Methods of Using FIX Polypeptides

Abstract

The present invention provides methods of administering long-acting Factor IX; methods of administering long-acting, chimeric and hybrid polypeptides comprising Factor IX; and methods of producing such chimeric and hybrid polypeptides using cells.

What is claimed is: 
     
         1 . A method of administering a long-acting Factor IX (FIX) polypeptide to a human subject in need thereof, comprising administering to the subject a dose of about 10 IU/kg to about 200 IU/kg of the long-acting FIX polypeptide at a dosing interval of about once a week or longer. 
     
     
         2 . The method of  claim 1 , wherein the subject is in need of treatment of a bleeding disorder. 
     
     
         3 . The method of  claim 1  or  2 , wherein the dose of the long-acting FIX polypeptide is about 10 IU/kg to about 50 IU/kg, about 10 IU/kg to about 100 IU/kg, about 25 IU/kg to about 50 IU/kg, about 25 IU/kg to about 75 IU/kg, about 25 IU/kg to about 100 IU/kg, about 25 IU/kg to about 125 IU/kg, about 25 IU/kg to about 150 IU/kg, about 50 IU/kg to about 100 IU/kg, about 50 IU/kg to about 150 IU/kg, about 100 IU/kg to about 150 IU/kg, about 150 IU/kg to about 200 IU/kg, or any combinations thereof. 
     
     
         4 . The method of any one of  claims 1  to  3 , wherein the dose of the long-acting FIX polypeptide is for prophylaxis of one or more bleeding episodes. 
     
     
         5 . The method of  claim 4 , wherein the dose is about 50 IU/kg. 
     
     
         6 . The method of  claim 4  or  5 , wherein an annualized bleeding rate of the bleeding episodes is less than 2, less than 3, less than 4, less than 5, less than 6, less than 7, less than 8, less than 9, or less than 10. 
     
     
         7 . The method of any one of  claims 1  to  3 , wherein the dose of the long-acting FIX polypeptide is for individualized interval prophylaxis of one or more bleeding episodes. 
     
     
         8 . The method of  claim 7 , wherein the dose of the long-acting FIX polypeptide is about 100 IU/kg. 
     
     
         9 . The method of  claim 7  or  8 , wherein an annualized bleeding rate of the bleeding episodes is less than 1, less than 2, less than 3, less than 4, less than 5, less than 6, less than 7, less than 8, or less than 9. 
     
     
         10 . The method of any one of  claims 1  to  3 , wherein the dose of the long-acting FIX polypeptide is for on-demand treatment of one or more bleeding episode. 
     
     
         11 . The method of  claim 10 , wherein an annualized bleeding rate of the bleeding episode is less than 10, less than 11, less than 12, less than 13, less than 14, less than 15, less than 16, less than 17, less than 18, less than 19, less than 20, less than 21, less than 22, less than 23, less than 24, less than 25, or less than 26. 
     
     
         12 . The method of any one of  claims 1  to  3 , wherein the dose of the long-acting FIX polypeptide is for perioperative management of a bleeding disorder. 
     
     
         13 . The method of any one of  claims 1  to  12 , wherein the dose is administered at a dosing frequency of about once a week to about once a month. 
     
     
         14 . The method of  claim 13 , wherein the dosing frequency is about once a week, about once in two weeks, about twice a month, about once in three weeks, about once in, four weeks, or about once a month. 
     
     
         15 . The method of  claim 14 , wherein the dosing frequency is about once a week. 
     
     
         16 . The method of  claim 14 , wherein the dosing frequency is about once in two weeks or about twice a month. 
     
     
         17 . The method of any one of  claims 1  to  14 , wherein the dosing interval is about every five days, about every six days, about every seven days, about every eight days, about every nine days, about every ten days, about every 11 days, about every 12 days, about every 13 days, about every 14 days, about every 15 days, about every 16 days, about every 17 days, about every 18 days, about every 19 days, about every 20 days, or about every 21 days. 
     
     
         18 . The method of  claim 17 , wherein the dosing interval is every 7 days to 14 days. 
     
