen was 25 IU / kg on day 1 and 50 IU / kg on day 4. The dosing and frequency of treatment could be adjusted based on the PK profile of the subject, and the goal was to maintain a sufficient factor trough level to prevent bleeding symptoms using a dosing of 25 IU / kg to 65 IU / kg and a treatment interval of 3 to 5 days.

[0252] More than half of the severe hemophilia patients in the United States still do not follow a prophylactic regimen, partly due to the treatment burden. Therefore, a once-weekly dosing regimen was included in the A-LONG study to evaluate whether this regimen would result in a lower ABR compared to the on-demand regimen. The median ABR in the once-weekly regimen was 3.59 (IQR 1.9, 8.4) compared to 33.6 (IQR 21.1, 48.7) for on-demand treatment.

[0253] All subjects had an initial PK assessment to characterize the PK of rFVIIIFc in a representative population of patients with hemophilia A.

[0254] Furthermore, a more extensive PK sampling was performed in a subset of subjects in the individualized prophylaxis arm (arm 1) at baseline, after a single dose of ADVATE® (50 IU / kg), and after a 4-day washout, followed by a single dose of rFVIIIFc (50 IU / kg). Blood samples were taken for ADVATE® over a 72-hour period. Blood samples were then taken for rFVIIIFc over a 120-hour period. The PK assessment of rFVIIIFc in this subset was repeated at 12 to 24 weeks with the same PK sampling schedule.

[0255] In the perioperative management, a single injection of rFVIIIFc was sufficient to maintain hemostasis until the end date / time of all major surgeries at a median dose of 51.4 IU / kg. The median rFVIIIFc consumption (summarized across all injections during each reference period) was 80.6 IU / kg on the day of major surgery, 161.3 IU / kg on days 1 - 3 following surgery, and 387.1 IU / kg on days 4 - 14 following surgery. Perioperative hemostasis with rFVIIIFc was evaluated as excellent or good for all 9 major surgeries. No subjects reported bleeding symptoms during the postoperative or rehabilitation period. Generally, 7 adverse events (AEs) were reported in 4 subjects (44.4%) in the surgical subgroup, 6 of which were of mild or moderate severity and 1 was considered severe. Two serious AEs (inguinal hernia and appendicitis) were reported in 2 subjects. All AEs during the perioperative period were evaluated by the researchers as unrelated to rFVIIIFc treatment.

[0256] Treating physicians evaluated the hemostatic efficacy of rFVIIIFc in 100% of surgeries as excellent or good on a 4 - point scale that included excellent, good, fair, and poor / non - evaluable.

[0257] rFVIIIFc resulted in a low median ABR of 3.59 in the weekly prophylaxis arm and 1.60 in the individualized interval prophylaxis arm. In contrast, the on - demand treatment arm had a median ABR of 33.57.

[0258] In the individualized prophylaxis arm, the median dosing interval was 3.5 days at a median dose of 77.7 IU / kg during the last 3 months of the study.

[0259] The terminal half - life was approximately 18.97 hours for rFVIIIFc and approximately 12.3 hours for ADVATE®.

[0260] 97.8% of bleeding symptoms were controlled with 1 or 2 injections.

[0261] The hemostatic efficacy of rFVIIIFc for perioperative management was evaluated by treating physicians as excellent or good in 100% of surgeries.

[0262] Arm 2 had a weekly dosing regimen. It was designed to investigate the benefit to patients in prophylactic therapy and compare subjects using the on-demand treatment regimen to these results. The median ABR in Arm 2 was 3.59 compared to 33.57 for the on-demand treatment regimen (Arm 3).

[0263] The median dose per injection required for bleeding resolution was 27.4 IU / kg, and the median total dose required was 28.2 IU / kg.

[0264] The median number of injections required for bleeding symptom resolution was consistently 1.0 when treatment was administered within 8 hours of bleeding onset, regardless of bleeding type or location. Generally, 78.8% of rFVIIIFc injections in Arm 1, 64.8% in Arm 2, and 79.7% of injections in Arm 3 were evaluated to produce an excellent or good response by the subjects. The median dosing interval of 3.5 days from Arm 1 represents a dosing interval achievable by the majority of patients: 30% of patients were able to achieve a dosing interval of at least 5 days. Some patients were able to achieve a regimen for weekly prophylaxis (Arm 2) with a median ABR of bleeding symptoms of 3.59.

[0265] Subjects in the individualized prophylaxis arm received an initial dose of 25 IU / kg on Day 1 and 50 IU / kg on Day 4. The study design allowed for adjustment of dosing intervals and amounts to maintain target factor trough levels and prevent bleeding. We believe that the median dosing intervals and amounts based on the last 6 months of the study most represent the individualized prophylaxis regimen.

