TW202334195A - Recombinant factor viii variants - Google Patents

Abstract

The present disclosure provides recombinant factor VIII polypeptides, polynucleotides encoding the recombinant factor VIII polypeptides, and compositions thereof, wherein the recombinant factor VIII polypeptides have been engineered to provide improved expression, activity/potency, clotting activity/potency, serum/plasma stability, and/or reduced immunogenicity. The present disclosure also relates to the use of the compositions comprising the recombinant factor VIII polypeptides or polynucleotides encoding the recombinant factor VIII polypeptides for therapeutic purposes.

Description

Recombinant factor VIII variants

Related applications

This application claims the benefit of U.S. Provisional Application No. 63/297,690, filed on January 7, 2022, under 35 U.S.C. § 119(e), which is incorporated herein by reference in its entirety. References to sequence listings, tables or computer programs

The file name of the sequence listing submitted at the same time is "CX7-224WO2_ST26.xml", the creation date is January 2, 2023 and the size is 11.46 million bytes. It is a part of this specification and is incorporated by reference. into this article. Field of invention

The present disclosure provides recombinant Factor VIII polypeptide variants and compositions thereof. Recombinant Factor VIII polypeptides have been engineered to have improved properties, such as improved performance, thermal stability, serum stability, plasma stability, stability under alkaline conditions, reduced immunogenicity, improved cofactor potency and improved blood coagulation. The present disclosure also relates to the use of compositions comprising recombinant Factor VIII polypeptides for therapeutic purposes.

Background of the invention

Human factor VIII ("FVIII"), also known as coagulation factor VIII or antihemophilic factor, is a protein cofactor that serves as an essential part of the blood's clotting pathway, which activates a series of enzymes in response to injury, leading to Blood clotting. Factor VIII directly supports Factor IX as a cofactor, increasing its enzymatic activity 10 ⁵ -fold (see, e.g., Mazurkiewicz-Pisarek et al., Acta Biochimica Polonica, 2016, 63.1:11-16). Hemophilia A (also known as haemophilia A) is an X-linked genetic deficiency in factor VIII that causes increased bleeding and a lack of blood clotting. There are three generally recognized forms of hemophilia A. People with mild hemophilia A bleed excessively after trauma or surgery but are otherwise healthy. People with moderate hemophilia A also experience joint and muscle bleeding after minor trauma. People with severe hemophilia A may suffer spontaneous joint and muscle bleeding even in the absence of trauma. All patients with hemophilia A require treatment, but those with severe forms and some with moderate forms require routine and frequent infusions of factor VIII to prevent excessive bleeding and death (see, e.g., Peters et al., Nature Reviews Drug Discovery, 2018, 17(7):493-508). The disease affects an estimated 1 in 5,000 men, but it also occurs in women. Treatment is generally supportive and not curative, so safe and effective treatments are still needed.

Summary of the invention

The present disclosure provides recombinant Factor VIII polypeptide variants and compositions thereof. Recombinant Factor VIII polypeptide variants have been engineered to have improved properties, including increased activity, thermal stability, serum stability, plasma stability, reduced immunogenicity, and/or improved blood coagulation. The present disclosure also relates to the use of recombinant Factor VIII polypeptides for therapeutic purposes.

In one aspect, the present disclosure provides a recombinant Factor VIII polypeptide, or a biologically active fragment thereof, comprising residue 20- corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072 The reference sequence of 1457 or has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more An amino acid sequence of sequence identity, wherein the amino acid sequence comprises the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072, or One or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072.

In some embodiments, the recombinant Factor VIII polypeptide comprises a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2 or is at least 70%, 75%, 80% identical to a reference sequence corresponding to SEQ ID NO: 2 , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98% or 99% or more sequence identity to an amino acid sequence, wherein the amino acid sequence comprises a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2 or a sequence corresponding to residues 20-1457 of SEQ ID NO: 2 One or more substitutions of the reference sequence of SEQ ID NO: 2.

In some embodiments, the recombinant Factor VIII polypeptide comprises a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072 or a reference sequence corresponding to SEQ ID NO: 252, The reference sequence of 504, 706, 840, 1200, 1514 or 2072 has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to an amino acid sequence, wherein the amino acid sequence comprises One or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution at the following amino acid positions: 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 37, 56, 57, 58, 59, 61, 63, 64, 65, 85, 96, 104, 110, 119, 127, 132, 158, 160, 164, 167, 168, 171, 175, 178, 186, 187, 205, 210, 211, 212, 215, 225, 246, 257, 260, 265, 281, 292, 294, 301, 303, 304, 306, 308, 310, 311, 312, 318, 320, 321, 325, 328, 332, 334, 335, 344, 346, 347, 349, 351, 353, 354, 355, 356, 358, 359, 362, 370, 387, 388, 390, 392, 393, 397, 407, 428, 429, 433, 435, 436, 481, 487, 488, 489, 491, 498, 499, 503, 514, 527, 533, 534, 535, 536, 538, 543, 550, 551, 555, 558, 569, 580, 581, 584, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 643, 646, 650, 665, 667, 676, 680, 681, 682, 683, 684, 686, 693, 696, 698, 700, 706, 707, 708, 712, 716, 724, 726, 729, 731, 732, 734, 735, 736, 737, 738, 739, 740, 744, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 759, 770, 773, 775, 785, 786, 787, 790, 791, 792, 793, 794, 795, 797, 798, 799, 800, 801, 802, 805, 806, 807, 808, 809, 810, 811, 812, 815, 816, 817, 818, 819, 821, 822, 824, 825, 826, 828, 830, 831, 836, 837, 838, 839, 840, 841, 844, 866, 873, 879, 892, 896, 897, 903, 907, 914, 916, 917, 918, 919, 921, 922, 923, 924, 926, 929, 938, 939, 945, 948, 951, 968, 999, 1037, 1040, 1041, 1042, 1043, 1049, 1050, 1051, 1053, 1054, 1076, 1078, 1080, 1084, 1088, 1089, 1092, 1093, 1094, 1095, 1098, 1099, 1100, 1101, 1104, 1108, 1109, 1113, 1114, 1116, 1127, 1129, 1138, 1145, 1148, 1151, 1156, 1158, 1160, 118 6. 1192, 1193, 1194, 1205, 1210, 1218, 1220, 1221, 1222, 1224, 1229, 1245, 1248, 1250, 1259, 1264, 1269, 1270, 1274, 1281, 1283, 1292, 1297, 1339, 135 2. 1373, 1377, 1378, 1380, 1389, 1414, 1422, 1427, 1428, 1439, 1440, 1442 or 1452 or a combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 .

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or amino acid residue 13G/Y, 14G/M/V, 15G/S, 16A/D/G/P/V, 17S /V, 18L/R, 19G, 20L, 21M, 24V, 37H, 56E/P, 57A/L/S/T/V, 58K, 59D/G/L/M/Q/R/S/V/W , 61Q/S, 63C/E/F/G/H/I/K/L/M/N/Q/R/S/T/W/Y, 64D/G, 65A, 85Q, 96I/L/R /V/W, 104S, 110H, 119G/V, 127V, 132P/R, 158G, 160E/T, 164E, 167G/H/R, 168G/R, 171A/D/E/F/H/K/N /M/P/Q/R/S/T/V/W/Y, 175A/G/H, 178C/M/Q/V/W, 186A, 187C/G, 205R, 210P, 211G/K/P /Q/R, 212A/F/G/K/L/M/N/P/R/S/V/Y, 215E/P/S, 225R, 246D, 257I, 260E/K/T, 265R/S /W, 281A/S, 292D, 294G, 301K/M, 303G/T, 304A/E/G, 306A/D/G/P/S/T, 308P, 310G/V, 311E/G, 312L, 318C /G/K/M/Q/R/T/V, 320G/I/L/P/Q/R/S/T/V, 321Y, 325Y, 328G/Q/S, 332A/F/G/H /N/P/Q/R/T/V/W/Y, 334G, 335E/P, 344A/E/L, 346R/V, 347N, 349G, 351S, 353S, 354R, 355C, 356L/N, 358K /S, 359E/G, 362E/G/N/P/T/V/W, 370A, 387A/G/I/L/Q/R/S/T/V/W, 388A/P, 390A/E /G/L/M/P/S/T/V, 392A, 393A/F/G/H/M/Q/R/W/Y, 397G/N, 407V, 428F, 429L/S, 433A/G , 435Q, 436D/G, 481V, 487N, 488G, 489E/F/Q/R, 491E, 498A, 499C/L, 503S, 514L, 527A, 533R/V/Y, 534F/I/L/M, 535C /G/N, 536A/G/K/L/M/P/R/W, 538V, 543G/L/P/W, 550G, 551W, 555E/G/R/T, 558A/E/G/L /P/Q/R/S/T/V/W, 569T, 580S/Y, 581K/V, 584A, 610H/K/L/R/S/T, 611D/E, 612G/S/T, 613S , 614A/F/H/I/N/T, 615D/N, 616D/E/G/Q/W, 617D/E/G/N, 618C/E/G/P/Y, 619E, 620N/P , 621K/M/R/T/W, 622D, 643N, 646I, 650M, 665G, 667I, 676Q, 680L/R, 681A/C/E/F/G/L/N/R/Q/S/T /W, 682R, 683W, 684G/K, 686W, 693T, 696E, 698D/S/W/Y, 700A/E/Q/T/V, 706A/G/M/Y, 707H, 708A/K/T /V, 712G/L/M/Q/S/T/Y, 716K/L/W, 724I, 726A/L, 729T, 731I/L/R, 732C/D/E/G/L/R/W /K/M/Q/S/T/V/Y, 734I/R/S, 735D/S/V, 736F/G, 737F/G/K/L/M/N/S/V, 738A/D /E/F/H/K/N/P/R/S/T, 739G/P/R, 740G, 744A/R, 746P, 747D, 748F/G/K/N/S/V, 749A/C /D/F/G/I/K/L/M/P/Q/R/S/T/V/Y、750A/D/G/K/M/N/P/Q/R/S/T /V/W, 751A/D/E/Y, 752D/E/F/G/L/M/Q/R/S/V, 753D, 754W, 755G/L/P/Q/S/Y, 756G /P/R/T, 757A/G/K/T, 759S, 770W, 773H, 775E/G/K/N/P/S/V, 785V, 786C/D/L, 787W, 790A/G/I /M/P/W, 791M/T, 792H/W/Y, 793G, 794R, 795C/N/S/T, 797C/G/P/Q/T/W/Y, 798C/R/T, 799A /E/G/M/P/Q/R/S/V, 800C/G/I/L/M/N/P/Q/R/T/V/W/Y, 801A/G/I/R , 802S/V, 805C/G/H/K/L/M/P/Q/R/S/V/W, 806E/G/R/S, 807C/L/P/R, 808K/S, 809A /C/G/I/K/L/N/Q/R/S/T/V/W, 810V, 811F, 812G/I/V, 815I, 816V/W, 817A/D, 818A/E/G /P/S/V/W, 819V, 821Q, 822S/T, 824W, 825L, 826G, 828K, 830M/V, 831W, 836G/R/V/W, 837G, 838G/T, 839A/G/K /P, 840G, 841W, 844H, 866A, 873L/Q/T, 879Q, 892T, 896L/V, 897G/K/R, 903E, 907D, 914V, 916C/E/I/K/L/M/Q /T, 917G, 918M/S, 919G, 921R/W, 922W, 923M, 924I/M/P/Q/W, 926T/V, 929A/Q/V, 938R/V, 939E/S, 945K, 948S , 951V, 968A/P, 999R, 1037H/M/N/P, 1040E, 1041G/H/N, 1042A/E/T, 1043E/R, 1049G/W, 1050E/P/Q/S/T, 1051E /H/K/R/S/W, 1053A/R, 1054C, 1076G/S, 1078S, 1080G/V, 1084L/N, 1088G/S, 1089F/S, 1092G, 1093A/E/G/M/N /P/Q/R/S/T/V/W/Y, 1094S, 1095G, 1098G, 1099G, 1100I/S, 1101G, 1104C/E, 1108M/S/V/W, 1109G, 1113F, 1114A/M , 1116A/G/I/L/R, 1127L/V, 1129P, 1138Q, 1145R, 1148A/C/E/L/M/N/Q/R/S, 1151S, 1156D/G/N/R, 1158N , 1160R, 1186E, 1192D, 1193A/E/G/I/L, 1194P, 1205L, 1210M/T/V/W, 1218A/G/L/M/P, 1220D/E/L/T, 1221G/S /T/V, 1222F, 1224P, 1229D, 1245R, 1248A/C/D/E/F/G/H/I/K/M/P/Q/R/S/T/W/Y, 1250A/P /S/T, 1259T, 1264L/R/W, 1269F, 1270V, 1274N/V, 1281H/K/P/Q/R/S, 1283E/H/L/S/T, 1292Y, 1297A, 1339C, 1352E , 1373S, 1377A/C/D/E/G/H/I/M/P/R/S/V/T/Y, 1378A/C/D/E, 1380A/G/W, 1389W, 1414A/C /D/E/G/H/N/P/T/W, 1422I, 1427Y, 1428G, 1439A/E/H/L/S/T, 1440A/C/E/G/S/T, 1442T or 1452L or a combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution at the following amino acid positions: 63, 171, 212, 260, 306, 317, 320, 332, 387, 390, 393, 534, 558, 681, 732, 738, 749, 750, 775, 1148, 1248, 1377 or 1439 or a combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide comprises at least one substitution or amino acid residue 63C/E/F/G/H/I/K/L/M/N/Q/R/S /T/W/Y, 171A/D/E/F/H/K/N/M/P/Q/R/S/T/V/W/Y, 212A/F/G/L/K/M /N/P/R/S/V/Y, 260E/K/T, 306A/D/G/P/S/T, 318C/G/K/L/M/Q/R/T/V, 320G /I/L/P/Q/R/S/T/V, 332A/F/G/H/N/P/Q/R/T/V/W/Y, 387A/G/I/L/Q /R/S/T/V/W, 390A/E/G/L/M/P/S/T/V, 393A/F/G/H/M/Q/R/W/Y, 499C/L , 534I/F/L/M, 558A/E/G/L/P/Q/R/S/T/V/W, 681A/C/E/F/G/L/N/R/Q/S /T/W, 700A/E/Q/T/V, 732C/D/E/G/L/R/W/K/M/Q/S/T/V/Y, 738A/D/E/F /H/K/N/P/R/S/T/Y, 749A/C/D/F/G/I/K/L/M/P/Q/R/S/T/V/Y, 750A /D/G/K/M/N/P/Q/R/S/T/V/W, 752D/E/F/G/L/M/Q/R/S/V, 775E/G/K /N/P/S/V, 800C/G/I/L/M/N/P/Q/R/T/V/W/Y, 805C/G/H/K/L/M/P/Q /R/S/V/W, 809A/C/G/I/K/L/N/Q/R/S/T/V/W, 1093A/E/G/M/N/P/Q/R /S/T/V/W/Y, 1148A/C/E/L/M/N/Q/R/S, 1248A/C/D/E/F/G/H/I/K/M/P /Q/R/S/T/W/Y, 1377A/C/D/E/G/H/I/M/P/R/S/V/T/Y, 1414A/C/D/E/G /H/N/P/T/W or 1439A/E/H/L/S/T or a combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or set of substitutions at the following amino acid positions: 1148, 85/708/1148, 798, 706, 171, 335, 63, 344 ,428,879,59,429,205,1264,797,1248,873,792,708,57,96,362,1292,346,433,65,795,684/798,353,332,1259,356 ,436,1245,1145,716,56,773,734,358,729,665,359,61,1250,726,37/96/171/429/708/799/1148,171/429/732/1148 ,96/171, 171/919/1151, 171/792/797/799/1148, 171/569/1148, 96/171/429/792/797/799, 171/429/708/1422, 171/429 ,321/362/732/797/799, 171/1100, 708/1148/1186, 429/724/732/1148, 171/362/795/797/799, 171/429/732, 96/171/643 /797/799, 171/792/797/799, 503, 171/732, 1148/1193, 96/171/362/429/732/795/797, 334, 732, 351, 1377, 999, 306, 349 , 257, 646, 435 or 58, wherein these positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide comprises at least one of Tables 4.1 , 4.2 , 5.1 , 5.2 , 6.1 , 6.2 , 7.1, 7.2 , 8.1 , 8.2 , 9.1 , 9.2 , 10.1 , 10.2 , 11.1 and 15.1 A substitution or set of substitutions as set forth in , wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the recombinant Factor VIII polypeptide comprises a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072 or a reference sequence corresponding to SEQ ID NO: 252, The reference sequence of 504, 706, 840, 1200, 1514 or 2072 has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity of the amino acid sequence.

In some embodiments, a recombinant Factor VIII polypeptide comprises a reference sequence corresponding to residues 20-1457 of even-numbered SEQ ID NO. in SEQ ID NO. 4-2346 and 2360-2870 or corresponding to SEQ ID NO. The reference sequences of the even-numbered SEQ ID NO. in 4-2346 and 2350-2870 have at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, Amino acid sequences with 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity.

In some embodiments, the recombinant Factor VIII polypeptide comprises a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072 or a reference sequence corresponding to SEQ ID NO: 252, The reference sequence of 504, 706, 840, 1200, 1514 or 2072 has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to an amino acid sequence, wherein the amino acid sequence comprises Relative to this reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, 504, 706, 840, 1200, 1514 or 2072 or relative to SEQ ID NO: 252, 504, 706, 840, 1200, One or more substitutions of 1514 or 2072 of the reference sequence.

In some of the foregoing embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution at the following positions: 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 37, 56, 57, 58, 59, 61, 63, 64, 65, 85, 96, 104, 110, 119, 127, 132, 158, 160, 164, 167, 168, 171, 175, 178, 186, 187, 205, 210, 211, 212, 215, 225, 246, 257, 260, 265, 281, 292, 294, 301, 303, 304, 306, 308, 310, 311, 312, 318, 320, 321, 325, 328, 332, 334, 335, 344, 346, 347, 349, 351, 353, 354, 355, 356, 358, 359, 362, 370, 387, 388, 390, 392, 393, 397, 407, 428, 429, 433, 435, 436, 481, 487, 488, 489, 491, 498, 499, 503, 514, 527, 533, 534, 535, 536, 538, 543, 550, 551, 555, 558, 569, 580, 581, 584, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 643, 646, 650, 665, 667, 676, 680, 681, 682, 683, 684, 686, 693, 696, 698, 700, 706, 707, 708, 712, 716, 724, 726, 729, 731, 732, 734, 735, 736, 737, 738, 739, 740, 744, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 759, 770, 773, 775, 785, 786, 787, 790, 791, 792, 793, 794, 795, 797, 798, 799, 800, 801, 802, 805, 806, 807, 808, 809, 810, 811, 812, 815, 816, 817, 818, 819, 821, 822, 824, 825, 826, 828, 830, 831, 836, 837, 838, 839, 840, 841, 844, 866, 873, 879, 892, 896, 897, 903, 907, 914, 916, 917, 918, 919, 921, 922, 923, 924, 926, 929, 938, 939, 945, 948, 951, 968, 999, 1037, 1040, 1041, 1042, 1043, 1049, 1050, 1051, 1053, 1054, 1076, 1078, 1080, 1084, 1088, 1089, 1092, 1093, 1094, 1095, 1098, 1099, 1100, 1101, 1104, 1108, 1109, 1113, 1114, 1116, 1127, 1129, 1138, 1145, 1148, 1151, 1156, 1158, 1160, 1186, 119 2. 1193,1194,1205,1210,1218,1220,1221,1222,1224,1229,1245,1248,1250,1259,1264,1269,1270,1274,1281,1283,1292,1297,1339,1352,137 3. 1377, 1378, 1380, 1389, 1414, 1422, 1427, 1428, 1439, 1440, 1442 or 1452 or a combination thereof, wherein the amino acid positions are relative to those corresponding to SEQ ID NO: 252, 504, 706, 840 , 1200, 1514 or 2072 of the reference sequence.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or amino acid residue 13G/Y, 14G/M/V, 15G/S, 16A/D/G/P/V, 17S /V, 18L/R, 19G, 20L, 21M, 24V, 37H, 56E/P, 57A/L/S/T/V, 58K, 59D/G/L/M/Q/R/S/V/W , 61Q/S, 63C/E/F/G/H/I/K/L/M/N/Q/R/S/T/W/Y, 64D/G, 65A, 85Q, 96I/L/R /V/W, 104S, 110H, 119G/V, 127V, 132P/R, 158G, 160E/T, 164E, 167G/H/R, 168G/R, 171A/D/E/F/H/K/N /M/P/Q/R/S/T/V/W/Y, 175A/G/H, 178C/M/Q/V/W, 186A, 187C/G, 205R, 210P, 211G/K/P /Q/R, 212A/F/G/L/K/M/N/P/R/S/V/Y, 215E/P/S, 225R, 246D, 257I, 260E/K/T, 265R/S /W, 281A/S, 292D, 294G, 301K/M, 303G/T, 304A/E/G, 306A/D/G/P/S/T, 308P, 310G/V, 311E/G, 312L, 318C /G/K/L/M/Q/R/T/V, 320G/I/L/P/Q/R/S/T/V, 321Y, 325Y, 328G/Q/S, 332A/F/G /H/N/P/Q/R/T/V/W/Y, 334G, 335E/P, 344A/E/L, 346R/V, 347N, 349G, 351S, 353S, 354R, 355C, 356L/N , 358K/S, 359E/G, 362E/G/N/P/T/V/W, 370A, 387A/G/I/L/Q/R/S/T/V/W, 388A/P, 390A /E/G/L/M/P/S/T/V, 392A, 393A/F/G/H/M/Q/R/W/Y, 397G/N, 407V, 428F, 429L/S, 433A /G, 435Q, 436D/G, 481V, 487N, 488G, 489E/F/Q/R, 491E, 498A, 499C/L, 503S, 514L, 527A, 533R/V/Y, 534I/F/L/M , 535C/G/N, 536A/G/K/L/M/P/R/W, 538V, 543G/L/P/W, 550G, 551W, 555E/G/R/T, 558A/E/G /L/P/Q/R/S/T/V/W, 569T, 580S/Y, 581K/V, 584A, 610H/K/L/R/S/T, 611D/E, 612G/S/T , 613S, 614A/F/H/I/N/T, 615D/N, 616D/E/G/Q/W, 617D/E/G/N, 618C/E/G/P/Y, 619E, 620N /P, 621K/M/R/T/W, 622D, 643N, 646I, 650M, 665G, 667I, 676Q, 680L/R, 681A/C/E/F/G/L/N/R/Q/S /T/W, 682R, 683W, 684G/K, 686W, 693T, 696E, 698D/S/W/Y, 700A/E/Q/T/V, 706A/G/M/Y, 707H, 708A/K /T/V, 712G/L/M/Q/S/T/Y, 716K/L/W, 724I, 726A/L, 729T, 731I/L/R, 732C/D/E/G/L/R /W/K/M/Q/S/T/V/Y, 734I/R/S, 735D/S/V, 736F/G, 737F/G/K/L/M/N/S/V, 738A /D/E/F/H/K/N/P/R/S/T/Y, 739G/P/R, 740G, 744A/R, 746P, 747D, 748F/G/K/N/S/V , 749A/C/D/F/G/I/K/L/M/P/Q/R/S/T/V/Y, 750A/D/G/K/M/N/P/Q/R /S/T/V/W, 751A/D/E/Y, 752D/E/F/G/L/M/Q/R/S/V, 753D, 754W, 755G/L/P/Q/S /Y, 756G/P/R/T, 757A/G/K/T, 759S, 770W, 773H, 775E/G/K/N/P/S/V, 785V, 786C/D/L, 787W, 790A /G/I/M/P/W, 791M/T, 792H/W/Y, 793G, 794R, 795C/N/S/T, 797C/G/P/Q/T/W/Y, 798C/R /T, 799A/E/G/M/P/Q/R/S/V, 800C/G/I/L/M/N/P/Q/R/T/V/W/Y, 801A/G /I/R, 802S/V, 805C/G/H/K/L/M/P/Q/R/S/V/W, 806E/G/R/S, 807C/L/P/R, 808K /S, 809A/C/G/I/K/L/N/Q/R/S/T/V/W, 810V, 811F, 812G/I/V, 815I, 816V/W, 817A/D, 818A /E/G/P/S/V/W, 819V, 821Q, 822S/T, 824W, 825L, 826G, 828K, 830M/V, 831W, 836G/R/V/W, 837G, 838G/T, 839A /G/K/P, 840G, 841W, 844H, 866A, 873L/Q/T, 879Q, 892T, 896L/V, 897G/K/R, 903E, 907D, 914V, 916C/E/I/K/L /M/Q/T, 917G, 918M/S, 919G, 921R/W, 922W, 923M, 924I/M/P/Q/W, 926T/V, 929A/Q/V, 938R/V, 939E/S , 945K, 948S, 951V, 968A/P, 999R, 1037H/M/N/P, 1040E, 1041G/H/N, 1042A/E/T, 1043E/R, 1049G/W, 1050E/P/Q/S /T, 1051E/H/K/R/S/W, 1053A/R, 1054C, 1076G/S, 1078S, 1080G/V, 1084L/N, 1088G/S, 1089F/S, 1092G, 1093A/E/G /M/N/P/Q/R/S/T/V/W/Y, 1094S, 1095G, 1098G, 1099G, 1100I/S, 1101G, 1104C/E, 1108M/S/V/W, 1109G, 1113F , 1114A/M, 1116A/G/I/L/R, 1127L/V, 1129P, 1138Q, 1145R, 1148A/C/E/L/M/N/Q/R/S, 1151S, 1156D/G/N /R, 1158N, 1160R, 1186E, 1192D, 1193A/E/G/I/L, 1194P, 1205L, 1210M/T/V/W, 1218A/G/L/M/P, 1220D/E/L/T , 1221G/S/T/V, 1222F, 1224P, 1229D, 1245R, 1248A/C/D/E/F/G/H/I/K/M/P/Q/R/S/T/W/Y , 1250A/P/S/T, 1259T, 1264L/R/W, 1269F, 1270V, 1274N/V, 1281H/K/P/Q/R/S, 1283E/H/L/S/T, 1292Y, 1297A , 1339C, 1352E, 1373S, 1377A/C/D/E/G/H/I/M/P/R/S/V/T/Y, 1378A/C/D/E, 1380A/G/W, 1389W , 1414A/C/D/E/G/H/N/P/T/W, 1422I, 1427Y, 1428G, 1439A/E/H/L/S/T, 1440A/C/E/G/S/T , 1442T or 1452L or a combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514 or 2072.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution at the following amino acid positions: 63, 171, 212, 260, 306, 317, 320, 332, 387, 390, 393, 534, 558, 681, 732, 738, 749, 750, 775, 1148, 1248, 1377 or 1439 or a combination thereof, wherein the amino acid positions are relative to those corresponding to SEQ ID NO: 252, 504, 706, 840 , 1200, 1514 or 2072 of the reference sequence.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide comprises at least one substitution or amino acid residue 63C/E/F/G/H/I/K/L/M/N/Q/R/S /T/W/Y, 171A/D/E/F/H/K/N/M/P/Q/R/S/T/V/W/Y, 212A/F/G/L/K/M /N/P/R/S/V/Y, 260E/K/T, 306A/D/G/P/S/T, 318C/G/K/L/M/Q/R/T/V, 320G /I/L/P/Q/R/S/T/V, 332A/F/G/H/N/P/Q/R/T/V/W/Y, 387A/G/I/L/Q /R/S/T/V/W, 390A/E/G/L/M/P/S/T/V, 393A/F/G/H/M/Q/R/W/Y, 499C/L , 534I/F/L/M, 558A/E/G/L/P/Q/R/S/T/V/W, 681A/C/E/F/G/L/N/R/Q/S /T/W, 700A/E/Q/T/V, 732C/D/E/G/L/R/W/K/M/Q/S/T/V/Y, 738A/D/E/F /H/K/N/P/R/S/T/Y, 749A/C/D/F/G/I/K/L/M/P/Q/R/S/T/V/Y, 750A /D/G/K/M/N/P/Q/R/S/T/V/W, 752D/E/F/G/L/M/Q/R/S/V, 775E/G/K /N/P/S/V, 800C/G/I/L/M/N/P/Q/R/T/V/W/Y, 805C/G/H/K/L/M/P/Q /R/S/V/W, 809A/C/G/I/K/L/N/Q/R/S/T/V/W, 1093A/E/G/M/N/P/Q/R /S/T/V/W/Y, 1148A/C/E/L/M/N/Q/R/S, 1248A/C/D/E/F/G/H/I/K/M/P /Q/R/S/T/W/Y, 1377A/C/D/E/G/H/I/M/P/R/S/V/T/Y, 1414A/C/D/E/G /H/N/P/T/W or 1439A/E/H/L/S/T or a combination thereof, wherein the amino acid positions are relative to those corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514 or 2072 of the reference sequence.

In some embodiments, the recombinant Factor VIII polypeptide comprises a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252 or is at least 70%, 75%, 80% identical to a reference sequence corresponding to SEQ ID NO: 252 , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98% or 99% or more sequence identity to an amino acid sequence, wherein the amino acid sequence comprises the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252 or relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252 One or more substitutions of the reference sequence of SEQ ID NO: 252.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or set of substitutions at the following amino acid positions: 1380, 388, 1043, 610, 1274, 1220, 1442, 1193, 621, 1054 ,755,1041,748,1049,1221,1051,612,1037,616,387,489,750,1224,618,1050,1053,896,897,749,260,481,613,756,63,265 ,752,1218,1378,614,1440,1439,1205,1222,1389,1269,390,397/1218,757,1373,346,63/1248,349,306/355/1422,1422,306/346 /708、1148、171/346/1248、63/797/799/1248/1422、171/799、362、1248、171/306/346/999/1248、171/1148、734/799/1248/1422 , 63/732, 63/171/306/346/732, 171, 171/1422, 63/362/732/799/1248, 999/1148/1422, 346/349, 306/349, 63/171/1148 /1151, 306, 344/349/354/1250, 1250, 63/306/344/349/354/503/1248, 59/63/332/349/503/706/708, 63/306/344/349 /354/706/1248, 59/63, 332/706/708/1248/1250, 503, 59/63/349/354, 59/349/708, 59/306/344/1248, 344/1248/1250 , 708, 63/344/349, 59, 63/344/349/354, 706, 59/306/770, 63/347/354/1248 or 354/503, where the amino acid positions are relative to the corresponding The reference sequence at SEQ ID NO: 252.

In some embodiments, the recombinant Factor VIII polypeptide comprises a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 504 or is at least 70%, 75%, 80% identical to a reference sequence corresponding to SEQ ID NO: 504 , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98% or 99% or more sequence identity to an amino acid sequence, wherein the amino acid sequence comprises the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 504 or relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 504 One or more substitutions of the reference sequence of SEQ ID NO: 504.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or set of substitutions at the following amino acid positions: 750, 1051, 260, 756, 1193, 332, 897, 387, 1037, 306 /749, 1270, 489, 487, 488, 903, 171/1248/1440, 260/1148/1378, 171/260/387/750/1248, 171/260, 171/260/387/750/1051/1148 ,171,750/1051/1148,171/260/756,171/1248,171/1148,59,1440,332/750/1248,756/1248,1248,171/387/1248/1440,750/1440 ,59/171/750,171/1193/1248,750/1148/1193/1440,1148,171/387/1051,748,306,306/387/390/749/1220,390,306/390/1037 ,306/612/749, 387/390/749, 63/390/503/749, 1220, 63/306, 749, 306/390/749/1037, 306/749/1220, 306/387/390/612 /749/1037 or 328, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 504.

In some embodiments, the recombinant Factor VIII polypeptide comprises a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 706 or is at least 70%, 75%, 80% identical to a reference sequence corresponding to SEQ ID NO: 706 , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98% or 99% or more sequence identity to an amino acid sequence, wherein the amino acid sequence comprises the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 706 or relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 706 One or more substitutions of the reference sequence of SEQ ID NO: 706.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or set of substitutions at the following amino acid positions: 1051, 1221, 1218, 387, 749/1218, 332, 897, 696, 816 ,824,826,393,825,698,332/387/390/1148,328/387/749,749,306/328/332/390/1051/1193/1218,63/306/332/1051/1148 , 63/306/332/387/1051, 387/390/1051, 186/306/328/332/390/749/1051/1148/1193, 387/390/756/1051, 306/390/1051, 306 /332/390/749, 306/387/390/1148, 306/328/332/387/390/749/1051/1148/1193, 332/387/390/749/1148, 387/390/749, 306 /387/749/1051/1148、387/749、306/945/1051/1193/1221、306/390/749/1051、63/306/332/390、306/387/390/749/1148/1193 /1221, 306/332/387/390/749/1218, 63/306/332/390/1051/1148/1193/1218, 924, 755, 752, 751, 923, 797, 754, 793, 753, 922 , 896, 938, 775, 732, 810, 747, 393/407, 819, 787, 802, 811, 392 or 397, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 706 .

In some embodiments, the recombinant Factor VIII polypeptide comprises a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 840 or is at least 70%, 75%, 80% identical to a reference sequence corresponding to SEQ ID NO: 840 , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98% or 99% or more sequence identity to an amino acid sequence, wherein the amino acid sequence comprises the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 840 or relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 840 One or more substitutions of the reference sequence of SEQ ID NO: 840.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or set of substitutions at the following amino acid positions: 620, 491, 1229, 615, 1283, 822, 328, 212, 818, 555 /684, 828, 550, 830, 839, 13, 14, 17, 836, 1210, 1281, 64, 682, 318, 20, 21, 325, 533, 19, 817, 831, 225, 1156, 393/747 ,753,393/732/751,63/797,622,617,907,1040,611,488,1192,1194,619,16,802,211,555,536,527,841,320,837,558 ,914,312,681,838,15,1427,18,683,24,534,840,1160,551,393,393/747/752/811/819/923/1148,393/751/775,63 /393/732/775/1148, 393/732/1148, 775/1148, 393/752/753/775/793/797, 393/923, 393/923/1148, 393/747/751, 393/747 /753/775/1148、752/923/1051、63/775、63/393/752/775/793/1051/1053、752/753/775/793/819、63/1051、63/747/752 /753/775/811/819/923/1051, 393/752/787, 751, 63/1148, 63/747/752/753/819/923/1148, 1148, 747/752/753/793/811 , 63, 797/1148, 751/752/753/1051, 63/732/1051, 752/797/815/923/1051/1148 or 747/753, wherein the amino acid positions are relative to those corresponding to SEQ. The reference sequence of ID NO: 840.

In some embodiments, the recombinant Factor VIII polypeptide comprises a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 1200 or at least 70%, 75%, 80% identical to a reference sequence corresponding to SEQ ID NO: 1200 , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98% or 99% or more sequence identity to an amino acid sequence, wherein the amino acid sequence comprises the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 1200 or relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 1200 One or more substitutions of the reference sequence of SEQ ID NO: 1200.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide comprises at least one substitution or set of substitutions at the following amino acid positions: 681, 622, 617, 558, 536, 211, 320, 839, 797, 533 ,751,303,951,164,818,807,799,735,794,926,755,921,801,1094,968,929,924,698,168,581,916,1428,805,1114,311 ,737,693,809,757,1099,676,740,791,211/536/622/681,536/558,211/536/555/617/732,211/681/732,211/536/555 ,555,211/536,211/212,211/536/617/681/732,211/536/558/617/681,555/732,536/555/558/617/732,536/617/681 /732, 536/555, 536/622/681, 732, 212/320/533/617/681, 211/312/533/534/617/619/681/839, 212, 211/212/320/619 /681/839、797/839、212/312/320/533/534/617/681、558/619/681、320/558/681、212/320/534/619/681/797/839、320 /558/619/732/839, 211/212/312/534/558/619/681/732/839, 320/534/558/619/732/797, 558/617/619/681, 211/534 /558/611/681、211/320/617/619/681、558/617/681/797、320/534/619/732、211/619/681、211/212/534/558/619/732 /839、212/320/619/681/797/839、212/320/611/839、320/558/619/681、312/533/617/681/797/839、558/611/681、312 ,211/320/558/619/681/732/797/839, 320/558/732/797, 533/558/611/619/716/732/839, 312/681, 320/534/619/681 ,211/320/533/536/619, 558/617/681/839, 212/320/534/558/681, 533/534/681/839, 212/534/619/681, 617/619/681 ,211/212/320/558/617/681, 212/534/558/681/797, 212/320/534, 211/312/619, 212/312/619/839, 210/212/320/558 /681, 212/312/534/681/797/839, 211/320/558/839, 294, 759, 538 or 1109, wherein the amino acid positions are relative to those corresponding to SEQ ID NO: 1200 Reference sequence.

In some embodiments, the recombinant Factor VIII polypeptide comprises a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 1514 or is at least 70%, 75%, 80% identical to a reference sequence corresponding to SEQ ID NO: 1514 , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% or more sequence identity to an amino acid sequence, wherein the amino acid sequence comprises the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 1514 or relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 1514 One or more substitutions of the reference sequence of SEQ ID NO: 1514.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or set of substitutions at the following amino acid positions: 1042, 1041, 1051, 1283, 1281, 916, 187, 215, 178, 1050 ,1210,104,615,610,536,618,621,614,110,301,1078,812,786,127,175,1093,1088,650,535,806,738,700,132,1076,1084 ,498,756,1108,746,160,543,310,584,918,1452,790,292,800,167,892,304,1116,752,281,917,712,1080,806/1352,1095 ,744,1098,948,119,785,1113,667,158,580,707,731,1100,1089,1092,939,1104,736,1297,1127,686,1129,1138,786/821,680 ,1101,808,739,308,536/555/619/732/799/805,555/619/801,555/619/799,619/799/801/805,536/555/619/732/799 ,799/805,801,246/619/799/801,536/555/619,619/839,619/799/805,536/801/805,555/619/799/805,619/801/805 ,555/619,536/619/801,801/805,536/619/799/805,555/619/732/799/801/805,749,616,611,370/614,1156,1158,555 /681/737/799, 536/619/809/1109, 536/555/619/737/799/809/839/916/921, 916/1109, 536/619/681/799/916/1109, 555 /619/681/807/1109, 555/619/681/737/799, 619/681/799/916/1109, 619/681, 555/921, 619/681/916, 555/619/681/799 , 555, 732, 619/799, 619, 681, 555/916, 839 or 799, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 1514.

In some embodiments, the recombinant Factor VIII polypeptide comprises a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2072 or at least 70%, 75%, 80% identical to a reference sequence corresponding to SEQ ID NO: 2072 , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% or more sequence identity to an amino acid sequence, wherein the amino acid sequence comprises the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2072 or relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2072 One or more substitutions of the reference sequence of SEQ ID NO: 2072.

In some embodiments, a recombinant Factor VIII polypeptide comprises an amino acid sequence having at least one substitution or set of substitutions at the following amino acid positions: 167, 167/738/1076/1093, 167/738/752/800/ 805/808/809/1116, 167/800/809/1076, 167/318, 167/318/800/805/808/809, 167/318/681, 167/318/681/700/805/808/ 809/1076/1116, 167/318/700, 167, 167/738/1076/1093, 167/738/752/800/805/808/809/1116, 167/800/809/1076, 167/318, 167/318/800/805/808/809, 167/318/681, 167/318/681/700/805/808/809/1076/1116, 167/318/700, 167/318/700/1076, 167/318/700/738/739/1076, 167/681/738/800/808/809, 167/178/700/752/1093, 167/700, 167/700/738/739/1093, 167/ 700/738/752/1093, 167/700/738/752/805/808/809/1093/1116, 808/809, 1076, 738, 738/808/809, 738/1076, 738/739, 738/ 739/809, 738/739/809/1093/1116, 738/739/1093, 738/739/752/1093, 738/739/799/800/1093, 738/739/1116, 738/800, 738/ 1093, 738/752, 738/752/1093, 738/752/799, 738/799/809/1093, 738/799/1093, 739, 739/800/809, 800, 800/805/809, 809, 809/1093, 809/1093/1116, 809/1116, 318, 318/1076, 318/1076/1116, 318/739/805/808/809/1076, 318/499/700/738/809, 318/ 1093, 318/752/1093, 318/1116, 318/543/739, 318/543/681/752/1116, 318/543/1093, 318/700/738/1093/1414, 681, 681/738, 681/738/739/1093, 681/738/739/752/799/800/809/1116/1339, 681/738/739/1116, 681/738/800, 681/800/805/809, 681/ 809/1093, 681/752/1093, 681/752/1093, 681/700, 681/700/738/739/1093/1116, 681/700/809, 681/700/1093, 681/700/752/ 808/809/1093/1116, 681/700/752/1093, 681/700/844, 681/700/805/809, 1093, 1093/1116, 752, 752/1076, 752/800/809, 752/ 800/1093, 752/809, 752/866, 752/1093, 752/1116, 178/318/739/752/809/1076/1116, 178/318/800/809, 178/318/681/800/ 808/809/1076, 178/681, 178/681/738/1116, 178/752/809, 178/700/800/805/808/809, 178/805/809/866, 1116, 543, 543/ 738/739/809, 543/738/809/1093, 543/739/1093, 543/800/1093/1116, 543/809/1093, 543/809/1093/1116, 543/681/738/739/ 752/809/1093, 543/681/738/739/752/1093, 543/681/739/752/800/805/809/1093, 543/681/700/738/739/800/1093/1116, 543/681/700/738/739/805/808/809, 543/681/700/752, 543/681/700/752/809/1093, 543/1093, 543/700, 543/700/738/ 739/752/800/1093/1116, 543/700/809, 543/700/1093, 543/700/752, 543/700/752/800/1093, 543/700/752/809/1116, 543/ 700/752/1093, 700, 700/738/739/800/1093, 700/738/739/799/1093, 700/738/800/1093, 700/738/1116, 700/809, 700/809/ 1093, 700/1093, 700/1093/1116, 700/752, 700/752/808/809/1093/1116, 700/752/809/1093, 700/752/799/1116, 700/752/805/ 809/1093, 700/799/800, 700/1116, 700/805/808/809/1076, 514/543/681/805/808/809, 805/808/809 or 805/809/1116, which Amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2072.

In some embodiments, a recombinant Factor VIII polypeptide comprises at least one of the polypeptides set forth in Tables 4.1 , 4.2 , 5.1 , 5.2 , 6.1 , 6.2 , 7.1, 7.2 , 8.1 , 8.2 , 9.1 , 9.2 , 10.1 , 10.2 , 11.1 , and 15.1 A substituted amino acid sequence, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072.

In some embodiments, a recombinant Factor VIII polypeptide comprises at least one of the polypeptides set forth in Tables 4.1 , 4.2 , 5.1 , 5.2 , 6.1 , 6.2 , 7.1, 7.2 , 8.1 , 8.2 , 9.1 , 9.2 , 10.1 , 10.2 , 11.1 , and 15.1 An amino acid sequence of a variant substitution or set of substitutions, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072.

In some embodiments, the recombinant Factor VIII polypeptide comprises an amino acid sequence comprising Table 4.1 , 4.2 , 5.1 , 5.2, 6.1 , 6.2 , 7.1 , 7.2 , 8.1 , 8.2 , 9.1 , 9.2 , 10.1 , 10.2 , residues 20-1457 of the even-numbered SEQ ID NO. sequences set forth in Tables 11.1 and 15.1 , or include Tables 4.1 , 4.2 , 5.1 , 5.2 , 6.1 , 6.2 , 7.1 , 7.2, 8.1 , 8.2 , 9.1 , 9.2 , The even-numbered SEQ ID NO. sequences set forth in 10.1 , 10.2 , 11.1 and 15.1 .

In some embodiments, a recombinant Factor VIII polypeptide comprises an amino acid sequence comprising residues 20-1457 of even-numbered SEQ ID NO. in SEQ ID NOs: 4-2346 and 2360-2870, or comprising SEQ ID NO: Even-numbered SEQ ID NO. among 4-2346 and 2350-2870. In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 amino acid substitutions. In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide optionally includes 1, 2, 3, 4, or 5 amino acid substitutions.

In some embodiments, the recombinant Factor VIII polypeptide comprises an amino acid sequence comprising residues 20-1457 of SEQ ID NO: 252, 504, 706, 840, 1200, 1514 or 2072, or comprising SEQ ID NO: 252, 504, 706, 840, 1200, 1514 or 2072. In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 amino acid substitutions. In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide optionally includes 1, 2, 3, 4, or 5 amino acid substitutions.

In the embodiments herein, the recombinant Factor VIII polypeptide has coagulation activity. In some embodiments, a recombinant Factor VIII polypeptide exhibits improved properties compared to a reference Factor VIII polypeptide. In some embodiments, the reference Factor VIII polypeptide has a sequence corresponding to residues 20-1457 of SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072 or corresponding to SEQ ID NO: 2, 252 , 504, 706, 840, 1200, 1514 or 2072 sequence. In some embodiments, the reference Factor VIII polypeptide has a sequence corresponding to residues 20-1457 of SEQ ID NO: 2 or a sequence corresponding to SEQ ID NO: 2.

In some embodiments, with a sequence having residues 20-1457 corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072 or having a sequence corresponding to SEQ ID NO: 2, 252, 504 , 706, 840, 1200, 1514 or 2072, the recombinant Factor VIII polypeptides described herein exhibit at least one improved property selected from: i) increased activity/potency; ii) increased Coagulation activity/potency; iii) increased plasma or serum stability; iv) increased thermal stability; v) reduced immunogenicity; vi) increased expression and/or secretion from cells transfected with the expression vector; vii) Increased transduction and/or secretion from cells transduced with gene therapy vectors (e.g. AAV, mRNA or lipid nanoparticles), or i), ii), iii), iv), v), vi) and a combination of any of vii). In some embodiments, the reference Factor VIII polypeptide has a sequence corresponding to residues 20-1457 of SEQ ID NO: 2 or has a sequence corresponding to SEQ ID NO: 2.

In some embodiments, recombinant Factor VIII polypeptides comprise "deimmunized" recombinant Factor VIII polypeptides, such as provided in Tables 12-1 to 12-8 and 15-2 .