     
         19 . The method of any one of  claims 1  to  18 , wherein the long-acting FIX polypeptide has a T 1/2beta  (activity) of at least about 40 hours, at least about 50 hours, at least about 60 hours, at least about 70 hours, at least about 80 hours, at least about 90 hours, at least about 100 hours, at least about 110 hours, at least about 120 hours, at least about 130 hours, at least about 140 hours, at least about 150 hours, at least about 160 hours, at least about 170 hours, at least about 180 hours, or at least about 190 hours. 
     
     
         20 . The method of  claim 19 , wherein the T 1/2beta  (activity) is about 40 to about 193 hours, about 50 hours to about 203 hours, about 30 hours to about 183 hours, about 40 hours to about 203 hours, about 40 hours to about 213 hours, about 40 hours to about 223 hours, about 30 hours to about 213 hours, about 30 hours to, about 223 hours, about 50 hours to about 213 hours, about 50 hours to about 223 hours, about 60 hours to about 203 hours, or about 60 hours to about 213 hours. 
     
     
         21 . The method of  claim 17  or  18 , wherein the T 1/2beta  (activity) is about 40 hours to about 193 hours. 
     
     
         22 . The method of any one of  claims 19  to  21 , wherein the mean of the T 1/2beta  (activity) is about 76 hours, about 77 hours, about 78 hours, about 79 hours, about 80 hours, about 81 hours, about 82 hours, about 83 hours, about 84 hours, about 85 hours, about 86 hours, about 87 hours, about 88 hours, about 89 hours, about 90 hours, about 91 hours, or about 92 hours. 
     
     
         23 . The method of any one of  claims 19  to  22 , wherein the mean of the T 1/2beta  (activity) is at least about 2.0 fold higher than a polypeptide consisting of amino acids 1 to 415 of SEQ ID NO:2 or BENEFIX®. 
     
     
         24 . The method of any one of  claim 23 , wherein the mean of the T 1/2beta  (activity) is at least about 2.0 fold, at least about 2.1 fold, at, least about 2.2 fold, at least about 2.3 fold, at least about 2.4 fold, at least about 2.5 fold, at least about 2.6 fold, at least about 2.7 fold, at least about 2.8 fold, at least about 2.9 fold, at least about 3.0 fold, at least about 3.1 fold, or at least about 3.2 fold higher than a polypeptide consisting of amino acids 1 to 415 of SEQ ID NO:2 or BENEFIX®. 
     
     
         25 . The method of any one of  claims 1  to  24 , wherein the long-acting FIX polypeptide has a T 1/2beta  (antigen) of at least about 60 hours, at least about 80 hours, at least about 100 hours, at least about 120 hours, at least about 140 hours, at least about 160 hours, at least about 180 hours, at least about 200 hours, at least about 220 hours, at least about 240 hours, at least about 260 hours, at least about 280 hours, at least about 300 hours, at least about 320 hours, at least about 340 hours, at least about 360 hours, or at least about 370 hours. 
     
     
         25 . The method of  claim 24 , wherein the T 1/2beta  (antigen) is about 62 to about 372 hours, about 63 hours to about 382 hours, about 63 hours to about 392 hours, about 63 hours to about 402 hours, about 63 hours to about 412 hours, about 53 hours to about 372 hours, about 53 hours to about 382 hours, about 53 hours to about 392 hours, about 53 hours to about 402 hours, about 53 hours to about 513 hours, about 70 hours to about 372 hours, about 70 hours to about 382 hours, about 70 hours to about 392 hours, about 70 hours to about 402 hours, or about 70 hours to about 412 hours. 
     
     
         27 . The method of  claim 25  or  26 , wherein the T 1/2beta  (antigen) is about 63 hours to about 372 hours. 
     
     
         28 . The method of any one of  claims 25  to  27 , wherein the mean of the T 1/2beta  (antigen) is about 115 hours, about 116 hours, about 117 hours, about 118 hours, about 119 hours, about 120 hours, about 121 hours, about 122 hours, about 123 hours, about 124 hours, about 125 hours, about 126 hours, about 127 hours, about 128 hours, about 129 hours, about 130 hours, or about 131 hours. 
     
     
         29 . The method of any one of  claims 25  to  28 , wherein the mean of the T 1/2beta  (antigen) is at least about 3.0 fold higher than a polypeptide consisting of amino acids 1 to 415 of SEQ ID NO:2 or BENEFIX®. 
     