[0266] 87.3% of bleeding episodes were resolved with a single injection of rFVIIIFc, and 97.8% were resolved with one or two injections. For the 96 (12.7%) bleeding episodes that required more than two injections for resolution, the median interval between the first and second injections was 30.9 hours.

[0267] The investigators' global assessment of the subject response to the rFVIIIFc regimen assigned to the subjects was rated as excellent or effective for 99.4%, 100%, and 98.1% of subject visits in Arms 1, 2, and 3, respectively.

[0268] In A-LONG, coagulation factor activity was measured using one-stage (aPTT) coagulation assays and chromogenic assays. The reported half-lives were based on the results of the one-stage coagulation assays. The half-lives of rFVIIIFc and ADVATE® were both slightly longer in the chromogenic assay, while the ratio between the products was consistent between the two assays.

[0269] Embodiments E1. A method of reducing the annualized bleeding rate in a subject having hemophilia, the method comprising administering to the subject an effective amount of a long-acting FVIII polypeptide at an administration interval of about three days or more.

[0270] E2. The method of embodiment E1, wherein the administration is prophylactic administration, individualized to the subject, and results in an annualized bleeding rate of less than about 5.0, less than about 4.9, less than about 4.8, less than about 4.7, less than about 4.6, or less than about 4.5.

[0271] E3. The method of embodiment E2, wherein the median annualized bleeding rate is about 1.6.

[0272] E4. The method of embodiment E1, wherein the administration is prophylactic administration and results in an annualized bleeding rate of less than about 9.0, less than about 8.9, less than about 8.8, less than about 8.7, less than about 8.6, less than about 8.5, or less than about 8.4 per week.

[0273] The method according to embodiment E4, wherein the median annualized bleeding rate is about 3.6.

[0274] The method according to embodiment E1, wherein the administration is on-demand or at the time of onset, resulting in an annualized bleeding rate of less than about 55, less than about 54, less than about 53, less than about 52, less than about 51, less than about 50, less than about 49, less than about 48, or less than about 47.

[0275] The method according to embodiment E6, wherein the median annualized bleeding rate is about 33.6.

[0276] The method according to any one of embodiments E1 to E7, wherein the effective amount is from about 20 IU / kg to about 90 IU / kg.

[0277] The method according to any one of embodiments E1 to E8, wherein the effective amount is 20 - 30 IU / kg, 30 - 40 IU / kg, 40 - 50 IU / kg, 50 - 60 IU / kg, 60 - 70 IU / kg, 70 - 80 IU / kg, or 80 - 90 IU / kg.

[0278] The method according to any one of embodiments E1 to E9, wherein the effective amount is 20 IU / kg, 25 IU / kg, 30 IU / kg, 35 IU / kg, 40 IU / kg, 45 IU / kg, 50 IU / kg, 55 IU / kg, 60 IU / kg, 65 IU / kg, 70 IU / kg, 75 IU / kg, 80 IU / kg, 85 IU / kg, or 90 IU / kg.

[0279] The method according to any of embodiments E8 to E10, wherein the administration is prophylactic and individualized with an effective amount of about 25 IU / kg to about 65 IU / kg twice a week or every three days, or about 50 IU / kg to about 65 IU / kg every four or five days.

[0280] The method according to embodiment E11, wherein the first dose of the long - acting FVIII polypeptide is about 25 IU / kg and the second dose of the long - acting FVIII polypeptide is about 50 IU / kg.

[0281] E13. The method according to any one of embodiments E8 - E10, wherein the administration is prophylactic administration, carried out weekly at an effective amount of 65 IU / kg per week.

[0282] E14. The method according to any one of embodiments E8 - E10, wherein the administration is on - demand or at - onset administration at an effective amount of 10 IU / kg to 75 IU / kg every 12 - 24 hours.

[0283] E15. The method according to any one of embodiments E1 - E7, wherein the effective amount is a fixed dosage that is standard across the total body weight.

[0284] E16. The method according to embodiment E15, wherein the fixed dosage is about 2,000 IU, about 2,500 IU, about 3,000 IU, about 3,500 IU, or about 4,000 IU per administration.

[0285] E17. The method according to any one of embodiments E1 - E7, wherein the effective amount is a stratified administration.

[0286] E18. The method according to any one of embodiments E1 - E17, wherein the trough level of the long - acting FVIII polypeptide is greater than 1%, greater than 2%, or greater than 3% of normal.

[0287] E19. The method according to any one of embodiments E1 - E18, wherein the long - acting FVIII polypeptide has a T 1 / 2ベータ (activity) of about 7 hours to about 48 hours, about 6 hours to about 49 hours, about 5 hours to about 50 hours.

[0288] E20. The method according to embodiment E19, wherein the long - acting FVIII has a T 1 / 2ベータ (activity) average of at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours.

[0289] E21.T 1 / 2ベータ The method according to embodiment E20, wherein the (active) average is about 19 hours.