In some other embodiments, recombinant Factor VIII is purified. In another aspect, the present disclosure also provides functional or biologically active fragments of the recombinant Factor VIII polypeptides disclosed herein.

In another aspect, the present disclosure further provides a recombinant polynucleotide encoding at least one recombinant Factor VIII polypeptide described herein, or a biologically active fragment thereof. In some embodiments, the polynucleotide is DNA, RNA, or mRNA.

In some embodiments, the recombinant polynucleotide comprises a reference polynucleotide sequence corresponding to nucleotide residues 58-4371 of odd-numbered SEQ ID NO. in SEQ ID NOs: 1-2345 and 2359-2869 Or have at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more A sequence identical polynucleotide sequence, wherein the recombinant polynucleotide encodes a recombinant Factor VIII polypeptide.

In some embodiments, recombinant polynucleotides comprise polynucleotide sequences that are codon-optimized for expression in a host cell. In some embodiments, recombinant polynucleotides comprise polynucleotide sequences that are codon-optimized for expression in mammalian cells, particularly human cells.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising the odd-numbered nucleotide residues of SEQ ID NO. in SEQ ID NOs: 1-2345 and 2359-2869 58-4371, or odd-numbered SEQ ID NO. including SEQ ID NOs: 1-2345 and 2349-2869.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising the nucleotide residues of SEQ ID NO: 1, 251, 503, 705, 839, 1199, 1513, or 2071 58-4371, or contains SEQ ID NO: 1, 251, 503, 705, 839, 1199, 1513 or 2071.

In another aspect, the present disclosure provides expression vectors comprising recombinant polynucleotides encoding recombinant Factor VIII polypeptides as described herein. In some embodiments, a recombinant polynucleotide is operably linked to one or more control sequences. In some embodiments, the control sequence includes a promoter. In some other embodiments, the promoter is a heterologous promoter.

In another aspect, the present disclosure provides a host cell comprising a recombinant polynucleotide encoding a recombinant Factor VIII polypeptide. In some embodiments, the host cell is a eukaryotic cell or a prokaryotic cell. In some embodiments, the host cell is a mammalian cell, particularly a human cell, such as a cultured human cell. In some embodiments, the host cells are human stem cells.

In another aspect, the present disclosure provides a method of producing a recombinant Factor VIII polypeptide described herein, comprising culturing a host comprising a recombinant polynucleotide encoding a recombinant Factor VIII polypeptide under conditions such that the recombinant Factor VIII polypeptide is produced. cells. In some embodiments, the method further comprises the step of recovering the expressed recombinant Factor VIII polypeptide, such as from the culture medium or cells. In some embodiments, the method further comprises the step of purifying the expressed recombinant Factor VIII polypeptide.

In another aspect, the present disclosure provides compositions comprising at least one recombinant Factor VIII polypeptide described herein. In some embodiments, recombinant Factor VIII polypeptides are provided in the form of pharmaceutical compositions. In some embodiments, a pharmaceutical composition includes a recombinant Factor VIII polypeptide and a pharmaceutically acceptable carrier and/or excipient. In some embodiments, the pharmaceutical composition is suitable for parenteral injection or infusion, particularly for administration to a human subject.

In some embodiments, pharmaceutical compositions comprise at least one recombinant polynucleotide encoding a recombinant Factor VIII polypeptide, particularly for use in gene therapy. In some embodiments, compositions for gene therapy comprise a recombinant polynucleotide encoding at least one of the recombinant Factor VIII polypeptides described herein. In some embodiments, a gene therapy composition comprises a viral vector comprising a recombinant polynucleotide encoding a recombinant Factor VIII polypeptide described herein.

In some embodiments, gene therapy compositions comprise a recombinant polynucleotide encoding at least one recombinant Factor VIII polypeptide described herein, wherein the polynucleotide is formulated in a liposome, a cationic polymer, or via a dendron polymers formulated or linked to cell-penetrating peptides. In some embodiments, recombinant polynucleotides of gene therapy compositions are provided as expression vectors for expression in mammalian cells.

In another aspect, recombinant Factor VIII polypeptides and corresponding recombinant polynucleotides encoding recombinant Factor VIII polypeptides are suitable for therapeutic use in treating a condition or disease characterized by Factor VIII deficiency. In some embodiments, methods for treating and/or preventing symptoms of Factor VIII deficiency in an individual comprise administering to an individual suffering from Factor VIII deficiency an effective amount of a recombinant Factor VIII polypeptide or a medicament comprising a recombinant Factor VIII polypeptide. composition. In some embodiments, methods for treating and/or preventing symptoms of Factor VIII deficiency in an individual comprise providing to an individual suffering from Factor VIII deficiency a recombinant polynucleotide comprising a recombinant Factor VIII polypeptide described herein. Gene therapy compositions.

In another aspect, the present disclosure also provides the use of a recombinant Factor VIII polypeptide, or a medicament thereof, or a gene therapy composition comprising a recombinant polynucleotide encoding a recombinant Factor VIII polypeptide, for treating Factor VIII deficiency in an individual. In some embodiments, use of a recombinant Factor VIII polypeptide or a gene therapy composition comprising a recombinant polynucleotide encoding a recombinant Factor VIII polypeptide is for the preparation of a medicament for treating Factor VIII deficiency in an individual. In some embodiments, the method or use is for treating hemophilia A in an individual.

Detailed description of preferred embodiments

The present disclosure relates to recombinant Factor VIII polypeptide (FVIII) variants and compositions thereof. Recombinant Factor VIII polypeptide variants are engineered to exhibit improved properties, such as improved activity, including blood coagulation activity, improved cofactor potency, improved performance, increased thermal stability, increased serum stability, increased plasma stability and/or reduced immunogenicity. The present disclosure also provides for the use of compositions comprising recombinant Factor VIII polypeptide variants or polynucleotides encoding such recombinant Factor VIII polypeptide variants for therapeutic purposes.

It should be understood that the foregoing general description, including the accompanying drawings, and the following detailed description are illustrative and explanatory only and do not limit the disclosure. Abbreviations and definitions

Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein and in the cell culture protocols, molecular genetics, microbiology, biochemistry, organic chemistry, analytical chemistry, and nucleic acid chemistry described below are those well known and commonly used in the art. , cell culture experimental procedures, molecular genetics, microbiology, biochemistry, organic chemistry, analytical chemistry and nucleic acid chemistry. Such techniques are well known and described in numerous textbooks and reference works familiar to those skilled in the art. Chemical synthesis and chemical analysis are performed using standard techniques or variations thereof.

Although any suitable methods and materials similar or equivalent to those described herein can be used in the practice of the invention, some methods and materials are described herein. It is to be understood that this invention is not limited to the particular methods, protocols and reagents described, as such methods, protocols and reagents may vary depending on the context in which they are used by those skilled in the art. Accordingly, the terms defined immediately below are more fully described by reference to the entire application.

Furthermore, as used herein, the singular "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

Numerical ranges include the numbers defining the range. Therefore, every numerical range disclosed herein is intended to include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. It is also intended that every maximum (or minimum) numerical limitation disclosed herein include every lower (or higher) numerical limitation, as if such lower (or higher) numerical limitations were expressly written herein.

The term "about" means an acceptable error for a particular value. In some cases, "about" means within 0.05%, 0.5%, 1.0% or 2.0% of a stated value range. In some cases, "about" means within 1, 2, 3 or 4 standard deviations of a given value.

As used herein, the term "comprising" and its cognates are used in its inclusive sense (i.e., equivalent to the term "includes" and its corresponding cognates).

It should also be understood that where the term "comprising" is used in the description of various embodiments, those skilled in the art will understand that in some specific cases, the phrase "consisting essentially of" or "consisting of" may be used. Composition" alternatively describes embodiments.

Additionally, the headings provided herein do not limit the various aspects or embodiments of the invention, which may be practiced with reference to the entire application. Accordingly, the terms defined immediately below are more fully defined by reference to the entire application. Nonetheless, in order to facilitate an understanding of the present invention, a number of terms are defined below.

"ATCC" refers to the American Type Culture Collection whose biobank collection includes genes and strains.

"NCBI" refers to the National Center for Biological Information and the sequence database provided therein.

"Protein", "polypeptide" and "peptide" are used interchangeably herein to mean a polymer of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modification (e.g. glycosylation) or phosphorylation).

"Mature protein" or "mature polypeptide" refers to the final processed biological protein or polypeptide or product.

"Proprotein", "propolypeptide" or "propeptide" refers to a precursor protein, polypeptide or peptide that is processed by post-translational modification to form a biologically active protein, polypeptide or peptide. In some embodiments, the post-translational modification is a cleavage reaction to form a protein, polypeptide or peptide.

"Preproprotein", "prepropolypeptide" or "prepropeptide" refers to a precursor protein, polypeptide or peptide, which includes a signal sequence or signal peptide and can be processed by post-translational modification, especially cleavage reaction, to produce a preprotein, prepropolypeptide or propeptide. Generally, cleavage reactions remove a signal sequence or signal peptide to produce a proprotein, propolypeptide or propeptide.

In the context of a protein or polypeptide, "full-length" refers to a protein or polypeptide that has not been processed to alter the amino acid sequence of the entire protein or polypeptide. For example, a full-length protein is the entire protein encoded in the corresponding mRNA.

"Amino acids" are referred to herein by their commonly known three-letter symbols or by their single-letter symbols recommended by the IUPAC-IUB Committee on Biochemical Nomenclature. The abbreviations for amino acids encoded by genes are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamic acid (Glu or E), glutamic acid (Gln or Q), glycine (Gly or G), histamine (His or H) , isoleucine (Ile or I), leucine (Leu or K), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y) and valine (Val or V) . When a three-letter abbreviation is used, the amino acid may be in the L configuration or the D configuration around the alpha carbon (C _α ) unless specifically preceded by an "L" or "D" or is obvious from the context in which the abbreviation is used. state. For example, whereas "Ala" indicates alanine without specifying the configuration around the alpha-carbon, "D-Ala" and "L-Ala" indicate D-alanine and L-alanine, respectively. When a polypeptide sequence is presented as a sequence of one-letter or three-letter abbreviations (or a mixture thereof), the sequence is presented in the amine (N) to carboxyl (C) direction according to common convention.

"Polynucleotide" is used herein to mean a polymer containing at least two nucleotides, where the nucleotides are deoxyribonucleotides or ribonucleotides or both deoxyribonucleotides and ribonucleosides A mixture of acids. In some embodiments, abbreviations for gene-encoded nucleosides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U ). Unless stated specifically, the abbreviation nucleoside may be ribonucleoside or 2'-deoxyribonucleoside. Nucleosides may be designated as ribonucleosides or 2'-deoxyribonucleosides, either on an individual basis or on a collective basis. When a nucleic acid sequence is presented as a string of single-letter abbreviations, according to common convention, the sequence is presented in the 5' to 3' direction, and the phosphates are not indicated. The term "DNA" refers to deoxyribonucleic acid. The term "RNA" refers to ribonucleic acid.

The terms "engineered," "recombinant," "non-naturally occurring," and "variant" when used with respect to cells, polynucleotides, or polypeptides mean that have been modified or modified in a manner that would not otherwise occur in nature. A substance consistent therewith but produced or derived from a synthetic substance and/or produced or derived by manipulation using recombinant techniques or a substance corresponding to a natural or natural form of that substance.

“Wild type” and “naturally occurring” refer to the form found in nature. For example, a wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and that has not been intentionally modified by human manipulation.

"Coding sequence" refers to a portion of a nucleic acid (eg, a gene) encoding the amino acid sequence of a protein or polypeptide.

"Percent sequence identity (%)" is used herein to refer to comparison results in polynucleotides and polypeptides, and is determined by comparing two optimally aligned sequences over a comparison window, where compared to a reference For sequences, the portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) for optimal alignment of the two sequences. This percentage can be calculated by determining the number of positions in the two sequences where the same nucleic acid base or amino acid residue exists to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window and multiplying the result Take 100 to generate percent sequence identity. Alternatively, the percentage can be calculated by determining the number of positions in the two sequences where the same nucleic acid base or amino acid residue exists or the number of positions where the nucleic acid base or amino acid residue aligns with a gap to produce a matching position. Number, divide the number of matching positions by the total number of positions in the comparison window and multiply the result by 100 to generate percent sequence identity. Those skilled in the art appreciate that there are many established algorithms that can be used to align two sequences. The optimal sequence alignment for comparison can be, for example, by Smith and Waterman's local homology algorithm (Smith and Waterman, Adv. Appl. Math., 1981, 2:482), by Needleman and Wunsch's homology Similarity comparison algorithm (Needleman and Wunsch, J. Mol. Biol., 1970, 48:443), similarity search method by Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 1988 , 85:2444), by computerized implementations of such algorithms (such as GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin software suite) or by visual inspection as is known in the art. Examples of algorithms suitable for determining percent sequence identity and sequence similarity include, but are not limited to, the BLAST and BLAST 2.0 algorithms described by Altschul et al. (see, respectively, Altschul et al., J. Mol. Biol., 1990, 215: 403-410; and Altschul et al., 1977, Nucleic Acids Res., 1977, 3389-3402). Software used to perform BLAST analyzes is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy some positive value when aligned with words of the same length in the database sequence. The threshold score T. T is called the neighborhood word score threshold (see Altschul et al., supra). These initial neighborhood word success results serve as seeds for initial searches to find longer HSPs containing them. Word matches are then extended in both directions along each sequence as long as the cumulative alignment score increases. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for a mismatched residue; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The extension of word hits in all directions is interrupted when: the cumulative alignment score decreases by the amount below; or to the end of any sequence. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the comparison. The BLASTN program (for nucleotide sequences) uses the following as default values: word length (W) of 11, expectation (E) of 10, M = 5, N = -4 and the result of the two-strand comparison. For amino acid sequences, the BLASTP program uses a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1989, 89:10915) as default parameters. Exemplary determinations of sequence alignment and % sequence identity can be performed using the BESTFIT or GAP programs in the GCG Wisconsin software suite (Accelrys, Madison WI) using the default parameters provided.

"Reference sequence" means a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of a larger sequence, such as a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence has a length of at least 20 nucleotides or amino acid residues, a length of at least 25 residues, a length of at least 50 residues, a length of at least 100 residues, or a nucleic acid or polypeptide its full length. Since two polynucleotides or polypeptides may each (1) comprise a sequence that is similar between the two sequences (i.e., a portion of the complete sequence) and (2) may further comprise a sequence that is divergent between the two sequences Therefore, sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing the sequences of the two polynucleotides or polypeptides over a "comparison window" to identify and compare local areas of sequence similarity. area to execute. In some embodiments, a "reference sequence" may be based on a primary amino acid sequence, where the reference sequence is a sequence that may have one or more changes in the primary sequence. "Comparison window" means a conceptual segment of at least about 20 contiguous nucleotide positions or amino acid residues within which a sequence can be compared to a reference sequence of at least 20 contiguous nucleotide or amino acid residues and The portion of the sequence in the comparison window may contain 20% or less of the additions or deletions (i.e., gaps) present for optimal alignment of the two sequences compared to the reference sequence (which contains no additions or deletions). missing)). Comparison windows can be longer than 20 contiguous residues, and optionally include windows of 30, 40, 50, 100, or longer.

"Corresponds to", "references" or "relative to" when used in the context of the numbering of a given amino acid or polynucleotide sequence means when the given amino acid or polynucleotide sequence is compared with a reference sequence When comparing, the residue number of the reference sequence specified. In other words, residue numbers or residue positions for a given polymer are assigned relative to a reference sequence rather than based on the actual numbered positions of the residues within a given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as that of a recombinant Factor VIII polypeptide, can be aligned to a reference sequence by introducing gaps to optimize the residue match between the two sequences. In these cases, notwithstanding the presence of gaps, the numbering of residues in a given amino acid or polynucleotide sequence is achieved relative to a reference sequence to which it is aligned.

"Mutation" refers to a change in a nucleic acid sequence. In some embodiments, mutations result in a change in the sequence encoding the polypeptide (i.e., compared to the original sequence without the mutation). In some embodiments, mutations comprise substitutions such that different amino acids are produced. In some alternative embodiments, mutations comprise additions such that an amino acid is added (eg, inserted) to the original polypeptide sequence. In some other embodiments, mutations comprise deletions such that an amino acid is deleted from the original polypeptide sequence. Any number of mutations can be present in a given sequence.

"Amino acid difference" or "residue difference" refers to the difference in an amino acid residue at a position in a polypeptide sequence relative to an amino acid residue at a corresponding position in a reference sequence. The positions where amino acids differ are generally referred to herein as "Xn", where n refers to the corresponding position in the reference sequence based on which the residue difference occurs. For example, "residue difference compared to position X59 of SEQ ID NO: 2" refers to the amino acid residue difference at the polypeptide position corresponding to position 59 of SEQ ID NO: 2. Therefore, if the reference polypeptide of SEQ ID NO: 2 has phenylalanine at position 59, then the "residue difference compared to position X59 of SEQ ID NO: 2" corresponds to SEQ ID NO: 2 except for phenylalanine. Amino acid substitution of any residue at position 59 of the polypeptide. In some instances herein, a specific amino acid residue difference at a position is designated "XnY", where "Xn" specifies the corresponding position as described above, and "Y" is in the engineered polypeptide The one-letter identifier of the amino acid found (ie, the residue that is different from the residue in the reference polypeptide). In some cases (for example, as shown in Tables 4.1 , 4.2 , 5.1 , 5.2 , 6.1 , 6.2, 7.1 , 7.2 , 8.1 , 8.2 , 9.1 , 9.2 , 10.1 , 10.2 , 11.1 , and 15.1 ), this disclosure is also provided by A specific amino acid difference represented by the conventional notation "AnB", where A is the one-letter identifier of the residue in the reference sequence, "n" is the number of residue positions in the reference sequence, and B is the engineered polypeptide A single-letter identifier for a residue substitution in the sequence. In some embodiments, amino acid differences such as substitutions are represented by the abbreviation "nB" without the identifier of the residue in the reference sequence. In some embodiments, the phrase "amino acid residue nB" indicates the presence of an amine residue in the engineered polypeptide, which may or may not be substituted in the case of the reference polypeptide.

In some cases, a polypeptide of the present disclosure may include one or more amino acid residue differences relative to a reference sequence, as indicated by a list of designated positions at which residue differences exist relative to the reference sequence, as described below. In some embodiments, when more than one amino acid can be used at a specific residue position in a polypeptide, the various amino acid residues that can be used are separated by a "/" (e.g., X61Q/X61S or X61Q/S or 61Q /S).

"Amino acid substitution set" and "substitution set" refer to a group of amino acid substitutions within a polypeptide sequence. In some embodiments, a set of substitutions includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions. In some embodiments, the set of substitutions refers to the set of amino acid substitutions present in any of the variant polypeptides listed in any of the tables in the Examples. Within a substitution set, individual substitutions are separated by a semicolon (";"; such as Q96R;L171P or abbreviated 96R;171P) or a slash ("/"; such as Q96R/L171P or abbreviated 96R/171P). In some embodiments, "substitution" includes deletion of an amino acid and may be represented by a "-" symbol.

"Conserved amino acid substitution" refers to residue substitution with different residues having similar side chains, and thus generally involves amines in a polypeptide with amino acids within the same or similar defined classes of amino acids. Acid substitution. By way of example and not limitation, accordingly, an amino acid having an aliphatic side chain may be substituted by another aliphatic amino acid (such as alanine, valine, leucine, and isoleucine); having a hydroxyl side chain The amino acid of the chain is replaced by another amino acid with a hydroxyl side chain (such as serine and threonine); the amino acid with an aromatic side chain is replaced by another amino acid with an aromatic side chain (such as serine and threonine). For example, phenylalanine, tyrosine, tryptophan, and histidine) are substituted; an amino acid with a basic side chain is substituted by another amino acid with a basic side chain (such as lysine and arginine) ;An amino acid with an acidic side chain is replaced by another amino acid with an acidic side chain (such as aspartic acid or glutamic acid); and/or a hydrophobic or hydrophilic amino acid is replaced by another hydrophobic or Hydrophilic amino acid replacement.

"Nonconservative substitution" refers to an amino acid substitution in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitutions can use amino acids between defined groups rather than within defined groups and affect (a) the structure of the peptide backbone in the substitution region (such as proline replacing glycine), (b) charge or hydrophobicity, or (c) side chain volume. By way of example and not limitation, exemplary non-conservative substitutions may be substitution of acidic amino acids with basic or aliphatic amino acids; substitution of aromatic amino acids with small amino acids; and substitution of hydrophilic amino acids with hydrophobic amines. Acid substitution.

"Deletion" refers to a modification of a polypeptide by removing one or more amino acids from a reference polypeptide. Deletions may include removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amines amino acids or 20 or more amino acids, up to 10% of the total amino acids constituting the reference polypeptide or up to 20% of the total amino acids constituting the reference polypeptide, while retaining activity and/or retaining the engineered Improved properties of polypeptides. Deletions may be directed to internal and/or terminal portions of the polypeptide. In various embodiments, deletions may comprise contiguous segments or may be non-contiguous.

"Insertion" refers to a modification of a polypeptide by the addition of one or more amino acids from a reference polypeptide. Insertion can occur within the internal portion of the polypeptide, or at the carboxyl or amine terminus. Inserts as used herein include fusion proteins as known in the art. Insertions may be contiguous stretches of amino acids or separated by one or more amino acids in a naturally occurring polypeptide.

"Functional fragment" or "biologically active fragment" as used interchangeably herein refers to a polypeptide that has amino-terminal and/or carboxyl-terminal deletions and/or internal deletions, but the remaining amino acid sequence is compared with that of the The corresponding positions in the sequence (eg, the full-length recombinant Factor VIII of the present disclosure) are identical and retain substantially all the activities of the full-length polypeptide. In some embodiments, "functional fragments" or "biologically active fragments" encompass recombinant Factor VIII that is processed into polypeptides by natural biological processes, such as cleavage into polypeptide fragments that form dimers and activated trimers.

"Isolated polypeptide" refers to a polypeptide (such as proteins, lipids, and polynucleotides) that is substantially separated from other contaminants that naturally accompany it. The term encompasses polypeptides that have been removed or purified from their naturally occurring environment or expression system (eg, host cells or in vitro synthesis). Recombinant Factor VIII polypeptides may be present intracellularly, in the cell medium, or prepared in various forms such as lysates or isolated preparations. Thus, in some embodiments, a recombinant Factor VIII polypeptide may be an isolated polypeptide.

"Substantially pure polypeptide" means a composition in which the polypeptide species is the dominant species present (i.e., it is more abundant on a molar concentration or weight basis than any other individual macromolecular species in the composition) and is A substantially purified composition is generally one in which the target species contains at least about 50% of the macromolecular species present on a molar or weight % basis. Generally speaking, substantially pure Factor VIII compositions comprise about 60% or more, about 70% or more, about 80% or more, about 90%, based on moles or weight % present in the composition. or more, about 95% or more, and about 98% or more of all macromolecular species. In some embodiments, the target species is purified to substantial homogeneity (i.e., contaminant species in the composition cannot be detected by conventional detection methods), wherein the composition consists primarily of a single macromolecular species. Solvent species, small molecules (<500 daltons), and elemental ionic species are not considered macromolecular species. In some embodiments, the isolated recombinant Factor VIII polypeptide is a substantially pure polypeptide composition.

"Improved protein properties" refers to a recombinant Factor VIII polypeptide that exhibits any improvement in protein properties compared to a reference FVIII polypeptide and/or a wild-type Factor VIII polypeptide or another recombinant Factor VIII polypeptide. Improved properties include (but are not limited to) properties such as: increased gene expression, increased protein production, increased thermal activity, increased thermal stability, increased activity at various pH levels, increased stability, increased Cofactor potency, increased specific activity, increased resistance to substrate and/or product inhibition, increased chemical stability, improved solvent stability, increased tolerance to acidic, neutral or alkaline pH properties, increased resistance to proteolytic activity (i.e., decreased susceptibility to proteolysis), decreased aggregation, increased solubility, decreased immunogenicity, increased secretion of polypeptide-producing cells, etc. .

"Increased activity", "increased cofactor potency" or "enhanced activity" refers to the improved properties of a recombinant Factor VIII polypeptide compared to a reference Factor VIII polypeptide, which can be measured by specific activity (e.g. product production/time/weight protein It is expressed as an increase in the percentage of substrate converted to product (e.g., the percentage of substrate converted to product using a specified amount of a Factor VIII polypeptide in combination with FIX over a specified period of time). Exemplary methods for determining protein activity are provided in the Examples. Any property related to protein activity can be affected. The improvement in protein activity may range from about 1.1-fold over the protein activity corresponding to the wild-type protein or the parent protein or polypeptide to another recombinant Factor VIII over naturally occurring Factor VIII or a derived Factor VIII polypeptide (e.g., one in which the B region is deleted) Factor VIII) up to 2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold or higher activity.

"Increased tolerance to alkaline pH" means that a recombinant Factor VIII according to the invention will have increased stability (e.g., over a specific period of time (1 hour)) compared to a reference Factor VIII or another recombinant Factor VIII polypeptide. up to 24 hours) higher retained activity at >pH 7) after exposure to alkaline pH).

As used herein, "physiological pH" means the pH range typically found in the blood of an individual (eg, a human). In some embodiments, "physiological pH" is about pH 7 to about 7.4.

"Alkaline pH" (eg used with reference to improved stability of alkaline pH conditions or increased tolerance of alkaline pH) means a pH range >pH 7, eg about 7 to 11.

"Acidic pH" (eg, used with reference to improved stability of acidic pH conditions or increased tolerance of acidic pH) means a pH 7>pH range, eg, about 1.5 to 4.5.

"Deimmunization" as used herein refers to manipulation of protein sequence to produce variants that are not predicted to be generally immunogenic as wild-type or reference proteins. In some embodiments, predicted deimmunization is complete because the variant protein is predicted to not stimulate an immune response in the patient to whom the variant protein is administered. This response can be measured by a variety of methods, including (but not limited to): the presence or abundance of anti-drug antibodies, the presence or abundance of neutralizing antibodies, the presence of allergic reactions, major histocompatibility complex II (MHC-II) The incidence or intensity of interleukin release following presentation of a peptide on a protein or administration of a protein. In some embodiments, the variant protein is less immunogenic than the wild-type or reference protein. In some embodiments, deimmunization involves modification of subsequences of proteins (eg, epitopes) recognized by human leukocyte antigen (HLA) receptors. In some embodiments, these epitopes are removed by altering their amino acid sequences to produce deimmunized variant proteins, in which such subsequences are no longer recognized by HLA receptors. In some other embodiments, these epitopes retain binding affinity for HLA receptors but do not exhibit it. In some embodiments, the deimmunized protein exhibits lower levels of response in biochemical and cellular biological predictors of human immune response, including dendritic cell T cell activation assays or (HLA) peptide binding assays. In some embodiments, these epitopes are removed by altering their amino acid sequence to produce deimmunized variant proteins, wherein the epitopes are no longer recognized by T cell receptors. In other embodiments, the deimmunized protein induces anergy in its corresponding T cell, activates T regulatory cells, or causes a pure-line deletion of recognizing B cells. In some embodiments, the total immunogenicity score (TIS) reflects the overall predicted immunogenicity of a variant (ie, a higher score indicates a higher predicted immunogenicity level).

In nucleic acid hybridization, "hybridization stringency" is related to hybridization conditions such as wash conditions. Generally, hybridization reactions are performed at lower stringency conditions, followed by wash changes at slightly higher stringency conditions (see, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001). The term "moderately stringent hybridization" refers to permitting target DNA binding with about 60% identity to the target DNA, preferably about 75% identity, about 85% identity, and greater than about 90% identity to the target polynucleotide. conditions for complementary nucleic acids. Exemplary moderately stringent conditions are equivalent to the following: hybridization in 50% formamide, 5×Denhart's solution, 5×SSPE, 0.2% SDS at 42°C, followed by Wash in 0.2 × SSPE, 0.2% SDS at 42°C. "High stringency hybridization" generally refers to conditions that are about 10°C or less from the thermal melting temperature T _m as determined for a defined polynucleotide sequence under solution conditions. In some embodiments, high stringency conditions refer to conditions that permit hybridization of only those nucleic acid sequences to form stable hybrids in 0.018 M NaCl at 65°C (i.e., as contemplated herein, if the hybrids are is unstable in 0.018 M NaCl, it will not be stable under high stringency conditions). High stringency conditions can be provided as follows: for example, hybridize under conditions equivalent to: 50% formamide, 5× Dunhart's solution, 5× SSPE, 0.2% SDS at 42°C, followed by 65°C Next, wash in 0.1×SSPE and 0.1% SDS. Another high stringency condition is to hybridize under conditions equivalent to: hybridization in 5X SSC containing 0.1% (w:v) SDS at 65°C and hybridization in 5X SSC containing 0.1% SDS at 65°C. Wash in 0.1x SSC. Other high stringency hybridization conditions as well as moderate stringency conditions are described in the references cited herein.

"Codon-optimized" refers to codon changes in a polynucleotide encoding a protein that result in more efficient expression of the encoded protein in the organism and/or cell of interest. Although the simplicity of the genetic code means that most amino acids are represented by a few codons, called "synonymous" or "synonymous" codons, it is known that the codon usage of a given organism is not random and is biased toward specific codons triplet. This codon usage preference may be higher for established genes, genes with a common function or ancestral origin, highly expressed proteins relative to low copy number proteins, and polymeric protein coding regions of biological genomes. In some embodiments, polynucleotides encoding Factor VIII polypeptides can be codon-optimized to self-select for expression, interpretation of GC content, cryptic splice sites, transcription termination signals, and motifs that can affect RNA stability. and nucleic acid secondary structure as well as any other factors of interest that are optimally produced by the host organism and/or cell type.

"Control sequences" as used herein are meant to include all components necessary for or advantageous for the performance of the polynucleotides and/or polypeptides of the present application. For nucleic acid sequences encoding polypeptides, each control sequence may be native or foreign. Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoter sequences, signal peptide sequences, initiation sequences, and transcription terminators. Control sequences include at least a promoter and transcription and translation termination signals.

"Operably linked" is defined herein as one in which the control sequence is suitably positioned relative to the polynucleotide of interest (ie, in a functional relationship) such that the control sequence directs or modulates the polynucleotide of interest. The configuration of expression of polynucleotides and/or polypeptides.

"Promoter sequence" refers to a nucleic acid sequence, such as a coding sequence, recognized by a host cell for expression of a polynucleotide of interest. The promoter sequence contains transcriptional control sequences that mediate the expression of the polynucleotide of interest. A promoter can be any nucleic acid sequence that exhibits transcriptional activity in the host cell of choice, including mutant promoters, truncated promoters, and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides. Homologous or heterologous to the host cell.

"Heterologous" or "recombinant" refers to the relationship between two or more nucleic acid or protein sequences (such as promoter sequences, signal peptides, terminator sequences, etc.) that are derived from different sources and are not related in nature.

"Suitable reaction conditions" refer to those conditions in a reaction solution (e.g., ranges of protein loading, temperature, pH, buffers, co-solvents, etc.) under which Factor VIII polypeptides of the present disclosure are able to function as factors The cofactor of IX is used to convert the substrate into the desired product compound. Exemplary "suitable reaction conditions" are provided in this application and illustrated in the Examples.

"Protein loading" refers to the concentration or amount of a component in the reaction mixture at the beginning of the reaction. "Substance", in the context of a reaction process involving Factor VIII, refers to the compound or molecule on which the Factor VIII-Factor IX polypeptide complex acts.

"Product" in the context of an enzymatic conversion process means a compound or molecule resulting from the action of the Factor VIII-Factor IX polypeptide complex on the substrate.

"Culture" as used herein refers to the growth of a population of microorganisms, mammalian or other suitable cells under any suitable conditions (eg, using liquid, gel, or solid media).

"Vector" refers to a polynucleotide construct used to introduce a polynucleotide sequence into a cell. In some embodiments, the vector system has an expression vector for a polynucleotide of interest operably linked to a control sequence suitable for affecting the expression of a polypeptide encoded in the polynucleotide sequence of interest. Performance in the host. In some embodiments, an "expression vector" has a promoter sequence operably linked to a polynucleotide sequence (e.g., a transgene) to drive expression in a host cell, and in some embodiments, also includes transcription termination subsequence.

"Gene therapy vector" as used herein refers to a vehicle or vehicle suitable for delivering a polynucleotide sequence to a cell. In some embodiments, vectors encapsulate genes (eg, therapeutic genes) or polynucleotide sequences for delivery to cells or tissues, including, but not limited to, adenovirus (AV) vectors, adeno-associated virus (AAV) ) vectors, lentiviral (LV) vectors, and non-viral vectors such as liposomes. It is contemplated that the present invention is not limited to any particular gene therapy vector, as any vehicle suitable for a given setting may be used herein. Gene therapy vectors may be designed to deliver genes to a specific species or host, or may have more general applicability.

"Performance" as used herein includes any step involved in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses cell-secreted polypeptides.

"Production" as used herein refers to the production of proteins and/or other compounds by cells. The term is intended to encompass any step involved in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses cell-secreted polypeptides.

"Host cell" and "host strain" refer to a suitable host for an expression vector containing a polynucleotide provided herein (eg, a polynucleotide encoding a recombinant Factor VIII variant). In some embodiments, the host cell is a prokaryotic or eukaryotic cell that has been transformed or transfected using vectors constructed using recombinant DNA techniques as known in the art.

"Analog" as used herein means having more than 70% sequence identity, but less than 100% sequence identity with a reference polypeptide (e.g., more than 75%, 78%, 80%, 83%, 85%, 88%, 90%). %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity). In some embodiments, "analog" means containing one or more non-naturally occurring amino acid residues including, but not limited to, homoarginine, ornithine, and norvaline as well as naturally occurring amine groups. acid peptides. In some embodiments, analogs also include one or more D-amino acid residues and non-peptide linkages between two or more amino acid residues.

"Therapeutic" as used herein refers to a compound or composition that has a beneficial or desirable medical effect that is administered to an individual exhibiting signs or symptoms of a disease or pathology.

"Pharmaceutical composition" as used herein refers to a composition suitable for medical use in a mammalian subject (eg, a human), which includes a pharmaceutically effective amount of a recombinant Factor VIII polypeptide and an acceptable carrier and/or excipient. agent.

"Compositions" and "formulations" encompass products containing at least one recombinant Factor VIII of the present disclosure intended for any suitable use (eg, pharmaceutical compositions, etc.).

"Gene therapy" refers to the delivery of one or more genes, polydeoxyribonucleotides or polynucleotide sequences together with a gene therapy vector to cells or tissues to modify those cells or tissues to treat or prevent disease. Gene therapy may include replacing a disease-causing mutated gene with a healthy copy of the gene; inactivating or "knocking out" a mutated gene that functions inappropriately; or providing a gene sufficient to treat and/or prevent a disease or condition. Functional replica. In some embodiments, gene therapy is used to treat a disease in a patient.

"mRNA therapy" refers to the delivery of mRNA polyribonucleotides to cells or tissues to modify those cells or tissues to treat or prevent disease. In some embodiments, mRNA polynucleotides for delivery to cells or tissues are formulated, for example, but not limited to, in liposomes. In some embodiments, mRNA therapy is used to treat a disease in a patient.

"Cell therapy" refers to the delivery of exogenously modified living cells to patients to provide missing genes to treat or prevent disease. The modified cells are then reintroduced into the body.

"Effective amount" means an amount sufficient to produce the desired result. One skilled in the art can determine the effective amount by using routine experimentation.

"Isolated" and "purified" are used to refer to a molecule (eg, isolated nucleic acid, polypeptide, etc.) or other component that has been removed from at least one other component with which it is naturally associated. The term "purified" does not require absolute purity, but rather is intended to be a relative definition.

"Individual" includes mammals, such as humans, non-human primates, livestock, companion animals, and laboratory animals (such as rodents and lagomorphs). Hopefully the term covers females as well as males.

"Patient" means any individual assessed for, treatment for, or experiencing a disease. In some embodiments, the patient is a human patient.

"Infant" means a child from the first month after birth to approximately one (1) year old. As used herein, the term "newborn" refers to a child from birth to the 28th day of life. The term "premature infant" refers to an infant born after the twentieth full week of pregnancy but before full term, with a birth weight generally ranging from about 500 to about 2499 grams. "Extremely low birth weight infants" are infants whose birth weight is less than 1500 g.

"Child" means an individual who has not reached the legal age to consent to treatment or research procedures. In some embodiments, the term refers to an individual between the time of birth and puberty.

"Adult" means an individual who has reached the age of majority in the relevant jurisdiction (for example, 18 years of age in the United States). In some embodiments, the term refers to any fully grown, mature organism. In some embodiments, the term "adolescent" refers to an individual under the age of 18 who has reached sexual maturity.

(e.g., administering) and "administering" a composition means providing a composition of the invention to an individual (e.g., to an individual affected by a factor VIII deficiency, such as hemophilia A).

"Pharmaceutically acceptable" means one that can be administered to an individual without causing any undesirable biological effects or interacting in a harmful manner with any of the components contained therein that have the desired biological activity. material.

"Excipient" refers to any pharmaceutically acceptable additive, carrier, diluent, adjuvant or other ingredient other than the active pharmaceutical ingredient (API; such as the recombinant Factor VIII polypeptide of the present disclosure). Excipients are typically included for purposes of formulation and/or administration.

"Carrier" when used in connection with a pharmaceutical composition means any of standard pharmaceutical carriers, buffers and excipients such as stabilizers, preservatives and adjuvants.

When used with reference to symptoms of a disease/condition, "therapeutically effective amount" means an amount of a compound (e.g., a recombinant Factor VIII polypeptide) that ameliorates, attenuates, or eliminates one or more symptoms of the disease/condition or prevents or delays the onset of symptoms. quantity and/or concentration. When used with reference to a disease or condition, a "therapeutically effective amount" refers to an amount and/or concentration of a composition (e.g., a recombinant Factor VIII polypeptide) that ameliorates, attenuates, or eliminates the disease/condition. In some embodiments, the term is used to refer to an amount of a composition sought by a researcher, physician, veterinarian, or other clinician to elicit a biological (eg, medical) response in a tissue, system, or individual animal.

As used herein, "treating" or "treatment" of a disease, condition, or syndrome includes (i) preventing the occurrence of the disease, condition, or syndrome in an individual, that is, rendering the disease, condition, or syndrome susceptible to the disease, condition, or syndrome in an individual; Animals that have a disease, disease or syndrome but have not yet experienced or shown symptoms of the disease, disease or syndrome do not develop clinical signs of the disease, disease or syndrome; (ii) inhibit the disease, disease or syndrome, that is, arrest its progression; and (iii) alleviate the disease, condition or syndrome, that is, cause the disease, condition or syndrome to subside. As such, the terms "treating", "treat" and "treatment" encompass preventive (eg preventive) treatment as well as palliative treatment. As is known in the art, adjustments to systemic delivery versus local delivery, age, weight, general health, sex, diet, timing of administration, drug interactions, and severity of condition may need to be made, and such adjustments may be made by Generally, it is determined by those who are familiar with this technology. Recombinant factor VIII polypeptide

As discussed above, blood coagulation factor VIII (FVIII) is a cofactor of activated coagulation factor IX (eg, FIXa), which in its proteolytically activated form interacts with FIXa to form a tight binding to and activated factor X (FX) Non-covalent complexes. Hemophilia A is a factor VIII deficiency caused by missing or defective FVIII protein. Treatment for patients with hemophilia A may include enzyme replacement therapy with preparations of the factor VIII protein. Standard enzyme replacement therapy uses a recombinant expression of wild-type human Factor VIII or a B-region deleted form of human Factor VIII (e.g., ^XYNTHA® Antihemophilic Factor (recombinant); Wyeth Pharmaceuticals LLC). Intravenously administered FVIII is known to circulate in the bloodstream and restore blood coagulation function. However, these drugs do not completely alleviate patient symptoms because additional doses are required after trauma or before surgery. Additionally, the protein is relatively unstable and cleared from the bloodstream within days. Therefore, the question remains as to available enzyme replacement therapy. Additionally, patients can develop immune responses (IgG and IgE antibodies targeting the administered drugs) and suffer severe allergic/anaphylactic reactions, severe infusion reactions, and even death.

The recombinant Factor VIII polypeptides or bioactive fragments thereof disclosed herein are intended to provide proteins suitable for the treatment of hemophilia A that are more stable and effective than currently available treatments, but with reduced side effects and improved outcomes. In some embodiments, recombinant Factor VIII polypeptides are characterized by increased stability in the blood (pH 7.4) where the enzymes are introduced into the bloodstream and where the enzymes are active during therapy. Recombinant Factor VIII polypeptides also exhibit, among other properties, improved coagulation function and, in some embodiments, reduced predicted immunogenicity (deimmunization).

The recombinant Factor VIII polypeptides of the present disclosure are derived from recombinant Factor VIII with a deletion of the B region (SEQ ID NO: 2; see, eg, U.S. Patent Publication No. 2017/0360900), where the factor VIII with a deletion of the B region is naturally occurring. Factor VIII amino acid sequence is dominant. Briefly, for purposes of explanation and not limitation, a Factor VIII polypeptide consists of five regions within the polypeptide: the signal peptide, the A1 region, the A2 region, the B region, and the light chain region. The recombinant Factor VIII in which the deletion of the B region is represented by SEQ ID NO: 2 has the same signal peptides A1, A2 and light chain regions as wild-type human Factor VIII, but its B region has been reduced to fourteen residues. In some embodiments, in the present disclosure, when a particular Factor VIII variant is referred to with reference to a Factor VIII deleted in the B region or to an amino acid difference in an amino acid residue in a Factor VIII polypeptide sequence, it will be understood that Variants of another Factor VIII polypeptide modified in equivalent positions (as determined from an optionally selected amino acid sequence alignment between respective amino acid sequences) are encompassed herein.

In addition, the present disclosure also provides biologically active fragments of the recombinant Factor VIII polypeptide variants disclosed herein. It is understood that Factor VIII is biologically processed into polypeptide fragments. By way of example, and not limitation, Factor VIII with a B region deletion can be processed by removing the signal sequence (e.g., amino acid residues 1 to 19), resulting in processed Factor VIII (e.g., about 20-1457 amines) amino acid residues). Factor VIII can subsequently be processed to produce a polypeptide encoding dimeric human Factor VIII (or a variant thereof) that undergoes one or more cleavages between residues 760 to 773 of SEQ ID NO: 2 and that does not contain a polypeptide corresponding to Some or all of the amino acid sequences of amino acids 760 to 773 of SEQ ID NO: 2. Factor VIII can be further processed to produce a polypeptide encoding trimeric human Factor VIII (or a variant thereof) with additional cleavage occurring between residues 391 and 392 of SEQ ID NO: 2 and which does not contain the polypeptide corresponding to SEQ ID NO : Some or all of the amino acid sequences of amino acids 760 to 773 of 2. Recombinant Factor VIII polypeptide variants may or may not be treated identically to a deleted Factor VIII having a naturally occurring sequence of the B region.

Therefore, references herein to "biologically active" or "functionally active" fragments of a recombinant Factor VIII polypeptide are intended to include expression by natural organisms when expressed in mammalian cells or patients, or after administration of a recombinant Factor VIII polypeptide to a patient. Polypeptides formed by processing, whether such "biologically active" or "functionally active" fragments are the product of processing similar to the deletion of Factor VIII with a naturally occurring sequence of the B region, or a deletion of the B region with a naturally occurring sequence Fragments of different processing results for Factor VIII.

In one aspect, the present disclosure provides a recombinant Factor VIII polypeptide, or a biologically active fragment thereof, comprising residue 20- corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072 The reference sequence of 1457 or has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more A sequence-identical amino acid sequence, wherein the amino acid sequence contains one or more substitutions relative to the reference sequence.

In some embodiments, the recombinant Factor VIII polypeptide or biologically active fragment thereof comprises a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072 or corresponding to The reference sequence of SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072 has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity of amino acids Sequence, wherein the amino acid sequence comprises the reference sequence relative to residues 20-1457 corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072 or relative to residues 20-1457 corresponding to SEQ ID NO. : One or more substitutions of 2, 252, 504, 706, 840, 1200, 1514 or 2072 of the reference sequence.

In some embodiments, the recombinant Factor VIII polypeptide, or biologically active fragment thereof, comprises a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2 or is at least 70% identical to a reference sequence corresponding to SEQ ID NO: 2. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , an amino acid sequence with 96%, 97%, 98% or 99% or more sequence identity, wherein the amino acid sequence comprises the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2 Or one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, a recombinant Factor VIII polypeptide or biologically active fragment thereof comprises a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, 504, 706, 840, 1200, 1514 or 2072 or corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514 or 2072. The reference sequence of ID NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072 has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, an amino acid sequence with 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the recombinant Factor VIII polypeptide or biologically active fragment thereof comprises a reference sequence corresponding to residues 20-1457 of even-numbered SEQ ID NO. in SEQ ID NO. 4-2346 and 2360-2870 or corresponding to The reference sequences of the even-numbered SEQ ID NO. in SEQ ID NO. 4-2346 and 2350-2870 have at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity of amino acids Sequence, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution at the following amino acid positions: 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 37, 56, 57, 58, 59, 61, 63, 64, 65, 85, 96, 104, 110, 119, 127, 132, 158, 160, 164, 167, 168, 171, 175, 178, 186, 187, 205, 210, 211, 212, 215, 225, 246, 257, 260, 265, 281, 292, 294, 301, 303, 304, 306, 308, 310, 311, 312, 318, 320, 321, 325, 328, 332, 334, 335, 344, 346, 347, 349, 351, 353, 354, 355, 356, 358, 359, 362, 370, 387, 388, 390, 392, 393, 397, 407, 428, 429, 433, 435, 436, 481, 487, 488, 489, 491, 498, 499, 503, 514, 527, 533, 534, 535, 536, 538, 543, 550, 551, 555, 558, 569, 580, 581, 584, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 643, 646, 650, 665, 667, 676, 680, 681, 682, 683, 684, 686, 693, 696, 698, 700, 706, 707, 708, 712, 716, 724, 726, 729, 731, 732, 734, 735, 736, 737, 738, 739, 740, 744, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 759, 770, 773, 775, 785, 786, 787, 790, 791, 792, 793, 794, 795, 797, 798, 799, 800, 801, 802, 805, 806, 807, 808, 809, 810, 811, 812, 815, 816, 817, 818, 819, 821, 822, 824, 825, 826, 828, 830, 831, 836, 837, 838, 839, 840, 841, 844, 866, 873, 879, 892, 896, 897, 903, 907, 914, 916, 917, 918, 919, 921, 922, 923, 924, 926, 929, 938, 939, 945, 948, 951, 968, 999, 1037, 1040, 1041, 1042, 1043, 1049, 1050, 1051, 1053, 1054, 1076, 1078, 1080, 1084, 1088, 1089, 1092, 1093, 1094, 1095, 1098, 1099, 1100, 1101, 1104, 1108, 1109, 1113, 1114, 1116, 1127, 1129, 1138, 1145, 1148, 1151, 1156, 1158, 1160, 118 6. 1192, 1193, 1194, 1205, 1210, 1218, 1220, 1221, 1222, 1224, 1229, 1245, 1248, 1250, 1259, 1264, 1269, 1270, 1274, 1281, 1283, 1292, 1297, 1339, 135 2. 1373, 1377, 1378, 1380, 1389, 1414, 1422, 1427, 1428, 1439, 1440, 1442 or 1452 or a combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2 .