     
         30 . The method of any one of  claim 39 , wherein the mean of the T 1/2beta  (antigen) is at least about 2.0 fold, at least about 2.5 fold, at least about 3.0 fold, at least about 3.5 fold, at least about 4.0 fold, at least about 4.5 told, at least about 5.0 fold higher than a polypeptide consisting of amino acids 1 to 415 of SEQ ID NO:2 or BENEFIX®. 
     
     
         31 . The method of any one of  claims 1  to  30 , further comprising measuring a baseline FIX activity of the subject prior to the administration of the long-acting FIX polypeptide. 
     
     
         32 . The method of any one of  claims 1  to  31 , wherein the plasma trough level of the long-acting FIX polypeptide is maintained at about 1% above the baseline in the subject after the administration. 
     
     
         33 . The method of  claim 32 , wherein the plasma trough level of the long-acting FIX polypeptide is maintained between about 1% and about 5%, between about 1% and about 6%, between about 1% and about 7%, between about 1% and about 8%, between about 1% and about 9%, between about 1% and about 10%, between about 1% and about 11%, between about 1% and about 12%, between about 1% and about 13%, between about 1% and about 14%, between about 1% and about 15% above the baseline in the subject. 
     
     
         34 . The method of any one of  claims 1  to  33 , wherein the dose comprises less than 25% of the long-acting Factor IX polypeptide in fully phosphorylated state and less than 25% of the long-acting Factor IX polypeptide in fully sulfated state. 
     
     
         35 . The method of  claim 34 , wherein the dose comprises less than about 10% of the long-acting Factor IX polypeptide in phosphorylated state and less than about 9% of the long-acting Factor IX polypeptide in the sulfated state. 
     
     
         36 . The method of any one of  claims 1  to  35 , wherein the dose is about 50 IU/kg, and the dosing interval is about 7 days. 
     
     
         37 . The method of any one of  claims 1  to  35 , wherein the dose is about 100 IU/kg and the dosing interval, is at least about 14 days. 
     
     
         38 . The method of any one of  claims 1  to  35 , wherein the dose is about 150 IU/kg and the dosing interval is at least about 21 days. 
     
     
         39 . The method of any one of  claims 1  to  38 , wherein the long-acting FIX polypeptide is a chimeric polypeptide comprising a FIX polypeptide and an FcRn binding partner. 
     
     
         40 . The method of  claim 39 , wherein the FcRn binding partner comprises an Fc region. 
     
     
         41 . The method of  claim 39  or  40 , wherein the long-acting FIX polypeptide further comprises a second FcRn binding partner. 
     
     
         42 . The method of  claim 41 , wherein the second FcRn binding partner comprises a second Fc region. 
     
     
         43 . The method of  claim 41  or  42 , wherein the FcRn binding partner and the second FcRn binding partner are associated. 
     
     
         44 . The method of  claim 43 , wherein the association is a covalent bond. 
     
     
         45 . The method of  claim 44  wherein the covalent bond is a disulfide bond. 
     
     
         46 . The method of any one of  claims 41  to  45 , wherein the second FcRn binding partner is not linked to an amino acid sequence by a peptide bond. 
     
     
         47 . The method of any one of  claims 1  to  46 , wherein the long-acting FIX polypeptide is FIX monomer dimer hybrid. 
     
     
         48 . The method of any of  claims 1  to  47 , wherein the subject is in need of control or prevention of bleeding or bleeding episodes. 
     
     
         49 . The method of  claim 48 , wherein the subject is in need of control or prevention of bleeding in minor hemorrhage, hemarthroses, superficial muscle hemorrhage, soft tissue hemorrhage, moderate hemorrhage, intramuscle or soft tissue hemorrhage with dissection, mucous membrane hemorrhage, hematuria, major hemorrhage, hemorrhage of the pharynx, hemorrhage of the retropharynx, hemorrhage of the retroperitonium, hemorrhage of the central nervous system, bruises, cuts, scrapes, joint hemorrhage, nose bleed, mouth bleed, gum bleed, intracranial bleeding, intraperitoneal bleeding, minor spontaneous hemorrhage, bleeding after major trauma, moderate skin bruising, or spontaneous hemorrhage into joints, muscles, internal organs or the brain. 
     
     
         50 . The method of  claim 48  or  49 , wherein the subject is in need of peri-operative management. 
     