[0290] E22.T 1 / 2ベータ The method according to embodiment E19 or E20, wherein the (active) average is at least about 1.5 times higher than the polypeptide consisting of amino acids 20 to 1457 of SEQ ID NO: 2 and amino acids 20 to 2351 of SEQ ID NO: 6, or ADVATE®.

[0291] E23. The method according to any one of embodiments E1 to E22, wherein the plasma trough level of the long-acting FVIII polypeptide is maintained at about 1% to about 5%, about 1% to about 6%, about 1% to about 7%, about 1% to about 8%, about 1% to about 9%, about 1% to about 10%, about 1% to about 11%, about 1% to about 12%, about 1% to about 13%, about 1% to about 14%, about 1% to about 15% above the baseline in the subject.

[0292] E24. The method according to any one of embodiments E1 to E23, wherein the long-acting FVIII polypeptide is a long-acting FVIII polypeptide comprising an FVIII polypeptide and a heterologous moiety.

[0293] E25. The method according to embodiment E24, wherein the heterologous moiety is an FcRn binding partner.

[0294] E26. The method according to embodiment E25, wherein the FcRn binding partner comprises an Fc region.

[0295] E27. The method according to embodiment E25 or E26, wherein the long-acting FVIII polypeptide further comprises a second FcRn binding partner.

[0296] E28. The method according to embodiment E27, wherein the second FcRn binding partner comprises a second Fc region.

[0297] E29. The method according to embodiment E27 or E28, wherein the FcRn binding partner and the second FcRn binding partner are bound.

[0298] E30. The method according to embodiment E29, wherein the bond is a covalent bond.

[0299] E31. The method according to embodiment E30, wherein the covalent bond is a disulfide bond.

[0300] E32. The method according to any one of embodiments E27 to E31, wherein the second FcRn binding partner is not bound to the amino acid sequence by a peptide bond.

[0301] E33. The method according to any one of embodiments E1 to E32, wherein the long-acting FVIII polypeptide is an FVIII monomer-dimer hybrid.

[0302] E34. The method according to any one of embodiments E24 to E33, wherein the FVIII polypeptide in the long-acting polypeptide is human FVIII.

[0303] E35. The method according to any one of embodiments E24 to E34, wherein the FVIII polypeptide in the long-acting polypeptide is full-length FVIII or B-domain-deleted FVIII.

[0304] E36. The method according to any one of embodiments E24 to E34, wherein the FVIII polypeptide is single-chain.

[0305] E37. The method according to any one of embodiments E24 to E34, wherein the FVIII polypeptide is unprocessed.

[0306] E38. The method according to any one of embodiments E24 to E34, wherein the FVIII polypeptide is within two chains, a first chain containing the heavy chain of the FVIII polypeptide and a second chain containing the light chain of the FVIII polypeptide.

[0307] The method according to any one of embodiments E25 to E38, wherein the FcRn binding partner in the chimeric polypeptide is human Fc.

[0308] The method according to any one of embodiments E24 to E39, wherein the FVIII polypeptide is at least 60%, 70%, 80%, 90%, 95%, 95%, 97%, 98%, 99%, or 100% identical to the FVIII amino acid sequence shown in Table 11A or 11B (amino acids 20 to 1457 of SEQ ID NO: 2 or amino acids 20 to 2351 of SEQ ID NO: 6) and does not contain a signal sequence.

[0309] The method according to any one of embodiments E25 to E40, wherein the FcRn binding partner is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the Fc amino acid sequence shown in Table 11B (amino acids 21 to 247 of SEQ ID NO: 4) and does not contain a signal sequence.

[0310] The method according to any one of embodiments E27 to E41, wherein the second FcRn binding partner in the chimeric polypeptide is human Fc.

[0311] The method according to embodiment E42, wherein the second FcRn binding partner is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the Fc amino acid sequence shown in Table 11B (amino acids 21 to 247 of SEQ ID NO: 4) and does not contain a signal sequence.

[0312] The method according to any one of embodiments E1 to E43, wherein the fixed dose of the coagulation factor is for the perioperative management of bleeding symptoms.

[0313] The method according to any one of embodiments E1 to E44, wherein the subject requires management or prevention of bleeding or bleeding symptoms.

[0314] E46. The method according to embodiment E45, wherein the subject requires management or prevention of bleeding in mild bleeding, hemarthrosis, superficial muscle bleeding, soft tissue bleeding, moderate bleeding, intramuscular or soft tissue bleeding by incision, mucosal bleeding, hematuria, massive bleeding, pharyngeal bleeding, posterior pharyngeal bleeding, retroperitoneal bleeding, central nervous system bleeding, contusion, laceration, abrasion, joint bleeding, epistaxis, oral bleeding, bleeding from the gums, intracranial bleeding, intra-abdominal bleeding, mild spontaneous bleeding, bleeding after major trauma, moderate skin injury, or spontaneous bleeding into joints, muscles, internal organs, or the brain.