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or amino acid residue 13G/Y, 14G/M/V, 15G/S, 16A/D/G/P/V, 17S /V, 18L/R, 19G, 20L, 21M, 24V, 37H, 56E/P, 57A/L/S/T/V, 58K, 59D/G/L/M/Q/R/S/V/W , 61Q/S, 63C/E/F/G/H/I/K/L/M/N/Q/R/S/T/W/Y, 64D/G, 65A, 85Q, 96I/L/R /V/W, 104S, 110H, 119G/V, 127V, 132P/R, 158G, 160E/T, 164E, 167G/H/R, 168G/R, 171A/D/E/F/H/K/N /M/P/Q/R/S/T/V/W/Y, 175A/G/H, 178C/M/Q/V/W, 186A, 187C/G, 205R, 210P, 211G/K/P /Q/R, 212A/F/G/K/L/M/N/P/R/S/V/Y, 215E/P/S, 225R, 246D, 257I, 260E/K/T, 265R/S /W, 281A/S, 292D, 294G, 301K/M, 303G/T, 304A/E/G, 306A/D/G/P/S/T, 308P, 310G/V, 311E/G, 312L, 318C /G/K/M/Q/R/T/V, 320G/I/L/P/Q/R/S/T/V, 321Y, 325Y, 328G/Q/S, 332A/F/G/H /N/P/Q/R/T/V/W/Y, 334G, 335E/P, 344A/E/L, 346R/V, 347N, 349G, 351S, 353S, 354R, 355C, 356L/N, 358K /S, 359E/G, 362E/G/N/P/T/V/W, 370A, 387A/G/I/L/Q/R/S/T/V/W, 388A/P, 390A/E /G/L/M/P/S/T/V, 392A, 393A/F/G/H/M/Q/R/W/Y, 397G/N, 407V, 428F, 429L/S, 433A/G , 435Q, 436D/G, 481V, 487N, 488G, 489E/F/Q/R, 491E, 498A, 499C/L, 503S, 514L, 527A, 533R/V/Y, 534F/I/L/M, 535C /G/N, 536A/G/K/L/M/P/R/W, 538V, 543G/L/P/W, 550G, 551W, 555E/G/R/T, 558A/E/G/L /P/Q/R/S/T/V/W, 569T, 580S/Y, 581K/V, 584A, 610H/K/L/R/S/T, 611D/E, 612G/S/T, 613S , 614A/F/H/I/N/T, 615D/N, 616D/E/G/Q/W, 617D/E/G/N, 618C/E/G/P/Y, 619E, 620N/P , 621K/M/R/T/W, 622D, 643N, 646I, 650M, 665G, 667I, 676Q, 680L/R, 681A/C/E/F/G/L/N/R/Q/S/T /W, 682R, 683W, 684G/K, 686W, 693T, 696E, 698D/S/W/Y, 700A/E/Q/T/V, 706A/G/M/Y, 707H, 708A/K/T /V, 712G/L/M/Q/S/T/Y, 716K/L/W, 724I, 726A/L, 729T, 731I/L/R, 732C/D/E/G/L/R/W /K/M/Q/S/T/V/Y, 734I/R/S, 735D/S/V, 736F/G, 737F/G/K/L/M/N/S/V, 738A/D /E/F/H/K/N/P/R/S/T, 739G/P/R, 740G, 744A/R, 746P, 747D, 748F/G/K/N/S/V, 749A/C /D/F/G/I/K/L/M/P/Q/R/S/T/V/Y、750A/D/G/K/M/N/P/Q/R/S/T /V/W, 751A/D/E/Y, 752D/E/F/G/L/M/Q/R/S/V, 753D, 754W, 755G/L/P/Q/S/Y, 756G /P/R/T, 757A/G/K/T, 759S, 770W, 773H, 775E/G/K/N/P/S/V, 785V, 786C/D/L, 787W, 790A/G/I /M/P/W, 791M/T, 792H/W/Y, 793G, 794R, 795C/N/S/T, 797C/G/P/Q/T/W/Y, 798C/R/T, 799A /E/G/M/P/Q/R/S/V, 800C/G/I/L/M/N/P/Q/R/T/V/W/Y, 801A/G/I/R , 802S/V, 805C/G/H/K/L/M/P/Q/R/S/V/W, 806E/G/R/S, 807C/L/P/R, 808K/S, 809A /C/G/I/K/L/N/Q/R/S/T/V/W, 810V, 811F, 812G/I/V, 815I, 816V/W, 817A/D, 818A/E/G /P/S/V/W, 819V, 821Q, 822S/T, 824W, 825L, 826G, 828K, 830M/V, 831W, 836G/R/V/W, 837G, 838G/T, 839A/G/K /P, 840G, 841W, 844H, 866A, 873L/Q/T, 879Q, 892T, 896L/V, 897G/K/R, 903E, 907D, 914V, 916C/E/I/K/L/M/Q /T, 917G, 918M/S, 919G, 921R/W, 922W, 923M, 924I/M/P/Q/W, 926T/V, 929A/Q/V, 938R/V, 939E/S, 945K, 948S , 951V, 968A/P, 999R, 1037H/M/N/P, 1040E, 1041G/H/N, 1042A/E/T, 1043E/R, 1049G/W, 1050E/P/Q/S/T, 1051E /H/K/R/S/W, 1053A/R, 1054C, 1076G/S, 1078S, 1080G/V, 1084L/N, 1088G/S, 1089F/S, 1092G, 1093A/E/G/M/N /P/Q/R/S/T/V/W/Y, 1094S, 1095G, 1098G, 1099G, 1100I/S, 1101G, 1104C/E, 1108M/S/V/W, 1109G, 1113F, 1114A/M , 1116A/G/I/L/R, 1127L/V, 1129P, 1138Q, 1145R, 1148A/C/E/L/M/N/Q/R/S, 1151S, 1156D/G/N/R, 1158N , 1160R, 1186E, 1192D, 1193A/E/G/I/L, 1194P, 1205L, 1210M/T/V/W, 1218A/G/L/M/P, 1220D/E/L/T, 1221G/S /T/V, 1222F, 1224P, 1229D, 1245R, 1248A/C/D/E/F/G/H/I/K/M/P/Q/R/S/T/W/Y, 1250A/P /S/T, 1259T, 1264L/R/W, 1269F, 1270V, 1274N/V, 1281H/K/P/Q/R/S, 1283E/H/L/S/T, 1292Y, 1297A, 1339C, 1352E , 1373S, 1377A/C/D/E/G/H/I/M/P/R/S/V/T/Y, 1378A/C/D/E, 1380A/G/W, 1389W, 1414A/C /D/E/G/H/N/P/T/W, 1422I, 1427Y, 1428G, 1439A/E/H/L/S/T, 1440A/C/E/G/S/T, 1442T or 1452L or a combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution L13G/Y, L14G/M/V, R15G/S, F16A/D/G/P/V, C17S/V, F18L/R , S19G, A20L, T21M, Y24V, Q37H, S56E/P, F57A/L/S/T/V, P58K, F59D/G/L/M/Q/R/S/V/W, T61Q/S, V63C /E/F/G/H/I/K/L/M/N/Q/R/S/T/W/Y, V64D/G, Y65A, P85Q, Q96I/L/R/V/W, V104S , M110H, A119G/V, A127V, E132P/R, Q158G, L160E/T, G164E, A167G/H/R, S168G/R, L171A/D/E/F/H/K/N/M/P/Q /R/S/T/V/W/Y, Y175A/G/H, L178C/M/Q/V/W, D186A, L187C/G, K205R, T210P, L211G/K/P/Q/R, H212A /F/G/L/K/M/N/P/R/S/V/Y, I215E/P/S, K225R, A246D, V257I, S260E/K/T, T265R/S/W, T281A/S , G292D, T294G, R301K/M, A303G/T, S304A/E/G, E306A/D/G/P/S/T, S308P, I310G/V, T311E/G, F312L, L318C/G/K/M /Q/R/T/V, M320G/I/L/P/Q/R/S/T/V, D321Y, F325Y, F328G/Q/S, S332A/F/G/H/N/P/Q /R/T/V/W/Y, H334G, Q335E/P, K344A/E/L, D346R/V, S347N, P349G, E351S, Q353S, L354R, R355C, M356L/N, N358K/S, N359E/G , A362E/G/N/P/T/V/W, T370A, F387A/G/I/L/Q/R/S/T/V/W, I388A/P, I390A/E/G/L/M /P/S/T/V, S392A, V393A/F/G/H/M/Q/R/W/Y, H397G/N, A407V, S428F, Q429L/S, N433A/G, P435Q, Q436D/G , L481V, Q487N, A488G, S489E/F/Q/R, P491E, G498A, I499C/L, R503S, V514L, I527A, T533R/V/Y, V534I/F/L/M, T535C/G/N, V536A /G/K/L/M/P/R/W, D538V, S543G/L/P/W, R550G, Y551W, F555E/G/R/T, M558A/E/G/L/P/Q/R /S/T/V/W, P569T, Q580S/Y, R581K/V, Q584A, I610H/K/L/R/S/T, Q611D/E, R612G/S/T, F613S, L614A/F/H /I/N/T, P615D/N, N616D/E/G/Q/W, P617D/E/G/N, A618C/E/G/P/Y, G619E, V620N/P, Q621K/M/R /T/W, L622D, S643N, L646I, L650M, T665G, F667I, T676Q, K680L/R, M681A/C/E/F/G/L/N/R/Q/S/T/W, V682R, Y683W , E684G/K, T686W, S693T, T696E, F698D/S/W/Y, S700A/E/Q/T/V, L706A/G/M/Y, W707H, I708A/K/T/V, H712G/L /M/Q/S/T/Y, F716K/L/W, L724I, K726A/L, S729T, D731I/L/R, K732C/D/E/G/L/R/W/K/M/Q /S/T/V/Y, T734I/R/S, G735D/S/V, D736F/G, Y737F/G/K/L/M/N/S/V, Y738A/D/E/F/H /K/N/P/R/S/T, E739G/P/R, D740G, D744A/R, A746P, A747D, Y748F/G/K/N/S/V, L749A/C/D/F/G /I/K/L/M/P/Q/R/S/T/V/Y, L750A/D/G/K/M/N/P/Q/R/S/T/V/W, A751A /D/E/Y, K752D/E/F/G/L/M/Q/R/S/V, N753D, N754W, A755G/L/P/Q/S/Y, I756G/P/R/T , E757A/G/K/T, R759S, R770W, R773H, I775E/G/K/N/P/S/V, E785V, E786C/D/L, I787W, D790A/G/I/M/P/W , D791M/T, T792H/W/Y, I793G, S794R, Y795C/N/S/T, M797C/G/P/Q/T/W/Y, K798C/R/T, K799A/E/G/M /P/Q/R/S/V, E800C/G/I/L/M/N/P/Q/R/T/V/W/Y, D801A/G/I/R, F802S/V, Y805C /G/H/K/L/M/P/Q/R/S/V/W, D806E/G/R/S, E807C/L/P/R, D808K/S, E809A/C/G/I /K/L/N/Q/R/S/T/V/W, N810V, Q811F, S812G/I/V, S815I, F816V/W, Q817A/D, K818A/E/G/P/S/V /W, K819V, R821Q, H822S/T, F824W, I825L, A826G, V828K, R830M/V, L831W, M836G/R/V/W, S837G, S838G/T, S839A/G/K/P, P840G, H841W , R844H, G866A, Y873L/Q/T, E879Q, V892T, I896L/V, M897G/K/R, Q903E, P907D, L914V, S916C/E/I/K/L/M/Q/T, Y917G, E918M /S, E919G, Q921R/W, R922W, Q923M, G924I/M/P/Q/W, E926T/V, K929A/Q/V, K938R/V, T939E/S, Q945K, M948S, T951V, L968A/P , Q999R, F1037H/M/N/P, N1040E, Y1041G/H/N, R1042A/E/T, F1043E/R, Y1049G/W, I1050E/P/Q/S/T, M1051E/H/K/R /S/W, T1053A/R, L1054C, E1076G/S, I1078S, S1080G/V, S1084L/N, F1088G/S, T1089F/S, K1092G, K1093A/E/G/M/N/P/Q/R /S/T/V/W/Y, E1094S, E1095G, M1098G, A1099G, L1100I/S, Y1101G, Y1104C/E, F1108M/S/V/W, E1109G, M1113F, L1114A/M, S1116A/G/I /L/R, I1127L/V, E1129P, L1138Q, K1145R, T1148A/C/E/L/M/N/Q/R/S, G1151S, H1156D/G/N/R, R1158N, F1160R, A1186E, P1192D , F1193A/E/G/I/L, S1194P, I1205L, K1210M/T/V/W, F1218A/G/L/M/P, S1220D/E/L/T, L1221G/S/T/V, Y1222F , S1224P, M1229D, T1245R, L1248A/C/D/E/F/G/H/I/K/M/P/Q/R/S/T/W/Y, V1250A/P/S/T, G1259T , I1264L/R/W, I1269F, I1270V, I1274N/V, Y1281H/K/P/Q/R/S, I1283E/H/L/S/T, M1292Y, N1297A, G1339C, K1352E, V1373S, L1377A/C /D/E/G/H/I/M/P/R/S/V/T/Y, T1378A/C/D/E, M1380A/G/W, S1389W, S1414A/C/D/E/G /H/N/P/T/W, L1422I, L1427Y, T1428G, V1439A/E/H/L/S/T, H1440A/C/E/G/S/T, I1442T or E1452L, or a combination thereof, where the Amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution at the following amino acid positions: 63, 171, 212, 260, 306, 317, 320, 332, 387, 390, 393, 534, 558, 681, 732, 738, 749, 750, 775, 1148, 1248, 1377 or 1439 or a combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide comprises at least one substitution or amino acid residue 63C/E/F/G/H/I/K/L/M/N/Q/R/S /T/W/Y, 171A/D/E/F/H/K/N/M/P/Q/R/S/T/V/W/Y, 212A/F/G/L/K/M /N/P/R/S/V/Y, 260E/K/T, 306A/D/G/P/S/T, 318C/G/K/L/M/Q/R/T/V, 320G /I/L/P/Q/R/S/T/V, 332A/F/G/H/N/P/Q/R/T/V/W/Y, 387A/G/I/L/Q /R/S/T/V/W, 390A/E/G/L/M/P/S/T/V, 393A/F/G/H/M/Q/R/W/Y, 499C/L , 534I/F/L/M, 558A/E/G/L/P/Q/R/S/T/V/W, 681A/C/E/F/G/L/N/R/Q/S /T/W, 700A/E/Q/T/V, 732C/D/E/G/L/R/W/K/M/Q/S/T/V/Y, 738A/D/E/F /H/K/N/P/R/S/T/Y, 749A/C/D/F/G/I/K/L/M/P/Q/R/S/T/V/Y, 750A /D/G/K/M/N/P/Q/R/S/T/V/W, 752D/E/F/G/L/M/Q/R/S/V, 775E/G/K /N/P/S/V, 800C/G/I/L/M/N/P/Q/R/T/V/W/Y, 805C/G/H/K/L/M/P/Q /R/S/V/W, 809A/C/G/I/K/L/N/Q/R/S/T/V/W, 1093A/E/G/M/N/P/Q/R /S/T/V/W/Y, 1148A/C/E/L/M/N/Q/R/S, 1248A/C/D/E/F/G/H/I/K/M/P /Q/R/S/T/W/Y, 1377A/C/D/E/G/H/I/M/P/R/S/V/T/Y, 1414A/C/D/E/G /H/N/P/T/W or 1439A/E/H/L/S/T or a combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide comprises at least one substitution or amino acid residue 63Q, 171P, 212A, 260T, 306G, 317G, 320R, 332P, 387I, 390A, 393G, 534I, 558W , 681G, 732W, 7338E, 749F, 750D, 775G, 1148N, 1248D, 1377D or 1439E or a combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution V63Q, L171P, H212A, S260T, E306G, L317G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681G, K732W, Y738E , L749F, L750D, I775G, T1148N, L1248D, L1377D or V1439E or a combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution at position 1377. In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution 1377D.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or set of substitutions at position 1439 or 1377/1439. In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or set of substitutions 1439E or 1377D/1439E.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide comprises at least one substitution or set of substitutions at positions 171, 260, 387, 750, 1248, or 171/260/387/750/1248. In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or set of substitutions 171P, 260T, 387I, 750D, 1248D, or 171P/260T/387I/750D/1248D.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide comprises at least one substitution or set of substitutions at positions 306, 332, 390, 749, or 306/332/390/749. In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or set of substitutions 306G, 332P, 390A, 749F, or 306G/332P/390A/749F.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide comprises at least one substitution or set of substitutions at positions 63, 393, 732, 775, 1148, or 63/393/732/775/1148. In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or set of substitutions 63Q, 393G, 732W, 775G, 1148N, or 63Q/393G/732W/775G/1148N.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or set of substitutions at positions 212, 320, 534, 558, 681, or 212/320/534/558/681. In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or set of substitutions 212A, 320R, 534I, 558W, 681G, or 212A/320R/534I/558W/681G.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide comprises at least one substitution or set of substitutions at positions 317, 738, or 317/738. In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide comprises at least one substitution or set of substitutions 317G, 738E, or 317G/738E.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or set of substitutions at the following amino acid positions: 1148, 85/708/1148, 798, 706, 171, 335, 63, 344 ,428,879,59,429,205,1264,797,1248,873,792,708,57,96,362,1292,346,433,65,795,684/798,353,332,1259,356 ,436,1245,1145,716,56,773,734,358,729,665,359,61,1250,726,37/96/171/429/708/799/1148,171/429/732/1148 ,96/171, 171/919/1151, 171/792/797/799/1148, 171/569/1148, 96/171/429/792/797/799, 171/429/708/1422, 171/429 ,321/362/732/797/799, 171/1100, 708/1148/1186, 429/724/732/1148, 171/362/795/797/799, 171/429/732, 96/171/643 /797/799, 171/792/797/799, 503, 171/732, 1148/1193, 96/171/362/429/732/795/797, 334, 732, 351, 1377, 999, 306, 349 , 257, 646, 435 or 58, wherein these positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or substitution set 1148N, 85Q/708T/1148N, 798T, 706G, 706A, 171Q, 335E, 63R, 344A, 428F, 879Q, 59L, 429S, 205R, 1264W, 797P, 1248S, 1264R, 873T, 792Y, 708K, 344L, 57V, 96L, 362W, 63G, 873L, 1292Y, 1148Q, 346V, 797G, 706Y, 1248R, 433A, 65A, 59W, 87 3Q. 795C、684K/798R、57L、353S、332Q、708A、1259T、63M、356L、1248G、1248P、436G、57A、59S、1245R、59R、96W、797Y、1145R、436D、716L、56E、773H、734I、 358S, 335P, 59Q, 729T, 716K, 665G, 362T, 57T, 359G, 61S, 1250A, 362V, 359E, 716W, 332W, 708V, 63H, 797C, 61Q, 356N, 1250S, 798C, 433G, 795N, 96I, 56P, 63N, 792W, 1264L, 1250P, 332P, 59V, 726L, 37H/96R/171P/429L/708T/799R/1148N, 171P, 171P/429L/732R/1148N, 96R/171P, 171P/919G/1151S , 171P/792Y/797Q/799R/1148N, 171P/569T/1148N, 96R/171P/429L/792Y/797T/799R, 171P/429L/708T/1422I, 171P/429L, 321Y/362P/732E/797T/7 99R、 171P/1100I, 708T/1148N/1186E, 429L/724I/732E/1148N, 171P/362E/795T/797Q/799M, 171P/429L/732E, 96R/171P/643N/797T/799R, 171P/792Y/ 797T/ 799M, 503S, 171P/732E, 1148N/1193L, 96R/171P/362E/429L/732R/795T/797Q, 726A, 59M, 734R, 362N, 334G, 732G, 63Q, 351S, 1377D, 795S, 358K, 999 R. 1250T, 306G, 349G, 1377G, 332F, 1377E, 257I, 646I, 346R, 435Q, 96V, 1377R, 1377S, 59G, 171M, 792H, 1248D, 306S, 706M, 171H, 1377A, 1377V, 734S, 11 48R, 57S, 59D, 344E, 362G or 58K, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or substitution set T1148N, P85Q/I708T/T1148N, K798T, L706G, L706A, L171Q, Q335E, V63R, K344A, S428F, E879Q, F59L, Q429S, K205R, I1264W, M797P, L1248S, I1264R, Y873T, T792Y, I708K, K344L, F57V, Q96L, A362W, V63G, Y873L, M1292Y, T1148Q, D346V, M797G, L706Y, L1 248R, N433A, Y65A, F59W, Y873Q, V795C, E684K/K798R, F57L, Q353S, S332Q, I708A, G1259T, V63M, M356L, L1248G, L1248P, Q436G, F57A, F59S, T1245R, F59R, Q96W, M797Y, K1145R, Q436D , F716L, S56E, R773H, T734I, N358S, Q335P, F59Q, S729T, F716K, T665G, A362T, F57T, N359G, T61S, V1250A, A362V, N359E, F716W, S332W, I708V, V63H, M797C, T61Q, M356N, V1250S, K 798C, N433G, V795N, Q96I, S56P, V63N, T792W, I1264L, V1250P, S332P, F59V, K726L, Q37H/Q96R/L171P/Q429L/I708T/K799R/T1148N, L171P, L171P/Q429L/K732R/T1148N, Q96R/ L171P, L171P/E919G/G1151S, L171P/T792Y/M797Q/K799R/T1148N, L171P/P569T/T1148N, Q96R/L171P/Q429L/T792Y/M797T/K799R, L171P/Q429L/I708T/L1422I, L171P/Q429L, D3 21Y/A362P/K732E/M797T/K799R、 L171P/L1100I, I708T/T1148N/A1186E, Q429L/L724I/K732E/T1148N, L171P/A362E/V795T/M797Q/K799M, L171P/Q429L/K732E, Q96R/L171P/S643N/M 797T/K799R, L171P/T792Y/M797T/ K799M, R503S, L171P/K732E, T1148N/F1193L, Q96R/L171P/A362E/Q429L/K732R/V795T/M797Q, K726A, F59M, T734R, A362N, H334G, K732G, V63Q, E351S , L1377D, V795S, N358K, Q999R, V1250T, E306G, P349G, L1377G, S332F, L1377E, V257I, L646I, D346R, P435Q, Q96V, L1377R, L1377S, F59G, L171M, T792H, L1248D, E306S, L706M, L171H, L1377A, L1377V, T734S, T1148R, F57S, F59D, K344E, A362G or P58K, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide comprises at least one substitution or set of substitutions at the following amino acid positions: 1377/1380, 388/1377, 1043/1377, 610/1377, 1274/1377 ,1220/1377,1377/1442,1193/1377,621/1377,1054/1377,755/1377,1041/1377,748/1377,1049/1377,1221/1377,1051/1377,612/1377,1037 /1377, 616/1377, 387/1377, 489/1377, 750/1377, 1224/1377, 618/1377, 1050/1377, 1053/1377, 896/1377, 897/1377, 749/1377, 260/1377 ,481/1377, 613/1377, 756/1377, 63/1377, 265/1377, 752/1377, 1218/1377, 1377/1378, 614/1377, 1378/1377, 1440/1377, 1377/1440, 1377 /1439、1205/1377、1222/1377、1377/1389、1269/1377、390/1377、397/1218/1377、757/1377、1373/1377、346/1377、63/1248/1377、349/1377 ,306/355/1377/1422, 1377/1422, 306/346/708/1377, 1148/1377, 171/346/1248/1377, 63/797/799/1248/1377/1422, 171/799/1377 ,362/1377, 1248/1377, 171/306/346/999/1248/1377, 171/1148/1377, 734/799/1248/1377/1422, 63/732/1377, 63/171/306/346 /732/1377、171/1377、171/1377/1422、63/362/732/799/1248/1377、999/1148/1377/1422、346/349/1377、306/349/1377、63/171 /1148/1377/1151、306/1377、344/349/354/1250/1377、1250/1377、63/306/344/349/354/503/1248/1377、59/63/332/349/503 /706/708/1377, 63/306/344/349/354/706/1248/1377, 59/63/1377, 332/706/708/1248/1250/1377, 503/1377, 59/63/349 /354/1377、59/349/708/1377、59/306/344/1248/1377、344/1248/1250/1377、708/1377、63/344/349/1377、59/1377、63/344 /349/354/1377, 706/1377, 59/306/770/1377, 63/347/354/1248/1377 or 354/503/1377, wherein the amino acid positions are relative to those corresponding to SEQ ID NO. : 2 of the reference sequence.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or substitution set 1377D/1380W, 388A/1377D, 1043E/1377D, 1043R/1377D, 610R/1377D, 1274N/1377D, 1220T/1377D, 1377D/1442T, 1193L/1377D, 621T/1377D, 1054C/1377D, 755Q/1377D, 1041G/1377D, 748S/1377D, 1049G/1377D, 1221V/1377D, 1051E/1377D, 612G/1 377D、1037H/1377D、616E/ 1377D, 1041H/1377D, 387S/1377D, 489R/1377D, 621M/1377D, 489Q/1377D, 750G/1377D, 1224P/1377D, 618C/1377D, 616G/1377D, 612T/1377D, 1050Q /1377D、1053A/1377D、 616W/1377D, 896V/1377D, 489F/1377D, 1050P/1377D, 897G/1377D, 749T/1377D, 1274V/1377D, 1220L/1377D, 260E/1377D, 481V/1377D, 613S/1377D ,387G/1377D,621R/ 1377D, 756R/1377D, 63R/1377D, 265S/1377D, 1221T/1377D, 1377D/1380G, 752G/1377D, 748K/1377D, 755L/1377D, 1218A/1377D, 748N/1377D, 1377D /1378E、614I/1377D、 749F/1377D, 387V/1377D, 750T/1377D, 1193G/1377D, 749V/1377D, 1378A/1377D, 750V/1377D, 387T/1377D, 387Q/1377D, 750A/1377D, 1037N/1377D ,1377D/1440A,1051S/ 1377D, 1221G/1377D, 1377D/1378D, 1377D/1440S, 387R/1377D, 1193A/1377D, 387I/1377D, 1218M/1377D, 265W/1377D, 1377D/1440E, 1377D/1439L, 1377D/1440T, 612S/1377D, 1049W/1377D, 1221S/1377D, 1220D/1377D, 749G/1377D, 749S/1377D, 1218G/1377D, 1205L/1377D, 1222F/1377D, 1193E/1377D, 1050S/1377D, 1377D /1389W、1377D/1439E、260T/ 74 8F/1377D, 390A/1377D, 13 73S/1377D, 1377D/1440G, 1193I/1377D, 1377D/1439T, 750D/1377D, 390G/1377D, 756G/1377D, 1220E/1377D, 1377D/1378C, 618G/1377D, 346R/1377D, 63Q/1248D/1377D, 349G /1377D、306G/355C/ 1377D/1422I, 1377D/1422I, 306G/346R/708T/1377D, 1148N/1377D, 171P/346R/1248D/1377D, 63Q/797T/799R/1248D/1377D/1422I, 171P/799R/13 77D、362P/1377D、 1248D/1377D, 63Q/1377D, 171P/306G/346R/999R/1248D/1377D, 171P/1148N/1377D, 734S/799R/1248D/1377D/1422I, 63Q/732E/1377D, 63Q/171P/ 306G/346R/ 732G/1377D, 171P/1377D, 171P/1377D/1422I, 63Q/362P/732G/799R/1248D/1377D, 999R/1148N/1377D/1422I, 346R/349G/1377D, 306G/349G/1377 D. 63Q/171P/ 1148N/1377D/1151S, 306G/1377D, 344E/349G/354R/1250T/1377D, 63G/1377D, 1250T/1377D, 63R/306G/344E/349G/354R/503S/1248R/1377D, 59D/ 63S/332P/ 349G/503S/706M/708K/1377D, 63S/306G/344A/349G/354R/706M/1248R/1377D, 1248R/1377D, 59D/63G/1377D, 332P/706M/708K/1248R/1250T/137 7D、503S/ 1377D, 59D/63R/349G/354R/1377D, 59D/349G/708K/1377D, 59D/306G/344E/1248R/1377D, 344E/1248R/1250T/1377D, 708K/1377D, 63R/344E/349G /1377D、 59D/1377D, 63S/344A/349G/354R/1377D, 706M/1377D, 59D/306G/770W/1377D, 63G/347N/354R/1248R/1377D or 354R/503S/1377D, wherein the amino acid position is Relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or set of substitutions at the following amino acid positions: 750/1377/1439, 1051/1377/1439, 260/1377/1439, 756 /1377/1439、1193/1377/1439、332/1377/1439、897/1377/1439、387/1377/1439、1037/1377/1439、306/749/1377/1439、1270/1377/1439、489 /1377/1439, 487/1377/1439, 488/1377/1439, 903/1377/1439, 171/1248/ 1377/1439/1440, 260/1148/1377/1378/1439, 171/260/387/750 /1248/1377/1439, 171/260/1377/1439, 171/260/387/750/1051/1148/1377/1439, 171/1377/1439, 750/1051/1148/1377/1439, 171/260 /756/1377/1439、171/1248/1377/1439、171/1148/1377/1439、59/1377/1439、1377/1439/1440、332/750/1248/1377/1439、756/1248/1377 /1439、1248/1377/1439、171/387/1248/1377/1440/1439、750/1377/1439/1440、59/171/750/1377/1439、171/1193/1248/1377/1439、750 /1148/1193/1377/1439/1440、1148/1377/1439、171/387/1051/1377/1439、748/1377/1439、306/1377/1439、306/387/390/749/1220/1377 /1439、390/1377/1439、306/390/1037/1377/1439、306/612/749/1377/1439、387/390/749/1377/1439、63/390/503/749/1377/1439 ,1220/1377/1439, 63/306/1377/1439, 749/1377/1439, 306/390/749/1037/1377/1439, 306/749/1220/1377/1439, 306/387/390/612 /749/1037/1377/1439 or 328/1377/1439, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or substitution set 750D/1377D/1439E, 1051K/1377D/1439E, 1051S/1377D/1439E, 260T/1377D/1439E, 756P/1377D/ 1439E, 1193A/1377D/1439E, 332P/1377D/1439E, 897R/1377D/1439E, 387Q/1377D/1439E, 1037N/1377D/1439E, 387R/1377D/1439E, 306G/749F/137 7D/1439E, 1270V/1377D/ 1439E, 489E/1377D/1439E, 487N/1377D/1439E, 488G/1377D/1439E, 903E/1377D/1439E, 171P/1248D/ 1377D/1439E/1440T, 260T/1148N/1377D/13 78D/1439E、171P/260T/ 387I/750D/1248D/1377D/1439E, 171P/260T/1377D/1439E, 171P/260T/387I/750D/1051S/1148N/1377D/1439E, 171P/1377D/1439E, 750D/1051S/1 148N/1377D/1439E、 171P/260T/756P/1377D/1439E, 171P/1248D/1377D/1439E, 171P/1148N/1377D/1439E, 387I/1377D/1439E, 59D/1377D/1439E, 1377D/1439E/1440S ,332P/750D/1248D/ 1377D/1439E, 756P/1248D/1377D/1439E, 1248D/1377D/1439E, 171P/387I/1248D/1377D/1440S/1439E, 750D/1377D/1439E/1440S, 59D/171P/750D /1377D/1439E、171P/ 3 06G/1377D/1439E, 306G/ 387R/390A/749F/1220D/1377D/1439E, 390A/1377D/1439E, 306G/390A/1037N/1377D/1439E, 306G/612S/749F/1377D/1439E, 387V/390A/749F/137 7D/1439E, 63S/ 390A/503S/749F/1377D/1439E, 387R/390A/749R/1377D/1439E, 1220D/1377D/1439E, 63S/306G/1377D/1439E, 749F/1377D/1439E, 306G/390A/749 F/1037N/1377D/ 1439E, 387V/1377D/1439E, 306G/749F/1220D/1377D/1439E, 306G/387V/390A/612S/749F/1037N/1377D/1439E or 328S/1377D/1439E, wherein the amino acid positions are relative to This reference sequence corresponds to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or set of substitutions at the following amino acid positions: 171/260/387/750/1051/1248/1377/1439, 171/260 /387/750/1221/1248/1377/1439, 171/260/387/750/1218/1248/1377/1439, 171/260/387/750/1248/1377/1439, 171/260/387/749 /750/1218/1248/1377/1439, 171/260/332/387/750/1248/1377/1439, 171/260/387/750/897/1248/1377/1439, 171/260/387/696 /750/1248/1377/1439, 171/260/387/750/816/1248/1377/1439, 171/260/387/750/824/1248/1377/1439, 171/260/387/750/826 /1248/1377/1439、171/260/387/393/750/1248/1377/1439、171/260/387/750/825/1248/1377/1439、171/260/387/698/750/1248 /1377/1439、171/260/332/387/390/750/1148/1248/1377/1439、171/260/328/387/749/750/1248/1377/1439、171/260/387/749 /750/1248/1377/1439, 171/260/306/328/332/387/390/750/1051/1193/1218/1248/1377/1439, 63/171/260/306/332/387/750 /1051/1148/1248/1377/1439, 63/171/260/306/332/387/750/1051/1248/1377/1439, 171/260/387/390/750/1051/1248/1377/1439 ,171/186/260/306/328/332/387/390/749/750/1051/1148/1193/1248/1377/1439, 171/260/387/390/750/756/1051/1248/1377 /1439、171/260/306/387/390//750/1051/1248/1377/1439、171/260/306/332/387/390/749/750/1248/1377/1439、171/260/ 306/387/390/750/1148/1248/1377/1439, 171/260/306/328/332/387/390/749/750/1051/1148/1193/1248/1377/1439, 171/260/ 332/387/390/749/750/1148/1248/1377/1439, 171/260/387/390/749/750/1248/1377/1439, 171/260/306/387/749/750/1051/ 1148/1248/1377/1439, 171/260/306/387/750/945/1051/1193/1221/1248/1377/1439, 171/260/306/387/390/749/750/1051/1248/ 1377/1439, 63/171/260/306/332/387/390/750/1248/1377/1439, 171/260/306/387/390/749/750/1148/1193/1221/1248/1377/ 1439、171/260/306/332/387//390/749/750/1218/1248/1377/1439、63/171/260/306/332/387/390/750/1051/1148/1193/1218 /1248/1377/1439、171/260/387/750/924/1248/1377/1439、171/260/387/750/755/1248/1377/1439、171/260/387/750/752/1248 /1377/1439、171/260/387/750/751/1248/1377/1439、171/260/387/750/923/1248/1377/1439、171/260/387/750/797/1248/1377 /1439、171/260/387/750/754/1248/1377/1439、171/260/387/750/793/1248/1377/1439、171/260/387/750/753/1248/1377/1439 ,171/260/387/750/922/1248/1377/1439, 171/260/387/750/896/1248/1377/1439, 171/260/387/750/938/1248/1377/1439, 171 /260/387/750/775/1248/1377/1439, 171/260/387/750/732/1248/1377/1439, 171/260/387/750/810/1248/1377/1439, 171/260 /387/750/747/1248/1377/1439, 171/260/387/393/407/750/1248/1377/1439, 171/260/387/750/819/1248/1377/1439, 171/260 /387/750/787/1248/1377/1439, 171/260/387/750/802/1248/1377/1439, 171/260/387/732/750/1248/1377/1439, 171/260/387 /750/811/1248/1377/1439, 171/260/387/392/750/1248/1377/1439 or 171/260/387/397/750/1248/1377/1439, where the amino acid positions Relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or set of substitutions 171P/260T/387I/750D/1051K/1248D/1377D/1439E, 171P/260T/387I/750D/1221G/1248D/ 1377D/1439E, 171P/260T/387I/750D/1218G/1248D/1377D/1439E, 171P/260T/387R/750D/1248D/1377D/1439E, 171P/260T/387I/750D/1051R/124 8D/1377D/1439E、 171P/260T/387I/749F/750D/1218G/1248D/1377D/1439E, 171P/260T/387T/750D/1248D/1377D/1439E, 171P/260T/332P/387I/750D/1248D/1377D /1439E、171P/ 260T/387I/750D/897K/1248D/1377D/1439E, 171P/260T/387I/696E/750D/1248D/1377D/1439E, 171P/260T/387I/750D/816W/1248D/1377D/1439E ,171P/260T/ 387I/750D/824W/1248D/1377D/1439E, 171P/260T/387I/750D/826G/1248D/1377D/1439E, 171P/260T/387I/393W/750D/1248D/1377D/1439E, 171P /260T/387I/ 171P/260T /387I/698W/ 171 P/260T/328S/ 387R/749F/750D/1248D/1377D/1439E, 171P/260T/387I/749F/750D/1248D/1377D/1439E, 171P/260T/306G/328S/332P/387I/390A/750D/1051R/1 193A/1218G/ 1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/750D/1051R/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387T/750D/1051K/1 248D/1377D/ 1439E、171P/260T/387T/390A/750D/1051R/1248D/1377D/1439E、171P/186A/260T/306G/328S/332P/387I/390A/749F/750D/1051K/1148N/1193A/ 1248D/1377D/ 1439E, 171P/260T/387T/390A/750D/756P/1051R/1248D/1377D/1439E, 171P/260T/306G/387I/390A//750D/1051R/1248D/1377D/1439E, 171P/260 T/306G/332P /387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/387R/390A/750D/1148N/1248D/1377D/1439E, 171P/260T/306G/328S/332P/387T/3 90A/749F /750D/1051R/1148N/1193A/1248D/1377D/1439E, 171P/260T/332P/387R/390A/749F/750D/1148N/1248D/1377D/1439E, 171P/260T/387R/390A/749 F/750D/1248D /1377D/1439E、171P/260T/306G/387T/749F/750D/1051K/1148N/1248D/1377D/1439E、171P/260T/387R/749F/750D/1248D/1377D/1439E、171P/260 T/306G/387I /750D/945K/1051R/1193A/1221G/1248D/1377D/1439E, 171P/260T/306G/387I/390A/749F/750D/1051K/1248D/1377D/1439E, 63Q/171P/260T/306G /332P/387I /390A/750D/1248D/1377D/1439E, 171P/260T/306G/387T/390A/749F/750D/1148N/1193A/1221G/1248D/1377D/1439E, 171P/260T/306G/332P/387R /390A/749F /750D/1218M/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/750D/1051R/1148N/1193A/1218G/1248D/1377D/1439E, 171P/260T/387 I/750D/924I /1248D/1377D/1439E、171P/260T/387I/750D/755S/1248D/1377D/1439E、171P/260T/387I/750D/752F/1248D/1377D/1439E、171P/260T/387I/750D /S751Y/1248D /1377D/1439E、171P/260T/387I/750D/923M/1248D/1377D/1439E、171P/260T/387I/750D/797Y/1248D/1377D/1439E、171P/260T/387I/750D/754W/ 1248D/1377D /1439E、171P/260T/387I/750D/793G/1248D/1377D/1439E、171P/260T/387I/750D/753D/1248D/1377D/1439E、171P/260T/387I/750D/922W/1248D/ 1377D/1439E , 171P/260T/387I/750D/896L/1248D/1377D/1439E, 171P/260T/387I/750D/938R/1248D/1377D/1439E, 171P/260T/387I/750D/775G/1248D/1377D /1439E、171P /260T/387I/750D/751E/1248D/1377D/1439E、171P/260T/387I/750D/732W/1248D/1377D/1439E、171P/260T/387I/750D/752G/1248D/1377D/1439E ,171P/260T /387I/750D/810V/1248D/1377D/1439E、171P/260T/387I/750D/938V/1248D/1377D/1439E、171P/260T/387I/750D/747D/1248D/1377D/1439E、171P /260T/387I /750D/755G/1248D/1377D/1439E、171P/260T/387I/393G/407V/750D/1248D/1377D/1439E、171P/260T/387I/750D/797W/1248D/1377D/1439E、171P /260T/387I /750D/819V/1248D/1377D/1439E、171P/260T/387I/393G/750D/1248D/1377D/1439E、171P/260T/387I/750D/787W/1248D/1377D/1439E、171P/260T /387I/393R /750D/1248D/1377D/1439E、171P/260T/387I/750D/802V/1248D/1377D/1439E、171P/260T/387I/750D/775E/1248D/1377D/1439E、171P/260T/387I /750D/924P /1248D/1377D/1439E、171P/260T/387I/732G/750D/1248D/1377D/1439E、171P/260T/387I/750D/811F/1248D/1377D/1439E、171P/260T/387I/750D /802S/1248D /1377D/1439E, 171P/260T/387I/750D/752L/1248D/1377D/1439E, 171P/260T/387I/392A/750D/1248D/1377D/1439E or 171P/260T/387I/397G/750D/ 1248D/1377D /1439E, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide comprises at least one substitution or set of substitutions at the following amino acid positions: 171/260/306/332/387/390/620/749/750/1248 /1377/1439、171/260/306/332/387/390/491/749/750/1248/1377/1439、171/260/306/332/387/390/749/750/1229/1248/1377 /1439、171/260/306/332/387/390/615/749/750/1248/1377/1439、171/260/306/332/387/390/749/750/1248/1283/1377/1439 ,171/260/306/332/387/390/749/750/822/1248/1377/1439, 171/260/306/328/332/387/390/749/750/1248/1377/1439, 171 /212/260/306/332/387/390/749/750/1248/1377/1439, 171/260/306/332/387/390/749/750/818/1248/1377/1439, 171/260 /306/332/387/390/555/684/749/750/1248/1377/1439, 171/260/306/332/387/390/749/750/828/1248/1377/1439, 171/260 /306/332/387/390/550/749/750/1248/1377/1439, 171/260/306/332/387/390/749/750/830/1248/1377/1439, 171/260/306 /332/387/390/749/750/839/1248/1377/1439, 13/171/260/306/332/387/390/749/750/1248/1377/1439, 14/171/260/306 /332/387/390/749/750/1248/1377/1439, 17/171/260/306/332/387/390/749/750/1248/1377/1439, 171/260/306/332/387 /390/749/750/836/1248/1377/1439, 171/260/306/332/387/390/749/750/1210/1248/1377/1439, 171/260/306/332/387/390 /749/750/1248/1281/1377/1439, 64/171/260/306/332/387/390/749/750/1248/1377/1439, 171/260/306/332/387/390/682 /749/750/1248/1377/1439, 171/260/306/318/332/387/390/749/750/1248/1377/1439, 20/171/260/306/332/387/390/749 /750/1248/1377/1439, 21/171/260/306/332/387/390/749/750/1248/1377/1439, 171/260/306/325/332/387/390/749/750 /1248/1377/1439、171/260/306/332/387/390/533/749/750/1248/1377/1439、19/171/260/306/332/387/390/749/750/1248 /1377/1439、171/260/306/332/387/390/749/750/817/1248/1377/1439、171/260/306/332/387/390/749/750/831/1248/1377 /1439、171/225/260/306/332/387/390/749/750/1248/1377/1439、171/260/306/332/387/390/749/750/1156/1248/1377/1439 ,171/260/306/332/387/390/393/747/749/750/1248/1377/1439, 171/260/306/332/387/390/749/750/753/1248/1377/1439 ,171/260/306/332/387/390/393/732/749/750/751/1248/1377/1439, 63/171/260/306/332/387/390/749/750/797/1248 /1377/1439、171/260/306/332/387/390/622/749/750/1248/1377/1439、171/260/306/332/387/390/617/749/750/1248/1377 /1439、171/260/306/332/387/390/749/750/907/1248/1377/1439、171/260/306/332/387/390/749/750/1040/1248/1377/1439 ,171/260/306/332/387/390/611/749/750/1248/1377/1439, 171/260/306/332/387/390/488/749/750/1248/1377/1439, 171 /260/306/332/387/390/749/750/1192/1248/1377/1439, 171/260/306/332/387/390/749/750/1194/1248/1377/1439, 171/260 /306/332/387/390/619/749/750/1248/1377/1439, 16/171/260/306/332/387/390/749/750/1248/1377/1439, 171/260/306 /332/387/390/749/750/802/1248/1377/1439, 171/211/260/306/332/387/390/749/750/1248/1377/1439, 171/260/306/332 /387/390/749/836/750/1248/1377/1439, 171/260/306/332/387/390/555/749/750/1248/1377/1439, 171/260/306/332/387 /390/536/749/750/1248/1377/1439, 171/260/306/332/387/390/527/749/750/1248/1377/1439, 171/260/306/332/387/390 /749/750/841/1248/1377/1439, 171/260/306/320/332/387/390/749/750/1248/1377/1439, 171/260/306/332/387/390/749 /750/837/1248/1377/1439, 171/260/306/332/387/390/558/749/750/1248/1377/1439, 171/260/306/332/387/390/749/750 /914/1248/1377/1439, 171/260/306/312/332/387/390/749/750/1248/1377/1439, 171/260/306/332/387/390/681/749/750 /1248/1377/1439, 171/260/306/332/387/390/749/750/838/1248/1377/1439, 15/171/260/306/332/387/390/749/750/1248 /1377/1439、171/260/306/332/387/390/749/750/1248/1377/1427/1439、18/171/260/306/332/387/390/749/750/1248/1377 /1439、171/260/306/332/387/390/683/749/750/1248/1377/1439、24/171/260/306/332/387/390/749/750/1248/1377/1439 ,171/260/306/332/387/390/534/749/750/1248/1377/1439, 171/260/306/332/387/390/749/750/840/1248/1377/1439, 171 /260/306/332/387/390/749/750/1160/1248/1377/1439, 171/260/306/332/387/390/551/749/750/1248/1377/1439, 171/260 /306/332/387/390/393/749/750/1248/1377/1439, 171/260/306/332/387/390/393/747/749/750/752/811/819/923/1148 1248/1377/1439, 171/260/306/332/387/390/393/749/750/751/775/1248/1377/1439, 63/171/260/306/332/387/390/393/ 732/749/750/775/1148/1248/1377/1439, 171/260/306/332/387/390/393/732/749/750/1148/1248/1377/1439, 171/260/306/ 332/387/390/749/750/775/1148/1248/1377/1439, 171/260/306/332/387/390/393/749/750/752/753/775/793/797/1248/ 1377/1439, 171/260/306/332/387/390/393/749/750/923/1248/1377/1439, 171/260/306/332/387/390/393/749/750/923/ 1148/1248/1377/1439, 171/260/306/332/387/390/393/747/749/750/751/1248/1377/1439, 171/260/306/332/387/390/393/ 747/749/750/753/775/1148/1248/1377/1439, 171/260/306/332/387/390/749/750/752/923/1051/1248/1377/1439, 63/171/ 260/306/332/387/390/749/750/775/1248/1377/1439, 63/171/260/306/332/387/390/393/749/750/752/775/793/1051/ 1053 1248/1377/1439, 171/260/306/332/387/390/749/750/752/753/775/793/819/1248/1377/1439, 63/171/260/306/332/387 /390/749/750/1051/1248/1377/1439, 63/171/260/306/332/387/390/747/749/750/752/753/775/811/819/923/1051 1248/ 1377/1439, 171/260/306/332/387/390/393/749/750/752/787/1248/1377/1439, 171/260/306/332/387/390/749/750/751/ 1248/1377/1439, 63/171/260/306/332/387/390/749/750/1148/1248/1377/1439, 63/171/260/306/332/387/390/747/749/ 750/752/753/819/923/1148 1248/1377/1439, 171/260/306/332/387/390/749/750/1148/1248/1377/1439, 171/260/306/332/387 /390/747/749/750/752/753/793/811/1248/1377/1439, 63/171/260/306/332/387/390/749/750/1248/1377/1439, 171/260 /306/332/387/390/749/750/797/1148/1248/1377/1439, 171/260/306/332/387/390/749/750/751/752/753/1051/1248/1377 /1439、63/171/260/306/332/387/390/732/749/750/1051/1248/1377/1439、171/260/306/332/387/390/749/750/752/797 /815/923/1051/1148 1248/1377/1439 or 171/260/306/332/387/390/747/749/750/753/1248/1377/1439, where the amino acid positions are relative to This reference sequence corresponds to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or substitution set 171P/260T/306G/332P/387I/390A/620N/749F/750D/1248D/1377D/1439E, 171P/260T/ 171P/260 T/306G/ 332P/387I/390A/615N/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/1248D/1283S/1377D/1439E, 171P/260T/3 06G/332P/ 387I/390A/749F/750D/822S/1248D/1377D/1439E, 171P/260T/306G/328G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/212L/260T/30 6G/332P/ 387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/818W/1248D/1377D/1439E, 171P/260T/306G/332P/38 7I/390A/ 749F/750D/1248D/1283L/1377D/1439E, 171P/260T/306G/332P/387I/390A/555G/684G/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/3 87I/390A/ 749F/750D/828K/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/550G/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/39 0A/749F/ 750D/830M/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/839A/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/74 9F/750D/ 822T/1248D/1377D/1439E, 13Y/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 14G/171P/260T/306G/332P/387I/390A/749F /750D/ 1248D/1377D/1439E, 17V/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/836 V/1248D/ 1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/839P/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/1210W/12 48D/1377D/ 1439E、171P/260T/306G/332P/387I/390A/749F/750D/1248D/1281R/1377D/1439E、64D/171P/260T/306G/332P/387I/390A/749F/750D/1248D/137 7D/1439E、 171P/212G/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/682R/749F/750D/1248D/1377D/143 9E、14M/ 171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/318C/332P/387I/390A/749F/750D/1248D/1377D/1439E, 17 S/171P/ 260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 20L/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260 T/306G/ 332P/387I/390A/749F/750D/818G/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/836G/1248D/1377D/1439E, 21M/171P/260 T/306G/ 332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/836R/1248D/1377D/1439E, 171P/260T/306G/32 5Y/332P/ 387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/533V/749F/750D/1248D/1377D/1439E, 19G/171P/260T/306G/332 P/387I/ 390A/749F/750D/1248D/1377D/1439E, 14V/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390 A/749F/ 750D/817D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/831W/1248D/1377D/1439E, 171P/225R/260T/306G/332P/387I/39 0A/749F/ 750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/1156R/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/7 50D/830V/ 1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/393G/A747D/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/7 50D/753D/ 1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/S751Y/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/39 0A/749F/ 750D/797W/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/622D/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/61 7E/749F/ 750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/617N/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/75 0D/907D/ 1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/1040E/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/617D/749F/7 50D/1248D/ 1377D/1439E, 171P/260T/306G/332P/387I/390A/611E/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/620P/749F/750D/124 8D/1377D/ 1439E、171P/260T/306G/332P/387I/390A/617G/749F/750D/1248D/1377D/1439E、171P/260T/306G/332P/387I/390A/488G/749F/750D/1248D/137 7D/1439E、 171P/260T/306G/332P/387I/390A/749F/750D/1192D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/1194P/1248D/1377D/1 439E、171P/ 260T/306G/332P/387I/390A/619E/749F/750D/1248D/1377D/1439E、171P/260T/306G/332P/387I/390A/615D/749F/750D/1248D/1377D/1439E、16 A/171P/ 260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/802S/1248D/1377D/1439E, 171P/26 0T/306G/ 332P/387I/390A/749F/750D/818V/1248D/1377D/1439E, 171P/L211K/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/3 06G/332P/ 387I/390A/749F/836W/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/555R/749F/750D/1248D/1377D/1439E, 171P/260T/306G/33 2P/387I/ 390A/536A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/527A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/38 7I/390A/ 555E/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/841W/1248D/1377D/1439E, 171P/260T/306G/320Q/332P/38 7I/390A/ 749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/837G/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/55 8R/749F/ 750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/555G/749F/750D/1248D/1377D/1439E, 171P/211R/260T/306G/332P/387I/390A/74 9F/750D/ 1248D/1377D/1439E, 64G/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/320R/332P/387I/390A/749F/750 D/1248D/ 1377D/1439E, 171P/260T/306G/332P/387I/390A/533Y/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/914V/124 8D/1377D/ 1439E、16V/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E、171P/260T/306G/332P/387I/390A/749F/750D/1248D/1283E/137 7D/1439E、 171P/260T/306G/332P/387I/390A/536M/749F/750D/1248D/1377D/1439E, 171P/260T/306G/312L/332P/387I/390A/749F/750D/1248D/1377D/143 9E、171P/ 17 1P/260T/ 306G/332P/387I/390A/536R/749F/750D/1248D/1377D/1439E, 15G/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260 T/306G/ 332P/387I/390A/536P/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/558W/749F/750D/1248D/1377D/1439E, 171P/260T/30 6G/332P/ 387I/390A/558E/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1427Y/1439E, 171P/260T/306G/3 32P/387I/ 390A/749F/750D/839G/1248D/1377D/1439E, 18L/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 13G/171P/260T/306G/332P /387I/ 390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/1210M/1248D/1377D/1439E, 171P/260T/306G/332P/387I/3 90A/749F/ 750D/1156G/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/558L/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/7 49F/750D/ 839K/1248D/1377D/1439E, 171P/211P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/211G/260T/306G/332P/387I/390A/74 9F/750D/ 1248D/1377D/1439E, 171P/211Q/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 16D/171P/260T/306G/332P/387I/390A/749F/750 D/1248D/ 1377D/1439E, 171P/260T/306G/M320G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/683W/749F/750D/12 48D/1377D/ 1439E、24V/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E、171P/260T/306G/332P/387I/390A/534I/749F/750D/1248D/1377 D/1439E、 171P/260T/306G/332P/387I/390A/681A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/840G/1248D/1377D/143 9E、171P/ 1 71P/260T/ 306G/328Q/332P/387I/390A/749F/750D/1248D/1377D/1439E, 15S/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260 T/306G/ 332P/387I/390A/536K/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/536G/749F/750D/1248D/1377D/1439E, 171P/260T/30 6G/332P/ 387I/390A/533R/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/681E/749F/750D/1248D/1377D/1439E, 171P/260T/306G/33 2P/387I/ 390A/536W/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/1156N/1248D/1377D/1439E, 16P/171P/260T/306G/33 2P/387I/ 390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/817A/1248D/1377D/1439E, 18R/171P/260T/306G/332P/387 I/390A/ 749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/551W/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/55 5T/749F/ 750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/681G/749F/750D/1248D/1377D/1439E, 171P/212A/260T/306G/332P/387I/390A/74 9F/750D/ 1248D/1377D/1439E, 16G/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/838 G/1248D/ 1377D/1439E, 171P/260T/306G/332P/387I/390A/681R/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/393G/749F/750D/124 8D/1377D/ 1439E、171P/260T/306G/332P/387I/390A/393G/747D/749F/750D/752G/811F/819V/923M/1148N/1248D/1377D/1439E、171P/260T/306G/332P/387 I/390A/ 393G/749F/750D/751Y/775G/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/1148N/1248D/1377D/143 9E、171P/ 260T/306G/332P/387I/390A/393G/732W/749F/750D/1148N/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/775G/1148N/124 8D/1377D/ 1439E, 171P/260T/306G/332P/387I/390A/393G/749F/750D/752L/753D/775G/793G/797W/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A /393G/ 749F/750D/923M/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/393G/749F/750D/923M/1148N/1248D/1377D/1439E, 171P/260T/306G/3 32P/387I/ 390A/393G/747D/749F/750D/751Y/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/393G/747D/749F/750D/753D/775G/1148N/1248D/137 7D/1439E、 171P/260T/306G/332P/387I/390A/749F/750D/752L/923M/1051R/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/749F/750D/775G/ 1248D/ 1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/749F/750D/752L/775G/793G/1051R/1053R 1248D/1377D/1439E, 171P/260T/306G/33 2P/387I/390A /749F/750D/752G/753D/775G/793G/819V/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/749F/750D/1051R/1248D/1377D/1439E, 63 Q/171P /260T/306G/332P/387I/390A/747D/749F/750D/752G/753D/775G/811F/819V/923M/1051R 1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A /393G/ 749F/750D/752G/787W/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/751Y/1248D/1377D/1439E, 63Q/171P/260T/306G/332 P/387I/ 390A/749F/750D/1148N/1248D/1377D/1439E, 63H/171P/260T/306G/332P/387I/390A/747D/749F/750D/752G/753D/819V/923M/1148N/1248D/137 7D/1439E、 171P/260T/306G/332P/387I/390A/749F/750D/1148N/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/747D/749F/750D/752L/753D/793G /811F/ 1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/797 W/1148N/ 1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/751Y/752L/753D/1051R/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/38 7I/390A/ 732W/749F/750D/1051R/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/752G/797W/815I/923M/1051R/1148N 1248D/1377D/ 1439E or 171P/260T /306G/332P/387I/390A/747D/749F/750D/753D/1248D/1377D/1439E, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide comprises at least one substitution or set of substitutions at the following amino acid positions: 63/171/260/306/332/387/390/393/681/732 /749/750/775/1148/1248/1377/1439, 63/171/260/306/332/387/390/393/622/732/749/750/775/1148/1248/1377/1439, 63 /171/260/306/332/387/390/393/617/732/749/750/775/1148/1248/1377/1439、63/171/260/306/332/387/390/393/558 /732/749/750/775/1148/1248/1377/1439, 63/171/260/306/332/387/390/393/536/732/749/750/775/1148/1248/1377/1439 , 63/171/211/260/306/332/387/390/393/732/749/750/775/1148/1248/1377/1439, 63/171/260/306/320/332/387/390 /393/732/749/750/775/1148/1248/1377/1439, 63/171/260/306/332/387/390/393/732/749/750/775/839/1148/1248/1377 /1439、63/171/260/306/332/387/390/393/732/749/750/775/797/1148/1248/1377/1439、63/171/260/306/332/387/390 /393/533/732/749/750/775/1148/1248/1377/1439, 63/171/260/306/332/387/390/393/732/749/750/751/775/1148/1248 /1377/1439、63/171/260/303/306/332/387/390/393/732/749/750/775/1148/1248/1377/1439、63/171/260/306/332/387 /390/393/732/749/750/951/775/1148/1248/1377/1439, 63/164/171/260/306/332/387/390/393/732/749/750/775/1148 /1248/1377/1439, 63/171/260/306/332/387/390/393/732/749/750/775/818/1148/1248/1377/1439, 63/171/260/306/332 /387/390/393/732/749/750/775/807/1148/1248/1377/1439, 63/171/260/306/332/387/390/393/732/749/750/775/799 /1148/1248/1377/1439, 63/171/260/306/332/387/390/393/732/735/749/750/775/1148/1248/1377/1439, 63/171/260/306 /332/387/390/393/732/749/750/775/794/1148/1248/1377/1439, 63/171/260/306/332/387/390/393/732/749/750/775 /926/1148/1248/1377/1439, 63/171/260/306/332/387/390/393/732/749/750/755/775/1148/1248/1377/1439, 63/171/260 /306/332/387/390/393/732/749/750/775/921/1148/1248/1377/1439, 63/171/260/306/332/387/390/393/732/749/750 /775/801/1148/1248/1377/1439, 63/171/260/306/332/387/390/393/732/749/750/775/1094/1148/1248/1377/1439, 63/171 /260/306/332/387/390/393/732/749/750/775/968/1148/1248/1377/1439, 63/171/260/306/332/387/390/393/732/749 /750/775/929/1148/1248/1377/1439, 63/171/260/306/332/387/390/393/732/749/750/775/924/1148/1248/1377/1439, 63 /171/260/306/332/387/390/393/698/732/749/750/775/1148/1248/1377/1439、63/168/171/260/306/332/387/390/393 /732/749/750/775/1148/1248/1377/1439, 63/171/260/306/332/387/390/393/581/732/749/750/775/1148/1248/1377/1439 , 63/171/260/306/332/387/390/393/732/749/750/775/916/1148/1248/1377/1439, 63/171/260/306/332/387/390/393 /732/749/750/775/1148/1248/1377/1428/1439, 63/171/260/306/332/387/390/393/732/749/750/775/805/1148/1248/1377 /1439、63/171/260/306/332/387/390/393/732/749/750/775/1114/1148/1248/1377/1439、63/171/260/306/311/332/387 /390/393/732/749/750/775/1148/1248/1377/1439, 63/171/260/306/332/387/390/393/732/737/749/750/775/1148/1248 /1377/1439、63/171/260/306/332/387/390/393/693/732/749/750/775/1148/1248/1377/1439、63/171/260/306/332/387 /390/393/732/749/750/775/809/1148/1248/1377/1439, 63/171/260/306/332/387/390/393/732/749/750/757/775/1148 /1248/1377/1439, 63/171/260/306/332/387/390/393/732/749/750/775/1099/1148/1248/1377/1439, 63/171/260/306/332 /387/390/393/676/732/749/750/775/1148/1248/1377/1439, 63/171/260/306/332/387/390/393/732/740/749/750/775 /1148/1248/1377/1439, 63/171/260/306/332/387/390/393/732/698/749/750/775/1148/1248/1377/1439, 63/171/260/306 /332/387/390/393/732/749/750/775/791/1148/1248/1377/1439、63/171/211/260/306/332/387/390/393/536/622/681 /732/749/750/775/1148/1248/1377/1439, 63/171/260/306/332/387/390/393/536/558/732/749/750/775/1148/1248/1377 /1439、63/171/211/260/306/332/387/390/393/536/555/617/749/750/775/1148/1248/1377/1439、63/171/211/260/306 /332/387/390/393/681/749/750/775/1148/1248/1377/1439, 63/171/211/260/306/332/387/390/393/536/555/732/749 /750/775/1148/1248/1377/1439, 63/171/260/306/332/387/390/393/555/732/749/750/775/1148/1248/1377/1439, 63/171 /211/260/306/332/387/390/393/536/732/749/750/775/1148/1248/1377/1439、63/171/211/212/260/306/332/387/390 /393/732/749/750/775/1148/1248/1377/1439, 63/171/211/260/306/332/387/390/393/536/617/681/749/750/775/1148 /1248/1377/1439, 63/171/211/260/306/332/387/390/393/536/558/617/681/732/749/750/775/1148/1248/1377/1439, 63 /171/260/306/332/387/390/393/555/749/750/775/1148/1248/1377/1439, 63/171/260/306/332/387/390/393/536/555 /558/617/749/750/775/1148/1248/1377/1439, 63/171/260/306/332/387/390/393/536/617/681/749/750/775/1148/1248 /1377/1439、63/171/260/306/332/387/390/393/536/555/732/749/750/775/1148/1248/1377/1439、63/171/260/306/332 /387/390/393/536/622/681/732/749/750/775/1148/1248/1377/1439, 63/171/260/306/332/387/390/393/749/750/775 /1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/533/617/681/732/749/750/775/1148/1248/1377/1439 , 63/171/211/260/306/312/332/387/390/393/533/534/617/619/681/732/749/750/775/839/1148/1248/1377/1439, 63 /171/212/260/306/332/387/390/393/732/749/750/775/1148/1248/1377/1439, 63/171/211/212/260/306/320/332/387 /390/393/619/681/732/749/750/775/839/1148/1248/1377/1439, 63/171/260/306/332/387/390/393/732/749/750/775 /797/839/1148/1248/1377/1439, 63/171/212/260/306/312/320/332/387/390/393/533/534/617/681/732/749/750/775 /1148/1248/1377/1439, 63/171/260/306/332/387/390/393/558/619/681/732/749/750/775/1148/1248/1377/1439, 63/171 /260/306/320/332/387/390/393/ 558/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387 /390/393/534/619/681/732/749/750/775/797/839/1148/1248/1377/1439, 63/171/260/306/320/332/387/390/393/558 /619/749/750/775/839/1148/1248/1377/1439, 63/171/211/212/260/306/312/332/387/390/393/534/558/619/681/749 /750/775/839/1148/1248/1377/1439, 63/171/260/306/320/332/387/390/393/534/558/619/749/750/775/797/1148/1248 /1377/1439, 63/171/260/306/332/387/390/393/558/617/619/681/732/749/750/775/1148/1248/1377/1439, 63/171/211 /260/306/332/387/390/393/534/558/611/681/732/749/750/775/1148/1248/1377/1439、63/171/211/260/306/320/332 /387/390/393/617/619/681/732/749/750/775/1148/1248/1377/1439, 63/171/260/306/332/387/390/393/558/617/681 /732/749/750/775/797/1148/1248/1377/1439、63/171/260/306/320/332/387/390/393/ 534/619//749/750/775/1148/ 1248/1377/1439, 63/171/211/260/306/332/387/390/393/ 619/681/732/749/750/775/1148/1248/1377/1439, 63/171/211/ 212/260/306/332/387/390/393/534/558/619/749/750/775/839/1148/1248/1377/1439, 63/171/212/260/306/320/332/ 387/390/393/619/681/732/749/750/775/797/839/1148/1248/1377/1439、63/171/212/260/306/320/332/387/390/393/ 611/732/749/750/775/839/1148/1248/1377/1439, 63/171/260/306/320/332/387/390/393/558/619/681/732/749/750/ 775/1148/1248/1377/1439, 63/171/260/306/312/332/387/390/393/533/617/681/732/749/750/775/797/839/1148/1248/ 1377/1439, 63/171/260/306/332/387/390/393/558/611/681/732/749/750/775/1148/1248/1377/1439, 63/171/260/306/ 312/332/387/390/393/732/749/750/775/1148/1248/1377/1439, 63/171/211/260/306/320/332/387/390/393/558/619/ 681/749/750/775/797/839/1148/1248/1377/1439, 63/171/260/306/320/332/387/390/393/558/749/750/775/797/1148/ 1248/1377/1439, 63/171/260/306/332/387/390/393/533/558/611/619/716//749/750/775/839/1148/1248/1377/1439, 63 /171/260/306/312/332/387/390/393/681/732/749/750/775/1148/1248/1377/1439, 63/171/260/306/320/332/387/390 /393/534/619/681/732/749/750/775/1148/1248/1377/1439, 63/171/211/260/306/320/332/387/390/393/533/536/619 /732/749/750/775/1148/1248/1377/1439, 63/171/260/306/332/387/390/393/558/617/681/732/749/750/775/839/1148 /1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439, 63 /171/260/306/332/387/390/393/533/534/681/732/749/750/775/839/1148/1248/1377/1439、63/171/212/260/306/332 /387/390/393/534/619/681/732/749/750/775/1148/1248/1377/1439, 63/171/260/306/332/387/390/393/617/619/681 /732/749/750/775/1148/1248/1377/1439, 63/171/211/212/260/306/320/332/387/390/393/558/617/681/732/749/750 /775/1148/1248/1377/1439, 63/171/212/260/306/332/387/390/393/534/558/681/732/749/750/775/797/1148/1248/1377 /1439、63/171/212/260/306/320/332/387/390/393/534/732/749/750/775/1148/1248/1377/1439、63/171/211/260/306 /312/332/387/390/393/619/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/312/332/387/390/393/619 /732/749/750/775/839/1148/1248/1377/1439, 63/171/210/212/260/306/320/332/387/390/393/558/681/732/749/750 /775/1148/1248/1377/1439, 63/171/212/260/306/312/332/387/390/393/534/681/732/749/750/775/797/839/1148/1248 /1377/1439, 63/171/211/260/306/320/332/387/390/393/558/732/749/750/775/839/1148/1248/1377/1439, 63/171/260 /294/306/332/387/390/393/732/749/750/775/1148/1248/1377/1439, 63/171/260/306/332/387/390/393/732/749/750 /759/775/1148/1248/1377/1439, 63/171/260/306/332/387/390/393/538/732/749/750/775/1148/1248/1377/1439 or 63/171 /260/306/332/387/390/393/732/749/750/775/1109/1148/1248/1377/1439, wherein the amino acid positions are relative to those corresponding to SEQ ID NO: 2 Reference sequence.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or substitution set 63Q/171P/260T/306G/332P/387I/390A/393G/681A/732W/749F/750D/775G/1148N/ 1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/622D/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306 G/332P/ 387I/390A/393G/617G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/558L/732W/749F/750D/ 775G/ 1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/536K/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/26 0T/306G/ 332P/387I/390A/393G/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/211Q/260T/306G/332P/387I/390A/393G/732W/749F/ 750D/ 775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/320R/332P/387I/390A/393G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/17 1P/260T/ 306G/332P/387I/390A/393G/558W/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/ 750D/ 775G/839K/1148N/1248D/1377D/1439E, 63Q/171P/211K/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 6 3Q/171P/ 260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/797W/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/533R/ 732W/ 749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/751A/775G/1148N/1248D/1377D/14 39E, 63Q/ 171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/751T/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/ 732W/ 749F/750D/951V/775G/1148N/1248D/1377D/1439E, 63Q/164E/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/1148N/1248D/137 7D/1439E、 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/818G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/3 93G/ 732W/749F/750D/775G/807L/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/799A/1148N/1248 D/1377D/ 1439E、63Q/171P/260T/306G/332P/387I/390A/393G/732W/735S/749F/750D/775G/1148N/1248D/1377D/1439E、63Q/171P/260T/306G/332P/387I/ 390A/ 393G/732W/749F/750D/775G/794R/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/926V/1148N /1248D/ 1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/755P/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P /387I/ 390A/393G/732W/749F/750D/775G/921W/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/801A/ 1148N/ 1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/1094S/1148N/1248D/1377D/1439E, 63Q/171P/260T/30 6G/332P/ 387I/390A/393G/732W/749F/750D/775G/968A/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/ 929Q/ 1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/924W/1148N/1248D/1377D/1439E, 63Q/171P/26 0T/306G/ 332P/387I/390A/393G/732W/749F/750D/775G/807P/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/698D/732W/749F/ 750D/ 775G/1148N/1248D/1377D/1439E, 63Q/168G/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/17 1P/260T/ 306G/332P/387I/390A/393G/732W/749F/750D/775G/929V/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/ 750D/ 775G/818A/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/818S/1148N/1248D/1377D/1439E, 6 3Q/171P/ 260T/306G/332P/387I/390A/393G/581K/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/ 749F/ 750D/775G/818P/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/916K/1148N/1248D/1377D/14 39E, 63Q/ 171P/260T/306G/332P/387I/390A/393G/698S/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/751G/306G/332P/387I/390A/ 393G/ 732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/916C/1148N/1248D/137 7D/1439E、 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/1148N/1248D/1377D/1428G/1439E, 63Q/171P/260T/306G/332P/387I/390A/ 393G/ 732W/749F/750D/775G/805L/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/924M/1148N/1248 D/1377D/ 1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/1114M/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/311G/332P /387I/ 390A/393G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/737N/749F/750D/775G/1148N /1248D/ 1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/805V/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P /387I/ 390A/393G/732W/749F/750D/775G/801I/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/751D/775G/ 1148N/ 1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/693T/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306 G/332P/ 387I/390A/393G/732W/749F/750D/775G/818E/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/ 929A/ 1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/311E/332P/387I/390A/393G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/26 0T/306G/ 332P/387I/390A/393G/732W/737L/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/ 775G/ 809W/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/757A/775G/1148N/1248D/1377D/1439E, 63Q/17 1P/260T/ 306G/332P/387I/390A/393G/732W/749F/750D/775G/1099G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F /750D/ 775G/799G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/916I/1148N/1248D/1377D/1439E, 6 3Q/171P/ 260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/805G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/ 737F/ 749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/737M/749F/750D/775G/1148N/1248D/1377D/14 39E, 63Q/ 171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/799P/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/ 732W/ 749F/750D/775G/807C/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/916E/1148N/1248D/137 7D/1439E、 63Q/171P/260T/306G/332P/387I/390A/393G/676Q/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/3 93G/ 581V/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/968P/1148N/1248 D/1377D/ 1439E、63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/755Y/775G/1148N/1248D/1377D/1439E、63Q/171P/260T/306G/332P/387I/ 390A/ 393G/732W/749F/750D/775G/916Q/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/735D/749F/750D/775G/1148N /1248D/ 1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/740G/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P /387I/ 390A/393G/732W/698Y/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/757G/775G/ 1148N/ 1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/791T/1148N/1248D/1377D/1439E, 63Q/171P/260T/306 G/332P/ 387I/390A/393G/732W/749F/750D/775G/1114A/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/735V/749F/750D /775G/ 1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/30 6G/332P/ 387I/390A/393G/536P/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/211R/260T/306G/332P/387I/390A/393G/536K/622D/681A/ 732W/ 749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/536K/558L/732W/749F/750D/775G/1148N/1248D/137 7D/1439E、 63Q/171P/211R/260T/306G/332P/387I/390A/393G/536R/555G/617G/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/211R/260T/306G/3 32P/ 387I/390A/393G/681R/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/211R/260T/306G/332P/387I/390A/393G/536K/555E/732W/749F/ 750D/ 775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/536R/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/17 1P/260T/ 306G/332P/387I/390A/393G/555E/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/211R/260T/306G/332P/387I/390A/393G/536P/ 732W/ 749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/211R/212P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/1148N/1248D/137 7D/1439E、 63Q/171P/211R/260T/306G/332P/387I/390A/393G/536K/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/211R/260T/306G/332P/3 87I/ 390A/393G/536K/617G/681A/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/211R/260T/306G/332P/387I/390A/393G/732W/749F/750D/ 775G/ 1148N/1248D/1377D/1439E, 63Q/171P/211R/260T/306G/332P/387I/390A/393G/536P/558L/617G/681A/732W/749F/750D/775G/1148N/1248D/137 7D/1439E、 63Q/171P/260T/306G/332P/387I/390A/393G/555G/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/5 36P/ 555E/558L/617G/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/536K/617G/681A/749F/750D/775G/ 1148N/ 1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/536K/555G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260 T/306G/ 332P/387I/390A/393G/536P/622D/681A/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/749F/ 750D/ 775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320Q/332P/387I/390A/393G/533R/617E/681G/732W/749F/750D/775G/1148N/1248 D/1377D/ 1439E、63Q/171P/211K/260T/306G/312L/332P/387I/390A/393G/533R/534I/617D/619E/681N/732W/749F/750D/775G/839K/1148N/1248D/1377D/ 1439E、 63Q/171P/212A/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/211K/212A/260T/306G/320Q/3 32P/ 387I/390A/393G/619E/681G/732W/749F/750D/775G/839K/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/ 750D/ 775G/797W/839K/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/312L/320Q/332P/387I/390A/393G/533R/534I/617E/681G/732W/749F/ 750D/ 775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/558W/619E/681G/732W/749F/750D/775G/1148N/1248D/1377D/14 39E, 63Q/ 171P/260T/306G/320Q/332P/387I/390A/393G/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320Q/ 332P/ 387I/390A/393G/534I/619E/681G/732W/749F/750D/775G/797W/839K/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/320R/332P/387I/390A/ 393G/ 558W/619E/749F/750D/775G/839K/1148N/1248D/1377D/1439E, 63Q/171P/211K/212A/260T/306G/312L/332P/387I/390A/393G/534I/558W/619E/ 681N/ 749F/750D/775G/839K/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/320R/332P/387I/390A/393G/534I/558R/619E/749F/750D/775G/797W/ 1148N/ 63 Q/171P/ 211Q/260T/306G/332P/387I/390A/393G/534I/558W/611E/681N/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/211K/260T/306G/ 320R/ 332P/387I/390A/393G/617E/619E/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/558W/ 617D/ 681G/732W/749F/750D/775G/797W/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/320R/332P/387I/390A/393G/534I/619E/749F/750D/775G/ 1148N/ 1248D/1377D/1439E, 63Q/171P/211K/260T/306G/332P/387I/390A/393G/619E/681N/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171 P/211K/ 212A/260T/306G/332P/387I/390A/393G/534I/558W/619E/749F/750D/775G/839K/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/ 332P/ 387I/390A/393G/619E/681N/732W/749F/750D/775G/797W/839K/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/ 393G/ 611E/732W/749F/750D/775G/839K/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/320Q/332P/387I/390A/393G/558W/619E/681N/732W/749F/ 750D/ 775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/312L/332P/387I/390A/393G/533R/617D/681N/732W/749F/750D/775G/797W/839K/1148N /1248D/ 1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/558W/611E/681N/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T /306G/ 312L/332P/387I/390A/393G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/211Q/260T/306G/320R/332P/387I/390A/393G/558W/ 619E/ 681G/749F/750D/775G/797W/839K/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/320Q/332P/387I/390A/393G/558W/749F/750D/775G/797W/ 1148N/ 1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/533R/558W/611E/619E/716L/749F/750D/775G/839K/1148N/1248D/1377D/143 9E, 63Q/ 171P/260T/306G/312L/332P/387I/390A/393G/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/320Q/332P/387I/ 390A/ 393G/534I/619E/681N/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/211K/260T/306G/320Q/332P/387I/390A/393G/533R/536L/ 619E/ 732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/558W/617E/681N/732W/749F/750D/775G/839K/ 1148N/ 1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/143 9E, 63Q/ 171P/260T/306G/332P/387I/390A/393G/533R/534I/681N/732W/749F/750D/775G/839K/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/ 332P/ 387I/390A/393G/534I/619E/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/617E/619E/ 681G/ 732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/211Q/212A/260T/306G/320R/332P/387I/390A/393G/558W/617E/681N/732W/749F/ 750D/ 775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/332P/387I/390A/393G/534I/558W/681N/732W/749F/750D/775G/797W/1148N/1248 D/1377D/ 