     
         51 . The method of  claim 48  or  49 , wherein the subject is in need of management of bleeding associated with surgery or dental extraction. 
     
     
         52 . The method of  claim 48  or  49 , wherein the subject will undergo, is undergoing, or has undergone major surgery. 
     
     
         53 . The method of  claim 52 , wherein the major surgery is orthopedic surgery, extensive oral surgery, urologic surgery, or hernia surgery. 
     
     
         54 . The method of  claim 52 , wherein the orthopedic surgery is replacement of knee, hip, or other major joint. 
     
     
         55 . The method of any one of  claims 1  to  54 , wherein the subject is in need of prophylactic treatment. 
     
     
         56 . The method of any one of  claims 1  to  54 , wherein the subject is in need of on-demand treatment. 
     
     
         57 . The method of any one of  claims 1  to  54 , wherein the subject is in need of treatment for a bleeding episode. 
     
     
         58 . The method of  claim 57 , wherein the subject is in need of treatment for hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, or bleeding in the illiopsoas sheath. 
     
     
         59 . The method of  claim 58 , wherein the subject is in need of surgical prophylaxis, peri-operative management, or treatment for surgery. 
     
     
         60 . The method of  claim 59 , wherein said surgery is minor surgery, major surgery, tooth extraction, tonsillectomy, inguinal herniotomy, synovectomy, total knee replacement, craniotomy, osteosynthesis, trauma surgery, intracranial surgery, intra-abdominal surgery, intrathoracic surgery, or joint replacement surgery. 
     
     
         61 . The method of any of  claims 39  to  60 , wherein the FIX polypeptide in the chimeric polypeptide is a human Factor IX. 
     
     
         62 . The method of any of  claims 39  to  61 , wherein the FIX polypeptide in the chimeric polypeptide is a mutant Factor IX. 
     
     
         63 . The method of any of  claims 39  to  62 , wherein the FcRn binding partner in the chimeric polypeptide is a human Fc. 
     
     
         64 . The method of any of  claims 39  to  63 , wherein the FcRn binding partner in the chimeric polypeptide is a mutant Fc. 
     
     
         65 . The method of  claim 64 , wherein the FIX polypeptide is at least 90% or 95% identical to a FIX amino acid sequence shown in Table 8A without a signal sequence and propeptide (amino acids 1 to 415 of SEQ ID NO:2). 
     
     
         66 . The method of  claim 63 , wherein said Factor IX is identical to a Factor IX amino acid sequence shown in Table 8A without a signal sequence and propeptide (amino acids 1 to 415 of SEQ ID NO:2). 
     
     
         67 . The method of any one of  claims 40  to  66  wherein the Fc is at least 90% or 95% identical to a Fc amino acid sequence shown in Table 8B without a signal sequence (amino acids 1 to 227 SEQ ID NO:4). 
     
     
         68 . The method of  claim 67 , wherein said Fc is identical to a Fc amino acid sequence shown in Table 8B without a signal sequence (amino acids 1 to 227 of SEQ ID NO:4). 
     
     
         69 . The method of any one of  claims 41  to  68 , wherein the second FcRn binding partner in the chimeric polypeptide is a human Fc. 
     
     
         70 . The method of any one of  claims 41  to  68 , wherein the second FcRn binding partner in the chimeric polypeptide is a mutant Fc. 
     
     
         71 . The method of  claim 69  or  70 , wherein the Fc is at least 90% or 95% identical to a Fc amino acid sequence shown in Table 8B without a signal sequence (amino acids 1 to 227 SEQ ID NO:4) 
     
     
         72 . The method of  claim 71 , wherein said Fc is identical to a Fc amino acid sequence shown in Table 6B without a signal sequence (amino acids 1 to 227 of SEQ ID NO:4). 
     
     
         73 . The method of any one of  claims 40  to  72 , wherein the chimeric polypeptide comprises a sequence at least 90% or 95% identical to the Factor IX and Fc amino acid sequence shown in Table 8A without a signal sequence and propeptide (amino acids 1 to 642 of SEQ ID NO:2). 
     
     
         74 . The method of  claim 73 , wherein said chimeric polypeptide comprises a sequence identical to the Factor IX and Fc amino acid sequence shown in Table 8A without a signal sequence and propeptide (amino acids 1 to 642 of SEQ ID NO:2). 
     