[0315] E47. The method according to embodiment E46, wherein the subject requires management of bleeding associated with surgery or tooth extraction.

[0316] E48. The method according to embodiment E47, wherein the subject will undergo, is undergoing, or has undergone major surgery.

[0317] E49. The method according to embodiment E48, wherein the major surgery is orthopedic surgery, extensive oral surgery, urological surgery, or hernia surgery.

[0318] E50. The method according to embodiment E49, wherein the orthopedic surgery is replacement of the knee, hip, or other major joint.

[0319] E51. The method according to any one of embodiments E1 to E50, wherein the subject requires long-term treatment.

[0320] E52. The method according to any one of embodiments E1 to E51, wherein the long-acting FVIII polypeptide is administered intravenously or subcutaneously.

[0321] E53. The long-acting FVIII polypeptide is (a) a long-acting FVIII polypeptide and (b) one or more stabilizers selected from the group consisting of sucrose, trehalose, raffinose, arginine, and mixtures thereof. (c) Sodium chloride (NaCl), and (d) L-Histidine, and (e) Calcium chloride, and (f) Polysorbate 20 or polysorbate 80, formulated into a pharmaceutical composition, the method according to any one of embodiments E1 to E52.

[0322] E54. The method according to embodiment E53, wherein the pharmaceutical composition does not contain mannitol, glycine, alanine, or hydroxyethyl starch.

[0323] E55. The method according to embodiment E53 or E54, wherein the pharmaceutical composition contains NaCl as the sole filler.

[0324] E56. The method according to any one of embodiments E53 to E55, wherein the pharmaceutical composition contains sucrose.

[0325] E57. The method according to embodiment E56, wherein the pharmaceutical composition contains about 1% (w / v) to about 2.5% (w / v) of sucrose.

[0326] E58. The method according to embodiment E57, wherein the pharmaceutical composition contains from about 1.3% (w / v) of sucrose to about 2.0% (w / v) of sucrose.

[0327] E59. The method according to embodiment E58, wherein the pharmaceutical composition contains about 1.33% (w / v) of sucrose or about 2.0% (w / v) of sucrose.

[0328] E60. The method according to embodiment E56, wherein the pharmaceutical composition contains from about 10 mg / ml to about 25 mg / ml of sucrose.

[0329] E61. The method according to embodiment E60, wherein the pharmaceutical composition contains from about 13 mg / ml to about 20 mg / ml of sucrose.

[0330] The method according to embodiment E61, wherein the pharmaceutical composition comprises sucrose at about 13.3 mg / ml or sucrose at about 20.0 mg / ml.

[0331] The method according to any one of embodiments E53 - E62, wherein the pharmaceutical composition comprises NaCl at about 150 mM to about 250 mM.

[0332] The method according to embodiment E63, wherein the pharmaceutical composition comprises NaCl at about 175 mM to about 225 mM.

[0333] The method according to embodiment E64, wherein the pharmaceutical composition comprises NaCl at about 200 mM to about 210 mM.

[0334] The method according to embodiment E65, wherein the pharmaceutical composition comprises NaCl at about 205 mM.

[0335] The method according to any one of embodiments E53 - E62, wherein the pharmaceutical composition comprises NaCl at about 8.8 mg / ml to about 14.6 mg / ml.

[0336] The method according to embodiment E67, wherein the pharmaceutical composition comprises NaCl at about 10 mg / ml to about 13 mg / ml.

[0337] The method according to embodiment E68, wherein the pharmaceutical composition comprises NaCl at about 12.0 mg / ml.

[0338] The method according to any one of embodiments E53 - E62, wherein the pharmaceutical composition comprises NaCl at about 250 mM to about 350 mM.

[0339] The method according to embodiment E70, wherein the pharmaceutical composition comprises NaCl at about 275 mM to about 325 mM.

[0340] The method according to embodiment E71, wherein the pharmaceutical composition comprises NaCl at about 308 mM.

[0341] The method according to any one of Embodiments E53 to E62, wherein the pharmaceutical composition contains NaCl at about 14.6 mg / ml to about 20.5 mg / ml.

[0342] The method according to Embodiment E73, wherein the pharmaceutical composition contains NaCl at about 16 mg / ml to about 19 mg / ml.

[0343] The method according to Embodiment E74, wherein the pharmaceutical composition contains NaCl at about 18.0 mg / ml.

[0344] The method according to any one of Embodiments E53 to E75, wherein the pharmaceutical composition contains L-histidine at about 5 mM to about 15 mM.

[0345] The method according to Embodiment E76, wherein the pharmaceutical composition contains L-histidine at about 6.64 mM or about 9.8 mM.

[0346] The method according to any one of Embodiments E53 to E75, wherein the pharmaceutical composition contains L-histidine at about 0.75 mg / ml to about 2.25 mg / ml.