1439E、63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/732W/749F/750D/775G/1148N/1248D/1377D/1439E、63Q/171P/211K/260T/ 306G/ 312L/332P/387I/390A/393G/619E/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/312L/332P/387I/390A/393G/ 619E/ 732W/749F/750D/775G/839K/1148N/1248D/1377D/1439E, 63Q/171P/210P/212A/260T/306G/320R/332P/387I/390A/393G/558W/681G/732W/749F/ 750D/ 775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/312L/332P/387I/390A/393G/534I/681N/732W/749F/750D/775G/797W/839K/1148N /1248D/ 1377D/1439E, 63Q/171P/211K/260T/306G/320R/332P/387I/390A/393G/558W/732W/749F/750D/775G/839K/1148N/1248D/1377D/1439E, 63Q/171P /260T/ 306G/332P/387I/390A/393G/732W/749F/750D/775G/924Q/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/ 750D/ 775G/916L/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/757T/775G/1148N/1248D/1377D/1439E, 6 3Q/171P/ 260T/306G/332P/387I/390A/393G/732W/737K/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/ 749F/ 750D/775G/818V/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/801G/1148N/1248D/1377D/14 39E, 63Q/ 171P/260T/294G/306G/332P/387I/390A/393G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/ 732W/ 749F/750D/775G/799S/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/799E/1148N/1248D/137 7D/1439E、 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/809G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/3 93G/ 732W/749F/750D/775G/791M/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/737V/749F/750D/775G/1148N/1248 D/1377D/ 1439E、63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/805Q/1148N/1248D/1377D/1439E、63Q/171P/260T/306G/332P/387I/ 390A/ 393G/732W/749F/750D/775G/799Q/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/737S/749F/750D/775G/1148N /1248D/ 1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/916M/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P /387I/ 390A/393G/732W/749F/750D/759S/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/799V/ 1148N/ 1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/538V/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306 G/332P/ 387I/390A/393G/732W/749F/750D/775G/1109G/1148N/1248D/1377D/1439E, 63Q/168R/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D /775G/ 1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/921R/1148N/1248D/1377D/1439E, 63Q/171P/26 0T/306G/ 332P/387I/390A/393G/732W/749F/750D/775G/926T/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/ 775G/ 916T/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/737G/749F/750D/775G/1148N/1248D/1377D/1439E or 63Q/17 1P/260T/ 306G/332P/387I/390A/393G/732W/749F/750D/775G/801R/1148N/1248D/1377D/1439E, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or set of substitutions at the following amino acid positions: 63/171/212/260/306/320/332/387/390/393 /534/558/681/732/749/750/775/1042/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681 /732/749/750/775/1041/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750 /775/1051/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248 /1283/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1281/1377/1439 , 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/916/1148/1248/1377/1439, 63/171/187 /212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439、63/171/212/215/260/306 /320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439、63/171/178/212/260/306/320/332/387 /390/393/534/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558 /681/732/749/750/775/1050/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749 /750/775/1148/1210/1248/1377/1439, 63/104/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775 /1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/615/681/732/749/750/775/1148/1248/1377 /1439、63/171/212/260/306/320/332/387/390/393/534/558/610/681/732/749/750/775/1148/1248/1377/1439、63/171 /212/260/306/320/332/387/390/393/534/536/558/681/732/749/750/775/1148/1248/1377/1439、63/171/212/260/306 /320/332/387/390/393/534/558/618/681/732/749/750/775/1148/1248/1377/1439、63/171/212/260/306/320/332/387 /390/393/534/558/621/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534 /558/614/681/732/749/750/775/1148/1248/1377/1439, 63/110/171/212/260/306/320/332/387/390/393/534/558/681 /732/749/750/775/1148/1248/1377/1439, 63/171/212/260/301/306/320/332/387/390/393/534/558/681/732/749/750 /775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1078/1148/1248 /1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/812/1148/1248/1377/1439, 63 /171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/786/1148/1248/1377/1439、63/127/171/212 /260/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439、63/171/175/212/260/306/320 /332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393 /534/558/681/732/749/750/775/1093/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681 /732/749/750/775/1088/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/650/681/732/749 /750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/535/558/681/732/749/750/775/1148 /1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/806/1148/1248/1377/1439 , 63/171/212/260/306/320/332/387/390/393/534/558/681/732/738/749/750/775/1148/1248/1377/1439, 63/171/212 /260/306/320/332/387/390/393/534/558/681/700/732/749/750/775/1148/1248/1377/1439、63/132/171/212/260/306 /320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439、63/171/212/260/306/320/332/387/390 /393/534/558/681/732/749/750/775/1076/1148/1248/1377/1439、63/171/212/260/306/320/332/387/390/393/534/558 /681/732/749/750/775/1084/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/498/534/558/681/732 /749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/756/775 /1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1108/1148/1248/1377 /1439、63/171/212/260/306/320/332/387/390/393/534/558/681/732/746/749/750/775/1148/1248/1377/1439、63/160 /171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439、63/171/212/260/306 /320/332/387/390/393/534/543/558/681/732/749/750/775/1148/1248/1377/1439、63/171/212/260/306/310/320/332 /387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534 /558/584/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732 /749/750/775/918/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775 /1148/1248/1377/1439/1452, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/790/1148/1248 /1377/1439, 63/171/212/260/292/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439, 63 /171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/800/1148/1248/1377/1439、63/167/171/212 /260/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332 /387/390/393/534/558/681/732/749/750/775/892/1148/1248/1377/1439、63/171/212/260/304/306/320/332/387/390 /393/534/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681 /732/749/750/775/1116/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750 /752/775/1148/1248/1377/1439, 63/171/212/260/281/306/320/332/387/390/393/534/558/681/732/749/750/775/1148 /1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/917/1148/1248/1377/1439 , 63/171/212/260/306/320/332/387/390/393/534/558/681/712/732/749/750/775/1148/1248/1377/1439, 63/171/212 /260/306/320/332/387/390/393/534/558/681/732/749/750/775/1080/1148/1248/1377/1439、63/171/212/260/306/320 /332/387/390/393/534/558/681/732/749/750/775/806/1148/1248/1352/1377/1439、63/171/212/260/306/320/332/387 /390/393/534/558/681/732/749/750/775/1095/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534 /558/681/732/744/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732 /749/750/775/1098/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775 /948/1148/1248/1377/1439, 63/119/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248 /1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/785/1148/1248/1377/1439, 63 /171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1113/1148/1248/1377/1439、63/171/212/260 /306/320/332/387/390/393/534/558/667/681/732/749/750/775/1148/1248/1377/1439、63/158/171/212/260/306/320 /332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393 /534/558/580/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681 /707/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/731/732/749 /750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1100/1148 /1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1089/1148/1248/1377/1439 , 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1092/1148/1248/1377/1439, 63/171/212 /260/306/320/332/387/390/393/534/558/681/732/749/750/775/939/1148/1248/1377/1439、63/171/212/260/306/320 /332/387/390/393/534/558/681/732/749/750/775/1104/1148/1248/1377/1439、63/171/212/260/306/320/332/387/390 /393/534/558/681/732/736/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558 /681/732/749/750/775/1148/1248/1297/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749 /750/775/1127/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/686/732/749/750/775 /1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1129/1248/1377 /1439、63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1138/1148/1248/1377/1439、63/171 /212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/786/821/1148/1248/1377/1439、63/171/212/260 /306/320/332/387/390/393/534/558/680/681/732/749/750/775/1148/1248/1377/1439、63/171/212/260/306/132/320 /332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393 /534/558/681/732/749/750/775/1101/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681 /732/749/750/775/808/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/739/749 /750/775/1148/1248/1377/1439, 63/171/212/260/306/308/320/332/387/390/393/534/558/681/732/749/750/775/1148 /1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/536/555/558/619/681/749/750/775/799/805/1148 /1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/555/558/619/681/732/749/750/775/801/1148/1248 /1377/1439, 63/171/212/260/306/320/332/387/390/393/534/555/558/619/681/732/749/750/775/799/1148/1248/1377 /1439, 63/171/212/260/306/320/332/387/390/393/534/558/619/681/732/749/750/775/799/801/805/1148/1248/1377 /1439, 63/171/212/260/306/320/332/387/390/393/534/536/555/558/619/681/749/750/775/799/1148/1248/1377/1439 , 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/799/805/1148/1248/1377/1439, 63/171 /212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/801/1148/1248/1377/1439、63/171/212/246/260 /306/320/332/387/390/393/534/558/619/681/732/749/750/775/799/801/1148/1248/1377/1439、63/171/212/260/306 /320/332/387/390/393/534/536/555/558/619/681/732/749/750/775/1148/1248/1377/1439、63/171/212/260/306/320 /332/387/390/393/534/558/619/681/732/749/750/775/839/1148/1248/1377/1439、63/171/212/260/306/320/332/387 /390/393/534/558/619/681/732/749/750/775/799/805/1148/1248/1377/1439、63/171/212/260/306/320/332/387/390 /393/534/536/558/681/732/749/750/775/801/805/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393 /534/555/558/619/681/732/749/750/775/799/805/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393 /534/558/619/681/732/749/750/775/801/805/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534 /555/558/619/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/536/558 /619/681/732/749/750/775/801/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732 /749/750/775/801/805/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/536/558/619/681/732 /749/750/775/799/805/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/555/558/619/681/749 /750/775/ 799/801/805/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/750/775 /1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/616/681/732/749/750/775/1148/1248/1377 /1439、63/171/212/260/306/320/332/387/390/393/534/558/611/681/732/749/750/775/1148/1248/1377/1439、63/171 /212/260/306/320/332/370/387/390/393/534/558/614/681/732/749/750/775/1148/1248/1377/1439、63/171/212/260 /306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1156/1248/1377/1439、63/171/212/260/306/320/332 /387/390/393/534/558/681/732/749/750/775/1148/1158/1248/1377/1439、63/171/212/260/306/320/332/387/390/393 /534/555/558/681/732/737/749/750/775/799/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534 /536/558/619/681/732/749/750/775/809/1109/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534 /536/555/558/619/681/732/737/749/750/775/799/809/839/916/921/1148/1248/1377/1439、63/171/212/260/306/320 /332/387/390/393/534/558/681/732/749/750/775/916/1109/1148/1248/1377/1439, 63/171/212/260/306/320/332/387 /390/393/534/536/558/619/681/732/749/750/775/799/916/1109/1148/1248/1377/1439、63/171/212/260/306/320/332 /387/390/393/534/555/558/619/681/732/749/750/775/807/1109/1148/1248/1377/1439、63/171/212/260/306/320/332 /387/390/393/534/555/558/619/681/749/750/775/799/1148/1248/1377/1439、63/171/212/260/306/320/332/387/390 /393/534/558/619/681/732/749/750/775/799/916/1109/1148/1248/1377/1439、63/171/212/260/306/320/332/387/390 /393/534/558/619/681/732/749/750/775/1148/1248/1377/1439、63/171/212/260/306/320/332/387/390/393/534/555 /558/681/732/749/750/775/921/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/619/681 /732/749/750/775/916/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/555/558/681/732/749 /750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/749/750/775/1148/1248/1377 /1439、63/171/212/260/306/320/332/387/390/393/534/558/619/681/732/749/750/775/799/1148/1248/1377/1439、63 /171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439、63/171/212/260/306 /320/332/387/390/393/534/555/558/681/732/749/750/775/916/1148/1248/1377/1439、63/171/212/260/306/320/332 /387/390/393/534/558/681/732/749/750/775/839/1148/1248/1377/1439 or 63/171/212/260/306/320/332/387/390/393 /534/558/681/732/749/750/775/799/1148/1248/1377/1439, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or substitution set 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/ 749F/750D/775G/1042E/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D /775G/ 1041N/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1051H/114 8N/1248D/ 1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1283H/1377D/143 9E, 63Q/ 171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1283T/1377D/1439E, 63Q/171P/212A /260T/ 306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1281Q/1377D/1439E, 63Q/171P/212A/260T/306G/320R /332P/ 387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1281P/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A /393G/ 534I/558W/681G/732W/749F/750D/775G/916E/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/ 681G/ 732W/749F/750D/775G/1051W/1148N/1248D/1377D/1439E, 63Q/171P/187C/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W /749F/ 750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/215E/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/ 1148N/ 1248D/1377D/1439E, 63Q/171P/212A/215P/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377 D/1439E、 63Q/171P/212A/215S/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/1 78C/ 212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/ 320R/ 332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1050T/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I /390A/ 393G/534I/558W/681G/732W/749F/750D/775G/1148N/1210T/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I /558W/ 681G/732W/749F/750D/775G/1148N/1248D/1281K/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W /749F/ 750D/775G/1042A/1148N/1248D/1377D/1439E, 63Q/104S/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D /775G/ 1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/615D/681G/732W/749F/750D/775G/1148N/1248 D/1377D/ 1439E、63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/610S/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E、63Q/ 171P/ 212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/610K/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/ 306G/ 320R/332P/387I/390A/393G/534I/558W/610T/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/ 387I/ 390A/393G/534I/536P/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/ 534I/ 558W/618Y/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/621W/ 681G/ 732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/618P/681G/732W/749F/ 750D/ 775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/614F/681G/732W/749F/750D/775G/1148N /1248D/ 1377D/1439E, 63Q/110H/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439 E. 63Q/ 171P/212A/260T/301K/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/ 260T/ 306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1078S/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R /332P/ 387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/812G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/ 393G/ 534I/558W/681G/732W/749F/750D/775G/786C/1148N/1248D/1377D/1439E, 63Q/127V/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/ 558W/ 681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/175G/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/ 749F/ 750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1093W /1148N/ 1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1088G/1148N/1248D/137 7D/1439E、 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1093T/1148N/1248D/1377D/1439E, 63Q/171P/ 212A/ 260T/306G/320R/332P/387I/390A/393G/534I/558W/650M/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/301M/ 306G/ 320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/ 390A/ 393G/534I/535N/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/ 558W/ 681G/732W/749F/750D/775G/806R/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/ 738E/ 749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/749F/750D/ 775G/ 1148N/1248D/1377D/1439E, 63Q/132R/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248 D/1377D/ 63Q /171P/ 212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1084L/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T /306G/ 320R/332P/387I/390A/393G/498A/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/ 387I/ 390A/393G/534I/558W/681G/732W/749F/750D/775G/786L/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/ 534I/ 558W/681G/732W/738F/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/535G/558W/ 681G/ 732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/ 775G/ 806E/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/756G/775G/1148N /1248D/ 1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1108S/1148N/1248D/1377D/143 9E, 63Q/ 171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1093V/1148N/1248D/1377D/1439E, 63Q/171P/212A /260T/ 306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/746P/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/160E/171P/212A/260T/306G/ 320R/ 332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/160T/171P/212A/260T/306G/320R/332P/387I/ 390A/ 393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/178V/212A/260T/306G/320R/332P/387I/390A/393G/534I/ 558W/ 681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/543L/558W/681G/732W/ 749F/ 750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/310G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/ 1148N/ 1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/700Q/732W/749F/750D/775G/1148N/1248D/1377 D/1439E、 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1093A/1148N/1248D/1377D/1439E, 63Q/171P/ 212A/ 260T/306G/320R/332P/387I/390A/393G/534I/558W/584A/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/ 320R/ 332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/918S/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/ 390A/ 393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E/1452L, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I /558W/ 681G/732W/738P/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/ 749F/ 750D/775G/790W/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/ 790I/ 1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/292D/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248 D/1377D/ 1439E、63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/800M/1148N/1248D/1377D/1439E、63Q/ 171P/ 212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1108M/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T /306G/ 320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1088S/1148N/1248D/1377D/1439E, 63Q/167G/171P/212A/260T/306G/320R /332P/ 387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/ 534I/ 558W/681G/732W/749F/750D/775G/892T/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/ 732W/ 749F/750D/775G/790P/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/ 775G/ 800R/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738K/749F/750D/775G/1148N /1248D/ 1377D/1439E, 63Q/171P/212A/260T/304G/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439 E. 63Q/ 171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1116G/1148N/1248D/1377D/1439E、63Q/171P/178Q /212A/ 260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/ 332P/ 387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1116A/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A /393G/ 534I/558W/681G/732W/749F/750D/752G/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/ 681G/ 732W/749F/750D/775G/1108W/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738D /749F/ 750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/281A/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/ 1148N/ 1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/917G/1148N/1248D/1377 D/1439E、 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/712M/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/2 12A/ 260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1080V/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G /320R/ 332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/806G/1148N/1248D/1352E/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P /387I/ 390A/393G/534I/558W/681G/732W/749F/750D/775G/1095G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G /534I/ 558W/681G/732W/749F/750D/775G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G /732W/ 744A/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/543G/558W/681G/732W/749F/ 750D/ 775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1098G/1148 N/1248D/ 1377D/1439E, 63Q/171P/212A/260T/281S/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439 E. 63Q/ 171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/756T/775G/1148N/1248D/1377D/1439E、63Q/171P/212A/ 260T/ 306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/948S/1148N/1248D/1377D/1439E, 63Q/119V/171P/212A/260T/306G/ 320R/ 332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/ 393G/ 534I/558W/681G/732W/749F/750D/775G/1116L/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W /681G/ 732W/749F/750D/775G/785V/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/ 750D/ 775G/1113F/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/700A/732W/749F/750D/775G /1148N/ 1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/786D/1148N/1248D/1377 D/1439E、 63Q/171P/175A/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/2 12A/ 260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/790M/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/ 320R/ 332P/387I/390A/393G/534I/558W/667I/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/158G/171P/212A/260T/306G/320R/332P/ 387I/ 390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/ 558W/ 580S/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/707H/ 732W/ 749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/731I/732W/749F/750D/ 775G/ 1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/918M/1148N/1248 D/1377D/ 1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1100S/1148N/1248D/1377D/1439E, 63Q /171P/ 212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1089S/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T /306G/ 320R/332P/387I/390A/393G/534I/558W/681G/712Y/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/175H/212A/260T/306G/320R/ 332P/ 387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/ 534I/ 558W/681G/732W/749F/750D/775G/1092G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G /732W/ 749F/750D/775G/939E/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/ 775G/ 1104E/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/800G/1148 N/1248D/ 1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/736F/749F/750D/775G/1148N/1248D/1377D/1439 E. 63Q/ 171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/543W/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/ 260T/ 306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1297A/1377D/1439E, 63Q/171P/212A/260T/306G/320R /332P/ 387I/390A/393G/534I/558W/681G/712L/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/ 393G/ 534I/558W/681G/732W/749F/750D/775G/1127V/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W /681G/ 732W/749F/750D/775G/939S/1148N/1248D/1377D/1439E, 63Q/171P/178M/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/ 749F/ 750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/731R/732W/749F/750D/775G/ 1148N/ 1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/686W/732W/749F/750D/775G/1148N/1248D/1377 D/1439E、 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/543P/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/2 12A/ 260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/736G/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/ 320R/ 332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1129P/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I /390A/ 393G/534I/558W/681G/732W/749F/750D/775G/1138Q/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I /558W/ 681G/732W/749F/750D/775G/812V/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/ 749F/ 750D/775G/786D/821Q/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/535C/558W/681G/732W/749F/ 750D/ 775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/680R/681G/732W/749F/750D/775G/1148N /1248D/ 1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1116I/1148N/1248D/1377D/143 9E, 63Q/ 171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1089F/1148N/1248D/1377D/1439E, 63Q/171P/212A /260T/ 304E/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/178W/212A/260T/306G/ 320R/ 332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/ 393G/ 534I/558W/681G/732W/749F/750D/775G/1104C/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W /681G/ 732W/744R/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738R/ 749F/ 750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1093S /1148N/ 1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/712S/732W/749F/750D/775G/1148N/1248D/1377 D/1439E、 63Q/171P/187G/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/2 12A/ 260T/306G/132P/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/ 320R/ 332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/812I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/ 390A/ 393G/534I/558W/681G/732W/749F/750D/775G/1080G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I /558W/ 681G/732W/749F/750D/775G/800C/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/ 749F/ 750D/775G/1101G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G /808K/ 1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/808S/1148N/1248 D/1377D/ 1439E、63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/790A/1148N/1248D/1377D/1439E、63Q/ 171P/ 212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/800V/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/ 306G/ 320R/332P/387I/390A/393G/534I/558W/680L/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/167R/171P/212A/260T/306G/320R/ 332P/ 387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/ 534I/ 558W/681G/732W/749F/750D/752E/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/ 732W/ 749F/750D/775G/1093N/1148N/1248D/1377D/1439E, 63Q/119G/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F /750D/ 775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/739G/749F/750D/775G/1148N /1248D/ 1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1116R/1148N/1248D/1377D/143 9E, 63Q/ 171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/580Y/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/ 260T/ 304A/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/ 332P/ 387I/390A/393G/534I/558W/681G/712Q/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/ 393G/ 534I/558W/681G/732W/749F/750D/775G/806S/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/ 681G/ 712G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/ 750D/ 775G/790G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1108V /1148N/ 1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/731L/732W/749F/750D/775G/1148N/1248D/1377 D/1439E、 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1084N/1148N/1248D/1377D/1439E, 63Q/171P/ 212A/ 260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1127L/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G /308P/ 320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/ 390A/ 393G/534I/558W/681G/732W/749F/750D/775G/1076S/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I /558W/ 681G/732W/749F/750D/775G/1093P/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W /749F/ 750D/775G/1093Y/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/739R/749F/750D /775G/ 1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/739P/749F/750D/775G/1148N/1248 D/1377D/ 1439E、63Q/171P/212A/260T/306G/310V/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E、63Q/ 171P/ 212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/712T/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/167H/171P/212A/ 260T/ 306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/ 387I/ 390A/393G/534I/558W/681G/732W/749F/750D/775G/1093R/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G /534I/ 536P/555E/558W/619E/681G/749F/750D/775G/799S/805Q/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/ 534I/ 555E/558W/619E/681G/732W/749F/750D/775G/801G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/ 555E/ 558W/619E/681G/732W/749F/750D/775G/799S/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/ 619E/ 681G/732W/749F/750D/775G/799S/801G/805Q/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/536P/ 555E/ 558W/619E/681G/749F/750D/775G/799S/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/ 732W/ 749F/750D/775G/799S/805Q/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/ 750D/ 775G/801G/1148N/1248D/1377D/1439E, 63Q/171P/212A/246D/260T/306G/320R/332P/387I/390A/393G/534I/558W/619E/681G/732W/749F/750D/ 775G/ 799S/801G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/536P/555E/558W/619E/681G/732W/749F/ 750D/ 775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/619E/681G/732W/749F/750D/775G/839K/ 1148N/ 1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/619E/681G/732W/749F/750D/775G/799S/805Q/1148N/ 1248D/ 1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/536P/558W/681G/732W/749F/750D/775G/801G/805Q/1148N/1248D/ 1377D/ 1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/555E/558W/619E/681G/732W/749F/750D/775G/799S/805Q/1148N/1248D/1 377D/ 1439E、63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/619E/681G/732W/749F/750D/775G/801G/805Q/1148N/1248D/1377D/ 1439E、 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/555E/558W/619E/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/1 71P/ 212A/260T/306G/320R/332P/387I/390A/393G/534I/536P/558W/619E/681G/732W/749F/750D/775G/801G/1148N/1248D/1377D/1439E, 63Q/171P/ 212A/ 260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/801G/805Q/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/ 306G/ 320R/332P/387I/390A/393G/534I/536P/558W/619E/681G/732W/749F/750D/775G/799S/805Q/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/ 306G/ 320R/332P/387I/390A/393G/534I/555E/558W/619E/681G/749F/750D/775G/799S/801G/805Q/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/ 306G/ 320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1281H/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P /387I/ 390A/393G/534I/558W/681G/732W/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/ 614H/ 681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/616D/681G/732W/ 749F/ 750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/621K/681G/732W/749F/750D/775G/ 1148N/ 1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1050E/1148N/1248D/137 7D/1439E、 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/610H/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/2 12A/ 260T/306G/320R/332P/387I/390A/393G/534I/558W/611D/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/ 320R/ 332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1042T/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I /390A/ 393G/534I/558W/616Q/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/ 558W/ 614N/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/370A/387I/390A/393G/534I/558W/614A/ 681G/ 732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/ 775G/ 1148N/1156D/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/121 0V/1248D/ 1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/614T/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439 E. 63Q/ 171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1158N/1248D/1377D/1439E, 63Q/171P/212A /260T/ 306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1281S/1377D/1439E, 63Q/171P/212A/260T/306G/320R /332P/ 387I/390A/393G/534I/558W/618E/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/ 393G/ 534I/555E/558W/681N/732W/737K/749F/750D/775G/799Q/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/ 534I/ 536P/558W/619E/681G/732W/749F/750D/775G/809G/1109G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G /534I/ 536K/555G/558W/619E/681G/732W/737K/749F/750D/775G/799Q/809G/839K/916T/921R/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/ 320R/ 332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/916T/1109G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P /387I/ 390A/393G/534I/536R/558W/619E/681N/732W/749F/750D/775G/799Q/916T/1109G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R /332P/ 387I/390A/393G/534I/555G/558W/619E/681N/732W/749F/750D/775G/807R/1109G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R /332P/ 387I/390A/393G/534I/555E/558W/619E/681N/749F/750D/775G/799Q/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/ 390A/ 393G/534I/558W/619E/681N/732W/749F/750D/775G/799S/916T/1109G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I /390A/ 393G/534I/558W/619E/681N/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/ 555G/ 558W/681G/732W/749F/750D/775G/921R/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/619E/ 681N/ 732W/749F/750D/775G/916T/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/555G/558W/619E/681N/ 732W/ 749F/750D/775G/799S/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/555E/558W/681G/732W/749F/ 750D/ 775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/749F/750D/775G/1148N/1248D/137 7D/1439E、 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/619E/681G/732W/749F/750D/775G/799S/1148N/1248D/1377D/1439E, 63Q/1 71P/ 212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/619E/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/ 306G/ 320R/332P/387I/390A/393G/534I/558W/681N/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/ 390A/ 393G/534I/555G/558W/681G/732W/749F/750D/775G/916T/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/ 534I/ 558W/681G/732W/749F/750D/775G/839K/1148N/1248D/1377D/1439E or 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/ 732W/ 749F/750D/775G/799Q/1148N/1248D/1377D/1439E, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or set of substitutions at the following amino acid positions: 63/167/171/212/260/306/318/320/332/387 /390/393/534/558/681/732/738/749/750/775/1148/1248/1377/1439, 63/167/171/212/260/306/318/320/332/387/390 /393/534/558/681/732/749/750/775/1076/1093/1148/1248/1377/1439, 63/167/171/212/260/306/318/320/332/387/390 /393/534/558/681/732/749/750/752/775/800/805/808/809/1116/1148/1248/1377/1439、63/167/171/212/260/306/318 /320/332/387/390/393/534/558/681/732/738/749/750/775/800/809/1076/1148/1248/1377/1439、63/167/171/212/260 /306/320/332/387/390/393/534/558/681/732/738/749/750/775/1148/1248/1377/1439、63/167/171/212/260/306/320 /332/387/390/393/534/558/681/732/738/749/750/775/800/805/808/809/1148/1248/1377/1439、63/167/171/212/260 /306/320/332/387/390/393/534/558/681/700/732/738/749/750/775/805/808/809/1076/1116/1148/1248/1377/1439, 63 /167/171/212/260/306/320/332/387/390/393/534/558/681/700/732/738/749/750/775/1148/1248/1377/1439, 63/167 /171/212/260/306/320/332/387/390/393/534/558/681/700/732/738/749/750/775/1076/1148/1248/1377/1439, 63/167 /171/212/260/306/320/332/387/390/393/534/558/681/700/732/739/749/750/775/1076/1148/1248/1377/1439, 63/167 /171/212/260/306/318/320/332/387/390/393/534/558/681/732/749/750/775/800/808/809/1148/1248/1377/1439, 63 /167/171/L178/212/260/306/318/320/332/387/390/393/534/558/681/700/732/738/749/750/752/775/1093/1148/1248 /1377/1439, 63/167/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/738/749/750/775/1148/1248 /1377/1439, 63/167/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/739/749/750/775/1093/1148 /1248/1377/1439, 63/167/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/749/750/752/775/1093 /1148/1248/1377/1439, 63/167/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/749/750/752/775 /805/808/809/1093/1116/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738 /749/750/775/808/809/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738 /749/750/775/1076/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/749/750 /775/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/749/750/775/808/809 /1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/749/750/775/1076/1148/1248 /1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/739/749/750/775/1148/1248/1377/1439 , 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/739/749/750/775/809/1148/1248/1377/1439, 63 /171/212/260/306/318/320/332/387/390/393/534/558/681/732/739/749/750/775/809/1093/1116/1148/1248/1377/1439 , 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/739/749/750/775/1093/1148/1248/1377/1439, 63 /171/212/260/306/318/320/332/387/390/393/534/558/681/732/739/749/750/752/775/1093/1148/1248/1377/1439, 63 /171/212/260/306/318/320/332/387/390/393/534/558/681/732/739/749/750/775/799/800/1093/1148/1248/1377/1439 , 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/739/749/750/775/1116/1148/1248/1377/1439, 63 /171/212/260/306/318/320/332/387/390/393/534/558/681/732/749/750/775/800/1148/1248/1377/1439, 63/171/212 /260/306/318/320/332/387/390/393/534/558/681/732/749/750/775/1093/1148/1248/1377/1439、63/171/212/260/306 /318/320/332/387/390/393/534/558/681/732/749/750/752/775/1148/1248/1377/1439、63/171/212/260/306/318/320 /332/387/390/393/534/558/681/732/749/750/752/775/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332 /387/390/393/534/558/681/732/749/750/752/775/799/1148/1248/1377/1439、63/171/212/260/306/318/320/332/387 /390/393/534/558/681/732/749/750/775/799/809/1093/1148/1248/1377/1439、63/171/212/260/306/318/320/332/387 /390/393/534/558/681/732/749/750/775/799/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390 /393/534/558/681/732/738/739/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393 /534/558/681/732/738/739/749/750/775/800/809/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390 /393/534/558/681/732/738/749/750/775/800/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393 /534/558/681/732/738/749/750/775/800/805/809/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390 /393/534/558/681/732/738/749/750/775/809/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393 /534/558/681/732/738/749/750/775/809/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393 /534/558/681/732/738/749/750/775/809/1093/1116/1148/1248/1377/1439、63/171/212/260/306/318/320/332/387/390 /393/534/558/681/732/738/749/750/775/809/1116/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393 /534/558/681/732/738/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681 /732/738/749/750/775/1076/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/738 /749/750/775/1076/1116/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/738/739 /749/750/775/805/808/809/1076/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/499/534/558/681 /700/732/749/750/775/809/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/738 /749/750/775/1093/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/738/749/750 /752/775/1093/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/738/749/750/775 /1116/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/543P/558/681/732/738/739/749/750/775 /1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/543/558/681/732/738/749/750/752/775/1116 /1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/543/558/681/732/738/749/750/775/1093/1148 /1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/700/732/749/750/775/1093/1148/1248/1377 /1414/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/775/1148/1248/1377/1439 , 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/212 /260/306/318/320/332/387/390/393/534/558/681/732/739/749/750/752/775/799/800/809/1116/1148/1248/1339/1377 /1439、63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/749/750/775/800/1148/1248/1377/1439、63 /171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/752/775/1093/1148/1248/1377/1439, 63 /171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/738/749/750/775/1148/1248/1377/1439, 63/171 /212/260/306/318/320/332/387/390/393/534/558/681/700/732/739/749/750/775/1093/1116/1148/1248/1377/1439, 63 /171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/738/749/750/775/809/1148/1248/1377/1439, 63 /171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/738/749/750/775/1093/1148/1248/1377/1439, 63 /171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/738/749/750/752/775/808/809/1093/1116/1148 /1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/738/749/750/752/775/1093 /1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/738/749/750/775/844 /1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/738/749/750/775/805 /809/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/775/1093 /1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/775/1093/1116 /1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/752/775/1148 /1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/752/775/1076/1148 /1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/752/775/800/809 /1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/752/775/800 /1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/752/775 /809/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/752/775 /866/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/752/775 /1116/1148/1248/1377/1439, 63/171/178/212/260/306/320/332/387/390/393/534/558/681/732/738/739/749/750/752 /775/809/1076/1116/1148/1248/1377/1439, 63/171/178/212/260/306/320/332/387/390/393/534/558/681/732/738/749 /750/775/800/809/1148/1248/1377/1439, 63/171/178/212/260/306/320/332/387/390/393/534/558/681/732/738/749 /750/775/800/808/809/1076/1148/1248/1377/1439, 63/171/178/212/260/306/318/320/332/387/390/393/534/558/681 /732/738/749/750/775/1148/1248/1377/1439, 63/171/178/212/260/306/318/320/332/387/390/393/534/558/681/732 /749/750/775/1116/1148/1248/1377/1439, 63/171/178/212/260/306/318/320/332/387/390/393/534/558/681/732/738 /749/750/752/775/809/1148/1248/1377/1439, 63/171/178/212/260/306/318/320/332/387/390/393/534/558/681/700 /732/738/749/750/775/800/805/808/809/1148/1248/1377/1439, 63/171/178/212/260/306/318/320/332/387/390/393 /534/558/681/732/738/749/750/775/805/809/866/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390 /393/534/558/681/732/738/749/750/775/1116/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393 /534/543/558/681/732/738/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534 /543/558/681/732/739/749/750/775/809/1148/1248/1377/1439、63/171/212/260/306/318/320/332/387/390/393/534 /543/558/681/732/749/750/775/809/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534 /543/558/681/732/738/739/749/750/775/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393 /534/543/558/681/732/738/749/750/775/800/1093/1116/1148/1248/1377/1439、63/171/212/260/306/318/320/332/387 /390/393/534/543/558/681/732/738/749/750/775/809/1093/1148/1248/1377/1439、63/171/212/260/306/318/320/332 /387/390/393/534/543/558/681/732/738/749/750/775/809/1093/1116/1148/1248/1377/1439、63/171/212/260/306/318 /320/332/387/390/393/534/543/558/681/732/739/749/750/752/775/809/1093/1148/1248/1377/1439、63/171/212/260 /306/318/320/332/387/390/393/534/543/558/681/732/739/749/750/752/775/1093/1148/1248/1377/1439, 63/171/212 /260/306/318/320/332/387/390/393/534/543/558/681/732/738/739/749/750/752/775/800/805/809/1093/1148/1248 /1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/543/558/681/700/732/739/749/750/775/800/1093 /1116/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/543/558/681/700/732/739/749/750 /775/805/808/809/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/543/558/681/700/732 /738/749/750/752/775/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/543/558/681/700 /732/738/749/750/752/775/809/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/543 /558/681/732/738/749/750/775/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/543 /558/681/700/732/738/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/543 /558/681/700/732/739/749/750/752/775/800/1093/1116/1148/1248/1377/1439、63/171/212/260/306/318/320/332/387 /390/393/534/543/558/681/700/732/738/749/750/775/809/1148/1248/1377/1439、63/171/212/260/306/318/320/332 /387/390/393/534/543/558/681/700/732/738/749/750/775/1093/1148/1248/1377/1439、63/171/212/260/306/318/320 /332/387/390/393/534/543/558/681/700/732/738/749/750/752/775/800/1093/1148/1248/1377/1439、63/171/212/260 /306/318/320/332/387/390/393/534/543/558/681/700/732/738/749/750/752/775/809/1116/1148/1248/1377/1439, 63 /171/212/260/306/318/320/332/387/390/393/534/543/558/681/700/732/738/749/750/752/775/1093/1148/1248/1377 /1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/739/749/750/775/800/1093/1148/1248 /1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/739/749/750/775/799/1093/1148 /1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/749/750/775/800/1093/1148 /1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/749/750/775/1116/1148/1248 /1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/738/749/750/775/809/1093/1148 /1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/738/749/750/775/1093/1116 /1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/738/749/750/752/775 /1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/738/749/750/752/775 /809/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/738/749/750 /752/775/799/1116/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/738 /749/750/752/775/805/809/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681 /700/732/738/749/750/775/799/800/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558 /681/700/732/738/749/750/775/1116/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558 /681/700/732/738/749/750/775/805/808/809/1076/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390 /393/514/534/543/558/681/732/738/749/750/775/805/808/809/1148/1248/1377/1439、63/171/212/260/306/318/320 /332/387/390/393/534/558/681/732/738/749/750/775/805/808/809/1148/1248/1377/1439 or 63/171/212/260/306/318 /320/332/387/390/393/534/558/681/732/738/749/750/775/805/809/1116/1148/1248/1377/1439, where the amino acid positions are relative To the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or substitution set 63Q/167G/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/ 681G/732W/738E/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/167G/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/ 681G/ 732W/749F/750D/775G/1076S/1093G/1148N/1248D/1377D/1439E, 63Q/167G/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558 W/681G/ 732W/749F/750D/752E/775G/800V/805Q/808S/809G/1116I/1148N/1248D/1377D/1439E, 63Q/167G/171P/212A/260T/306G/318G/320R/332P/387I /390A/ 393G/534I/558W/681G/732W/738E/749F/750D/775G/800V/809G/1076S/1148N/1248D/1377D/1439E, 63Q/167G/171P/212A/260T/306G/320R/332P /387I/ 390A/393G/534I/558W/681G/732W/738E/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/167G/171P/212A/260T/306G/320R/332P/387I/390A/ 393G/ 534I/558W/681G/732W/738E/749F/750D/775G/800V/805Q/808S/809G/1148N/1248D/1377D/1439E, 63Q/167G/171P/212A/260T/306G/320R/332P/ 387I/ 390A/393G/534I/558W/681N/732W/738E/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/167G/171P/212A/260T/306G/320R/332P/387I/390A/ 393G/ 534I/558W/681N/700E/732W/738E/749F/750D/775G/805Q/808S/809G/1076S/1116I/1148N/1248D/1377D/1439E, 63Q/167G/171P/212A/260T/306 G/320R/ 332P/387I/390A/393G/534I/558W/681G/700E/732W/738E/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/167G/171P/212A/260T/306G/320R/ 332P/ 387I/390A/393G/534I/558W/681G/700E/732W/738E/749F/750D/775G/1076S/1148N/1248D/1377D/1439E, 63Q/167G/171P/212A/260T/306G/320R /332P/ 387I/390A/393G/534I/558W/681G/700E/732W/739G/749F/750D/775G/1076S/1148N/1248D/1377D/1439E, 63Q/167G/171P/212A/260T/306G/318G /320R/ 332P/387I/390A/393G/534I/558W/681N/732W/749F/750D/775G/800V/808S/809G/1148N/1248D/1377D/1439E, 63Q/167G/171P/178Q/212A/260T/ 306G/ 318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/738E/749F/750D/752E/775G/1093G/1148N/1248D/1377D/1439E, 63Q/167G/171P /212A/ 260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/738E/749F/750D/775G/1148N/1248D/1377D/1439E、63Q/167G/171P/ 212A/ 260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/739G/749F/750D/775G/1093G/1148N/1248D/1377D/1439E, 63Q/167G /171P/ 212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/749F/750D/752E/775G/1093G/1148N/1248D/1377D/1439E, 63Q /167G/ 171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/749F/750D/752E/775G/805Q/808S/809G/1093A/1116I/1 148N/ 1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/775G/808S/809G/1 148N/ 1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/775G/1076S/1148N /1248D/ 1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439 E. 63Q/ 171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/808S/809G/1148N/1248D/1377D/1439E, 63Q/ 171P/ 212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1076S/1148N/1248D/1377D/1439E, 63Q/171P/212A /260T/ 306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/739G/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/ 318G/ 320R/332P/387I/390A/393G/534I/558W/681G/732W/739G/749F/750D/775G/809G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/ 320R/ 332P/387I/390A/393G/534I/558W/681G/732W/739G/749F/750D/775G/809G/1093G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306 G/318G/ 320R/332P/387I/390A/393G/534I/558W/681G/732W/739G/749F/750D/775G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G /320R/ 332P/387I/390A/393G/534I/558W/681G/732W/739G/749F/750D/752E/775G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G /320R/ 332P/387I/390A/393G/534I/558W/681G/732W/739G/749F/750D/775G/799S/800V/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G /318G/ 320R/332P/387I/390A/393G/534I/558W/681G/732W/739G/749F/750D/775G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G /320R/ 332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/800V/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/ 387I/ 390A/393G/534I/558W/681G/732W/749F/750D/775G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A /393G/ 534I/558W/681G/732W/749F/750D/752E/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/ 558W/ 681G/732W/749F/750D/752E/775G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W /681G/ 732W/749F/750D/752E/775G/799S/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/ 732W/ 749F/750D/775G/799S/809G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G /732W/ 749F/750D/775G/799S/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W /738E/ 739G/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/ 739G/ 749F/750D/775G/800V/809G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/ 738E/ 749F/750D/775G/800V/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/ 749F/ 750D/775G/800V/805Q/809G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/ 738E/ 749F/750D/775G/809G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/ 749F/ 750D/775G/809G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E /749F/ 750D/775G/809G/1093G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732 W/738E/ 749F/750D/775G/809G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E /749F/ 750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/775G/ 1076S/ 1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/775G/1076S/1116 I/1148N/ 1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/739G/749F/750D/775G/805Q/808S/8 09G/ 1076S/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/I499L/534I/558W/681G/700E/732W/749F/750D/775 G/809G/ 1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/775G/1093A/1148 N/1248D/ 1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/752E/775G/1093A/1148N/1248D /1377D/ 1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/775G/1116I/1148N/1248D/1377D/1439 E. 63Q/ 171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/543P/558W/681G/732W/738E/739G/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/ 171P/ 212A/260T/306G/320R/332P/387I/390A/393G/534I/543P/558W/681N/732W/738E/749F/750D/752E/775G/1116I/1148N/1248D/1377D/1439E, 63Q /171P/ 212A/260T/306G/320R/332P/387I/390A/393G/534I/543P/558W/681G/732W/738E/749F/750D/775G/1093A/1148N/1248D/1377D/1439E, 63Q/171P /212A/ 260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/749F/750D/775G/1093A/1148N/1248D/1377D/1414N/1439E, 63Q/171P/212 A/260T/ 306G/318G/320R/332P/387I/390A/393G/534I/558W/681N/732W/738E/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/ 318G/ 320R/332P/387I/390A/393G/534I/558W/681N/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/ 387I/ 390A/393G/534I/558W/681N/732W/739G/749F/750D/775G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I /390A/ 393G/534I/558W/681N/732W/739G/749F/750D/752E/775G/799S/800V/809G/1116I/1148N/1248D/1339C/1377D/1439E, 63Q/171P/212A/260T/306 G/318G/ 320R/332P/387I/390A/393G/534I/558W/681N/732W/739G/749F/750D/775G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G /320R/ 332P/387I/390A/393G/534I/558W/681N/732W/749F/750D/775G/800V/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/ 387I/ 390A/393G/534I/558W/681N/732W/738E/749F/750D/775G/800V/805Q/809G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/ 332P/ 387I/390A/393G/534I/558W/681N/732W/738E/749F/750D/775G/809G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R /332P/ 387I/390A/393G/534I/558W/681N/732W/738E/749F/750D/752E/775G/1093A/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R /332P/ 387I/390A/393G/534I/558W/681N/732W/738E/749F/750D/752E/775G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R /332P/ 387I/390A/393G/534I/558W/681N/700E/732W/738E/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/ 387I/ 390A/393G/534I/558W/681N/700E/732W/739G/749F/750D/775G/1093G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320 R/332P/ 387I/390A/393G/534I/558W/681N/700E/732W/738E/749F/750D/775G/809G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/ 332P/ 387I/390A/393G/534I/558W/681N/700E/732W/738E/749F/750D/775G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R /332P/ 387I/390A/393G/534I/558W/681N/700E/732W/738E/749F/750D/752E/775G/808S/809G/1093G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212 A/260T/ 306G/318G/320R/332P/387I/390A/393G/534I/558W/681N/700E/732W/738E/749F/750D/752E/775G/1093G/1148N/1248D/1377D/1439E, 63Q/171P /212A/ 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63Q/171P/178Q/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/775G/800V/809G/1148N/1248D/1377D/1 439E. 63Q/171P/178Q/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681N/732W/738E/749F/750D/775G/800V/808S/809G/1076S/1148N/12 48D/ 1377D/1439E, 63Q/171P/178Q/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681N/732W/738E/749F/750D/775G/1148N/1248D/ 1377D/ 1439E、63Q/171P/178Q/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681N/732W/749F/750D/775G/1116I/1148N/1248D/1377D /1439E、 63Q/171P/178Q/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/752E/775G/809G/1148N/1248D/13 77D/ 1439E, 63Q/171P/178Q/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/738E/749F/750D/775G/800V/805Q/808 S/ 809G/1148N/1248D/1377D/1439E, 63Q/171P/178Q/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/ 775G/ 805Q/809G/866A/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/ 750D/ 775G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681G/732W/738E/749F /750D/ 775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681G/732W/739G/749F/750D/ 775G/ 809G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681G/732W/749F/750D/775G/ 809G/ 1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681G/732W/738E/739G/749F /750D/ 775G/1093A/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681G/732W/738E/749F /750D/ 775G/800V/1093G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681G/732 W/738E/ 749F/750D/775G/809G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681G /732W/ 738E/749F/750D/775G/809G/1093G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543 P/558W/ 681N/732W/739G/749F/750D/752E/775G/809G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G /534I/ 543P/558W/681N/732W/739G/749F/750D/752E/775G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A /393G/ 534I/543P/558W/681N/732W/738E/739G/749F/750D/752E/775G/800V/805Q/809G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G /318G/ 320R/332P/387I/390A/393G/534I/543P/558W/681N/700E/732W/739G/749F/750D/775G/800V/1093G/1116I/1148N/1248D/1377D/1439E, 63Q/171 P/212A/ 260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681N/700E/732W/739G/749F/750D/775G/805Q/808S/809G/1148N/1248D/1377D/ 1439E、 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681N/700E/732W/738E/749F/750D/752E/775G/1148N/1248D/13 77D/ 1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681N/700E/732W/738E/749F/750D/752E/775G/809G/109 3G/ 1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681G/732W/738E/749F/750D/775G/ 1093G/ 1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681G/700E/732W/738E/749F/750D/ 775G/ 1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681G/700E/732W/739G/749F/750D/ 752E/ 775G/800V/1093G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681G/700 E/732W/ 738E/749F/750D/775G/809G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681G/ 700E/ 732W/738E/749F/750D/775G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W /681G/ 700E/732W/738E/749F/750D/752E/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/ 558W/ 681G/700E/732W/738E/749F/750D/752E/775G/800V/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A /393G/ 534I/543P/558W/681G/700E/732W/738E/749F/750D/752E/775G/809G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R /332P/ 387I/390A/393G/534I/543P/558W/681G/700E/732W/738E/749F/750D/752E/775G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G /318G/ 320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/738E/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/ 320R/ 332P/387I/390A/393G/534I/558W/681G/700E/732W/739G/749F/750D/775G/800V/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G /318G/ 320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/739G/749F/750D/775G/799S/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T /306G/ 318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/749F/750D/775G/800V/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T /306G/ 318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/749F/750D/775G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G /318G/ 320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/738E/749F/750D/775G/809G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/ 318G/ 320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/738E/749F/750D/775G/809G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T /306G/ 318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/738E/749F/750D/775G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T /306G/ 318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/738E/749F/750D/775G/1093G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212 A/260T/ 306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/738E/749F/750D/752E/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/ 260T/ 306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/738E/749F/750D/752E/775G/808S/809G/1093G/1116I/1148N/1248D/1377 D/1439E、 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/738E/749F/750D/752E/775G/809G/1093G/1148N/12 48D/ 1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/738E/749F/750D/752E/775G/799S/11 16I/ 1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/738E/749F/750D/752E/ 775G/ 805Q/809G/1093A/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/738E /749F/ 750D/775G/799S/800V/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/ 738E/ 749F/750D/775G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W /738E/ 749F/750D/775G/805Q/808S/809G/1076S/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/514L/534I /543P/ 558W/681N/732W/738E/749F/750D/775G/805Q/808S/809G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/ 393G/ 534I/558W/681G/732W/738E/749F/750D/775G/805Q/808S/809G/1148N/1248D/1377D/1439E or 63Q/171P/212A/260T/306G/318G/320R/332P/387I/ 390A/ 393G/534I/558W/681G/732W/738E/749F/750D/775G/805Q/809G/1116I/1148N/1248D/1377D/1439E, wherein the amino acid positions are relative to those corresponding to SEQ ID NO: 2 the reference sequence.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide comprises at least one of the amino acid sequences in Tables 4.1 , 4.2 , 5.1, 5.2 , 6.1 , 6.2 , 7.1 , 7.2 , 8.1 , 8.2 , 9.1 , 9.2 , 10.1, 10.2 , 11.1 and Substitutions at the positions set forth in 15.1 , wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide comprises at least one of Tables 4.1 , 4.2 , 5.1 , 5.2 , 6.1 , 6.2 , 7.1, 7.2 , 8.1 , 8.2 , 9.1 , 9.2 , 10.1 , 10.2 , 11.1 and 15.1 The set of substitutions set forth in , wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide comprises at least one of the amino acid sequences in Tables 4.1 , 4.2 , 5.1, 5.2 , 6.1 , 6.2 , 7.1 , 7.2 , 8.1 , 8.2 , 9.1 , 9.2 , 10.1, 10.2 , 11.1 and A substitution or set of substitutions at the positions set forth in 15.1 , wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide comprises at least one of Tables 4.1 , 4.2 , 5.1 , 5.2 , 6.1 , 6.2 , 7.1, 7.2 , 8.1 , 8.2 , 9.1 , 9.2 , 10.1 , 10.2 , 11.1 and 15.1 A substitution or set of substitutions of a variant set forth in , wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the recombinant Factor VIII polypeptide comprises a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072 or a reference sequence corresponding to SEQ ID NO: 252, The reference sequence of 504, 706, 840, 1200, 1514 or 2072 has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity of the amino acid sequence.