     
         75 . The method of any one of  claims 1  to  74 , wherein the subject is in need of long-term treatment. 
     
     
         76 . The method of any one of  claims 1  to  75 , wherein the long-acting FIX polypeptide is administered as part of a pharmaceutical composition comprising at least one excipient. 
     
     
         77 . The method of any one of  claims 1  to  76 , wherein the dose is administered intravenously or subcutaneously. 
     
     
         78 . The method of any one of  claims 19  to  77 , wherein the T 1/2beta  (activity) is measured by one stage clotting assay. 
     
     
         79 . The method of any one of  claims 1  to  78 , wherein the long-acting FIX polypeptide has a Mean Residence Time (MRT) of at least about 50 hours, at least about 60 hours, at least about 70 hours, at least about 80 hours, at least about 90 hours, at least about 100 hours, at least about 110 hours, at least about 120 hours, at least about 130 hours, at least about 140 hours, at least about 150 hours, at least about 160 hours, at least about 170 hours, at least about 180 hours, or at least about 190 hours. 
     
     
         80 . The method of  claim 79 , wherein the MRT is about 50 to about 200 hours, about 60 hours to about 210 hours, about 60 hours to about 183 hours, about 70 hours to about 150 hours, about 70 hours to about 140 hours, about 70 hours to about 130 hours, about 80 hours to about 120 hours, about 80 hours to about 110 hours, or about 88 hours to about 110 hours. 
     
     
         81 . The method of any one of  claims 19  to  22 , wherein the MRT is at least about 2.0 fold higher than a polypeptide consisting of amino acids 1 to 415 of SEQ ID NO:2 or BENEFIX®. 
     
     
         82 . The method of any one of  claims 1  to  81 , wherein the long-acting FIX polypeptide is formulated in a pharmaceutical composition comprising:
 (a) the long-acting FIX polypeptide; 
 (b) a carbohydrate mixture comprising sucrose and mannitol; 
 (c) sodium chloride (NaCl); 
 (d) L-histidine; and 
 (e) polysorbate 20 or polysorbate 80. 
 
     
     
         83 . The method of  claim 82 , wherein the pharmaceutical composition comprises about 1% (w/v) to about 2% (w/v) sucrose. 
     
     
         84 . The method of  claim 83 , wherein the pharmaceutical composition comprises about 1.2% (w/v) sucrose or about 1.7% (w/v) sucrose. 
     
     
         85 . The method of  claim 82 , wherein the pharmaceutical composition comprises about 10 mg/ml to about 20 mg/ml sucrose. 
     
     
         86 . The method of  claim 85 , wherein the pharmaceutical composition comprises about 11.9 mg/ml sucrose or about 16.7 mg/ml sucrose. 
     
     
         87 . The method of any one of  claims 82  to  86 , wherein the pharmaceutical composition comprises about 2% (w/v) to about 4% (w/v) mannitol. 
     
     
         88 . The method of  claim 87 , wherein the pharmaceutical composition comprises about 2.4% (w/v) mannitol or about 3.3% (w/v) mannitol. 
     
     
         89 . The method of any one of  claims 82  to  86 , wherein the pharmaceutical composition comprises about 20 mg/ml to about 40 mg/ml mannitol. 
     
     
         90 . The method of  claim 89 , wherein the pharmaceutical composition comprises about 23.8 mg/ml mannitol or about 33.3 mg/ml mannitol. 
     
     
         91 . The method of  claim 82 , wherein the pharmaceutical composition comprises about 1.0% to about 2.0% sucrose and about 2.0% (w/v) to about 4.0% (w/v) mannitol. 
     
     
         92 . The method of  claim 91 , wherein the pharmaceutical composition comprises about 1.2% (w/v) sucrose and about 2.4% (w/v) mannitol. 
     
     
         93 . The method of  claim 92 , wherein the pharmaceutical composition comprises about 1.7% (w/v) sucrose and about 3.3% (w/v) mannitol. 
     
     
         94 . The method of  claim 82 , wherein the pharmaceutical composition comprises about 10 mg/ml to about 20 mg/ml sucrose and about 20 mg/ml to about 40 mg/ml mannitol. 
     