[0347] The method according to Embodiment E78, wherein the pharmaceutical composition contains L-histidine at about 1.03 mg / ml or about 1.55 mg / ml.

[0348] The method according to any one of Embodiments E53 to E79, wherein the pharmaceutical composition contains calcium chloride at about 5 mM to about 10 mM.

[0349] The method according to Embodiment E80, wherein the pharmaceutical composition contains calcium chloride at about 5.4 mM or about 8 mM.

[0350] The method according to any one of Embodiments E53 to E79, wherein the pharmaceutical composition contains calcium chloride dihydrate at about 0.75 mg / ml to about 1.5 mg / ml.

[0351] The method according to embodiment E82, wherein the pharmaceutical composition comprises about 0.8 mg / ml of calcium chloride dihydrate or about 1.18 mg / ml of calcium chloride dihydrate.

[0352] The method according to any one of embodiments E53 to E83, wherein the pharmaceutical composition comprises about 0.008% (w / v) to about 0.025% (w / v) of polysorbate 20 or polysorbate 80.

[0353] The method according to embodiment E84, wherein the pharmaceutical composition comprises about 0.013% (w / v) of polysorbate 20 or polysorbate 80, or about 0.02% (w / v) of polysorbate 20 or polysorbate 80.

[0354] The method according to any one of embodiments E53 to E83, wherein the pharmaceutical composition comprises about 0.08 mg / ml to about 0.25 mg / ml of polysorbate 20 or polysorbate 80.

[0355] The method according to embodiment E86, wherein the pharmaceutical composition comprises about 0.13% mg / ml of polysorbate 20 or polysorbate 80 or about 0.20 mg / ml of polysorbate 20 or polysorbate 80.

[0356] The method according to any one of embodiments E53 to E87, wherein the rFVIIIFc polypeptide comprises a first subunit comprising an amino acid sequence that is at least 90% or 95% identical to amino acids 20 to 1684 of SEQ ID NO: 2 or amino acids 20 to 2578 of SEQ ID NO: 6, and a second subunit comprising an amino acid sequence that is at least 90% to 95% identical to amino acids 21 to 247 of SEQ ID NO: 4.

[0357] The method according to embodiment E88, wherein the rFVIIIFc polypeptide comprises a first subunit comprising amino acids 20 to 1684 of SEQ ID NO: 2 or amino acids 20 to 2578 of SEQ ID NO: 6, and a second subunit comprising amino acids 21 to 247 of SEQ ID NO: 4.

[0358] E90. The method according to any one of Embodiments E53 to E89, wherein the long-acting FVIII polypeptide is present at a concentration of about 50 IU / ml to about 2500 IU / ml.

[0359] E91. The method according to Embodiment E90, wherein the pharmaceutical composition comprises a long-acting FVIII polypeptide at 83 IU / ml, 167 IU / ml, 250 IU / ml, 333 IU / ml, 500 IU / ml, 667 IU / ml, 1000 IU / ml, 1333 IU / ml, 1667 IU / ml, or 2000 IU / ml.

[0360] E92. The method according to any one of Embodiments E53 to E89, wherein the long-acting FVIII polypeptide is present at a concentration of about 100 IU / ml to about 4000 IU / ml.

[0361] E93. The method according to Embodiment E92, wherein the pharmaceutical composition comprises a long-acting FVIII polypeptide at 150 IU / ml, 287.5 IU / ml, 431.25 IU / ml, 575 IU / ml, 862.5 IU / ml, 1150 IU / ml, 1725 IU / ml, 2300 IU / ml, 2875 IU / ml, or 3450 IU / ml.

[0362] E94. The pharmaceutical composition comprises (a) a long-acting FVIII polypeptide of about 50 IU / ml to about 2500 IU / ml, and (b) sucrose of about 1% (w / v) to about 2.5% (w / v), and (c) NaCl of about 150 mM to about 250 mM, and (d) L-histidine of about 5 mM to about 15 mM, and (e) calcium chloride of about 5 mM to about 10 mM, and (f) polysorbate 20 or polysorbate 80 of about 0.008% (w / v) to about 0.025%, and the method according to any one of Embodiments E53 to E56.

[0363] The method according to embodiment E94, wherein the pharmaceutical composition comprises from about 175 mM to about 225 mM of NaCl.

[0364] The method according to embodiment E95, wherein the pharmaceutical composition comprises from about 200 mM to about 210 mM of NaCl.

[0365] E97. The pharmaceutical composition comprises (a) a long-acting FVIII polypeptide of about 83 IU / ml, about 167 IU / ml, about 250 IU / ml, about 333 IU / ml, about 500 IU / ml, about 667 IU / ml, about 1000 IU / ml, about 1333 IU / ml, about 1667 IU / ml, or about 2000 IU / ml; and (b) about 1.33% (w / v) of sucrose; and (c) about 205 mM of NaCl; and (d) about 6.64 mM of L-histidine; and (e) about 5.4 mM of calcium chloride; and (f) about 0.013% (w / v) of polysorbate 20 or polysorbate 80, and the method according to embodiment E96.