In some embodiments, the recombinant Factor VIII polypeptide comprises a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072 or a reference sequence corresponding to SEQ ID NO: 252, The reference sequence of 504, 706, 840, 1200, 1514 or 2072 has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity of the amino acid sequence, the restriction being the recombination factor VIII polypeptide does not include the amino acid sequence of SEQ ID NO: 2.

In some embodiments, a recombinant Factor VIII polypeptide comprises a reference sequence corresponding to residues 20-1457 of even-numbered SEQ ID NO. in SEQ ID NO. 4-2346 and 2360-2870 or corresponding to SEQ ID NO. The reference sequences of the even-numbered SEQ ID NO. in 4-2346 and 2350-2870 have at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, Amino acid sequences with 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity.

In some embodiments, the recombinant Factor VIII polypeptide comprises a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072 or a reference sequence corresponding to SEQ ID NO: 252, The reference sequence of 504, 706, 840, 1200, 1514 or 2072 has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to an amino acid sequence, wherein the amino acid sequence comprises Relative to this reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, 504, 706, 840, 1200, 1514 or 2072 or relative to SEQ ID NO: 252, 504, 706, 840, 1200, One or more substitutions of 1514 or 2072 of the reference sequence.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution at the following amino acid positions: 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 37, 56, 57, 58, 59, 61, 63, 64, 65, 85, 96, 104, 110, 119, 127, 132, 158, 160, 164, 167, 168, 171, 175, 178, 186, 187, 205, 210, 211, 212, 215, 225, 246, 257, 260, 265, 281, 292, 294, 301, 303, 304, 306, 308, 310, 311, 312, 318, 320, 321, 325, 328, 332, 334, 335, 344, 346, 347, 349, 351, 353, 354, 355, 356, 358, 359, 362, 370, 387, 388, 390, 392, 393, 397, 407, 428, 429, 433, 435, 436, 481, 487, 488, 489, 491, 498, 499, 503, 514, 527, 533, 534, 535, 536, 538, 543, 550, 551, 555, 558, 569, 580, 581, 584, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 643, 646, 650, 665, 667, 676, 680, 681, 682, 683, 684, 686, 693, 696, 698, 700, 706, 707, 708, 712, 716, 724, 726, 729, 731, 732, 734, 735, 736, 737, 738, 739, 740, 744, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 759, 770, 773, 775, 785, 786, 787, 790, 791, 792, 793, 794, 795, 797, 798, 799, 800, 801, 802, 805, 806, 807, 808, 809, 810, 811, 812, 815, 816, 817, 818, 819, 821, 822, 824, 825, 826, 828, 830, 831, 836, 837, 838, 839, 840, 841, 844, 866, 873, 879, 892, 896, 897, 903, 907, 914, 916, 917, 918, 919, 921, 922, 923, 924, 926, 929, 938, 939, 945, 948, 951, 968, 999, 1037, 1040, 1041, 1042, 1043, 1049, 1050, 1051, 1053, 1054, 1076, 1078, 1080, 1084, 1088, 1089, 1092, 1093, 1094, 1095, 1098, 1099, 1100, 1101, 1104, 1108, 1109, 1113, 1114, 1116, 1127, 1129, 1138, 1145, 1148, 1151, 1156, 1158, 1160, 118 6. 1192, 1193, 1194, 1205, 1210, 1218, 1220, 1221, 1222, 1224, 1229, 1245, 1248, 1250, 1259, 1264, 1269, 1270, 1274, 1281, 1283, 1292, 1297, 1339, 135 2. 1373, 1377, 1378, 1380, 1389, 1414, 1422, 1427, 1428, 1439, 1440, 1442 or 1452 or a combination thereof, wherein the amino acid positions are relative to those corresponding to SEQ ID NO: 252, 504, 706 , 840, 1200, 1514 or 2072 of the reference sequence.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or amino acid residue 13G/Y, 14G/M/V, 15G/S, 16A/D/G/P/V, 17S /V, 18L/R, 19G, 20L, 21M, 24V, 37H, 56E/P, 57A/L/S/T/V, 58K, 59D/G/L/M/Q/R/S/V/W , 61Q/S, 63C/E/F/G/H/I/K/L/M/N/Q/R/S/T/W/Y, 64D/G, 65A, 85Q, 96I/L/R /V/W, 104S, 110H, 119G/V, 127V, 132P/R, 158G, 160E/T, 164E, 167G/H/R, 168G/R, 171A/D/E/F/H/K/N /M/P/Q/R/S/T/V/W/Y, 175A/G/H, 178C/M/Q/V/W, 186A, 187C/G, 205R, 210P, 211G/K/P /Q/R, 212A/F/G/L/K/M/N/P/R/S/V/Y, 215E/P/S, 225R, 246D, 257I, 260E/K/T, 265R/S /W, 281A/S, 292D, 294G, 301K/M, 303G/T, 304A/E/G, 306A/D/G/P/S/T, 308P, 310G/V, 311E/G, 312L, 318C /G/K/L/M/Q/R/T/V, 320G/I/L/P/Q/R/S/T/V, 321Y, 325Y, 328G/Q/S, 332A/F/G /H/N/P/Q/R/T/V/W/Y, 334G, 335E/P, 344A/E/L, 346R/V, 347N, 349G, 351S, 353S, 354R, 355C, 356L/N , 358K/S, 359E/G, 362E/G/N/P/T/V/W, 370A, 387A/G/I/L/Q/R/S/T/V/W, 388A/P, 390A /E/G/L/M/P/S/T/V, 392A, 393A/F/G/H/M/Q/R/W/Y, 397G/N, 407V, 428F, 429L/S, 433A /G, 435Q, 436D/G, 481V, 487N, 488G, 489E/F/Q/R, 491E, 498A, 499C/L, 503S, 514L, 527A, 533R/V/Y, 534I/F/L/M , 535C/G/N, 536A/G/K/L/M/P/R/W, 538V, 543G/L/P/W, 550G, 551W, 555E/G/R/T, 558A/E/G /L/P/Q/R/S/T/V/W, 569T, 580S/Y, 581K/V, 584A, 610H/K/L/R/S/T, 611D/E, 612G/S/T , 613S, 614A/F/H/I/N/T, 615D/N, 616D/E/G/Q/W, 617D/E/G/N, 618C/E/G/P/Y, 619E, 620N /P, 621K/M/R/T/W, 622D, 643N, 646I, 650M, 665G, 667I, 676Q, 680L/R, 681A/C/E/F/G/L/N/R/Q/S /T/W, 682R, 683W, 684G/K, 686W, 693T, 696E, 698D/S/W/Y, 700A/E/Q/T/V, 706A/G/M/Y, 707H, 708A/K /T/V, 712G/L/M/Q/S/T/Y, 716K/L/W, 724I, 726A/L, 729T, 731I/L/R, 732C/D/E/G/L/R /W/K/M/Q/S/T/V/Y, 734I/R/S, 735D/S/V, 736F/G, 737F/G/K/L/M/N/S/V, 738A /D/E/F/H/K/N/P/R/S/T/Y, 739G/P/R, 740G, 744A/R, 746P, 747D, 748F/G/K/N/S/V , 749A/C/D/F/G/I/K/L/M/P/Q/R/S/T/V/Y, 750A/D/G/K/M/N/P/Q/R /S/T/V/W, 751A/D/E/Y, 752D/E/F/G/L/M/Q/R/S/V, 753D, 754W, 755G/L/P/Q/S /Y, 756G/P/R/T, 757A/G/K/T, 759S, 770W, 773H, 775E/G/K/N/P/S/V, 785V, 786C/D/L, 787W, 790A /G/I/M/P/W, 791M/T, 792H/W/Y, 793G, 794R, 795C/N/S/T, 797C/G/P/Q/T/W/Y, 798C/R /T, 799A/E/G/M/P/Q/R/S/V, 800C/G/I/L/M/N/P/Q/R/T/V/W/Y, 801A/G /I/R, 802S/V, 805C/G/H/K/L/M/P/Q/R/S/V/W, 806E/G/R/S, 807C/L/P/R, 808K /S, 809A/C/G/I/K/L/N/Q/R/S/T/V/W, 810V, 811F, 812G/I/V, 815I, 816V/W, 817A/D, 818A /E/G/P/S/V/W, 819V, 821Q, 822S/T, 824W, 825L, 826G, 828K, 830M/V, 831W, 836G/R/V/W, 837G, 838G/T, 839A /G/K/P, 840G, 841W, 844H, 866A, 873L/Q/T, 879Q, 892T, 896L/V, 897G/K/R, 903E, 907D, 914V, 916C/E/I/K/L /M/Q/T, 917G, 918M/S, 919G, 921R/W, 922W, 923M, 924I/M/P/Q/W, 926T/V, 929A/Q/V, 938R/V, 939E/S , 945K, 948S, 951V, 968A/P, 999R, 1037H/M/N/P, 1040E, 1041G/H/N, 1042A/E/T, 1043E/R, 1049G/W, 1050E/P/Q/S /T, 1051E/H/K/R/S/W, 1053A/R, 1054C, 1076G/S, 1078S, 1080G/V, 1084L/N, 1088G/S, 1089F/S, 1092G, 1093A/E/G /M/N/P/Q/R/S/T/V/W/Y, 1094S, 1095G, 1098G, 1099G, 1100I/S, 1101G, 1104C/E, 1108M/S/V/W, 1109G, 1113F , 1114A/M, 1116A/G/I/L/R, 1127L/V, 1129P, 1138Q, 1145R, 1148A/C/E/L/M/N/Q/R/S, 1151S, 1156D/G/N /R, 1158N, 1160R, 1186E, 1192D, 1193A/E/G/I/L, 1194P, 1205L, 1210M/T/V/W, 1218A/G/L/M/P, 1220D/E/L/T , 1221G/S/T/V, 1222F, 1224P, 1229D, 1245R, 1248A/C/D/E/F/G/H/I/K/M/P/Q/R/S/T/W/Y , 1250A/P/S/T, 1259T, 1264L/R/W, 1269F, 1270V, 1274N/V, 1281H/K/P/Q/R/S, 1283E/H/L/S/T, 1292Y, 1297A , 1339C, 1352E, 1373S, 1377A/C/D/E/G/H/I/M/P/R/S/V/T/Y, 1378A/C/D/E, 1380A/G/W, 1389W , 1414A/C/D/E/G/H/N/P/T/W, 1422I, 1427Y, 1428G, 1439A/E/H/L/S/T, 1440A/C/E/G/S/T or 1442T or a combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514 or 2072.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution at the following amino acid positions: 63, 171, 212, 260, 306, 317, 320, 332, 387, 390, 393, 534, 558, 681, 732, 738, 749, 750, 775, 1148, 1248, 1377 or 1439 or combinations thereof, wherein the amino acid positions are relative to those corresponding to SEQ ID NO: 252, 504, 706, 840 , 1200, 1514 or 2072 of the reference sequence.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide comprises at least one substitution or amino acid residue 63C/E/F/G/H/I/K/L/M/N/Q/R/S /T/W/Y, 171A/D/E/F/H/K/N/M/P/Q/R/S/T/V/W/Y, 212A/F/G/L/K/M /N/P/R/S/V/Y, 260E/K/T, 306A/D/G/P/S/T, 318C/G/K/L/M/Q/R/T/V, 320G /I/L/P/Q/R/S/T/V, 332A/F/G/H/N/P/Q/R/T/V/W/Y, 387A/G/I/L/Q /R/S/T/V/W, 390A/E/G/L/M/P/S/T/V, 393A/F/G/H/M/Q/R/W/Y, 499C/L , 534I/F/L/M, 558A/E/G/L/P/Q/R/S/T/V/W, 681A/C/E/F/G/L/N/R/Q/S /T/W, 700A/E/Q/T/V, 732C/D/E/G/L/R/W/K/M/Q/S/T/V/Y, 738A/D/E/F /H/K/N/P/R/S/T/Y, 749A/C/D/F/G/I/K/L/M/P/Q/R/S/T/V/Y, 750A /D/G/K/M/N/P/Q/R/S/T/V/W, 752D/E/F/G/L/M/Q/R/S/V, 775E/G/K /N/P/S/V, 800C/G/I/L/M/N/P/Q/R/T/V/W/Y, 805C/G/H/K/L/M/P/Q /R/S/V/W, 809A/C/G/I/K/L/N/Q/R/S/T/V/W, 1093A/E/G/M/N/P/Q/R /S/T/V/W/Y, 1148A/C/E/L/M/N/Q/R/S, 1248A/C/D/E/F/G/H/I/K/M/P /Q/R/S/T/W/Y, 1377A/C/D/E/G/H/I/M/P/R/S/V/T/Y, 1414A/C/D/E/G /H/N/P/T/W or 1439A/E/H/L/S/T or a combination thereof, wherein the amino acid positions are relative to those corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514 or 2072 of the reference sequence.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide comprises at least one substitution or amino acid residue 63Q, 171P, 212A, 260T, 306G, 317G, 320R, 332P, 387I, 390A, 393G, 534I, 558W , 681G, 732W, 7338E, 749F, 750D, 775G, 1148N, 1248D, 1377D or 1439E or combinations thereof, wherein the amino acid positions are relative to those corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514 or 2072 of the reference sequence.