     
         95 . The method of  claim 94 , wherein the pharmaceutical composition comprises (i) about 11.9 mg/ml sucrose and about 23.8 mg/ml mannitol or (ii) about 16.7 mg/ml sucrose and about 33.3 mg/ml mannitol. 
     
     
         96 . The method of any one of  claims 82  to  95 , wherein the pharmaceutical composition comprises between about 50 mM and about 60 mM NaCl. 
     
     
         97 . The method of  claim 96 , wherein the pharmaceutical composition comprises about 55.6 mM NaCl. 
     
     
         98 . The method of any one of  claims 82  to  95 , wherein the pharmaceutical composition comprises between about 3 mg/ml and about 4 mg/ml NaCl. 
     
     
         99 . The method of  claim 98 , wherein the pharmaceutical composition comprises about 3.25 mg/ml NaCl. 
     
     
         100 . The method of any one of  claims 82  to  99 , wherein the pharmaceutical composition comprises between about 20 mM and about 40 mM L-histidine. 
     
     
         101 . The method of  claim 100 , wherein the pharmaceutical composition comprises about 25 mM L-histidine or about 35 mM L-histidine. 
     
     
         102 . The method of any one of  claims 82  to  99 , wherein the pharmaceutical composition comprises between about 3 mg/ml and about 6 mg/ml L-histidine. 
     
     
         103 . The method of  claim 102 , the pharmaceutical composition comprises about 3.88 mg/ml L-histidine or about 5.43 mg/ml L-histidine. 
     
     
         104 . The method of any one of  claims 82  to  103 , wherein the pharmaceutical composition comprises between about 0.008% (w/v) and about 0.020% (w/v) polysorbate 20 or polysorbate 80. 
     
     
         105 . The method of  claim 104 , wherein the pharmaceutical composition comprises about 0.010% (w/v) polysorbate 20 or polysorbate 80 or about 0.014% (w/v) polysorbate 20 or polysorbate 80. 
     
     
         106 . The method of any one of  claims 82  to  103 , wherein the pharmaceutical composition comprises between about 0.08 mg/ml and about 0.2 mg/ml polysorbate 20 or polysorbate 80. 
     
     
         107 . The method of  claim 106 , wherein the pharmaceutical composition comprises about 0.10% mg/ml polysorbate 20 or polysorbate 80 or about 0.14 mg/ml polysorbate 20 or polysorbate 80. 
     
     
         108 . The method of any one of  claims 1  to  35 , wherein the rFIXFc polypeptide comprises a first subunit comprising an amino acid sequence at least 90% or 95% identical to amino acids 1 to 642 of SEQ ID NO:2, and a second subunit comprising an amino acid sequence at least 90% to 95% identical to amino acids 1 to 227 of SEQ ID NO:4. 
     
     
         109 . The method of  claim 108 , wherein the rFIXFc polypeptide comprises a first subunit comprising amino acids 1 to 642 of SEQ ID NO:2, and a second subunit comprising amino acids 1 to 227 of SEQ ID NO:4. 
     
     
         110 . The method of any one of  claims 82  to  109 , wherein the long-acting FIX polypeptide is present at a concentration of between about 25 IU/ml and about 1200 IU/ml. 
     
     
         111 . The method of  claim 110 , wherein the pharmaceutical composition comprises 50 IU/ml, 100 IU/ml, 200 IU/ml, 400 IU/ml, or 600 IU/ml of the long-acting FIX polypeptide. 
     
     
         112 . The method of  claim 111 , wherein the pharmaceutical composition comprises 50 IU/ml, 100 IU/ml, 200 IU/ml, or 400 IU/ml of the long-acting FIX polypeptide. 
     
     
         113 . The method of  claim 111 , wherein the pharmaceutical composition comprises 600 IU/ml of the long-acting FIX polypeptide. 
     
     
         114 . The method of  claim 82 , wherein the pharmaceutical composition comprises:
 (a) between about 25 IU/ml and about 700 IU/ml of the long-acting FIX polypeptide;   (b) between about 1% (w/v) and about 2% (w/v) of sucrose;   (c) between about 2% (w/v) and about 4% (w/v) of mannitol;   (d) between about 50 mM and about 60 mM NaCl;   (e) between about 20 mM and about 40 mM L-histidine; and   (f) between about 0.008% (w/v) and about 0.015% of polysorbate 20 or polysorbate 80.   
     