[0366] E98. The pharmaceutical composition comprises (a) a long-acting FVIII polypeptide of about 100 IU / ml to about 4000 IU / ml; and (b) about 1% (w / v) to about 2.5% (w / v) of sucrose; and (c) about 250 mM to about 350 mM of NaCl; and (d) about 5 mM to about 15 mM of L-histidine; and (e) about 5 mM to about 10 mM of calcium chloride; and (f) about 0.008% (w / v) to about 0.025% of polysorbate 20 or polysorbate 80, and the method according to any one of embodiments E53 - E56.

[0367] The method according to embodiment E98, wherein the pharmaceutical composition comprises from about 275 mM to about 325 mM of NaCl.

[0368] E100. The pharmaceutical composition comprises (a) a long-acting FVIII polypeptide of about 150 IU / ml, about 287.5 IU / ml, about 431.25 IU / ml, about 575 IU / ml, about 862.5 IU / ml, about 1150 IU / ml, about 1725 IU / ml, about 2300 IU / ml, about 2875 IU / ml, or 3450 IU / ml, and (b) about 2.0% (w / v) sucrose, and (c) about 308 mM NaCl, and (d) about 9.8 mM L-histidine, and (e) about 8 mM calcium chloride, and (f) about 0.020% (w / v) polysorbate 20 or polysorbate 80, and is the method according to embodiment E99.

[0369] E101. The pharmaceutical composition comprises (a) a long-acting FVIII polypeptide of about 50 IU / ml to about 2500 IU / ml, and (b) about 10 mg / ml to about 25 mg / ml sucrose, and (c) about 8.8 mg / ml to about 14.6 mg / ml NaCl, and (d) about 0.75 mg / ml to about 2.25 mg / ml L-histidine, and (e) about 0.75 mg / ml to about 1.5 mg / ml calcium chloride dihydrate, and (f) about 0.08 mg / ml to about 0.25 mg / ml polysorbate 20 or polysorbate 80, and is the method according to any one of embodiments E53 to E56.

[0370] E102. The pharmaceutical composition according to embodiment E101, wherein the pharmaceutical composition comprises about 10 mg / ml to 13 mg / ml NaCl.

[0371] E103. The pharmaceutical composition comprises (a) About 83 IU / ml, about 167 IU / ml, about 250 IU / ml, about 333 IU / ml, about 500 IU / ml, about 667 IU / ml, about 1000 IU / ml, about 1333 IU / ml, about 1667 IU / ml, or about 2000 IU / ml of a long-acting FVIII polypeptide, and (b) About 13.3 mg / ml of sucrose, and (c) About 12.0 mg / ml of NaCl, and (d) About 1.03 mg / ml of L-histidine, and (e) About 0.8 mg / ml of calcium chloride dihydrate, and (f) About 0.13 mg / ml of polysorbate 20 or polysorbate 80, a method according to embodiment E102.

[0372] E104. The pharmaceutical composition is (a) About 100 IU / ml to about 4000 IU / ml of a long-acting FVIII polypeptide, and (b) About 10 mg / ml to about 25 mg / ml of sucrose, and (c) About 14.6 mg / ml to about 20.5 mg / ml of NaCl, and (d) About 0.75 mg / ml to about 2.25 mg / ml of L-histidine, and (e) About 0.75 mg / ml to about 1.5 mg / ml of calcium chloride dihydrate, and (f) About 0.08 mg / ml to about 0.25 mg / ml of polysorbate 20 or polysorbate 80, a method according to any one of embodiments E53 - E56.

[0373] E105. The pharmaceutical composition contains about 16 mg / ml to about 19 mg / ml of NaCl, a method according to embodiment E104.

[0374] E106. The pharmaceutical composition is (a) about 150 IU / ml, about 287.5 IU / ml, about 431.25 IU / ml, about 575 IU / ml, about 862.5 IU / ml, about 1150 IU / ml, about 1725 IU / ml, about 2300 IU / ml, about 2875 IU / ml, or 3450 IU / ml of a long-acting FVIII polypeptide, and (b) about 20.0 mg / ml of sucrose, and (c) about 18.0 mg / ml of NaCl, and (d) about 1.55 mg / ml of L-histidine, and (e) about 1.18 mg / ml of calcium chloride dihydrate, and (f) about 0.20 mg / ml of polysorbate 20 or polysorbate 80, comprising the method according to embodiment E105.