In some embodiments, the recombinant Factor VIII polypeptide comprises a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252 or is at least 70%, 75%, 80% identical to a reference sequence corresponding to SEQ ID NO: 252 , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98% or 99% or more sequence identity to an amino acid sequence, wherein the amino acid sequence comprises the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252 or relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252 One or more substitutions of the reference sequence of SEQ ID NO: 252.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or set of substitutions at the following amino acid positions: 1380, 388, 1043, 610, 1274, 1220, 1442, 1193, 621, 1054 ,755,1041,748,1049,1221,1051,612,1037,616,387,489,750,1224,618,1050,1053,896,897,749,260,481,613,756,63,265 ,752,1218,1378,614,1440,1439,1205,1222,1389,1269,390,397/1218,757,1373,346,63/1248,349,306/355/1422,1422,306/346 /708、1148、171/346/1248、63/797/799/1248/1422、171/799、362、1248、171/306/346/999/1248、171/1148、734/799/1248/1422 , 63/732, 63/171/306/346/732, 171, 171/1422, 63/362/732/799/1248, 999/1148/1422, 346/349, 306/349, 63/171/1148 /1151, 306, 344/349/354/1250, 1250, 63/306/344/349/354/503/1248, 59/63/332/349/503/706/708, 63/306/344/349 /354/706/1248, 59/63, 332/706/708/1248/1250, 503, 59/63/349/354, 59/349/708, 59/306/344/1248, 344/1248/1250 , 708, 63/344/349, 59, 63/344/349/354, 706, 59/306/770, 63/347/354/1248 or 354/503, where the amino acid positions are relative to the corresponding The reference sequence at SEQ ID NO: 252.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or set of substitutions 1380W, 388A, 1043E, 1043R, 610R, 1274N, 1220T, 1442T, 1193L, 621T, 1054C, 755Q, 1041G, 748S, 1049G, 1221V, 1051E, 612G, 1037H, 616E, 1041H, 387S, 489R, 621M, 489Q, 750G, 1224P, 618C, 616G, 612T, 1050Q, 1053A, 616W, 896V, 489F, 1050P, 897G ,749T,1274V, 1220L, 260E, 481V, 613S, 387G, 621R, 756R, 63R, 265S, 1221T, 1380G, 752G, 748K, 755L, 1218A, 748N, 1378E, 614I, 749F, 387V, 750T, 1193G, 749V, 137 8A, 750V, 387T, 387Q, 750A, 1037N, 1440A, 1051S, 1221G, 1378D, 1440S, 387R, 1193A, 387I, 1218M, 265W, 1440E, 1439L, 1440T, 612S, 1049W, 1221S, 1220D, 749G ,749S,1218G,1205L, 1222F, 1193E, 1050S, 1389W, 1439E, 260T, 1037P, 1051K, 1269F, 265R, 1051R, 1218L, 897R, 388P, 748G, 756P, 748F, 390A, 610L, 755G, 749R, 748V, 138 0A, 1440C, 397N/ 1218M, 1037M, 1218P, 757K, 1373S, 1440G, 1193I, 1439T, 750D, 390G, 756G, 1220E, 1378C, 618G, 346R, 63Q/1248D, 349G, 306G/355C/1422I, 1422I, 30 6G/346R/708T, 1148N, 171P/346R/1248D, 63Q/797T/799R/1248D/1422I, 171P/799R, 362P, 1248D, 63Q, 171P/306G/346R/999R/1248D, 171P/1148N, 734S/799R/124 8D/1422I、 63Q/732E, 63Q/171P/306G/346R/732G, 171P, 171P/1422I, 63Q/362P/732G/799R/1248D, 999R/1148N/1422I, 346R/349G, 306G/349G, 63Q/171P/114 8N/ 1151S, 306G, 344E/349G/354R/1250T, 63G, 1250T, 63R/306G/344E/349G/354R/503S/1248R, 59D/63S/332P/349G/503S/706M/708K, 63S/306G/344A / 349G/354R/706M/1248R, 1248R, 59D/63G, 332P/706M/708K/1248R/1250T, 503S, 59D/63R/349G/354R, 59D/349G/708K, 59D/306G/344E/1248R, 344E / 1248R/1250T, 708K, 63R/344E/349G, 59D, 63S/344A/349G/354R, 706M, 59D/306G/770W, 63G/347N/354R/1248R or 354R/503S, where the amino acid position is Relative to the reference sequence corresponding to SEQ ID NO: 252.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or substitution set M1380W, I388A, F1043E, F1043R, I610R, I1274N, S1220T, I1442T, F1193L, Q621T, L1054C, A755Q, Y1041G, Y748S, Y1049G, L1221V, M1051E, R612G, F1037H, N616E, Y1041H, F387S, S489R, Q621M, S489Q, L750G, S1224P, A618C, N616G, R612T, I1050Q, T1053A, N616W, I8 96V, S489F, I1050P, M897G, L749T, I1274V, S1220L, S260E, L481V, F613S, F387G, Q621R, I756R, V63R, I265S, L1221T, M1380G, K752G, Y748K, A755L, F1218A, Y748N, T1378E, L614I, L749F, F387V, L750T, F1193G, L749V, T1378A, L750V, F387T, F387Q, L750A, F1037N, H1440A, M1051S, L1221G, T1378D, H1440S, F387R, F1193A, F387I, F1218M, I265W, H1440E, V1439L, H1440T, R612S, Y1049 W, L1221S, S1220D, L749G, L749S, F1218G, I1205L, Y1222F, F1193E, I1050S, S1389W, V1439E, S260T, F1037P, M1051K, I1269F, I265R, M1051R, F1218L, M897R, I388P, Y748G, I756P, Y748F, I390A, I610L, A755G, L749R, Y748V, M1380A, H1440C, H397N/ F1218M, F1037M, F1218P, E757K, V1373S, H1440G, F1193I, V1439T, L750D, I390G, I756G, S1220E, T1378C, A618G, D346R, V63Q/L1248D, P349G, E306G/R 355C/L1422I, L1422I, E306G/D346R/I708T, T1148N, L171P/D346R/L1248D, V63Q/M797T/K799R/L1248D/L1422I, L171P/K799R, A362P, L1248D, V63Q, L171P/E306G/D346R/Q999R/L1248D, L171P/ T1148N, T734S/K799R/L1248D/L1422I, V63Q/K732E, V63Q/L171P/E306G/D346R/K732G, L171P, L171P/L1422I, V63Q/A362P/K732G/K799R/L1248D, Q999R/T1148N/L1422I, D346R/P349G, E30 6G/P349G, V63Q/L171P/T1148N/ G1151S, E306G, K344E/P349G/L354R/V1250T, V63G, V1250T, V63R/E306G/K344E/P349G/L354R/R503S/L1248R, F59D/V63S/S332P/P349G/R503S/L706 M/I708K, V63S/E306G/K344A/ P349G/L354R/L706M/L1248R, L1248R, F59D/V63G, S332P/L706M/I708K/L1248R/V1250T, R503S, F59D/V63R/P349G/L354R, F59D/P349G/I708K, F59D/ E306G/K344E/L1248R, K344E/ L1248R/V1250T, I708K, V63R/K344E/P349G, F59D, V63S/K344A/P349G/L354R, L706M, F59D/E306G/R770W, V63G/S347N/L354R/L1248R or L354R/R503S, Wherein the amino acid positions are Relative to the reference sequence corresponding to SEQ ID NO: 252.

In some embodiments, the recombinant Factor VIII polypeptide comprises a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 504 or is at least 70%, 75%, 80% identical to a reference sequence corresponding to SEQ ID NO: 504 , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98% or 99% or more sequence identity to an amino acid sequence, wherein the amino acid sequence comprises the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 504 or relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 504 One or more substitutions of the reference sequence of SEQ ID NO: 504.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or set of substitutions at the following amino acid positions: 750, 1051, 260, 756, 1193, 332, 897, 387, 1037, 306 /749, 1270, 489, 487, 488, 903, 171/1248/1440, 260/1148/1378, 171/260/387/750/1248, 171/260, 171/260/387/750/1051/1148 ,171,750/1051/1148,171/260/756,171/1248,171/1148,59,1440,332/750/1248,756/1248,1248,171/387/1248/1440,750/1440 ,59/171/750,171/1193/1248,750/1148/1193/1440,1148,171/387/1051,748,306,306/387/390/749/1220,390,306/390/1037 ,306/612/749, 387/390/749, 63/390/503/749, 1220, 63/306, 749, 306/390/749/1037, 306/749/1220, 306/387/390/612 /749/1037 or 328, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 504.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or substitution set 750D, 1051K, 1051S, 260T, 756P, 1193A, 332P, 897R, 387Q, 1037N, 387R, 306G/749F, 1270V, 489E, 487N, 488G, 903E, 171P/1248D/1440T, 260T/1148N/1378D, 171P/260T/387I/750D/1248D, 171P/260T, 171P/260T/387I/750D/1051S/1148N, 1 71P, 750D/ 1051S/1148N, 171P/260T/756P, 171P/1248D, 171P/1148N, 387I, 59D, 1440S, 332P/750D/1248D, 756P/1248D, 1248D, 171P/387I/1248D/1440S, 750D /1440S、59D/ 171P/750D, 171P/1193A/1248D, 750D/1148N/1193A/1440S, 1148N, 171P/387I/1051K, 748G, 306G, 306G/387R/390A/749F/1220D, 390A, 306G/390A/ 1037N, 306G/ 612S/749F, 387V/390A/749F, 63S/390A/503S/749F, 387R/390A/749R, 1220D, 63S/306G, 749F, 306G/390A/749F/1037N, 387V, 306G/749F/1220D, 30 6G/ 387V/390A/612S/749F/1037N or 328S, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 504.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or substitution set L750D, M1051K, M1051S, S260T, I756P, F1193A, S332P, M897R, F387Q, F1037N, F387R, E306G/L749F, I1270V, S489E, Q487N, A488G, Q903E, L171P/L1248D/H1440T, S260T/T1148N/T1378D, L171P/S260T/F387I/L750D/L1248D, L171P/S260T, L171P/S260T/F387I /L750D/M1051S/T1148N, L171P, L750D/ M1051S/T1148N, L171P/S260T/I756P, L171P/L1248D, L171P/T1148N, F387I, F59D, H1440S, S332P/L750D/L1248D, I756P/L1248D, L1248D, L171P/F38 7I/L1248D/H1440S, L750D/H1440S, F59D/ L171P/L750D, L171P/F1193A/L1248D, L750D/T1148N/F1193A/H1440S, T1148N, L171P/F387I/M1051K, Y748G, E306G, E306G/F387R/I390A/L749F/S12 20D, I390A, E306G/I390A/F1037N, E306G/ F387 V、E306G/L749F/S1220D、E306G/ F387V/I390A/R612S/L749F/F1037N or F328S, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 504.

In some embodiments, the recombinant Factor VIII polypeptide comprises a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 706 or is at least 70%, 75%, 80% identical to a reference sequence corresponding to SEQ ID NO: 706 , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98% or 99% or more sequence identity to an amino acid sequence, wherein the amino acid sequence comprises the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 706 or relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 706 One or more substitutions of the reference sequence of SEQ ID NO: 706.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or set of substitutions at the following amino acid positions: 1051, 1221, 1218, 387, 749/1218, 332, 897, 696, 816 ,824,826,393,825,698,332/387/390/1148,328/387/749,749,306/328/332/390/1051/1193/1218,63/306/332/1051/1148 , 63/306/332/387/1051, 387/390/1051, 186/306/328/332/390/749/1051/1148/1193, 387/390/756/1051, 306/390/1051, 306 /332/390/749, 306/387/390/1148, 306/328/332/387/390/749/1051/1148/1193, 332/387/390/749/1148, 387/390/749, 306 /387/749/1051/1148、387/749、306/945/1051/1193/1221、306/390/749/1051、63/306/332/390、306/387/390/749/1148/1193 /1221, 306/332/387/390/749/1218, 63/306/332/390/1051/1148/1193/1218, 924, 755, 752, 751, 923, 797, 754, 793, 753, 922 , 896, 938, 775, 732, 810, 747, 393/407, 819, 787, 802, 811, 392 or 397, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 706 .

In some embodiments, a recombinant Factor VIII polypeptide comprises at least one substitution or set of substitutions 1051K, 1221G, 1218G, 387R, 1051R, 749F/1218G, 387T, 332P, 897K, 696E, 816W, 824W, 826G, 393W, 825L, 897R, 816V, 698W, 393Q, 332P/387R/390A/1148N, 328S/387R/749F, 749F, 306G/328S/332P/390A/1051R/1193A/1218G, 63Q/306G/332P/1051R/1148 N、63Q/ 306G/332P/387T/1051K, 387T/390A/1051R, 186A/306G/328S/332P/390A/749F/1051K/1148N/1193A, 387T/390A/756P/1051R, 306G/390A/1051R, 3 06G/332P/ 390A/749F, 306G/387R/390A/1148N, 306G/328S/332P/387T/390A/749F/1051R/1148N/1193A, 332P/387R/390A/749F/1148N, 387R/390A/749F, 306 G/387T/ 749F/1051K/1148N, 387R/749F, 306G/945K/1051R/1193A/1221G, 306G/390A/749F/1051K, 63Q/306G/332P/390A, 306G/387T/390A/749F/1148N/11 93A/1221G、 306G/332P/387R/390A/749F/1218M, 63Q/306G/332P/390A/1051R/1148N/1193A/1218G, 924I, 755S, 752F, 751Y, 923M, 797Y, 754W, 793G, 753D, 922W ,896L, 938R, 775G, 751E, 732W, 752G, 810V, 938V, 747D, 755G, 393G/407V, 797W, 819V, 393G, 787W, 393R, 802V, 775E, 924P, 732G, 811F, 802S, 752L, 392A or 397 G of An amino acid sequence, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 706.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or substitution set M1051K, L1221G, F1218G, I387R, M1051R, L749F/F1218G, I387T, S332P, M897K, T696E, F816W, F824W, A826G, V393W, I825L, M897R, F816V, F698W, V393Q, S332P/I387R/I390A/T1148N, F328S/I387R/L749F, L749F, E306G/F328S/S332P/I390A/M1051R/F1193A/F 1218G, V63Q/E306G/S332P/M1051R/ T1148N, V63Q/E306G/S332P/I387T/M1051K, I387T/I390A/M1051R, D186A/E306G/F328S/S332P/I390A/L749F/M1051K/T1148N/F1193A, I387T/I390A /I756P/M1051R, E306G/I390A/M1051R, E306G/S332P/I390A/L749F, E306G/I387R/I390A/T1148N, E306G/F328S/S332P/I387T/I390A/L749F/M1051R/T1148N/F1193A, S332P/I387R/I390A/ L749F/T1148N, I387R/I390A/L749F, E306G/I387T/L749F/M1051K/T1148N, I387R/L749F, E306G/Q945K/M1051R/F1193A/L1221G, E306G/I390A/L749F/M1051K, V63Q/E306G/S332P/I390A ,E306G/I387T/I390A/L749F/T1148N/ F1193A/L1221G, E306G/S332P/I387R/I390A/L749F/F1218M, V63Q/E306G/S332P/I390A/M1051R/T1148N/F1193A/F1218G, G924I, A755S, K752F, S751 Y, Q923M, M797Y, N754W, I793G, N753D, R922W, I896L, K938R, I775G, S751E, K732W, K752G, N810V, K938V, A747D, A755G, V393G/A407V, M797W, K819V, V393G, I787W, V393R, F802V, I775E, G924 P, K732G, Q811F, F802S, K752L, S392A or H397G, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 706.

In some embodiments, the recombinant Factor VIII polypeptide comprises a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 840 or is at least 70%, 75%, 80% identical to a reference sequence corresponding to SEQ ID NO: 840 , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98% or 99% or more sequence identity to an amino acid sequence, wherein the amino acid sequence comprises the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 840 or relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 840 One or more substitutions of the reference sequence of SEQ ID NO: 840.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or set of substitutions at the following amino acid positions: 620, 491, 1229, 615, 1283, 822, 328, 212, 818, 555 /684, 828, 550, 830, 839, 13, 14, 17, 836, 1210, 1281, 64, 682, 318, 20, 21, 325, 533, 19, 817, 831, 225, 1156, 393/747 ,753,393/732/751,63/797,622,617,907,1040,611,488,1192,1194,619,16,802,211,555,536,527,841,320,837,558 ,914,312,681,838,15,1427,18,683,24,534,840,1160,551,393,393/747/752/811/819/923/1148,393/751/775,63 /393/732/775/1148, 393/732/1148, 775/1148, 393/752/753/775/793/797, 393/923, 393/923/1148, 393/747/751, 393/747 /753/775/1148、752/923/1051、63/775、63/393/752/775/793/1051/1053、752/753/775/793/819、63/1051、63/747/752 /753/775/811/819/923/1051, 393/752/787, 751, 63/1148, 63/747/752/753/819/923/1148, 1148, 747/752/753/793/811 , 63, 797/1148, 751/752/753/1051, 63/732/1051, 752/797/815/923/1051/1148 or 747/753, wherein the amino acid positions are relative to those corresponding to SEQ. The reference sequence of ID NO: 840.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or substitution set 620N, 491E, 1229D, 615N, 1283S, 822S, 328G, 212L, 818W, 1283L, 555G/684G, 828K, 550G, 830M, 839A, 822T, 13Y, 14G, 17V, 836V, 839P, 1210W, 1281R, 64D, 212G, 682R, 14M, 318C, 17S, 20L, 818G, 836G, 21M, 836R, 325Y, 533V, 19G, 14V, 817D, 831W, 225R, 1156R, 830V, 393G/747D, 753D, 393G/732W/751Y, 63Q/797W, 622D, 617E, 617N, 907D, 1040E, 617D, 611E, 620P, 617G, 488G, 1192D, 1 194P、 619E、615D、16A、802S、818V、211K、836W、555R、536A、527A、555E、841W、320Q、837G、558R、555G、211R、64G、320R、533Y、914V、16V、1283E、536M、312L、 681N、838T、536R、15G、536P、558W、558E、1427Y、839G、18L、13G、1210M、1156G、558L、839K、211P、211G、211Q、16D、320G、683W、24V、534I、681A、840G、 1160R、681C、328Q、15S、536K、536G、533R、681E、536W、1156N、16P、817A、18R、551W、555T、681G、212A、16G、838G、681R、393G、393G/747D/752G/811F/ 819V/923M/1148N, 393G/751Y/775G, 63Q/393G/732W/775G/1148N, 393G/732W/1148N, 775G/1148N, 393G/752L/753D/775G/793G/797W, 393G/923M, 393G/ 923M/1148N, 393G/747D/751Y, 393G/747D/753D/775G/1148N, 752L/923M/1051R, 63Q/775G, 63Q/393G/752L/775G/793G/1051R/1053R, 752G/753D/ 775G/ 793G/819V, 63Q/1051R, 63Q/747D/752G/753D/775G/811F/819V/923M/1051R, 393G/752G/787W, 751Y, 63Q/1148N, 63H/747D/752G/753D/819V/923M / 1148N, 1148N, 747D/752L/753D/793G/811F, 63Q, 797W/1148N, 751Y/752L/753D/1051R, 63Q/732W/1051R, 752G/797W/815I/923M/1051R/1148N or 747 D/753D, The amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 840.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or substitution set V620N, P491E, M1229D, P615N, I1283S, H822S, F328G, H212L, K818W, I1283L, F555G/E684G, V828K, R550G, R830M, S839A, H822T, L13Y, L14G, C17V, M836V, S839P, K1210W, Y1281R, V64D, H212G, V682R, L14M, L318C, C17S, A20L, K818G, M836G, T21M, M836R, F325 Y, T533V, S19G, L14V, Q817D, L831W, K225R, H1156R, R830V, V393G/A747D, N753D, V393G/K732W/S751Y, V63Q/M797W, L622D, P617E, P617N, P907D, N1040E, P617D, Q611E, V62 0P, P617G, A488G, P1192D, S1194P, G619E, P615D, F16A, F802S, K818V, L211K, M836W, F555R, V536A, I527A, F555E, H841W, M320Q, S837G, M558R, F555G, L211R, V64G, M320R, T533Y, L914V, F16V, I1283E, V536M, F312L, M681N, S838T, V536R, R15G, V536P, M558W, M558E, L1427Y, S839G, F18L, L13G, K1210M, H1156G, M558L, S839K, L211P, L211G, L211Q, F16D, M320G, Y683W , Y24V, V534I, M681A, P840G, F1160R, M681C, F328Q, R15S, V536K, V536G, T533R, M681E, V536W, H1156N, F16P, Q817A, F18R, Y551W, F555T, M681G, H212A, F16G, S838G, M681R, V393G, V393G/A747D/K752G/Q811F/ K819V/Q923M/T1148N, V393G/S751Y/I775G, V63Q/V393G/K732W/I775G/T1148N, V393G/K732W/T1148N, I775G/T1148N, V393G/K752L/N753D/I775G/I 793G/M797W, V393G/Q923M, V393G/ Q923M/T1148N, V393G/A747D/S751Y, V393G/A747D/N753D/I775G/T1148N, K752L/Q923M/M1051R, V63Q/I775G, V63Q/V393G/K752L/I775G/I793G/M10 51R/T1053R, K752G/N753D/I775G/ I793G/K819V, V63Q/M1051R, V63Q/A747D/K752G/N753D/I775G/Q811F/K819V/Q923M/M1051R, V393G/K752G/I787W, S751Y, V63Q/T1148N, V63H/A747D /K752G/N753D/K819V/Q923M/ T1148N, T1148N, A747D/K752L/N753D/I793G/Q811F, V63Q, M797W/T1148N, S751Y/K752L/N753D/M1051R, V63Q/K732W/M1051R, K752G/M797W/S815I/Q 923M/M1051R/T1148N or A747D/N753D, The amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 840.

In some embodiments, the recombinant Factor VIII polypeptide comprises a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 1200 or at least 70%, 75%, 80% identical to a reference sequence corresponding to SEQ ID NO: 1200 , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98% or 99% or more sequence identity to an amino acid sequence, wherein the amino acid sequence comprises the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 1200 or relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 1200 One or more substitutions of the reference sequence of SEQ ID NO: 1200.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide comprises at least one substitution or set of substitutions at the following amino acid positions: 681, 622, 617, 558, 536, 211, 320, 839, 797, 533 ,751,303,951,164,818,807,799,735,794,926,755,921,801,1094,968,929,924,698,168,581,916,1428,805,1114,311 ,737,693,809,757,1099,676,740,791,211/536/622/681,536/558,211/536/555/617/732,211/681/732,211/536/555 ,555,211/536,211/212,211/536/617/681/732,211/536/558/617/681,555/732,536/555/558/617/732,536/617/681 /732, 536/555, 536/622/681, 732, 212/320/533/617/681, 211/312/533/534/617/619/681/839, 212, 211/212/320/619 /681/839、797/839、212/312/320/533/534/617/681、558/619/681、320/558/681、212/320/534/619/681/797/839、320 /558/619/732/839, 211/212/312/534/558/619/681/732/839, 320/534/558/619/732/797, 558/617/619/681, 211/534 /558/611/681、211/320/617/619/681、558/617/681/797、320/534/619/732、211/619/681、211/212/534/558/619/732 /839、212/320/619/681/797/839、212/320/611/839、320/558/619/681、312/533/617/681/797/839、558/611/681、312 ,211/320/558/619/681/732/797/839, 320/558/732/797, 533/558/611/619/716/732/839, 312/681, 320/534/619/681 ,211/320/533/536/619, 558/617/681/839, 212/320/534/558/681, 533/534/681/839, 212/534/619/681, 617/619/681 ,211/212/320/558/617/681, 212/534/558/681/797, 212/320/534, 211/312/619, 212/312/619/839, 210/212/320/558 /681, 212/312/534/681/797/839, 211/320/558/839, 294, 759, 538 or 1109, wherein the amino acid positions are relative to those corresponding to SEQ ID NO: 1200 Reference sequence.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or substitution set 681A, 622D, 617G, 558L, 536K, 681G, 211Q, 320R, 558W, 839K, 211K, 797W, 533R, 751A, 303T、951V、164E、818G、807L、799A、735S、794R、926V、755P、921W、801A、1094S、968A、929Q、924W、807P、698D、168G、929V、818A、818S、581K、818P、91 6K, 698S, 303G, 916C, 1428G, 805L, 924M, 1114M, 311G, 737N, 805V, 801I, 751D, 693T, 818E, 929A, 311E, 737L, 809W, 757A, 1099G, 799G, 916I, 805G, 737F, 737M, 799P, 807C, 916E, 676Q, 581V, 968P, 755Y, 916Q, 735D, 740G, 698Y, 757G, 791T, 1114A, 735V, 536P, 211R/536K/622D/681A, 536K/558L, 211R/536R/55 5G/ 617G/732K, 211R/681R/732K, 211R/536K/555E, 536R, 555E, 211R/536P, 211R/212P, 211R/536K, 211R/536K/617G/681A/732K, 211R, 211R/536P/558 L/ 617G/681A, 555G/732K, 536P/555E/558L/617G/732K, 536K/617G/681A/732K, 536K/555G, 536P/622D/681A, 732K, 212A/320Q/533R/617E/681G, 211 K/ 312L/533R/534I/617D/619E/681N/839K, 212A, 211K/212A/320Q/619E/681G/839K, 797W/S839K, 212A/312L/320Q/533R/534I/617E/681G, 558W/61 9E/ 681G, 320Q/558W/681G, 212A/320Q/534I/619E/681G/797W/839K, 320R/558W/619E/732K/839K, 211K/212A/312L/534I/558W/619E/681N/732K/839 K. 320R/534I/558R/619E/732K/797W, 558W/617D/619E/681N, 211Q/534I/558W/611E/681N, 211K/320R/617E/619E/681G, 558W/617D/681G/797W, 320 R/ 534I/619E/732K, 211K/619E/681N, 211K/212A/534I/558W/619E/732K/839K, 212A/320R/619E/681N/797W/839K, 212A/320R/611E/839K, 320Q/558 W/ 533 R/ 558W/611E/619E/716L/732K/839K, 312L/681G, 320Q/534I/619E/681N, 211K/320Q/533R/536L/619E, 558W/617E/681N/839K, 212A/320R/534I/558 W/ 681G、533R/534I/681N/839K、212A/534I/619E/681G、617E/619E/681G、211Q/212A/320R/558W/617E/681N、212A/534I/558W/681N/797W、212A/320 R/ 534I, 211K/312L/619E, 212A/312L/619E/839K, 210P/212A/320R/558W/681G, 212A/312L/534I/681N/797W/839K, 211K/320R/558W/839K, 924Q, 916 L. 757T, 737K, 818, 801G, 294G, 799S, 799E, 809G, 791M, 737V, 805Q, 799Q, 737S, 916M, 759S, 799V, 538V, 1109G, 168R, 926T, 916T, 737G, or 801R, of which this of which is this. Amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 1200.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or substitution set M681A, L622D, P617G, M558L, V536K, M681G, L211Q, M320R, M558W, S839K, L211K, M797W, T533R, S751A, A303T, T951V, G164E, K818G, E807L, K799A, G735S, S794R, E926V, A755P, Q921W, D801A, E1094S, L968A, K929Q, G924W, E807P, F698D, S168G, K929V, K81 8A, K818S, R581K, K818P, S916K, F698S, A303G, S916C, T1428G, Y805L, G924M, L1114M, T311G, Y737N, Y805V, D801I, S751D, S693T, K818E, K929A, T311E, Y737L, E809W, E757A, A1099G, K 799G, S916I, Y805G, Y737F, Y737M, K799P, E807C, S916E, T676Q, R581V, L968P, A755Y, S916Q, G735D, D740G, F698Y, E757G, D791T, L1114A, G735V, V536P, L211R/V536K/L622D/M681A, V53 6K/M558L, L211R/V536R/F555G/ P617G/W732K, L211R/M681R/W732K, L211R/V536K/F555E, V536R, F555E, L211R/V536P, L211R/H212P, L211R/V536K, L211R/V536K/P617G/M681A/W732 K, L211R, L211R/V536P/M558L/ P617G/M681A, F555G/W732K, V536P/F555E/M558L/P617G/W732K, V536K/P617G/M681A/W732K, V536K/F555G, V536P/L622D/M681A, W732K, H212A/M320 Q/T533R/P617E/M681G, L211K/ F312L/T533R/V534I/P617D/G619E/M681N/S839K, H212A, L211K/H212A/M320Q/G619E/M681G/S839K, M797W/S839K, H212A/F312L/M320Q/T533R/V534 I/P617E/M681G, M558W/G619E/ M681G, M320Q/M558W/M681G, H212A/M320Q/V534I/G619E/M681G/M797W/S839K, M320R/M558W/G619E/W732K/S839K, L211K/H212A/F312L/V534I/M558 W/G619E/M681N/W732K/S839K, M320R/V534I/M558R/G619E/W732K/M797W, M558W/P617D/G619E/M681N, L211Q/V534I/M558W/Q611E/M681N, L211K/M320R/P617E/G619E/M681G, M558 W/P617D/M681G/M797W, M320R/ V534I/G619E/W732K, L211K/G619E/M681N, L211K/H212A/V534I/M558W/G619E/W732K/S839K, H212A/M320R/G619E/M681N/M797W/S839K, H212A/M320 R/Q611E/S839K, M320Q/M558W/ G619E/M681N, F312L/T533R/P617D/M681N/M797W/S839K, M558W/Q611E/M681N, F312L, L211Q/M320R/M558W/G619E/M681G/W732K/M797W/S839K, M320 Q/M558W/W732K/M797W, T533R/ M558W/Q611E/G619E/F716L/W732K/S839K, F312L/M681G, M320Q/V534I/G619E/M681N, L211K/M320Q/T533R/V536L/G619E, M558W/P617E/M681N/S839 K. H212A/M320R/V534I/M558W/ M681G, T533R/V534I/M681N/S839K, H212A/V534I/G619E/M681G, P617E/G619E/M681G, L211Q/H212A/M320R/M558W/P617E/M681N, H212A/V534I/M558 W/M681N/M797W, H212A/M320R/ V534I, L211K/F312L/G619E, H212A/F312L/G619E/S839K, T210P/H212A/M320R/M558W/M681G, H212A/F312L/V534I/M681N/M797W/S839K, L211K/M320 R/M558W/S839K, G924Q, S916L, E757T, Y737K, K818V, D801G, T294G, K799S, K799E, E809G, D791M, Y737V, Y805Q, K799Q, Y737S, S916M, R759S, K799V, D538V, E1109G, S168R, Q921R, E92 6T, S916T, Y737G or D801R, which Amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 1200.

In some embodiments, the recombinant Factor VIII polypeptide comprises a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 1514 or is at least 70%, 75%, 80% identical to a reference sequence corresponding to SEQ ID NO: 1514 , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% or more sequence identity to an amino acid sequence, wherein the amino acid sequence comprises the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 1514 or relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 1514 One or more substitutions of the reference sequence of SEQ ID NO: 1514.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or set of substitutions at the following amino acid positions: 1042, 1041, 1051, 1283, 1281, 916, 187, 215, 178, 1050 ,1210,104,615,610,536,618,621,614,110,301,1078,812,786,127,175,1093,1088,650,535,806,738,700,132,1076,1084 ,498,756,1108,746,160,543,310,584,918,1452,790,292,800,167,892,304,1116,752,281,917,712,1080,806/1352,1095 ,744,1098,948,119,785,1113,667,158,580,707,731,1100,1089,1092,939,1104,736,1297,1127,686,1129,1138,786/821,680 ,1101,808,739,308,536/555/619/732/799/805,555/619/801,555/619/799,619/799/801/805,536/555/619/732/799 ,799/805,801,246/619/799/801,536/555/619,619/839,619/799/805,536/801/805,555/619/799/805,619/801/805 ,555/619,536/619/801,801/805,536/619/799/805,555/619/732/799/801/805,749,616,611,370/614,1156,1158,555 /681/737/799, 536/619/809/1109, 536/555/619/737/799/809/839/916/921, 916/1109, 536/619/681/799/916/1109, 555 /619/681/807/1109, 555/619/681/737/799, 619/681/799/916/1109, 619/681, 555/921, 619/681/916, 555/619/681/799 , 555, 732, 619/799, 619, 681, 555/916, 839 or 799, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 1514.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or set of substitutions 1042E, 1041N, 1051H, 1283H, 1283T, 1281Q, 1281P, 916E, 1051W, 187C, 215E, 215P, 215S, 178C, 1050T, 1210T, 1281K, 1042A, 104S, 615D, 610S, 610K, 610T, 536P, 618Y, 621W, 618P, 614F, 110H, 301K, 1078S, 812G, 786C, 127V, 175G, 1093W, 1088G, 1 093T、650M、 301M, 535N, 806R, 738E, 700E, 132R, 1076G, 1084L, 498A, 786L, 738F, 535G, 806E, 756G, 1108S, 1093V, 746P, 160E, 160T, 178V, 543L, 310G, 700Q, 1093 A、584A、 918S, 1452L, 738P, 790W, 790I, 292D, 800M, 1108M, 1088S, 167G, 892T, 790P, 800R, 738K, 304G, 1116G, 178Q, 1116A, 752G, 1108W, 738D, 281A, 917G, 71 2M, 1080V, 806G/1352E、1095G、1093G、744A、543G、1098G、281S、756T、948S、119V、1116L、785V、1113F、700A、786D、175A、790M、667I、158G、580S、707H、731I、91 8M, 1100S, 5 35C, 680R, 1116I, 1089F, 304E, 178W, 1104C, 744R, 738R, 1093S, 712S, 187G, 132P, 812I, 1080G, 800C, 1101G, 808K, 808S, 790A, 800V, 680L, 167R, 752E, 1093N, 1 19G, 739G, 1116R, 580Y, 304A, 712Q, 806S, 712G, 790G, 1108V, 731L, 1084N, 1127L, 308P, 1076S, 1093P, 1093Y, 739R, 739P, 310V, 712T, 167H, 1093R, 536P/555E/ 619E/732K/ 536 P/ 555E/619E, 619E/839K, 619E/799S/805Q, 536P/801G/805Q, 555E/619E/799S/805Q, 619E/801G/805Q, 555E/619E, 536P/619E/801G, 801G/805Q, 536 P/ 619E/799S/805Q, 555E/619E/732K/799S/801G/805Q, 1281H, 749L, 614H, 616D, 621K, 1050E, 610H, 611D, 1042T, 616Q, 614N, 370A/614A, 1156D, 1210 V、614T、 1158N, 1281S, 618E, 555E/681N/737K/799Q, 536P/619E/809G/1109G, 536K/555G/619E/737K/799Q/809G/839K/916T/921R, 916T/1109G, 536R/619E /681N/ 799Q/916T/1109G, 555G/619E/681N/807R/1109G, 555E/619E/681N/737K/799Q, 619E/681N/799S/916T/1109G, 619E/681N, 555G/921R, 619E/681N/ 916T、 555G/619E/681N/799S, 555E, 732K, 619E/799S, 619E, 681N, 555G/916T, 839K or 799Q, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 1514.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or substitution set R1042E, Y1041N, M1051H, I1283H, I1283T, Y1281Q, Y1281P, S916E, M1051W, L187C, I215E, I215P, I215S, L178C, I1050T, K1210T, Y1281K, R1042A, V104S, P615D, I610S, I610K, I610T, V536P, A618Y, Q621W, A618P, L614F, M110H, R301K, I1078S, S812G, E786C, A127V , Y175G, K1093W, F1088G, K1093T, L650M, R301M, T535N, D806R, Y738E, S700E, E132R, E1076G, S1084L, G498A, E786L, Y738F, T535G, D806E, I756G, F1108S, K1093V, S746P, L160E, L160T, L178V, S543L, I310G, S700Q, K1093A, Q584A, E918S, E1452L, Y738P, D790W, D790I, G292D, E800M, F1108M, F1088S, A167G, V892T, D790P, E800R, Y738K, S304G, S1116G, L178Q, S1116A, K752G, F1108 W, Y738D, T281A, Y917G, H712M, S1080V, D806G/K1352E, E1095G, K1093G, D744A, S543G, M1098G, T281S, I756T, M948S, A119V, S1116L, E785V, M1113F, S700A, E786D, Y175A, D790M, F667I, Q158 G, Q580S, W707H, D731I, E918M, L1100S, T1089S, H712Y, Y175H, K1092G, T939E, Y1104E, E800G, D736F, S543W, N1297A, H712L, I1127V, T939S, L178M, D731R, T686W, S543P, D736G, E1129P, L113 8Q, S812V, E786D/R821Q, T535C, K680R, S1116I, T1089F, S304E, L178W, Y1104C, D744R, Y738R, K1093S, H712S, L187G, E132P, S812I, S1080G, E800C, Y1101G, D808K, D808S, D790A, E800V, K680 L, A167R, K752E, K1093N, A119G, E739G, S1116R, Q580Y, S304A, H712Q, D806S, H712G, D790G, F1108V, D731L, S1084N, I1127L, S308P, E1076S, K1093P, K1093Y, E739R, E739P, I310V, H712T, A16 7H, K1093R, V536P/F555E/G619E/W732K/ K799S/Y805Q, F555E/G619E/D801G, F555E/G619E/K799S, G619E/K799S/D801G/Y805Q, V536P/F555E/G619E/W732K/K799S, K799S/Y805Q, D801G, A246 D/G619E/K799S/D801G, V536P/ F555E/G619E, G619E/S839K, G619E/K799S/Y805Q, V536P/D801G/Y805Q, F555E/G619E/K799S/Y805Q, G619E/D801G/Y805Q, F555E/G619E, V536P/G619 E/D801G, D801G/Y805Q, V536P/ G619E/K799S/Y805Q, F555E/G619E/W732K/K799S/D801G/Y805Q, Y1281H, F749L, L614H, N616D, Q621K, I1050E, I610H, Q611D, R1042T, N616Q, L614N, T 370A/L614A, H1156D, K1210V, L614T, R1158N, Y1281S, A618E, F555E/G681N/Y737K/K799Q, V536P/G619E/E809G/E1109G, V536K/F555G/G619E/Y737K/K799Q/E809G/S839K/S916T/Q921R, S 916T/E1109G, V536R/G619E/G681N/ K799Q/S916T/E1109G, F555G/G619E/G681N/E807R/E1109G, F555E/G619E/G681N/Y737K/K799Q, G619E/G681N/K799S/S916T/E1109G, G619E/G681N, F 555G/Q921R, G619E/G681N/S916T, F555G/G619E/G681N/K799S, F555E, W732K, G619E/K799S, G619E, G681N, F555G/S916T, S839K or K799Q, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 1514.

In some embodiments, the recombinant Factor VIII polypeptide comprises a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2072 or at least 70%, 75%, 80% identical to a reference sequence corresponding to SEQ ID NO: 2072 , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98% or 99% or more sequence identity to an amino acid sequence, wherein the amino acid sequence comprises the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2072 or relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2072 One or more substitutions of the reference sequence of SEQ ID NO: 2072.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or set of substitutions at the following amino acid positions: 167, 167/738/1076/1093, 167/738/752/800/805 /808/809/1116, 167/800/809/1076, 167/318, 167/318/800/805/808/809, 167/318/681, 167/318/681/700/805/808/809 /1076/1116, 167/318/700, 167, 167/738/1076/1093, 167/738/752/800/805/808/809/1116, 167/800/809/1076, 167/318, 167 /318/800/805/808/809, 167/318/681, 167/318/681/700/805/808/809/1076/1116, 167/318/700, 167/318/700/1076, 167 /318/700/738/739/1076, 167/681/738/800/808/809, 167/178/700/752/1093, 167/700, 167/700/738/739/1093, 167/700 /738/752/1093, 167/700/738/752/805/808/809/1093/1116, 808/809, 1076, 738, 738/808/809, 738/1076, 738/739, 738/739 /809、738/739/809/1093/1116、738/739/1093、738/739/752/1093、738/739/799/800/1093、738/739/1116、738/800、738/1093 ,738/752, 738/752/1093, 738/752/799, 738/799/809/1093, 738/799/1093, 739, 739/800/809, 800, 800/805/809, 809, 809 /1093, 809/1093/1116, 809/1116, 318, 318/1076, 318/1076/1116, 318/739/805/808/809/1076, 318/499/700/738/809, 318/1093 ,318/752/1093, 318/1116, 318/543/739, 318/543/681/752/1116, 318/543/1093, 318/700/738/1093/1414, 681, 681/738, 681 /738/739/1093、681/738/739/752/799/800/809/1116/1339、681/738/739/1116、681/738/800、681/800/805/809、681/809 /1093、681/752/1093、681/752/1093、681/700、681/700/738/739/1093/1116、681/700/809、681/700/1093、681/700/752/808 /809/1093/1116, 681/700/752/1093, 681/700/844, 681/700/805/809, 1093, 1093/1116, 752, 752/1076, 752/800/809, 752/800 /1093、752/809、752/866、752/1093、752/1116、178/318/739/752/809/1076/1116、178/318/800/809、178/318/681/800/808 /809/1076, 178/681, 178/681/738/1116, 178/752/809, 178/700/800/805/808/809, 178/805/809/866, 1116, 543, 543/738 /739/809、543/738/809/1093、543/739/1093、543/800/1093/1116、543/809/1093、543/809/1093/1116、543/681/738/739/752 /809/1093、543/681/738/739/752/1093、543/681/739/752/800/805/809/1093、543/681/700/738/739/800/1093/1116、543 /681/700/738/739/805/808/809, 543/681/700/752, 543/681/700/752/809/1093, 543/1093, 543/700, 543/700/738/739 /752/800/1093/1116, 543/700/809, 543/700/1093, 543/700/752, 543/700/752/800/1093, 543/700/752/809/1116, 543/700 /752/1093, 700, 700/738/739/800/1093, 700/738/739/799/1093, 700/738/800/1093, 700/738/1116, 700/809, 700/809/1093 ,700/1093, 700/1093/1116, 700/752, 700/752/808/809/1093/1116, 700/752/809/1093, 700/752/799/1116, 700/752/805/809 /1093, 700/799/800, 700/1116, 700/805/808/809/1076, 514/543/681/805/808/809, 805/808/809 or 805/809/1116, among which Amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2072.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or substitution set 167G, 167G/738Y/1076S/1093G, 167G/738Y/752E/800V/805Q/808S/809G/1116I, 167G/ 800V/809G/1076S, 167G/318L, 167G/318L/800V/805Q/808S/809G, 167G/318L/681N, 167G/318L/681N/700E/805Q/808S/809G/1076S/1116I, 167G/ 318L/ 700E, 167G/318L/700E/1076S, 167G/318L/700E/738Y/739G/1076S, 167G/681N/738Y/800V/808S/809G, 167G/178Q/700E/752E/1093G, 167G/700E, 167G/ 700E/738Y/739G/1093G, 167G/700E/738Y/752E/1093G, 167G/700E/738Y/752E/805Q/808S/809G/1093A/1116I, 808S/809G, 1076S, 738Y, 738Y/808 S/809G, 738Y/1076S, 738Y/739G, 738Y/739G/809G, 738Y/739G/809G/1093G/1116I, 738Y/739G/1093G, 738Y/739G/752E/1093G, 738Y/739G/799S/800V/109 3G、738Y/ 739G/1116I, 738Y/800V, 738Y/1093G, 738Y/752E, 738Y/752E/1093G, 738Y/752E/799S, 738Y/799S/809G/1093G, 738Y/799S/1093G, 739G, 739G/800 V/809G, 800V, 800V/805Q/809G, 809G, 809G/1093G, 809G/1093G/1116I, 809G/1116I, 318L, 318L/1076S, 318L/1076S/1116I, 318L/739G/805Q/808S/809G/1 076S, 318L/ 499L/700E/738Y/809G, 318L/1093A, 318L/752E/1093A, 318L/1116I, 318L/543P/739G, 318L/543P/681N/752E/1116I, 318L/543P/1093A, 318L/700 E/738Y/ 1093A/1414N, 681N, 681N/738Y, 681N/738Y/739G/1093G, 681N/738Y/739G/752E/799S/800V/809G/1116I/1339C, 681N/738Y/739G/1116I, 681N/73 8Y/800V, 681N/800V/805Q/809G, 681N/809G/1093G, 681N/752E/1093A, 681N/752E/1093G, 681N/700E, 681N/700E/738Y/739G/1093G/1116I, 681N/700E/809 G、681N/ 700E/1093G, 681N/700E/752E/808S/809G/1093G/1116I, 681N/700E/752E/1093G, 681N/700E/844H, 681N/700E/805Q/809G, 1093A, 1093G, 1093G/1 116I, 752E, 17 8Q/318L/ 800V/809G, 178Q/318L/681N/800V/808S/809G/1076S, 178Q/681N, 178Q/681N/738Y/1116I, 178Q/752E/809G, 178Q/700E/800V/805Q/808S/809G, 1 78Q/ 805Q/809G/866A, 1116I, 543P, 543P/738Y/739G/809G, 543P/738Y/809G/1093G, 543P/739G/1093A, 543P/800V/1093G/1116I, 543P/809G/1093G, 54 3P/809G/ 1093G/1116I, 543P/681N/738Y/739G/752E/809G/1093G, 543P/681N/738Y/739G/752E/1093G, 543P/681N/739G/752E/800V/805Q/809G/1093G, 543 P/681N/ 700E/738Y/739G/800V/1093G/1116I, 543P/681N/700E/738Y/739G/805Q/808S/809G, 543P/681N/700E/752E, 543P/681N/700E/752E/809G/1093G, 543P/ 1093G, 543P/700E, 543P/700E/738Y/739G/752E/800V/1093G/1116I, 543P/700E/809G, 543P/700E/1093G, 543P/700E/752E, 543P/700E/752E/800V /1093G、 543P/700E/752E/809G/1116I, 543P/700E/752E/1093G, 700E, 700E/738Y/739G/800V/1093G, 700E/738Y/739G/799S/1093G, 700E/738Y/800V/1093 G、700E/ 738Y/1116I, 700E/809G, 700E/809G/1093G, 700E/1093G, 700E/1093G/1116I, 700E/752E, 700E/752E/808S/809G/1093G/1116I, 700E/752E/809G/1 093G、700E/ 752E/799S/1116I, 700E/752E/805Q/809G/1093A, 700E/799S/800V, 700E/1116I, 700E/805Q/808S/809G/1076S, 514L/543P/681N/805Q/808S/809G ,805Q/ 808S/809G or 805Q/809G/1116I, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2072.