     
         115 . The method of  claim 114 , wherein the pharmaceutical composition comprises:
 (a) about 50 IU/ml, about 100 IU/ml, about 200 IU/ml, or 400 IU/ml of the long-acting FIX polypeptide;   (b) about 1.2% (w/v) of sucrose;   (c) about 2.4% (w/v) of mannitol;   (d) about 55.6 mM NaCl;   (e) about 25 L-histidine; and   (f) about 0.010% (w/v) of polysorbate 20 or polysorbate 80.   
     
     
         116 . The method of  claim 114 , wherein the pharmaceutical composition comprises:
 (a) about 600 IU/ml of the long-acting FIX polypeptide;   (b) about 1.7% (w/v) of sucrose;   (c) about 3.3% (w/v) of mannitol;   (d) about 55.6 mM NaCl;   (e) about 35 mM L-histidine; and   (f) about 0.014% (w/v) of polysorbate 20 or polysorbate 80.   
     
     
         117 . The method of  claim 82 , wherein the pharmaceutical composition comprises:
 (a) between about 25 IU/ml and about 700 IU/ml of the long-acting FIX polypeptide;   (b) between about 10 mg/ml and about 20 mg/ml of sucrose;   (c) between about 20 mg/ml and about 40 mg/ml of mannitol;   (d) between about 3 mg/ml and about 4 mg/ml NaCl;   (e) between about 3 mg/ml and about 6 mg/ml L-histidine; and   (f) between about 0.08 mg/ml and about 0.15 mg/ml of polysorbate 20 or polysorbate 80.   
     
     
         118 . The method of  claim 117 , wherein the pharmaceutical composition comprises:
 (a) about 50 IU/ml, about 100 IU/ml, about 200 IU/ml, or about 400 IU/ml of the long-acting FIX polypeptide;   (b) about 11.9 mg/ml of sucrose;   (c) about 23.8 mg/ml of mannitol;   (d) about 3.25 mg/ml NaCl;   (e) about 3.88 mg/ml L-histidine; and   (f) about 0.10 mg/ml of polysorbate 20 or polysorbate 80.   
     
     
         119 . The method of  claim 117 , wherein the pharmaceutical composition comprises:
 (a) about 600 IU/ml of the long-acting FIX polypeptide;   (b) about 16.7 mg/ml of sucrose;   (c) about 33.3 mg/ml of mannitol;   (d) about 3.25 mg/ml NaCl;   (e) about 5.43 mg/ml L-histidine; and   (f) about 0.14 mg/ml of polysorbate 20 or polysorbate 80.   
     
     
         120 . The method of any one of  claims 1  to  81 , wherein the long-acting FIX polypeptide is packaged in a pharmaceutical, kit comprising:
 (a) a first container comprising a lyophilized powder, where the powder comprises
 (i) the long-acting FIX polypeptide, 
 (ii) sucrose; 
 (iii) mannitol; 
 (iv) L-histidine; and 
 (v) polysorbate 20 or polysorbate 80; and 
 
 (b) a second container comprising 0.325% (w/v) NaCl to be combined with the lyophilized powder of the first container. 
 
     
     
         121 . The method of  claim 120 , wherein the pharmaceutical kit comprises:
 (a) a first container comprising a lyophilized powder, where the powder comprises
 (i) about 250 IU, about 500 IU, about 1000 IU, or about 2000 IU of the long-acting FIX polypeptide, 
 (ii) about 59.5 mg of sucrose; 
 (iii) about 119 mg of mannitol; 
 (iv) about 19.4 mg of L-histidine; and 
 (v) about 0.50 mg of polysorbate 20 or polysorbate 80; and 
   (b) a second container comprising 0.325% (w/v) NaCl at a volume sufficient to produce, when combined with the lyophilized powder of the first container, a solution comprising:
 (i) about 50 IU/ml, about 100 IU/ml, about 200 IU/ml, or about 400 IU/ml of the long-acting FIX polypeptide, respectively; 
 (ii) about 1.2% (w/v) of sucrose; 
 (iii) about 2.4% (w/v) of mannitol; 
 (iv) about 55.6 mM NaCl; 
 (v) about 25 mM L-histidine; and 
 (vi) about 0.01% (w/v) of polysorbate 20 or polysorbate 80. 
   