[0375] E107. A long-acting FVIII polypeptide is a pharmaceutical kit, (a) a first container containing a lyophilized powder, the powder comprising (i) a long-acting FVIII polypeptide, and (ii) one or more stabilizers selected from the group consisting of sucrose, trehalose, raffinose, arginine, and mixtures thereof, and (iii) sodium chloride (NaCl), and (iv) L-histidine, and (v) calcium chloride, and (vi) polysorbate 20 or polysorbate 80, the first container; and (b) a second container containing sterile water for injection to be combined with the lyophilized powder of the first container, packed in a pharmaceutical kit, the method according to any one of embodiments E1 - E52.

[0376] E108. The pharmaceutical kit does not contain mannitol, glycine, alanine, or hydroxyethyl starch, the method according to embodiment E107.

[0377] The method according to embodiment E107 or E108, wherein the pharmaceutical kit contains NaCl as the sole filler.

[0378] E110. The pharmaceutical kit comprises (a) A first container containing a lyophilized powder, the powder comprising (i) a long-acting FVIII polypeptide of about 250 IU, about 500 IU, about 750 IU, about 1000 IU, about 1500 IU, about 2000 IU, about 3000 IU, about 4000 IU, about 5000 IU, or about 6000 IU; (ii) about 40 mg of sucrose; (iii) about 36 mg of sodium chloride; (iv) about 3.1 mg of L-histidine; (v) about 2.40 mg of calcium chloride dihydrate; (v) about 0.40 mg of polysorbate 20 or polysorbate 80, and a first container; (b) A second container containing sterile water for injection, which, when combined with the lyophilized powder in the first container, (i) respectively, a long-acting FVIII polypeptide of about 83 IU / ml, about 167 IU / ml, about 250 IU / ml, about 333 IU / ml, about 500 IU / ml, about 667 IU / ml, about 1000 IU / ml, about 1333 IU / ml, or about 1667 IU / ml, or about 2000 IU / ml; (ii) about 1.33% (w / v) of sucrose; (iii) about 205 mM of NaCl; (iv) about 6.64 mM of L-histidine; (v) about 5.4 mM of calcium chloride; (vi) about 0.013% (w / v) of polysorbate 20 or polysorbate 80, and a second container containing sterile water for injection in a volume sufficient to produce a solution, according to any one of embodiments E107 to E109.

[0379] E111. The pharmaceutical composition comprises (a) A first container containing a lyophilized powder, the powder comprising (i) a long-acting FVIII polypeptide of about 250 IU, about 500 IU, about 750 IU, about 1000 IU, about 1500 IU, about 2000 IU, about 3000 IU, about 4000 IU, about 5000 IU, or about 6000 IU; and (ii) about 40 mg of sucrose; and (iii) about 36 mg of sodium chloride; and (iv) about 3.1 mg of L-histidine; and (v) about 2.40 mg of calcium chloride dihydrate; and (v) about 0.40 mg of polysorbate 20 or polysorbate 80, the first container; and (b) A second container containing sterile water for injection, which, when combined with the lyophilized powder of the first container, (i) respectively, a long-acting FVIII polypeptide of about 83 IU / ml, about 167 IU / ml, about 250 IU / ml, about 333 IU / ml, about 500 IU / ml, about 667 IU / ml, about 1000 IU / ml, about 1333 IU / ml, about 1667 IU / ml, or about 2000 IU / ml; and (ii) about 13.3 mg / ml of sucrose; and (iii) about 12.0 mg / ml of NaCl; and (iv) about 1.03 mg / ml of L-histidine; and (v) about 0.80 mg / ml of calcium chloride; and (vi) about 0.13 mg / ml of polysorbate 20 or polysorbate 80, in a volume sufficient to produce a solution, the second container containing sterile water for injection, a method according to any one of embodiments E107 - E109.

[0380] E112. The method according to any one of embodiments E107 - E111, wherein the first container is a glass vial with a rubber stopper.

[0381] E113. The method according to any one of embodiments E107 - E112, wherein the second container is a syringe body, and the syringe body is associated with a plunger.

[0382] E114. The method according to embodiment E113, wherein the pharmaceutical kit further comprises an adapter for connecting a glass vial to the syringe body.

[0383] E115. The method according to embodiment E113 or embodiment E114, wherein the pharmaceutical kit further comprises an infusion tube that is connected to a needle suitable for intravenous injection.

[0384] E116. The method according to any one of embodiments E70 to E75, E92, E93, E98 to E100, and E104 to E106, wherein the pharmaceutical composition is the composition before lyophilization.

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[0385] The foregoing description of specific embodiments will enable others skilled in the art to make and use the invention without undue experimentation and without departing from the general concept of the invention, by applying knowledge within the skill of the art, and will fully disclose the general nature of the invention which can be readily modified and / or adapted for various applications of such specific embodiments. Accordingly, such adaptations and modifications are intended to be within the meaning and scope of the equivalents of the disclosed embodiments, based on the teachings and guidance presented herein. It is to be understood that the terminology or phraseology herein is for the purpose of description and not of limitation, as such terminology or phraseology would be understood by those skilled in the art in light of the teachings and guidance.