In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or substitution set A167G, A167G/E738Y/E1076S/K1093G, A167G/E738Y/K752E/E800V/Y805Q/D808S/E809G/S1116I, A167G/ E800V/E809G/E1076S, A167G/G318L, A167G/G318L/E800V/Y805Q/D808S/E809G, A167G/G318L/G681N, A167G/G318L/G681N/S700E/Y805Q/D808S/E80 9G/E1076S/S1116I, A167G/G318L/ S700E, A167G/G318L/S700E/E1076S, A167G/G318L/S700E/E738Y/E739G/E1076S, A167G/G681N/E738Y/E800V/D808S/E809G, A167G/L178Q/S700E/K7 52E/K1093G, A167G/S700E, A167G/ S700E/E738Y/E739G/K1093G, A167G/S700E/E738Y/K752E/K1093G, A167G/S700E/E738Y/K752E/Y805Q/D808S/E809G/K1093A/S1116I, D808S/E809G, E1076S, E738Y, E738Y/D808S/E809G, E738Y/E1076S, E738Y/E739G, E738Y/E739G/E809G, E738Y/E739G/E809G/K1093G/S1116I, E738Y/E739G/K1093G, E738Y/E739G/K752E/K1093G, E738Y /E739G/K799S/E800V/K1093G, E738Y/ E739G/S1116I, E738Y/E800V, E738Y/K1093G, E738Y/K752E, E738Y/K752E/K1093G, E738Y/K752E/K799S, E738Y/K799S/E809G/K1093G, E738Y/K799S/ K1093G, E739G, E739G/E800V/E809G, E800V, E800V/Y805Q/E809G, E809G, E809G/K1093G, E809G/K1093G/S1116I, E809G/S1116I, G318L, G318L/E1076S, G318L/E1076S/S1116I, G318L/E73 9G/Y805Q/D808S/E809G/E1076S, G318L/ I499L/S700E/E738Y/E809G, G318L/K1093A, G318L/K752E/K1093A, G318L/S1116I, G318L/S543P/E739G, G318L/S543P/G681N/K752E/S1116I, G318L/ S543P/K1093A, G318L/S700E/E738Y/ K1093A/S1414N, G681N, G681N/E738Y, G681N/E738Y/E739G/K1093G, G681N/E738Y/E739G/K752E/K799S/E800V/E809G/S1116I/G1339C, G681N/E738Y /E739G/S1116I、G681N/E738Y/E800V、 G681N/E800V/Y805Q/E809G, G681N/E809G/K1093G, G681N/K752E/K1093A, G681N/K752E/K1093G, G681N/S700E, G681N/S700E/E738Y/E739G/K1093G/ S1116I, G681N/S700E/E809G, G681N/ S700E/K1093G, G681N/S700E/K752E/D808S/E809G/K1093G/S1116I, G681N/S700E/K752E/K1093G, G681N/S700E/R844H, G681N/S700E/Y805Q/E809G, K1093A, K1093G, K1093G/S1116I, K752E, K752E/E1076S, K752E/E800V/E809G, K752E/E800V/K1093G, K752E/E809G, K752E/G866A, K752E/K1093G, K752E/S1116I, L178Q/G318L/E739G/K752E/ E809G/E1076S/S1116I, L178Q/G318L/ E800V/E809G, L178Q/G318L/G681N/E800V/D808S/E809G/E1076S, L178Q/G681N, L178Q/G681N/E738Y/S1116I, L178Q/K752E/E809G, L178Q/S700E/E8 00V/Y805Q/D808S/E809G, L178Q/ Y805Q/E809G/G866A, S1116I, S543P, S543P/E738Y/E739G/E809G, S543P/E738Y/E809G/K1093G, S543P/E739G/K1093A, S543P/E800V/K1093G/S1116I ,S543P/E809G/K1093G,S543P/E809G/ K1093G/S1116I, S543P/G681N/E738Y/E739G/K752E/E809G/K1093G, S543P/G681N/E738Y/E739G/K752E/K1093G, S543P/G681N/E739G/K752E/E800V/ Y805Q/E809G/K1093G, S543P/G681N/ S700E/E738Y/E739G/E800V/K1093G/S1116I, S543P/G681N/S700E/E738Y/E739G/Y805Q/D808S/E809G, S543P/G681N/S700E/K752E, S543P/G681N/S7 00E/K752E/E809G/K1093G, S543P/ K1093G, S543P/S700E, S543P/S700E/E738Y/E739G/K752E/E800V/K1093G/S1116I, S543P/S700E/E809G, S543P/S700E/K1093G, S543P/S700E/K752E, S543P/S700E/K752E/E800V/K1093G, S543P/S700E/K752E/E809G/S1116I, S543P/S700E/K752E/K1093G, S700E, S700E/E738Y/E739G/E800V/K1093G, S700E/E738Y/E739G/K799S/K1093G, S700E/E738Y/E800V/K1093G, S700E/ E738Y/S1116I, S700E/E809G, S700E/E809G/K1093G, S700E/K1093G, S700E/K1093G/S1116I, S700E/K752E, S700E/K752E/D808S/E809G/K1093G/S111 6I, S700E/K752E/E809G/K1093G, S700E/ K752E/K799S/S1116I, S700E/K752E/Y805Q/E809G/K1093A, S700E/K799S/E800V, S700E/S1116I, S700E/Y805Q/D808S/E809G/E1076S, V514L/S543P/ G681N/Y805Q/D808S/E809G, Y805Q/ D808S/E809G or Y805Q/E809G/S1116I, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2072.

In some embodiments, a recombinant Factor VIII polypeptide comprises at least one of the peptides listed in Tables 4.1 , 4.2 , 5.1 , 5.2 , 6.1 , 6.2 , 7.1 , 7.2, 8.1 , 8.2 , 9.1 , 9.2 , 10.1 , 10.2 , 11.1 , and 15.1 Substituted amino acid sequences at set forth amine positions relative to the reference sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072.

In some embodiments, a recombinant Factor VIII polypeptide comprises at least one of the compounds described in Tables 4.1 , 4.2 , 5.1 , 5.2 , 6.1 , 6.2 , 7.1 , 7.2 , 8.1 , 8.2 , 9.1 , 9.2 , 10.1 , 10.2 , 11.1 , and 15.1 The amino acid sequence of the substitution set, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072.

In some embodiments, a recombinant Factor VIII polypeptide comprises at least one of the compounds described in Tables 4.1 , 4.2 , 5.1 , 5.2 , 6.1 , 6.2 , 7.1 , 7.2 , 8.1 , 8.2 , 9.1 , 9.2 , 10.1 , 10.2 , 11.1 , and 15.1 An amino acid sequence of a substitution or set of substitutions, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072.

In some embodiments, a recombinant Factor VIII polypeptide comprises at least one of the compounds described in Tables 4.1 , 4.2 , 5.1 , 5.2 , 6.1 , 6.2 , 7.1 , 7.2 , 8.1 , 8.2 , 9.1 , 9.2 , 10.1 , 10.2 , 11.1 , and 15.1 Amino acid sequences of substitutions or sets of substitutions of variants, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072.

4-4 -The amino acid sequence of the even-numbered SEQ ID NO. in 2346 and 2350-2870, wherein optionally, the amino acid sequence includes 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 Amino acid substitution, insertion or deletion. In some embodiments, the amino acid sequence optionally includes 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions. In some of the foregoing embodiments, the amino acid sequence optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 amino acid substitutions, or 1, 2, 3, 4 or 5 substitutions. In some embodiments, substitutions include non-conservative substitutions. In some embodiments, substitutions include conservative substitutions.

In some embodiments, a recombinant Factor VIII polypeptide comprises an amino acid sequence comprising residues 20-1457 of even-numbered SEQ ID NO. in SEQ ID NO: 4-316 or comprising one of SEQ ID NO: 4-316 Amino acid sequence of even-numbered SEQ ID NO. In some embodiments, a recombinant Factor VIII polypeptide comprises an amino acid sequence comprising residues 20-1457 of even-numbered SEQ ID NO. of SEQ ID NO: 318-410 or 412-666 or comprising SEQ ID NO: 318 -The amino acid sequence of the even-numbered SEQ ID NO. 410 or 412-666. In some embodiments, a recombinant Factor VIII polypeptide comprises an amino acid sequence comprising residues 20-1457 of even-numbered SEQ ID NO. of SEQ ID NO: 668-700 or 702-780 or comprising SEQ ID NO: 668 -The amino acid sequence of the even-numbered SEQ ID NO. 700 or 702-780. In some embodiments, a recombinant Factor VIII polypeptide comprises an amino acid sequence comprising residues 20-1457 of even-numbered SEQ ID NO. of SEQ ID NO: 782-818 or 820-934 or comprising SEQ ID NO: 782 -The amino acid sequence of the even-numbered SEQ ID NO. 818 or 820-934. In some embodiments, a recombinant Factor VIII polypeptide comprises an amino acid sequence comprising residues 20-1457 of even-numbered SEQ ID NO. of SEQ ID NO: 936-1028 or 1030-1250 or comprising SEQ ID NO: 936 -The amino acid sequence of the even-numbered SEQ ID NO. 1028 or 1030-1250. In some embodiments, a recombinant Factor VIII polypeptide comprises an amino acid sequence comprising residues 20-1457 of even-numbered SEQ ID NO. of SEQ ID NO: 1252-1408 or 1410-1588 or comprising SEQ ID NO: 1252 -The amino acid sequence of the even-numbered SEQ ID NO. 1408 or 1410-1588. In some embodiments, a recombinant Factor VIII polypeptide comprises an amino acid sequence comprising residues 20-1457 of even-numbered SEQ ID NO. of SEQ ID NO: 1590-1648 or 1650-2068 or comprising SEQ ID NO: 1590 -The amino acid sequence of an even-numbered SEQ ID NO. from 1648 or 1650-2068. In some embodiments, a recombinant Factor VIII polypeptide comprises an amino acid sequence comprising residues 20-1457 of even-numbered SEQ ID NO. in SEQ ID NO: 2070-2346 or comprising one of SEQ ID NO: 2070-2346 Amino acid sequence of even-numbered SEQ ID NO. In some embodiments, a recombinant Factor VIII polypeptide comprises an amino acid sequence comprising residues 20-1457 of even-numbered SEQ ID NO. in SEQ ID NO: 2360-2870 or comprising one of SEQ ID NO: 2350-2870 Amino acid sequence of even-numbered SEQ ID NO. In some embodiments, the amino acid sequence optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 amino acid substitutions, insertions, or deletions. In some embodiments, the amino acid sequence of the recombinant Factor VIII polypeptide optionally includes 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions. In some of the foregoing embodiments, the amino acid sequence optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 amino acid substitutions, or 1, 2, 3, 4 or 5 amino acid substitutions. In some embodiments, substitutions include non-conservative substitutions. In some embodiments, substitutions include conservative substitutions.

In some embodiments, a recombinant Factor VIII polypeptide comprises an amino acid sequence comprising residues 20-1457 of SEQ ID NO: 252, 504, 706, 840, 1200, 1514 or 2072 or comprising SEQ ID NO: 252, 504 , 706, 840, 1200, 1514 or 2072 amino acid sequences. In some embodiments, the amino acid sequence optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 amino acid substitutions, insertions, or deletions. In some embodiments, the amino acid sequence of the optionally recombinant Factor VIII polypeptide includes 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions. In some of the foregoing embodiments, the amino acid sequence optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 amino acid substitutions, or 1, 2, 3, 4 or 5 amino acid substitutions. In some embodiments, substitutions include non-conservative substitutions. In some embodiments, substitutions include conservative substitutions.

In some embodiments, recombinant Factor VIII polypeptides described herein have coagulation activity. In some embodiments, a recombinant Factor VIII polypeptide has improved properties compared to a reference Factor VIII polypeptide, wherein the reference Factor VIII polypeptide has a property corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072 The sequence of residues 20-1457 or the sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072.

In some embodiments, a recombinant Factor VIII polypeptide has increased activity compared to a reference Factor VIII polypeptide, wherein the reference Factor VIII polypeptide has an activity corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072 The sequence of residues 20-1457 or the sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072.

In some embodiments, with a sequence having residues 20-1457 corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072 or corresponding to SEQ ID NO: 2, 252, 504, Compared with the reference Factor VIII polypeptide of the sequence 706, 840, 1200, 1514 or 2072, the increased activity is coagulation activity, especially the increased potency.

In some embodiments, with a sequence having residues 20-1457 corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072 or corresponding to SEQ ID NO: 2, 252, 504, Compared with the reference Factor VIII polypeptide of the sequence 706, 840, 1200, 1514 or 2072, the recombinant Factor VIII polypeptide has increased stability to serum and/or plasma exposure, especially serum or plasma from hemophilia A patients.

In some embodiments, the recombinant Factor VIII polypeptide has a sequence corresponding to residues 20-1457 of SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072 at about pH 7.4 or corresponds to SEQ The reference Factor VIII polypeptide with the sequence of ID NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072 is more stable.

In some embodiments, the recombinant Factor VIII polypeptide has increased thermal stability compared to a reference Factor VIII polypeptide, wherein the reference Factor VIII polypeptide has a polypeptide corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or The sequence of residues 20-1457 of 2072 or the sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072.

In some embodiments, a recombinant Factor VIII polypeptide is characterized by having a sequence corresponding to residues 20-1457 of SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072 or corresponding to SEQ ID NO. : Compared with the reference Factor VIII polypeptide of the sequence 2, 252, 504, 706, 840, 1200, 1514 or 2072, the expression and/or secretion of cells transfected with the expression vector is increased.

In some embodiments, a recombinant Factor VIII polypeptide is characterized by having a sequence corresponding to residues 20-1457 of SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072 or corresponding to SEQ ID NO. : 2, 252, 504, 706, 840, 1200, 1514 or 2072 sequence of reference Factor VIII polypeptide compared to the transduction of cells transduced with gene therapy compositions (such as AAV, mRNA or lipid nanoparticles) and /or increased secretion.

In some embodiments, the improved properties are compared to a reference Factor VIII polypeptide having a sequence corresponding to residues 20-1457 of SEQ ID NO: 2 or a sequence corresponding to SEQ ID NO: 2.

In some embodiments, with a sequence having residues 20-1457 corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072 or having a sequence corresponding to SEQ ID NO: 2, 252, 504 , 706, 840, 1200, 1514 or 2072, the recombinant Factor VIII polypeptide of the present disclosure exhibits at least one improved property selected from the following: i) increased activity/potency; ii) increased Coagulation activity/potency; iii) increased plasma or serum stability; iv) increased thermal stability; v) reduced immunogenicity; vi) increased expression and/or secretion from cells transfected with the expression vector; vii) Increased transduction and/or secretion from cells transduced with gene therapy vectors (e.g. AAV, mRNA or lipid nanoparticles), or i), ii), iii), iv), v), vi) and/or a combination of any of vii). In some embodiments, the reference recombinant Factor VIII polypeptide has a sequence corresponding to residues 20-1457 of SEQ ID NO: 2 or has a sequence corresponding to SEQ ID NO: 2.

In some embodiments, the recombinant Factor VIII polypeptide is a "deimmunized" recombinant Factor VIII polypeptide (e.g., as presented in the examples in Tables 12.1 , 12.2 , 12.3 , 12.4 , 12.5 , 12.6 , 12.7 , 12.8 , and 15.2 ). In some embodiments, a recombinant Factor VIII polypeptide is characterized by a reduced total immunogenicity score (TIS) and/or immunogenic hit count (IHC) compared to a reference Factor VIII polypeptide, wherein the reference Factor VIII polypeptide has a property corresponding to Sequences of SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072, such as those provided in Tables 12.1 , 12.2 , 12.3 , 12.4 , 12.5 , 12.6 , 12.7 , 12.8 and 15.2 in the Examples.

In some embodiments, a "deimmunized" recombinant Factor VIII polypeptide comprises an amino acid sequence comprising residues 20-1457 or residues 1-1457 selected from the following sequence: SEQ ID NO: 4 ,6,8,10,12,14,16,18,20,26,28,32,34,36,38,40,42,44,46,48,50,52,54,56,60,64 ,66,68,72,74,76,78,82,84,86,88,90,92,94,96,98,100,102,104,106,108,110,112,114,116,118 ,120,122,130,132,134,136,138,140,142,146,148,150,152,154,156,158,160,162,164,166,168,170,172,174,176 ,178,180,182,184,186,188,190,192,194,196,198,200,202,204,206,208,210,212,214,216,218,220,222,224,226 ,228,230,232,234,236,238,242,244,248,252,260,262,266,270,274,278,280,282,284,286,288,290,292,296,298 ,300,302,310,312,320,322,324,326,328,330,332,334,336,338,340,342,344,346,348,350,352,354,356,358,360 ,362,364,366,368,370,372,374,376,378,380,382,384,386,388,390,392,394,396,398,400,402,404,406,408,410 ,412,414,416,418,420,424,426,428,430,432,434,436,438,442,444,446,448,452,454,456,458,460,462,464,466 ,468,470,472,474,476,478,480,482,484,486,488,490,492,498,500,502,504,506,508,510,512,514,516,518,520 ,522,524,526,530,532,534,536,538,540,542,544,546,548,550,552,554,556,558,560,562,564,566,568,570,574 ,578,580,582,584,586,588,590,592,594,596,598,600,602,604,606,608,610,612,614,618,620,622,624,626,628 ,630,632,634,636,638,640,642,644,646,648,650,652,654,656,658,660,662,664,666,668,670,672,674,676,678 ,680,682,684,686,688,690,692,694,696,698,700,702,704,706,708,710,712,714,716,718,720,722,724,728,730 ,732,734,736,738,740,742,744,746,748,750,752,754,756,758,760,762,764,766,768,770,772,774,776,778,780 ,782,784,786,788,790,792,794,796,798,800,802,804,806,808,810,812,814,816,818,820,822,824,826,828,830 ,832,834,836,838,840,842,844,846,848,850,852,854,856,858,860,862,864,868,876,878,880,882,884,886,890 ,892,896,900,904,906,908,910,912,914,922,932,934,936,938,940,942,944,946,948,952,956,958,960,962,964 ,966,970,974,976,978,980,982,986,988,990,996,998,1002,1006,1008,1012,1014,1018,1020,1022,1024,1026,1028,1030,1032 ,1034,1036,1038,1040,1042,1044,1046,1048,1050,1052,1054,1056,1058,1064,1066,1068,1070,1072,1074,1076,1078,1080,1082,1084,10 86 ,1088,1090,1092,1096,1098,1100,1102,1104,1108,1110,1112,1114,1116,1120,1124,1126,1128,1130,1132,1134,1136,1138,1140,1142,11 44 ,1146,1148,1150,1152,1154,1156,1158,1160,1162,1164,1166,1168,1170,1172,1174,1176,1178,1180,1182,1184,1186,1188,1190,1192,11 94 ,1196,1198,1200,1202,1204,1206,1208,1210,1212,1214,1216,1218,1220,1222,1224,1226,1228,1232,1234,1236,1238,1240,1242,1244,12 46 ,1248,1250,1252,1254,1256,1258,1260,1262,1264,1266,1268,1270,1272,1274,1276,1278,1280,1286,1300,1306,1310,1314,1316,1320,13 22 ,1324,1326,1328,1330,1332,1334,1336,1344,1352,1356,1368,1370,1372,1380,1384,1386,1388,1390,1392,1394,1400,1406,1410,1412,14 14 ,1416,1418,1420,1422,1424,1426,1428,1430,1432,1434,1436,1438,1440,1442,1444,1446,1448,1450,1452,1454,1456,1458,1460,1462,14 64 ,1466,1468,1470,1472,1474,1476,1478,1480,1482,1484,1486,1488,1490,1492,1494,1496,1498,1500,1502,1504,1506,1508,1510,1512,15 14 ,1516,1518,1520,1522,1524,1526,1528,1530,1532,1534,1536,1540,1550,1552,1554,1564,1568,1570,1584,1590,1592,1594,1596,1598,16 00 ,1602,1604,1606,1608,1610,1612,1614,1616,1618,1620,1622,1624,1626,1628,1630,1632,1634,1636,1638,1640,1642,1644,1646,1648,16 50 ,1654,1658,1660,1662,1664,1666,1670,1676,1690,1694,1698,1706,1710,1712,1714,1720,1724,1726,1728,1732,1734,1740,1746,1748,17 50 ,1752,1754,1756,1760,1762,1772,1778,1782,1784,1786,1788,1792,1794,1796,1798,1806,1810,1814,1820,1826,1846,1854,1858,1860,18 68 , 1870, 1872, 1878, 1880, 1882, 1898, 1906, 1908, 1910, 1920, 1924, 1926, 1930, 1932, 1940, 1942, 1950, 1954, 1956, 1958, 1960, 1962, 1964, 1966, 19 70 ,1972,1974,1976,1978,1980,1982,1984,1986,1990,1992,1994,1998,2000,2002,2004,2006,2008,2010,2012,2014,2016,2018,2020,2022,20 24 , 2026, 2028, 2030, 2032, 2034, 2038, 2040, 2042, 2044, 2046, 2050, 2052, 2054, 2056, 2058, 2060, 2064, 2066, 2068, 2070, 2098, 2102, 2104, 2106, 21 22 ,2138,2142,2190,2204,2206,2210,2212,2214,2216,2220,2226,2228,2234,2236,2242,2250,2252,2254,2266,2268,2272,2276,2278,2280,22 82 ,2284,2288,2290,2302,2306,2308,2310,2314,2316,2318,2320,2322,2324,2326,2330,2334,2336,2338,2340,2370,2372,2374,2380,2382,23 86 ,2390,2392,2394,2396,2398,2402,2406,2408,2412,2420,2422,2432,2436,2440,2446,2448,2450,2454,2456,2460,2464,2466,2468,2472,24 76 ,2478,2484,2486,2488,2494,2496,2498,2500,2504,2506,2510,2518,2520,2526,2530,2534,2542,2544,2554,2556,2560,2562,2564,2570,25 74 ,2576,2578,2586,2588,2590,2592,2594,2598,2600,2602,2604,2610,2614,2618,2622,2628,2632,2638,2644,2646,2648,2650,2652,2654,26 56 ,2662,2664,2672,2674,2676,2682,2684,2688,2690,2694,2702,2704,2708,2710,2712,2714,2728,2734,2736,2738,2742,2748,2750,2754,27 60 ,2762,2764,2766,2768,2770,2774,2776,2778,2780,2782,2784,2790,2794,2798,2800,2802,2804,2806,2808,2810,2818,2820,2824,2826,28 28 , 2830, 2832, 2834, 2836, 2840, 2842, 2844, 2848, 2850, 2852, 2854, 2856, 2858, 2860, 2862, 2864, 2866, 2868 or 2870.

In some embodiments, for any of the recombinant Factor VIII polypeptides disclosed herein, the recombinant Factor VIII polypeptide is purified or is a purified preparation.

In some embodiments, a recombinant Factor VIII polypeptide comprises a full-length recombinant Factor VIII polypeptide of any of the recombinant Factor VIII polypeptides described herein. In some embodiments, a recombinant Factor VIII polypeptide comprises a "preproprotein" or "prepropolypeptide" of any of the recombinant Factor VIII polypeptides described herein. In some embodiments, the signal sequence of a full-length recombinant Factor VIII polypeptide or "preproprotein" or "prepropolypeptide" can comprise any compatible signal sequence, including synthetic, mouse (eg, IgG), or human signal sequences. In some embodiments, a signal sequence present in an exemplary recombinant Factor VIII polypeptide is replaced with a compatible signal sequence (eg, synthetic or mouse). In some embodiments, the signal sequence comprises a signal sequence in a naturally occurring human Factor VIII polypeptide.

In some embodiments, the recombinant Factor VIII polypeptide is processed or cleaved, for example, into a pro-polypeptide or pro-protein form lacking a signal sequence. In some embodiments, the recombinant Factor VIII polypeptide is processed or cleaved to the mature form of the recombinant Factor VIII polypeptide, such as described herein. In some embodiments, mature forms of recombinant Factor VIII polypeptides comprise active forms of recombinant Factor VIII polypeptides, particularly polypeptides that interact with FIX (e.g., equivalent forms of the light and heavy chains of Factor VIII) (see, e.g., Orlova et al. Human, Acta Naturae, 2013, 5(2): 19-39:3983-3996; Lenting et al., Blood, 1998, 92(11); incorporated herein by reference).

Therefore, for each recombinant Factor VIII polypeptide described herein, the present disclosure provides the pre-polypeptide, pro-polypeptide or mature biologically active form of the recombinant Factor VIII polypeptide, including Tables 4.1 , 4.2 , 5.1 , 5.2 , 6.1 , 6.2 , 7.1 , 7.2 , 8.1 , 8.2 , 9.1 , 9.2 , 10.1 , 10.2 , 11.1 and 15.1 each of the recombinant Factor VIII polypeptides disclosed.

In some embodiments, the present disclosure further provides functional or biologically active fragments of recombinant Factor VIII polypeptides described herein. In some embodiments, the functional or biologically active fragment has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In some embodiments, the functional or biologically active fragment comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, of the parental sequence of the recombinant Factor VIII polypeptide. 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In some embodiments, the functional fragments are truncated to less than 5, less than 10, less than 15, less than 10, less than 25, less than 30, less than 35, less than 40 , less than 45 and less than 50 amino acids. Exemplary biological or functional fragments of recombinant Factor VIII polypeptides include the amino acid sequence comprising SEQ ID NO: 2350, 2352, 2354, 2356, or 2358, the activity of which is shown in Figure 3 .

In some embodiments, functional or biologically active fragments of recombinant Factor VIII polypeptides described herein include at least one mutation or set of mutations in the amino acid sequence of recombinant Factor VIII described herein. Thus, in some embodiments, a functional or biologically active fragment of recombinant Factor VIII exhibits enhanced or improved properties associated with a mutation or set of mutations in the parent recombinant Factor VIII polypeptide.

In some other embodiments, as discussed herein, functional fragments or biologically active fragments encompass biologically processed polypeptides of recombinant Factor VIII polypeptides, such as after expression in mammalian cells or in a patient or when the recombinant Factor VIII polypeptide is administered to a patient Functional or biologically active fragments produced. In some embodiments, the biologically processed functional or biologically active fragment of the recombinant Factor VIII polypeptide resulting from one or more cleavages occurs between residues 760 to 773, and in some embodiments, the additional one or more Cleavage occurs between residues 391 and 392, where these positions are relative to the reference sequence of SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072. In some embodiments, functional or biologically active fragments are derived from recombinants presented in Tables 4.1 , 4.2 , 5.1 , 5.2 , 6.1, 6.2 , 7.1 , 7.2 , 8.1 , 8.2 , 9.1 , 9.2 , 10.1 , 10.2 , 11.1 , and 15.1 Factor VIII polypeptide. In some embodiments, the functional or biologically active fragment is derived from a recombinant Factor VIII polypeptide comprising an amino acid sequence of an even-numbered SEQ ID NO. selected from SEQ ID NOs: 4-2346 and 2350-2870. Polynucleotides, expression vectors and host cells encoding recombinant polypeptides

In another aspect, the present disclosure provides polynucleotides encoding engineered Factor VIII polypeptides described herein. In some embodiments, a polynucleotide is operably linked to one or more heterologous or homologous regulatory sequences that control gene expression to produce a recombinant polynucleotide capable of expressing a polypeptide. Expression constructs containing heterologous polynucleotides encoding recombinant Factor VIII polypeptides can be introduced into appropriate host cells to express the corresponding recombinant Factor VIII polypeptides.

As will be apparent to those skilled in the art, the availability of protein sequences and knowledge of the codons corresponding to various amino acids provide a description of all polynucleotides capable of encoding the polypeptides of the invention. The simplicity of the genetic code, in which identical amino acids are encoded by alternative or synonymous codons, allows the production of extremely large numbers of nucleic acids, all of which encode recombinant Factor VIII polypeptides. Thus, given knowledge of a particular amino acid sequence, one skilled in the art can make any number of different codons by simply modifying the sequence of one or more codons in a manner that does not alter the amino acid sequence of the protein. nucleic acids. In this regard, the present invention specifically considers every possible variation in the polynucleotides that can be made that encodes the polypeptides described herein by selecting combinations based on possible codon options, and all such variations will be deemed to be specifically disclosed for any polypeptide described herein, including Tables 4.1 , 4.2 , 5.1 , 5.2 , 6.1 , 6.2 , 7.1 , 7.2, 8.1 , 8.2 , 9.1 , 9.2 , 10.1 , 10.2 , 11.1 and 15.1 Variants provided in .

In some embodiments, it is preferred to select codons that are appropriate for the host cell in which the protein is being produced. For example, the preferred codon systems for use in bacteria are for expression in bacteria, the preferred codon systems for fungi are typically for expression in fungi, and the preferred codon systems for mammals are Sublines are designed for expression in mammals and mammalian cells. In some embodiments, the codon-optimized polynucleotide encoding the engineered factor polypeptide is located at about 40%, 50%, 60%, 70%, 80%, or 40% of the codon positions of the full-length coding region. More than 90% of the sites contain better codons. In some embodiments, the invention provides recombinant polynucleotide sequences in which codons are optimized for expression in human cells or tissues.

As discussed above, it is to be understood that the present disclosure provides recombinant polynucleotides encoding each of the recombinant Factor VIII polypeptides described herein, or biologically or functionally active fragments thereof.

Thus, by way of example and not limitation, in some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide or a biologically active recombinant Factor VIII fragment thereof, which includes and corresponds to SEQ ID NO: 2. A reference sequence for residues 20-1457 of 252, 504, 706, 840, 1200, 1514 or 2072 or a reference sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072 Have at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, An amino acid sequence with 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity, wherein the amino acid sequence comprises an amino acid sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072 of the reference sequence or one or relative to the reference sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072 Multiple substitutions.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2 or corresponding to The reference sequence of SEQ ID NO: 2 has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, An amino acid sequence with 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity, wherein the amino acid sequence contains an amino acid sequence corresponding to SEQ. The reference sequence for residues 20-1457 of ID NO: 2 or one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising a sequence corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072 The reference sequence of residues 20-1457 or has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or an amino acid sequence with greater sequence identity, wherein the amino acid sequence comprises the reference sequence relative to residues 20-1457 corresponding to SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2 One or more substitutions of the reference sequence.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising an even numbered SEQ ID corresponding to SEQ ID NOs. 4-2346 and 2360-2870. The reference sequence of residues 20-1457 of SEQ ID NO. or has at least 70%, 75%, 80%, or 81% similarity with the reference sequence corresponding to the even-numbered SEQ ID NO. in SEQ ID NO. 4-2346 and 2350-2870 , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 % or 99% or more sequence identity to an amino acid sequence, wherein the amino acid sequence comprises the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2 : One or more substitutions of the reference sequence in 2.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising an amino acid sequence comprising at least one substitution at the following amino acid position: 13 ,14,15,16,17,18,19,20,21,24,37,56,57,58,59,61,63,64,65,85,96,104,110,119,127,132 ,158,160,164,167,168,171,175,178,186,187,205,210,211,212,215,225,246,257,260,265,281,292,294,301,303 ,304,306,308,310,311,312,318,320,321,325,328,332,334,335,344,346,347,349,351,353,354,355,356,358,359 ,362,370,387,388,390,392,393,397,407,428,429,433,435,436,481,487,488,489,491,498,499,503,514,527,533 ,534,535,536,538,543,550,551,555,558,569,580,581,584,610,611,612,613,614,615,616,617,618,619,620,621 ,622,643,646,650,665,667,676,680,681,682,683,684,686,693,696,698,700,706,707,708,712,716,724,726,729 ,731,732,734,735,736,737,738,739,740,744,746,747,748,749,750,751,752,753,754,755,756,757,759,770,773 ,775,785,786,787,790,791,792,793,794,795,797,798,799,800,801,802,805,806,807,808,809,810,811,812,815 ,816,817,818,819,821,822,824,825,826,828,830,831,836,837,838,839,840,841,844,866,873,879,892,896,897 ,903,907,914,916,917,918,919,921,922,923,924,926,929,938,939,945,948,951,968,999,1037,1040,1041,1042,1043 ,1049,1050,1051,1053,1054,1076,1078,1080,1084,1088,1089,1092,1093,1094,1095,1098,1099,1100,1101,1104,1108,1109,1113,1114,11 16 ,1127,1129,1138,1145,1148,1151,1156,1158,1160,1186,1192,1193,1194,1205,1210,1218,1220,1221,1222,1224,1229,1245,1248,1250,12 59 , 1264, 1269, 1270, 1274, 1281, 1283, 1292, 1297, 1339, 1352, 1373, 1377, 1378, 1380, 1389, 1414, 1422, 1427, 1428, 1439, 1440, 1442 or 1452 or combinations thereof, The amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising at least one substitution or amino acid residue 13G/Y, 14G/M/V , 15G/S, 16A/D/G/P/V, 17S/V, 18L/R, 19G, 20L, 21M, 24V, 37H, 56E/P, 57A/L/S/T/V, 58K, 59D /G/L/M/Q/R/S/V/W, 61Q/S, 63C/E/F/G/H/I/K/L/M/N/Q/R/S/T/W /Y, 64D/G, 65A, 85Q, 96I/L/R/V/W, 104S, 110H, 119G/V, 127V, 132P/R, 158G, 160E/T, 164E, 167G/H/R, 168G /R, 171A/D/E/F/H/K/N/M/P/Q/R/S/T/V/W/Y, 175A/G/H, 178C/M/Q/V/W , 186A, 187C/G, 205R, 210P, 211G/K/P/Q/R, 212A/F/G/K/L/M/N/P/R/S/V/Y, 215E/P/S , 225R, 246D, 257I, 260E/K/T, 265R/S/W, 281A/S, 292D, 294G, 301K/M, 303G/T, 304A/E/G, 306A/D/G/P/S /T, 308P, 310G/V, 311E/G, 312L, 318C/G/K/M/Q/R/T/V, 320G/I/L/P/Q/R/S/T/V, 321Y , 325Y, 328G/Q/S, 332A/F/G/H/N/P/Q/R/T/V/W/Y, 334G, 335E/P, 344A/E/L, 346R/V, 347N , 349G, 351S, 353S, 354R, 355C, 356L/N, 358K/S, 359E/G, 362E/G/N/P/T/V/W, 370A, 387A/G/I/L/Q/R /S/T/V/W, 388A/P, 390A/E/G/L/M/P/S/T/V, 392A, 393A/F/G/H/M/Q/R/W/Y , 397G/N, 407V, 428F, 429L/S, 433A/G, 435Q, 436D/G, 481V, 487N, 488G, 489E/F/Q/R, 491E, 498A, 499C/L, 503S, 514L, 527A , 533R/V/Y, 534F/I/L/M, 535C/G/N, 536A/G/K/L/M/P/R/W, 538V, 543G/L/P/W, 550G, 551W , 555E/G/R/T, 558A/E/G/L/P/Q/R/S/T/V/W, 569T, 580S/Y, 581K/V, 584A, 610H/K/L/R /S/T, 611D/E, 612G/S/T, 613S, 614A/F/H/I/N/T, 615D/N, 616D/E/G/Q/W, 617D/E/G/N , 618C/E/G/P/Y, 619E, 620N/P, 621K/M/R/T/W, 622D, 643N, 646I, 650M, 665G, 667I, 676Q, 680L/R, 681A/C/E /F/G/L/N/R/Q/S/T/W, 682R, 683W, 684G/K, 686W, 693T, 696E, 698D/S/W/Y, 700A/E/Q/T/V , 706A/G/M/Y, 707H, 708A/K/T/V, 712G/L/M/Q/S/T/Y, 716K/L/W, 724I, 726A/L, 729T, 731I/L /R, 732C/D/E/G/L/R/W/K/M/Q/S/T/V/Y, 734I/R/S, 735D/S/V, 736F/G, 737F/G /K/L/M/N/S/V, 738A/D/E/F/H/K/N/P/R/S/T, 739G/P/R, 740G, 744A/R, 746P, 747D , 748F/G/K/N/S/V, 749A/C/D/F/G/I/K/L/M/P/Q/R/S/T/V/Y, 750A/D/G /K/M/N/P/Q/R/S/T/V/W, 751A/D/E/Y, 752D/E/F/G/L/M/Q/R/S/V, 753D , 754W, 755G/L/P/Q/S/Y, 756G/P/R/T, 757A/G/K/T, 759S, 770W, 773H, 775E/G/K/N/P/S/V , 785V, 786C/D/L, 787W, 790A/G/I/M/P/W, 791M/T, 792H/W/Y, 793G, 794R, 795C/N/S/T, 797C/G/P /Q/T/W/Y, 798C/R/T, 799A/E/G/M/P/Q/R/S/V, 800C/G/I/L/M/N/P/Q/R /T/V/W/Y, 801A/G/I/R, 802S/V, 805C/G/H/K/L/M/P/Q/R/S/V/W, 806E/G/R /S, 807C/L/P/R, 808K/S, 809A/C/G/I/K/L/N/Q/R/S/T/V/W, 810V, 811F, 812G/I/V , 815I, 816V/W, 817A/D, 818A/E/G/P/S/V/W, 819V, 821Q, 822S/T, 824W, 825L, 826G, 828K, 830M/V, 831W, 836G/R /V/W, 837G, 838G/T, 839A/G/K/P, 840G, 841W, 844H, 866A, 873L/Q/T, 879Q, 892T, 896L/V, 897G/K/R, 903E, 907D , 914V, 916C/E/I/K/L/M/Q/T, 917G, 918M/S, 919G, 921R/W, 922W, 923M, 924I/M/P/Q/W, 926T/V, 929A /Q/V, 938R/V, 939E/S, 945K, 948S, 951V, 968A/P, 999R, 1037H/M/N/P, 1040E, 1041G/H/N, 1042A/E/T, 1043E/R , 1049G/W, 1050E/P/Q/S/T, 1051E/H/K/R/S/W, 1053A/R, 1054C, 1076G/S, 1078S, 1080G/V, 1084L/N, 1088G/S , 1089F/S, 1092G, 1093A/E/G/M/N/P/Q/R/S/T/V/W/Y, 1094S, 1095G, 1098G, 1099G, 1100I/S, 1101G, 1104C/E , 1108M/S/V/W, 1109G, 1113F, 1114A/M, 1116A/G/I/L/R, 1127L/V, 1129P, 1138Q, 1145R, 1148A/C/E/L/M/N/Q /R/S, 1151S, 1156D/G/N/R, 1158N, 1160R, 1186E, 1192D, 1193A/E/G/I/L, 1194P, 1205L, 1210M/T/V/W, 1218A/G/L /M/P, 1220D/E/L/T, 1221G/S/T/V, 1222F, 1224P, 1229D, 1245R, 1248A/C/D/E/F/G/H/I/K/M/P /Q/R/S/T/W/Y, 1250A/P/S/T, 1259T, 1264L/R/W, 1269F, 1270V, 1274N/V, 1281H/K/P/Q/R/S, 1283E /H/L/S/T, 1292Y, 1297A, 1339C, 1352E, 1373S, 1377A/C/D/E/G/H/I/M/P/R/S/V/T/Y, 1378A/C /D/E, 1380A/G/W, 1389W, 1414A/C/D/E/G/H/N/P/T/W, 1422I, 1427Y, 1428G, 1439A/E/H/L/S/T , 1440A/C/E/G/S/T, 1442T or 1452L, or the amino acid sequence of a combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising an amino acid sequence comprising at least one substitution at the following amino acid position: 63 , 171, 212, 260, 306, 317, 320, 332, 387, 390, 393, 534, 558, 681, 732, 738, 749, 750, 775, 1148, 1248, 1377 or 1439 or a combination thereof, wherein the Amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising at least one substitution or amino acid residue 63C/E/F/G/H /I/K/L/M/N/Q/R/S/T/W/Y, 171A/D/E/F/H/K/N/M/P/Q/R/S/T/V /W/Y, 212A/F/G/L/K/M/N/P/R/S/V/Y, 260E/K/T, 306A/D/G/P/S/T, 318C/G /K/L/M/Q/R/T/V, 320G/I/L/P/Q/R/S/T/V, 332A/F/G/H/N/P/Q/R/T /V/W/Y, 387A/G/I/L/Q/R/S/T/V/W, 390A/E/G/L/M/P/S/T/V, 393A/F/G /H/M/Q/R/W/Y, 499C/L, 534I/F/L/M, 558A/E/G/L/P/Q/R/S/T/V/W, 681A/C /E/F/G/L/N/R/Q/S/T/W, 700A/E/Q/T/V, 732C/D/E/G/L/R/W/K/M/Q /S/T/V/Y, 738A/D/E/F/H/K/N/P/R/S/T/Y, 749A/C/D/F/G/I/K/L/M /P/Q/R/S/T/V/Y, 750A/D/G/K/M/N/P/Q/R/S/T/V/W, 752D/E/F/G/L /M/Q/R/S/V, 775E/G/K/N/P/S/V, 800C/G/I/L/M/N/P/Q/R/T/V/W/Y , 805C/G/H/K/L/M/P/Q/R/S/V/W, 809A/C/G/I/K/L/N/Q/R/S/T/V/W , 1093A/E/G/M/N/P/Q/R/S/T/V/W/Y, 1148A/C/E/L/M/N/Q/R/S, 1248A/C/D /E/F/G/H/I/K/M/P/Q/R/S/T/W/Y, 1377A/C/D/E/G/H/I/M/P/R/S /V/T/Y, 1414A/C/D/E/G/H/N/P/T/W or 1439A/E/H/L/S/T or the amino acid sequence of a combination thereof, wherein Amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising an amino acid comprising at least one substitution or set of substitutions at the following amino acid positions: Sequence: 1148, 85/708/1148, 798, 706, 171, 335, 63, 344, 428, 879, 59, 429, 205, 1264, 797, 1248, 873, 792, 708, 57, 96, 362, 1292, 346, 433, 65, 795, 684/798, 353, 332, 1259, 356, 436, 1245, 1145, 716, 56, 773, 734, 358, 729, 665, 359, 61, 1250, 726, 37/96/171/429/708/799/1148, 171/429/732/1148, 96/171, 171/919/1151, 171/792/797/799/1148, 171/569/1148, 96/ 171/429/792/797/799, 171/429/708/1422, 171/429, 321/362/732/797/799, 171/1100, 708/1148/1186, 429/724/732/1148, 171/362/795/797/799, 171/429/732, 96/171/643/797/799, 171/792/797/799, 503, 171/732, 1148/1193, 96/171/362/ 429/732/795/797, 334, 732, 351, 1377, 999, 306, 349, 257, 646, 435 or 58, where these positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising a polynucleotide sequence contained in Tables 4.1 , 4.2 , 5.1 , 5.2 , 6.1 , 6.2 , 7.1 , 7.2 , Substituted amino acid sequences at the amino acid positions set forth in 8.1 , 8.2 , 9.1 , 9.2 , 10.1 , 10.2 , 11.1 and 15.1 , wherein the amino acid positions are relative to those corresponding to SEQ ID NO: 2 the reference sequence.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising at least one of Tables 4.1 , 4.2 , 5.1 , 5.2 , 6.1 , 6.2 , 7.1 , 7.2 , 8.1 , 8.2 , 9.1 , 9.2 , 10.1 , 10.2 , 11.1 and 15.1 , wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising at least one of the peptides in Tables 4.1 , 4.2 , 5.1 , 5.2 , 6.1 , 6.2 , 7.1 , Amino acid sequences of substitutions or sets of substitutions at the amine positions set forth in 7.2 , 8.1 , 8.2 , 9.1 , 9.2 , 10.1 , 10.2 , 11.1 and 15.1 , wherein the amino acid positions are relative to those corresponding to SEQ ID NO: 2 of this reference sequence.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising at least one of Tables 4.1 , 4.2 , 5.1 , 5.2 , 6.1 , 6.2 , 7.1 , 7.2 , 8.1 , 8.2 , 9.1 , 9.2 , 10.1 , 10.2 , 11.1 and 15.1 , amino acid sequences of substitutions or sets of substitutions of the variants set forth in SEQ ID NO: 2 the reference sequence.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising a sequence corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072 The reference sequence of residues 20-1457 or has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or amino acid sequences with greater sequence identity.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising an even numbered SEQ ID corresponding to SEQ ID NOs. 4-2346 and 2360-2870 The reference sequence of residues 20-1457 of SEQ ID NO. or has at least 70%, 75%, 80%, or 81% similarity with the reference sequence corresponding to the even-numbered SEQ ID NO. in SEQ ID NO. 4-2346 and 2350-2870 , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 % or amino acid sequences with 99% or more sequence identity.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising a sequence corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072 The reference sequence of residues 20-1457 or has at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more Sequence identity amino acid sequence, wherein the amino acid sequence comprises relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, 504, 706, 840, 1200, 1514 or 2072 or relative to One or more substitutions of the reference sequence corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514 or 2072.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising an amino acid sequence comprising at least one substitution at the following amino acid position: 13 ,14,15,16,17,18,19,20,21,24,37,56,57,58,59,61,63,64,65,85,96,104,110,119,127,132 ,158,160,164,167,168,171,175,178,186,187,205,210,211,212,215,225,246,257,260,265,281,292,294,301,303 ,304,306,308,310,311,312,318,320,321,325,328,332,334,335,344,346,347,349,351,353,354,355,356,358,359 ,362,370,387,388,390,392,393,397,407,428,429,433,435,436,481,487,488,489,491,498,499,503,514,527,533 ,534,535,536,538,543,550,551,555,558,569,580,581,584,610,611,612,613,614,615,616,617,618,619,620,621 ,622,643,646,650,665,667,676,680,681,682,683,684,686,693,696,698,700,706,707,708,712,716,724,726,729 ,731,732,734,735,736,737,738,739,740,744,746,747,748,749,750,751,752,753,754,755,756,757,759,770,773 ,775,785,786,787,790,791,792,793,794,795,797,798,799,800,801,802,805,806,807,808,809,810,811,812,815 ,816,817,818,819,821,822,824,825,826,828,830,831,836,837,838,839,840,841,844,866,873,879,892,896,897 ,903,907,914,916,917,918,919,921,922,923,924,926,929,938,939,945,948,951,968,999,1037,1040,1041,1042,1043 ,1049,1050,1051,1053,1054,1076,1078,1080,1084,1088,1089,1092,1093,1094,1095,1098,1099,1100,1101,1104,1108,1109,1113,1114,11 16 ,1127,1129,1138,1145,1148,1151,1156,1158,1160,1186,1192,1193,1194,1205,1210,1218,1220,1221,1222,1224,1229,1245,1248,1250,12 59 , 1264, 1269, 1270, 1274, 1281, 1283, 1292, 1297, 1339, 1352, 1373, 1377, 1378, 1380, 1389, 1414, 1422, 1427, 1428, 1439, 1440, 1442 or 1452 or combinations thereof, The amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514 or 2072.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising at least one substitution or amino acid residue 13G/Y, 14G/M/V , 15G/S, 16A/D/G/P/V, 17S/V, 18L/R, 19G, 20L, 21M, 24V, 37H, 56E/P, 57A/L/S/T/V, 58K, 59D /G/L/M/Q/R/S/V/W, 61Q/S, 63C/E/F/G/H/I/K/L/M/N/Q/R/S/T/W /Y, 64D/G, 65A, 85Q, 96I/L/R/V/W, 104S, 110H, 119G/V, 127V, 132P/R, 158G, 160E/T, 164E, 167G/H/R, 168G /R, 171A/D/E/F/H/K/N/M/P/Q/R/S/T/V/W/Y, 175A/G/H, 178C/M/Q/V/W , 186A, 187C/G, 205R, 210P, 211G/K/P/Q/R, 212A/F/G/L/K/M/N/P/R/S/V/Y, 215E/P/S , 225R, 246D, 257I, 260E/K/T, 265R/S/W, 281A/S, 292D, 294G, 301K/M, 303G/T, 304A/E/G, 306A/D/G/P/S /T, 308P, 310G/V, 311E/G, 312L, 318C/G/K/L/M/Q/R/T/V, 320G/I/L/P/Q/R/S/T/V , 321Y, 325Y, 328G/Q/S, 332A/F/G/H/N/P/Q/R/T/V/W/Y, 334G, 335E/P, 344A/E/L, 346R/V , 347N, 349G, 351S, 353S, 354R, 355C, 356L/N, 358K/S, 359E/G, 362E/G/N/P/T/V/W, 370A, 387A/G/I/L/Q /R/S/T/V/W, 388A/P, 390A/E/G/L/M/P/S/T/V, 392A, 393A/F/G/H/M/Q/R/W /Y, 397G/N, 407V, 428F, 429L/S, 433A/G, 435Q, 436D/G, 481V, 487N, 488G, 489E/F/Q/R, 491E, 498A, 499C/L, 503S, 514L , 527A, 533R/V/Y, 534I/F/L/M, 535C/G/N, 536A/G/K/L/M/P/R/W, 538V, 543G/L/P/W, 550G , 551W, 555E/G/R/T, 558A/E/G/L/P/Q/R/S/T/V/W, 569T, 580S/Y, 581K/V, 584A, 610H/K/L /R/S/T, 611D/E, 612G/S/T, 613S, 614A/F/H/I/N/T, 615D/N, 616D/E/G/Q/W, 617D/E/G /N, 618C/E/G/P/Y, 619E, 620N/P, 621K/M/R/T/W, 622D, 643N, 646I, 650M, 665G, 667I, 676Q, 680L/R, 681A/C /E/F/G/L/N/R/Q/S/T/W, 682R, 683W, 684G/K, 686W, 693T, 696E, 698D/S/W/Y, 700A/E/Q/T /V, 706A/G/M/Y, 707H, 708A/K/T/V, 712G/L/M/Q/S/T/Y, 716K/L/W, 724I, 726A/L, 729T, 731I /L/R, 732C/D/E/G/L/R/W/K/M/Q/S/T/V/Y, 734I/R/S, 735D/S/V, 736F/G, 737F /G/K/L/M/N/S/V, 738A/D/E/F/H/K/N/P/R/S/T/Y, 739G/P/R, 740G, 744A/R , 746P, 747D, 748F/G/K/N/S/V, 749A/C/D/F/G/I/K/L/M/P/Q/R/S/T/V/Y, 750A /D/G/K/M/N/P/Q/R/S/T/V/W、751A/D/E/Y、752D/E/F/G/L/M/Q/R/S /V, 753D, 754W, 755G/L/P/Q/S/Y, 756G/P/R/T, 757A/G/K/T, 759S, 770W, 773H, 775E/G/K/N/P /S/V, 785V, 786C/D/L, 787W, 790A/G/I/M/P/W, 791M/T, 792H/W/Y, 793G, 794R, 795C/N/S/T, 797C /G/P/Q/T/W/Y, 798C/R/T, 799A/E/G/M/P/Q/R/S/V, 800C/G/I/L/M/N/P /Q/R/T/V/W/Y, 801A/G/I/R, 802S/V, 805C/G/H/K/L/M/P/Q/R/S/V/W, 806E /G/R/S, 807C/L/P/R, 808K/S, 809A/C/G/I/K/L/N/Q/R/S/T/V/W, 810V, 811F, 812G /I/V, 815I, 816V/W, 817A/D, 818A/E/G/P/S/V/W, 819V, 821Q, 822S/T, 824W, 825L, 826G, 828K, 830M/V, 831W , 836G/R/V/W, 837G, 838G/T, 839A/G/K/P, 840G, 841W, 844H, 866A, 873L/Q/T, 879Q, 892T, 896L/V, 897G/K/R , 903E, 907D, 914V, 916C/E/I/K/L/M/Q/T, 917G, 918M/S, 919G, 921R/W, 922W, 923M, 924I/M/P/Q/W, 926T /V, 929A/Q/V, 938R/V, 939E/S, 945K, 948S, 951V, 968A/P, 999R, 1037H/M/N/P, 1040E, 1041G/H/N, 1042A/E/T , 1043E/R, 1049G/W, 1050E/P/Q/S/T, 1051E/H/K/R/S/W, 1053A/R, 1054C, 1076G/S, 1078S, 1080G/V, 1084L/N , 1088G/S, 1089F/S, 1092G, 1093A/E/G/M/N/P/Q/R/S/T/V/W/Y, 1094S, 1095G, 1098G, 1099G, 1100I/S, 1101G , 1104C/E, 1108M/S/V/W, 1109G, 1113F, 1114A/M, 1116A/G/I/L/R, 1127L/V, 1129P, 1138Q, 1145R, 1148A/C/E/L/M /N/Q/R/S, 1151S, 1156D/G/N/R, 1158N, 1160R, 1186E, 1192D, 1193A/E/G/I/L, 1194P, 1205L, 1210M/T/V/W, 1218A /G/L/M/P, 1220D/E/L/T, 1221G/S/T/V, 1222F, 1224P, 1229D, 1245R, 1248A/C/D/E/F/G/H/I/K /M/P/Q/R/S/T/W/Y, 1250A/P/S/T, 1259T, 1264L/R/W, 1269F, 1270V, 1274N/V, 1281H/K/P/Q/R /S, 1283E/H/L/S/T, 1292Y, 1297A, 1339C, 1352E, 1373S, 1377A/C/D/E/G/H/I/M/P/R/S/V/T/Y , 1378A/C/D/E, 1380A/G/W, 1389W, 1414A/C/D/E/G/H/N/P/T/W, 1422I, 1427Y, 1428G, 1439A/E/H/L /S/T, 1440A/C/E/G/S/T, 1442T or 1452L or the amino acid sequence of a combination thereof, wherein the amino acid positions are relative to those corresponding to SEQ ID NO: 252, 504, 706 , 840, 1200, 1514 or 2072 of the reference sequence.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising an amino acid comprising at least one substitution or set of substitutions at the following amino acid positions: Sequence: 63, 171, 212, 260, 306, 317, 320, 332, 387, 390, 393, 534, 558, 681, 732, 738, 749, 750, 775, 1148, 1248, 1377 or 1439 or combinations thereof , wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514 or 2072.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252 or corresponding to The reference sequence of SEQ ID NO: 252 has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, An amino acid sequence with 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity, wherein the amino acid sequence contains an amino acid sequence corresponding to SEQ. The reference sequence for residues 20-1457 of ID NO: 252 or one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 252.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising an amino acid comprising at least one substitution or set of substitutions at the following amino acid positions: Sequence: 1380, 388, 1043, 610, 1274, 1220, 1442, 1193, 621, 1054, 755, 1041, 748, 1049, 1221, 1051, 612, 1037, 616, 387, 489, 750, 1224, 618, 1050, 1053, 896, 897, 749, 260, 481, 613, 756, 63, 265, 752, 1218, 1378, 614, 1440, 1439, 1205, 1222, 1389, 1269, 390, 397/1218, 757, 1373, 346, 63/1248, 349, 306/355/1422, 1422, 306/346/708, 1148, 171/346/1248, 63/797/799/1248/1422, 171/799, 362, 1248, 171/306/346/999/1248, 171/1148, 734/799/1248/1422, 63/732, 63/171/306/346/732, 171, 171/1422, 63/362/732/799/ 1248, 999/1148/1422, 346/349, 306/349, 63/171/1148/1151, 306, 344/349/354/1250, 1250, 63/306/344/349/354/503/1248, 59/63/332/349/503/706/708, 63/306/344/349/354/706/1248, 59/63, 332/706/708/1248/1250, 503, 59/63/349/ 354, 59/349/708, 59/306/344/1248, 344/1248/1250, 708, 63/344/349, 59, 63/344/349/354, 706, 59/306/770, 63/ 347/354/1248 or 354/503, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 252.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 504 or corresponding to The reference sequence of SEQ ID NO: 504 has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, An amino acid sequence with 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity, wherein the amino acid sequence contains an amino acid sequence corresponding to SEQ. The reference sequence for residues 20-1457 of ID NO: 504 or one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 504.