     
     
         122 . The method of  claim 120 , wherein the pharmaceutical kit comprises:
 (a) a first container comprising a lyophilized powder, where the powder comprises
 (i) about 3000 IU of the long-acting FIX polypeptide, 
 (ii) about 83.3 mg of sucrose; 
 (iii) about 167 mg of mannitol; 
 (iv) about 27.2 mg of L-histidine; and 
 (v) about 0.7 mg of polysorbate 20 or polysorbate 80; and 
   (b) a second container comprising 0.325% (w/v) NaCl at a volume sufficient to produce, when combined with the lyophilized powder of the first container, a solution comprising:
 (i) about 600 IU/ml of the long-acting FIX polypeptide; 
 (ii) about 1.7% (w/v) of sucrose; 
 (iii) about 3.3% (w/v) of mannitol; 
 (iv) about 55.6 mM NaCl; 
 (v) about 35 mM L-histidine; and 
 (vi) about 0.014% (w/v) of polysorbate 20 or polysorbate 80. 
   
     
     
         123 . The method of  claim 120 , wherein the pharmaceutical composition comprises:
 (a) a first container comprising a lyophilized powder, where the powder comprises
 (i) about 250 IU, about 500 IU, about 1000 IU, or about 2000 IU of the long-acting FIX polypeptide, 
 (ii) about 59.5 mg of sucrose; 
 (iii) about 119 mg of mannitol; 
 (iv) about 19.4 mg of L-histidine; and 
 (v) about 0.50 mg of polysorbate 20 or polysorbate 80; and 
   (b) a second container comprising 0.325% (w/v) NaCl at a volume sufficient to produce, when combined with the lyophilized powder of the first container, a solution comprising:
 (i) about 50 IU/ml, about 100 IU/ml, about 200 IU/ml, or about 400 IU/ml of the long-acting FIX polypeptide, respectively; 
 (ii) about 11.9 mg/ml of sucrose; 
 (iii) about 23.8 mg/ml of mannitol; 
 (iv) about 3.25 mg/ml NaCl; 
 (v) about 3.88 mg/ml L-histidine; and 
 (vi) about 0.10 mg/ml of polysorbate 20 or polysorbate 80. 
   
     
     
         124 . The method of  claim 120 , wherein the pharmaceutical kit comprises:
 (a) a first container comprising a lyophilized powder, where the powder comprises
 (i) about 3000 IU of a rFIXFc polypeptide, 
 (ii) about 83.3 mg of sucrose; 
 (iii) about 167 mg of mannitol; 
 (iv) about 27.2 mg of L-histidine; and 
 (v) about 0.7 mg of polysorbate 20 or polysorbate 80; and 
   (b) a second container comprising 0.325% (w/v) NaCl at a volume sufficient to produce, when combined with the lyophilized powder of the first container, a solution comprising:
 (i) about 600 IU/ml of a rFIXFc polypeptide; 
 (ii) about 16.7 mg/ml of sucrose; 
 (iii) about 33.3 mg/ml of mannitol; 
 (iv) about 3.25 mg/ml NaCl; 
 (v) about 5.43 mg/ml L-histidine; and 
 (vi) about 0.14 mg/ml of polysorbate 20 or polysorbate 80. 
   
     
     
         125 . The method of any one of  claims 120  to  124 , wherein the first container is a glass vial comprising a rubber stopper. 
     
     
         126 . The method of any one of  claims 120  to  125 , wherein the second container is a syringe body, and wherein the syringe body is associated with a plunger. 
     
     
         127 . The method of  claim 126 , the pharmaceutical kit further comprises an adaptor to connect the glass vial to the syringe body. 
     
     
         128 . The method of  claim 126  or  claim 127 , wherein the pharmaceutical kit further comprises infusion tubing associated with a needle to be connected to the syringe, suitable for intravenous infusion. 
     
     
         129 . The method of any one of  claims 1  to  128 , wherein the human subject does not produce an inhibitor against FIX after administration of the long-acting FIX polypeptide. 
     
     
         130 . The method of  claim 129 , wherein the inhibitor is a neutralizing antibody against FIX.