[0386] Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. The specification and examples are to be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

[0387] All patents and publications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A pharmaceutical composition comprising: (a) a chimeric polypeptide comprising a Factor VIII (FVIII) polypeptide and an Fc region; (b) sucrose at about 10 mg / mL to about 25 mg / mL; (c) sodium chloride (NaCl) at about 10 mg / mL to about 13 mg / mL; (d) L-histidine at about 0.75 mg / mL to about 2.25 mg / mL; (e) calcium chloride at about 5 mM to about 10 mM; (f) polysorbate 20 or polysorbate 80 at about 0.08 mg / mL to about 0.25 mg / mL. A pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, wherein the Fc region is a first Fc region comprising an amino acid sequence that is at least 95% identical to amino acids 21 to 247 of SEQ ID NO:

4.

3. The pharmaceutical composition according to claim 1 or 2, comprising 13.3 mg / mL of sucrose.

4. The pharmaceutical composition according to any one of claims 1 to 3, comprising 12.0 mg / mL of NaCl.

5. The pharmaceutical composition according to any one of claims 1 to 4, comprising 1.03 mg / mL of L-histidine.

6. The pharmaceutical composition according to any one of claims 1 to 5, comprising 5.4 mM of calcium chloride.

7. The pharmaceutical composition according to any one of claims 1 to 6, comprising 0.13 mg / mL of polysorbate 20.

8. The pharmaceutical composition according to any one of claims 1 to 6, comprising 0.13 mg / mL of polysorbate 80.

9. The pharmaceutical composition according to any one of claims 1 to 8, comprising about 50 IU / mL to about 2500 IU / mL of the chimeric polypeptide.

10. The pharmaceutical composition according to any one of claims 1 to 9, comprising 83 IU / mL, 167 IU / mL, 250 IU / mL, 333 IU / mL, 500 IU / mL, 667 IU / mL, 1000 IU / mL, 1333 IU / mL, 1667 IU / mL, or 2000 IU / mL of the chimeric polypeptide.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein mannitol, glycine, alanine, or hydroxyethyl starch is not included as a filler in the pharmaceutical composition.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the chimeric polypeptide is an rFVIIIFc monomer-dimer hybrid. ​ **Claim 13**: The pharmaceutical composition according to claim 12, wherein the rFVIII Fc monomer-dimer hybrid comprises a second Fc region comprising an amino acid sequence that is at least 95% identical to amino acids 21 to 247 of SEQ ID NO:

4. **Claim 14**: The pharmaceutical composition according to claim 12 or 13, wherein the first Fc region comprises an Fc domain having a hinge region, the second Fc region comprises an Fc domain having a hinge region, and the first Fc region is covalently linked to the second Fc region via a disulfide bond. **Claim 15**: The pharmaceutical composition according to any one of claims 1 to 10, wherein the first Fc region comprises an amino acid sequence that is at least 99% identical to amino acids 21 to 247 of SEQ ID NO:

4. **Claim 16**: The pharmaceutical composition according to any one of claims 13 to 15, wherein the second Fc region comprises an amino acid sequence that is at least 99% identical to amino acids 21 to 247 of SEQ ID NO:

4. **Claim 17** The pharmaceutical composition according to any one of claims 1 to 16, comprising a combination of chimeric polypeptides of (i) a first rFVIII Fc monomer-dimer hybrid wherein the FVIII polypeptide is single-chain FVIII and (ii) a second rFVIII Fc monomer-dimer hybrid wherein the FVIII polypeptide is processed FVIII, in an amount of about 50 IU / mL to about 2500 IU / mL. **Claim 18** The pharmaceutical composition according to claim 17, wherein the single-chain FVIII comprises an amino acid sequence that is at least 95% identical to amino acids 20 to 1457 of SEQ ID NO:

2. **Claim 19** The pharmaceutical composition according to claim 17 or 18, wherein the single-chain FVIII comprises an amino acid sequence that is at least 99% identical to amino acids 20 to 1457 of SEQ ID NO: 2, and the first Fc region comprises an amino acid sequence that is at least 99% identical to amino acids 21 to 247 of SEQ ID NO:

4. **Claim 20**: (a) about 13.3 mg / mL of sucrose; (b) about 12.0 mg / mL of sodium chloride (NaCl); (c) about 1.03 mg / mL of L-histidine; (d) about 5.4 mM of calcium chloride; (e) about 0.13 mg / mL of polysorbate 20 or polysorbate 80; The pharmaceutical composition according to any one of claims 1 to 19, comprising the same. **Claim 21** The pharmaceutical composition for use according to any one of claims 1 to 20, wherein the polysorbate 20 or polysorbate 80 is polysorbate 20.

22. The pharmaceutical composition for use according to any one of claims 1 to 21, wherein the polysorbate 20 or polysorbate 80 is polysorbate 80.

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