In some embodiments, a recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising an amino acid comprising at least one substitution or set of substitutions at the following amino acid positions: Sequences: 750, 1051, 260, 756, 1193, 332, 897, 387, 1037, 306/749, 1270, 489, 487, 488, 903, 171/1248/1440, 260/1148/1378, 171/260/ 387/750/1248, 171/260, 171/260/387/750/1051/1148, 171, 750/1051/1148, 171/260/756, 171/1248, 171/1148, 59, 1440, 332/ 750/1248, 756/1248, 1248, 171/387/1248/1440, 750/1440, 59/171/750, 171/1193/1248, 750/1148/1193/1440, 1148, 171/387/1051, 748, 306, 306/387/390/749/1220, 390, 306/390/1037, 306/612/749, 387/390/749, 63/390/503/749, 1220, 63/306, 749, 306/390/749/1037, 306/749/1220, 306/387/390/612/749/1037 or 328, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 504.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 706 or corresponding to The reference sequence of SEQ ID NO: 706 has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, An amino acid sequence with 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity, wherein the amino acid sequence contains an amino acid sequence corresponding to SEQ. The reference sequence for residues 20-1457 of ID NO: 706 or one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 706.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising an amino acid comprising at least one substitution or set of substitutions at the following amino acid positions: Sequence: 1051, 1221, 1218, 387, 749/1218, 332, 897, 696, 816, 824, 826, 393, 825, 698, 332/387/390/1148, 328/387/749, 749, 306/ 328/332/390/1051/1193/1218, 63/306/332/1051/1148, 63/306/332/387/1051, 387/390/1051, 186/306/328/332/390/749/ 1051/1148/1193, 387/390/756/1051, 306/390/1051, 306/332/390/749, 306/387/390/1148, 306/328/332/387/390/749/1051/ 1148/1193, 332/387/390/749/1148, 387/390/749, 306/387/749/1051/1148, 387/749, 306/945/1051/1193/1221, 306/390/749/ 1051, 63/306/332/390, 306/387/390/749/1148/1193/1221, 306/332/387/390/749/1218, 63/306/332/390/1051/1148/1193/ 1218, 924, 755, 752, 751, 923, 797, 754, 793, 753, 922, 896, 938, 775, 732, 810, 747, 393/407, 819, 787, 802, 811, 392 or 397, The amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 706.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 840 or corresponding to The reference sequence of SEQ ID NO: 840 has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, An amino acid sequence with 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity, wherein the amino acid sequence contains an amino acid sequence corresponding to SEQ. The reference sequence for residues 20-1457 of ID NO: 840 or one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 840.

In some embodiments, a recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising an amino acid comprising at least one substitution or set of substitutions at the following amino acid positions: Sequence: 620, 491, 1229, 615, 1283, 822, 328, 212, 818, 555/684, 828, 550, 830, 839, 13, 14, 17, 836, 1210, 1281, 64, 682, 318, 20, 21, 325, 533, 19, 817, 831, 225, 1156, 393/747, 753, 393/732/751, 63/797, 622, 617, 907, 1040, 611, 488, 1192, 1194, 619, 16, 802, 211, 555, 536, 527, 841, 320, 837, 558, 914, 312, 681, 838, 15, 1427, 18, 683, 24, 534, 840, 1160, 551, 393, 393/747/752/811/819/923/1148, 393/751/775, 63/393/732/775/1148, 393/732/1148, 775/1148, 393/752/753/775/793/ 797, 393/923, 393/923/1148, 393/747/751, 393/747/753/775/1148, 752/923/1051, 63/775, 63/393/752/775/793/1051/ 1053, 752/753/775/793/819, 63/1051, 63/747/752/753/775/811/819/923/1051, 393/752/787, 751, 63/1148, 63/747/ 752/753/819/923/1148, 1148, 747/752/753/793/811, 63, 797/1148, 751/752/753/1051, 63/732/1051, 752/797/815/923/ 1051/1148 or 747/753, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 840.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 1200 or corresponding to The reference sequence of SEQ ID NO: 1200 has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, An amino acid sequence with 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity, wherein the amino acid sequence contains an amino acid sequence corresponding to SEQ. The reference sequence of residues 20-1457 of ID NO: 1200 or one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 1200.

In some embodiments, a recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising an amino acid comprising at least one substitution or set of substitutions at the following amino acid positions: Sequence: 681, 622, 617, 558, 536, 211, 320, 839, 797, 533, 751, 303, 951, 164, 818, 807, 799, 735, 794, 926, 755, 921, 801, 1094, 968, 929, 924, 698, 168, 581, 916, 1428, 805, 1114, 311, 737, 693, 809, 757, 1099, 676, 740, 791, 211/536/622/681, 536/558, 211/536/555/617/732, 211/681/732, 211/536/555, 555, 211/536, 211/212, 211/536/617/681/732, 211/536/558/617/ 681, 555/732, 536/555/558/617/732, 536/617/681/732, 536/555, 536/622/681, 732, 212/320/533/617/681, 211/312/ 533/534/617/619/681/839, 212, 211/212/320/619/681/839, 797/839, 212/312/320/533/534/617/681, 558/619/681, 320/558/681, 212/320/534/619/681/797/839, 320/558/619/732/839, 211/212/312/534/558/619/681/732/839, 320/ 534/558/619/732/797, 558/617/619/681, 211/534/558/611/681, 211/320/617/619/681, 558/617/681/797, 320/534/ 619/732、211/619/681、211/212/534/558/619/732/839、212/320/619/681/797/839、212/320/611/839、320/558/619/ 681, 312/533/617/681/797/839, 558/611/681, 312, 211/320/558/619/681/732/797/839, 320/558/732/797, 533/558/ 611/619/716/732/839, 312/681, 320/534/619/681, 211/320/533/536/619, 558/617/681/839, 212/320/534/558/681, 533/534/681/839, 212/534/619/681, 617/619/681, 211/212/320/558/617/681, 212/534/558/681/797, 212/320/534, 211/312/619、212/312/619/839、210/212/320/558/681、212/312/534/681/797/839、211/320/558/839、294、759、538 or 1109, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 1200.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 1514 or corresponding to The reference sequence of SEQ ID NO: 1514 has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, An amino acid sequence with 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity, wherein the amino acid sequence contains an amino acid sequence corresponding to SEQ. The reference sequence for residues 20-1457 of ID NO: 1514 or one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 1514.

In some embodiments, a recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising an amino acid sequence having at least one substitution or set of substitutions at the following amino acid positions: : 1042, 1041, 1051, 1283, 1281, 916, 187, 215, 178, 1050, 1210, 104, 615, 610, 536, 618, 621, 614, 110, 301, 1078, 812, 786, 127, 175 ,1093,1088,650,535,806,738,700,132,1076,1084,498,756,1108,746,160,543,310,584,918,1452,790,292,800,167,892 ,304,1116,752,281,917,712,1080,806/1352,1095,744,1098,948,119,785,1113,667,158,580,707,731,1100,1089,1092,939 ,1104,736,1297,1127,686,1129,1138,786/821,680,1101,808,739,308,536/555/619/732/799/805,555/619/801,555/619 /799, 619/799/801/805, 536/555/619/732/799, 799/805, 801, 246/619/799/801, 536/555/619, 619/839, 619/799/805 ,536/801/805,555/619/799/805,619/801/805,555/619,536/619/801,801/805,536/619/799/805,555/619/732/799 /801/805, 749, 616, 611, 370/614, 1156, 1158, 555/681/737/799, 536/619/809/1109, 536/555/619/737/799/809/839/916 /921, 916/1109, 536/619/681/799/916/1109, 555/619/681/807/1109, 555/619/681/737/799, 619/681/799/916/1109, 619 /681, 555/921, 619/681/916, 555/619/681/799, 555, 732, 619/799, 619, 681, 555/916, 839 or 799, where the amino acid positions are relative To the reference sequence corresponding to SEQ ID NO: 1514.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2072 or corresponding to The reference sequence of SEQ ID NO: 2072 has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, An amino acid sequence with 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity, wherein the amino acid sequence contains an amino acid sequence corresponding to SEQ. The reference sequence for residues 20-1457 of ID NO: 2072 or one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 2072.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising an amino acid comprising at least one substitution or set of substitutions at the following amino acid positions: Sequence: 167, 167/738/1076/1093, 167/738/752/800/805/808/809/1116, 167/800/809/1076, 167/318, 167/318/800/805/808/ 809、167/318/681、167/318/681/700/805/808/809/1076/1116、167/318/700、167、167/738/1076/1093、167/738/752/800/ 805/808/809/1116, 167/800/809/1076, 167/318, 167/318/800/805/808/809, 167/318/681, 167/318/681/700/805/808/ 809/1076/1116, 167/318/700, 167/318/700/1076, 167/318/700/738/739/1076, 167/681/738/800/808/809, 167/178/700/ 752/1093, 167/700, 167/700/738/739/1093, 167/700/738/752/1093, 167/700/738/752/805/808/809/1093/1116, 808/809, 1076, 738, 738/808/809, 738/1076, 738/739, 738/739/809, 738/739/809/1093/1116, 738/739/1093, 738/739/752/1093, 738/ 739/799/800/1093, 738/739/1116, 738/800, 738/1093, 738/752, 738/752/1093, 738/752/799, 738/799/809/1093, 738/799/ 1093, 739, 739/800/809, 800, 800/805/809, 809, 809/1093, 809/1093/1116, 809/1116, 318, 318/1076, 318/1076/1116, 318/739/ 805/808/809/1076, 318/499/700/738/809, 318/1093, 318/752/1093, 318/1116, 318/543/739, 318/543/681/752/1116, 318/ 543/1093, 318/700/738/1093/1414, 681, 681/738, 681/738/739/1093, 681/738/739/752/799/800/809/1116/1339, 681/738/ 739/1116, 681/738/800, 681/800/805/809, 681/809/1093, 681/752/1093, 681/752/1093, 681/700, 681/700/738/739/1093/ 1116, 681/700/809, 681/700/1093, 681/700/752/808/809/1093/1116, 681/700/752/1093, 681/700/844, 681/700/805/809, 1093, 1093/1116, 752, 752/1076, 752/800/809, 752/800/1093, 752/809, 752/866, 752/1093, 752/1116, 178/318/739/752/809/ 1076/1116, 178/318/800/809, 178/318/681/800/808/809/1076, 178/681, 178/681/738/1116, 178/752/809, 178/700/800/ 805/808/809, 178/805/809/866, 1116, 543, 543/738/739/809, 543/738/809/1093, 543/739/1093, 543/800/1093/1116, 543/ 809/1093, 543/809/1093/1116, 543/681/738/739/752/809/1093, 543/681/738/739/752/1093, 543/681/739/752/800/805/ 809/1093、543/681/700/738/739/800/1093/1116、543/681/700/738/739/805/808/809、543/681/700/752、543/681/700/ 752/809/1093, 543/1093, 543/700, 543/700/738/739/752/800/1093/1116, 543/700/809, 543/700/1093, 543/700/752, 543/ 700/752/800/1093, 543/700/752/809/1116, 543/700/752/1093, 700, 700/738/739/800/1093, 700/738/739/799/1093, 700/ 738/800/1093, 700/738/1116, 700/809, 700/809/1093, 700/1093, 700/1093/1116, 700/752, 700/752/808/809/1093/1116, 700/ 752/809/1093, 700/752/799/1116, 700/752/805/809/1093, 700/799/800, 700/1116, 700/805/808/809/1076, 514/543/681/ 805/808/809, 805/808/809 or 805/809/1116, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2072.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising a polynucleotide sequence contained in Tables 4.1 , 4.2 , 5.1 , 5.2 , 6.1 , 6.2 , 7.1 , 7.2 , Substituted amino acid sequences at the amine positions set forth in 8.1 , 8.2 , 9.1 , 9.2 , 10.1 , 10.2 , 11.1 and 15.1 , wherein the amino acid positions are relative to those corresponding to SEQ ID NO: 2, 252 , 504, 706, 840, 1200, 1514 or 2072 of the reference sequence.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising at least one of Tables 4.1 , 4.2 , 5.1 , 5.2 , 6.1 , 6.2 , 7.1 , 7.2 , 8.1 , 8.2 , 9.1 , 9.2 , 10.1 , 10.2 , 11.1 and 15.1 , wherein the amino acid positions are relative to those corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072 of the reference sequence.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising at least one of Tables 4.1 , 4.2 , 5.1 , 5.2 , 6.1 , 6.2 , 7.1 , 7.2 , 8.1 , 8.2 , 9.1 , 9.2 , 10.1 , 10.2 , 11.1 and 15.1 , amino acid sequences of substitutions or sets of substitutions, wherein the amino acid positions are relative to those corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072 of the reference sequence.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising at least one of Tables 4.1 , 4.2 , 5.1 , 5.2 , 6.1 , 6.2 , 7.1 , 7.2 , 8.1 , 8.2 , 9.1 , 9.2 , 10.1 , 10.2 , 11.1 and 15.1 , amino acid sequences of substitutions or sets of substitutions of the variants set forth in SEQ ID NO: 2 , 252, 504, 706, 840, 1200, 1514 or 2072 of the reference sequence.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising Table 4.1 , 4.2 , 5.1 , 5.2 , 6.1 , 6.2 , 7.1 , 7.2 , 8.1 , 8.2 , 9.1 , 9.2 , 10.1 , 10.2 , 11.1 and 15.1 , residues 20-1457 of the even-numbered SEQ ID NO. sequences set forth in Tables 4.1 , 4.2 , 5.1 , 5.2, 6.1 , 6.2 , 7.1 , 7.2 , 8.1 , 8.2 , 9.1 , 9.2 , 10.1 , 10.2 , 11.1 and 15.1 , the amino acid sequence of the even-numbered SEQ ID NO. sequence.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising an even-numbered SEQ ID NO: 4-2346 and 2360-2870. . or residues 20-1457 of SEQ ID NOs: 4-2346 and 2350-2870 including the amino acid sequences of even-numbered SEQ ID NO.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide comprising SEQ ID NO: 252, 504, 706, 840, 1200, 1514 or 2072. Residues 20-1457 or comprise the amino acid sequence of SEQ ID NO: 252, 504, 706, 840, 1200, 1514 or 2072.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a full-length polypeptide or preproprotein or prepropolypeptide of a recombinant Factor VIII polypeptide described herein.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a proprotein or propolypeptide or mature form of a recombinant Factor VIII polypeptide described herein.

In some embodiments, a recombinant polynucleotide comprises a polynucleotide sequence encoding a biologically or functionally active fragment of a recombinant VIII polypeptide described herein. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a biologically or functionally active fragment with one or more cleavages between amino acid residues 760 to 773, and the polypeptide does not contain a fragment corresponding to Some or all of the amino acid sequences of amino acids 760-773, wherein these positions are relative to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a biologically or functionally active fragment having cleavage between amino acid residues 391 to 392, wherein these positions are relative to SEQ ID NO: 2. Reference sequence of 252, 504, 706, 840, 1200, 1514 or 2072.

In some embodiments, the recombinant polynucleotide comprises a reference polynucleotide sequence corresponding to nucleotide residues 58-4371 of odd-numbered SEQ ID NO. in SEQ ID NOs: 3-2345 and 2359-2869 Or have at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more A sequence identical polynucleotide sequence, wherein the recombinant polynucleotide encodes a recombinant Factor VIII polypeptide.

In some embodiments, the recombinant polynucleotide comprises a reference polynucleotide sequence corresponding to nucleotide residues 58-4371 of SEQ ID NO: 1, 251, 503, 705, 839, 1199, 1513, or 2071 Or have at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more A sequence identical polynucleotide sequence, wherein the recombinant polynucleotide encodes a recombinant Factor VIII polypeptide.

In some embodiments, a recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant Factor VIII polypeptide described herein, wherein the polynucleotide sequence is codon-optimized for expression of encoded recombinant Factor VIII Peptides. In some embodiments, polynucleotide sequences are codon-optimized for expression in prokaryotic or eukaryotic cells. In some embodiments, polynucleotide sequences are codon-optimized for expression in mammalian cells. In some embodiments, the polynucleotide sequence is codon-optimized for expression in human cells.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising the odd-numbered nucleotide residues of SEQ ID NO. in SEQ ID NOs: 3-2345 and 2359-2869 58-4371, or odd-numbered SEQ ID NO. including SEQ ID NO: 3-2345 and 2349-2869.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising the nucleotide residues of SEQ ID NO: 1, 251, 503, 705, 839, 1199, 1513, or 2071 58-4371, or contains SEQ ID NO. 1, 251, 503, 705, 839, 1199, 1513 or 2071.

In some embodiments, the present disclosure provides a recombinant polynucleotide capable of hybridizing under highly stringent conditions to a reference polynucleotide encoding recombinant Factor VIII described herein, such as Tables 4.1 , 4.2 , 5.1 , 5.2 , The recombinant polynucleotides or their reverse complements provided in 6.1 , 6.2 , 7.1 , 7.2 , 8.1 , 8.2 , 9.1 , 9.2 , 10.1 , 10.2 , 11.1 and 15.1 . In some embodiments, the present disclosure provides a recombinant polynucleotide capable of hybridizing under highly stringent conditions to the reverse complement of a reference polynucleotide encoding a recombinant Factor VIII polypeptide described herein, wherein under stringent conditions The hybridized recombinant polynucleotide encoding a polynucleotide selected from Table 4.1 , 4.2 , 5.1 , 5.2 , 6.1 , 6.2 , 7.1 , 7.2 compared to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072. , 8.1 , 8.2 , 9.1 , 9.2 , 10.1 , 10.2 , 11.1 and 15.1 , a recombinant Factor VIII polypeptide having an amino acid sequence that differs by one or more residues at any residue position.

In some embodiments, the recombinant polynucleotide that hybridizes under highly stringent conditions comprises nucleotide residues 58-4371 corresponding to SEQ ID NO: 1, 251, 503, 705, 839, 1199, 1513, or 2071 The reference polynucleotide sequence or is at least 70%, 75%, 80%, 81%, 82%, 83% identical to the reference sequence of SEQ ID NO: 1, 251, 503, 705, 839, 1199, 1513 or 2071 , 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more Multiple sequence identity polynucleotide sequences. In some other embodiments, the recombinant polynucleotides that hybridize under highly stringent conditions include those corresponding to Tables 4.1 , 4.2 , 5.1 , 5.2 , 6.1, 6.2 , 7.1 , 7.2 , 8.1 , 8.2 , 9.1 , 9.2 , 10.1 , At least one polynucleotide reference sequence for nucleotide residues 58-4371 of the polynucleotide sequences provided in 10.2 , 11.1 and 15.1 or Tables 4.1 , 4.2 , 5.1 , 5.2 , 6.1 , 6.2 , 7.1 , 7.2 , The polynucleotide sequences provided in 8.1 , 8.2 , 9.1 , 9.2 , 10.1 , 10.2 , 11.1 and 15.1 have at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, Polynucleotide sequences with 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity , wherein the recombinant polynucleotide that hybridizes under stringent conditions encodes a recombinant Factor VIII polypeptide.

In some embodiments, an isolated polynucleotide encoding any of the recombinant Factor VIII polypeptides provided herein is manipulated in various ways to provide polypeptide expression. In some embodiments, a polynucleotide encoding a polypeptide is provided in an expression vector in which one or more control sequences are present to modulate the expression of the polynucleotide and/or polypeptide. Manipulation of the isolated polynucleotide prior to its insertion into the vector may be desirable or necessary depending on the expression vector. Techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art.

In some embodiments, the control sequence also includes, among other sequences, a promoter sequence, a Kozak sequence, a leader sequence, a polyadenylation sequence, a propeptide sequence, a signal peptide sequence, a DNA-based sequence for gene therapy maintenance Regulatory elements and transcription terminators. As is known in the art, a suitable promoter can be selected based on the host cell used. Exemplary promoters for use in filamentous fungal host cells include promoters obtained from the genes Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic acid protease, Aspergillus oryzae ( Aspergillus niger ) neutral α-amylase, Kojima niger acid-stable α-amylase, Kojima niger or Aspergillus awamori ( Aspergillus awamori ) glucoamylase (FVIIIA), Rhizomucor oryzae lipase, Kojima oryzae Alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetaminase and Fusarium oxysporum trypsin-like protease (see for example WO 96/00787), and NA2- The tpi promoter (a promoter hybrid from the genes of Kojima niger neutral α-amylase and Kojima oryzae triose phosphate isomerase), as well as its mutated, truncated and hybridized promoters. Exemplary yeast cell promoters may be derived from genes for the following enzymes: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde- 3-Phosphate dehydrogenase (ADH2/GAP) and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other promoters useful in yeast host cells are known in the art (see, eg, Romanos et al., 1992, Yeast 8:423-488). Exemplary promoters for use in mammalian cells include, but are not limited to, promoters from cytomegalovirus (CMV), chicken beta-actin promoter fused to the CMV enhancer, monkey vacuolating virus 40 (SV40) promoters from Homo sapiens phosphoglycerate kinase, β-actin, elongation factor-1a or glyceraldehyde-3-phosphate dehydrogenase or promoters from chicken ( Gallus gallus ) β-actin.

In some embodiments, the control sequence is a suitable transcription termination sequence that is a sequence recognized by the host cell for terminating transcription. The termination sequence is operably linked to the 3' end of the nucleic acid sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice can be used in the present invention. For example, exemplary transcription terminators for use in filamentous fungal host cells can be obtained from genes for: M. oryzae TAKA amylase, M. nidulans amylase, M. nidulans anthranilate synthesis enzymes, A. niger alpha-glucosidase and A. malleus trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other suitable promoters for use in yeast host cells are known in the art (see, eg, Romanos et al., supra). Exemplary terminators for use in mammalian cells include, but are not limited to, terminators from cytomegalovirus (CMV), terminators from simian vacuolating virus 40 (SV40), terminators from Homo sapiens growth hormone hGH, terminators from bovine Terminator for the growth hormone BGH and terminator from human or rabbit beta-globulin.

In some embodiments, the control sequence is a suitable leader sequence, i.e. 5'-cap modification, 5' UTR, etc. In some embodiments, these regulatory sequence elements mediate binding to molecules involved in mRNA transport and translation, inhibit 5'-exonucleolytic degradation and confer resistance to decapping. The leader sequence is operably linked to the 5' end of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice can be used. Exemplary leader sequences for use in filamentous fungal host cells are obtained from the genes for M. oryzae TAKA amylase and M. nidulans triose phosphate isomerase. Suitable leaders for use in yeast host cells include, but are not limited to, those obtained from genes for the following enzymes: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha. -factor and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP). Suitable leader sequences for use in mammalian host cells include, but are not limited to, 5'-UTR elements present in orthopoxvirus mRNA.

In some embodiments, the control sequence includes a 3' non-translated nucleic acid region and a polyadenylation tail nucleic acid sequence operably linked to the 3' end of the protein-encoding nucleic acid sequence that mediates binding to proteins involved in mRNA transport and translation. and sequences of mRNA half-lives. Any polyadenylation sequence and 3' UTR that is functional in the host cell of choice can be used in the present invention. Exemplary polyadenylation sequences for use in filamentous fungal host cells include, but are not limited to, those from genes for the following enzymes: M. oryzae TAKA amylase, M. niger amylase, M. nidulans Koji mold anthranilate synthase, A malleus trypsin-like protease, and Koji mold alpha-glucosidase. Suitable polyadenylation sequences for yeast host cells are also known in the art (see, eg, Guo and Sherman, Mol. Cell. Biol., 1995, 15:5983-5990). Polyadenylation and 3' UTR sequences useful in mammalian host cells include, but are not limited to, alpha-hemoglobin mRNA and beta-hemoglobin mRNA 3 that have several sequence elements that increase mRNA stability and increase mRNA translation. '-UTR.

In some embodiments, the control sequence is a signal peptide coding region that encodes an amino acid sequence linked to the amino terminus of the polypeptide and directs the encoded polypeptide into the secretory pathway of the cell. The 5' end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in the translation reading frame to the coding segment encoding the secreted polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding region foreign to the coding sequence. Any signal peptide coding region in the secretory pathway that directs the expressed polypeptide to the host cell of choice can be used for expression of the recombinant Factor VIII polypeptides provided herein. Effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to, signal peptide coding regions obtained from the following genes: M. oryzae TAKA amylase, M. niger neutral amylase, M. niger amylase , Rhizomucor miehei aspartic acid protease, Humicola insolens cellulase and Humicola lanuginosa lipase. Signal peptides suitable for use in yeast host cells include, but are not limited to, signal peptides from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Signal peptides suitable for use in mammalian host cells include, but are not limited to, signal peptides from the gene for immunoglobulin gamma (IgG). In some embodiments, the signal peptide is that of a protein expressed in human cells.

In another aspect, the present disclosure also provides a recombinant expression vector comprising a polynucleotide encoding a recombinant Factor VIII polypeptide, and one or more expression regulatory regions, such as a promoter and terminator, an origin of replication, etc., as appropriate. Depends on the type of host being introduced. In some embodiments, the various nucleic acids and control sequences described above are joined together to produce a recombinant expression vector that includes one or more convenient restriction sites to allow insertion or substitution of a recombinant Factor VIII polypeptide encoding at such sites. The nucleic acid sequence. Alternatively, the polynucleotide sequences of the present disclosure are expressed by inserting the polynucleotide sequence or a nucleic acid construct comprising the polynucleotide sequence into an appropriate vector for expression. In creating an expression vector, the coding sequence is positioned in the vector such that the coding sequence is operably linked to control sequences suitable for expression.

The recombinant expression vector can be any vector (e.g., plasmids or viruses including, but not limited to, adenovirus (AV), adeno-associated virus (AAV), lentivirus (LV), and non-viral vectors such as liposomes), which can Recombinant DNA procedures are conveniently performed and can result in the expression of recombinant Factor VIII polynucleotide sequences. The choice of vector will generally depend on the compatibility of the vector with the host cell into which the vector is introduced. The carrier can be a linear or closed circular plastid.

In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extrachromosomal entity that replicates independently of chromosomal replication, such as a plastid, extrachromosomal element, minichromosome, or artificial chromosome). The vector may contain any means for ensuring self-replication. In some alternative embodiments, the vector may be one that, when introduced into a host cell, integrates into the genome and replicates with the chromosome into which it has been integrated. Additionally, a single vector or plasmid, or two or more vectors or plasmids that together contain the total DNA to be introduced into the host cell genome, or transposons, can be used.

In some embodiments, the expression vector preferably contains one or more selectable markers that allow the transformed cells to be readily selected. "Selectable markers" are genes whose products provide biocide or viral resistance, resistance to heavy metals, auxotrophic prototrophy, and the like. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1 and URA3. Selectable markers for use in filamentous fungal host cells include (but are not limited to) amdS (acetaminase), argB (ornithine amine formyltransferase), bar (glufosinate acetyltransferase ), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotic acid nucleoside-5'-phosphate decarboxylase), sC (adenosyl sulfate chelate transferase) and trpC ( anthranilate synthase) and its equivalents.

In another aspect, the invention provides a host cell comprising a polynucleotide encoding at least one recombinant Factor VIII polypeptide described herein operably linked to one or more cells for use in Control sequences for expression of recombinant Factor VIII polypeptides in host cells. Host cells for expressing polypeptides encoded by expression vectors of the invention are well known in the art and include, but are not limited to, fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae and Pichia pastoris ) , such as ATCC accession number 201178); insect cells (such as Drosophila S2 cells and Spodoptera exigua Sf9 cells), plant cells, animal cells (such as CHO, CHO-K1, COS and BHK) and human cells (such as HEK293T, human fibroblast cells, THP-1, Jurkat and Bowes melanoma cell lines). In some embodiments, the host cells are human cells obtained or derived from an individual with hemophilia A.

Accordingly, in another aspect, the present disclosure provides a method for producing a recombinant Factor VIII polypeptide, wherein the method comprises culturing a polynucleotide capable of expressing a polynucleotide encoding a recombinant Factor VIII polypeptide under conditions suitable for expressing the polypeptide. host cell. In some embodiments, the method further comprises the step of isolating the recombinant Factor VIII polypeptide, such as from culture medium or cells. In some embodiments, the method further comprises the step of purifying the expressed recombinant Factor VIII polypeptide as described herein.

Suitable media and growth conditions for the host cells described above are well known in the art. Polynucleotides for expression of recombinant Factor VIII polypeptides can be introduced into cells by various methods known in the art. Techniques include, inter alia, electroporation, gene gun particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion.

Recombinant Factor VIII polypeptides having the properties disclosed herein can be produced by subjecting polynucleotides encoding naturally occurring or recombinant Factor VIII polypeptides to mutagenesis and/or directed evolution methods known in the art and described herein. obtain. Exemplary directed evolution techniques are mutagenesis and/or DNA shuffling (see, eg, Stemmer, Proc. Natl. Acad. Sci. USA, 1994, 91:10747-10751; WO 95/22625; WO 97/0078; WO 97/35966 ; WO 98/27230; WO 00/42651; WO 01/75767 and US Patent 6,537,746). Other directed evolution procedures that can be used include inter alia the staggered extension method (StEP), in vitro recombination (see, e.g., Zhao et al., Nat. Biotechnol., 1998, 16:258-261), mutation-induced PCR (see, e.g., Caldwell et al., PCR Methods Appl., 1994, 3:S136-S140) and cassette mutagenesis (see, e.g., Black et al., Proc. Natl. Acad. Sci. USA, 1996, 93:3525-3529).

For example, mutagenesis and directed evolution methods can be readily applied to polynucleotides to generate libraries of variants that can be expressed, screened, and analyzed. Mutation induction and directed evolution methods are well known in the art (see, for example, U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548, 6,117,679, No. 6,132,970, No. 6,165,793, No. 6,180,406, No. 6,251,674, No. 6,277,638, No. 6,287,861, No. 6,287,862, No. 6,291,242, No. 6,297,053, No. 6,303,344, No. 6,3 No. 09,883, No. 6,319,713, No. 6,319,714 No. 6,323,030, 6,326,204, 6,335,160, 6,335,198, 6,344,356, 6,352,859, 6,355,484, 6,358,740, 6,358,742, 6,365,377, 6 , No. 365,408, No. 6,368,861, No. 6,372,497, No. 6,376,246, No. 6,379,964, No. 6,387,702, No. 6,391,552, No. 6,391,640, No. 6,395,547, No. 6,406,855, No. 6,406,910, No. 6,413,745, No. 6,4 No. 13,774, No. 6,420,175, No. 6,423,542 No. 6,426,224, 6,436,675, 6,444,468, 6,455,253, 6,479,652, 6,482,647, 6,489,146, 6,506,602, 6,506,603, 6,519,065, 6 , No. 521,453, No. 6,528,311, No. 6,537,746, No. 6,573,098, No. 6,576,467, No. 6,579,678, No. 6,586,182, No. 6,602,986, No. 6,613,514, No. 6,653,072, No. 6,716,631, No. 6,946,296, No. 6,9 No. 61,664, No. 6,995,017, No. 7,024,312 No. 7,058,515, 7,105,297, 7,148,054, 7,288,375, 7,421,347, 7,430,477, 7,534,564, 7,620,500, 7,620,502, 7,629,170, 7 , No. 702,464, No. 7,747,391, No. 7,747,393, No. 7,751,986, No. 7,776,598, No. 7,783,428, No. 7,795,030, No. 7,853,410, No. 7,868,138, No. 7,873,499, No. 7,904,249, No. 7,957,912, No. 8,3 No. 83,346, No. 8,504,498, No. 8,849,575 No. 8,876,066, 8,768,871, 9,593,326, and all related non-U.S. counterparts; Ling et al., Anal. Biochem . , 1997, 254(2):157-78; Dale et al., Meth. Mol. Biol . , 1996, 57:369-74; Smith, Ann. Rev. Genet . , 1985, 19:423-462; Botstein et al., Science, 1985, 229:1193-1201; Carter, Biochem. J. , 1986 , 237:1-7; Kramer et al., 1984, Cell, 38:879-887; Wells et al., Gene, 1985, 34:315-323; Minshull et al., Curr. Op. Chem. Biol., 1999, 3:284-290; Christians et al., Nat. Biotechnol., 1999, 17:259-264; Crameri et al., Nature, 1998, 391:288-291; Crameri et al., Nat. Biotechnol., 1997, 15: 436-438; Zhang et al., Proc. Nat. Acad. Sci. USA, 1997, 94:4504-4509; Crameri et al., Nat. Biotechnol. , 1996, 14:315-319; Stemmer, Nature, 1994, 370 :389-391; Stemmer, Proc. Nat. Acad. Sci. USA, 1994, 91:10747-10751; U.S. Patent Publication No. 2008/0220990, No. US 2009/0312196, No. US2014/0005057, No. US2014 /0214391, US2014/0221216; US2015/0050658, US2015/0133307, US2015/0134315 and all related non-US counterparts; WO 95/22625, WO 97/0078, WO 97/35966, WO 98 /27230, WO 00/42651, WO 01/75767 and WO 2009/152336; all of which are incorporated herein by reference).

In some embodiments, protein variants obtained after a mutagenesis treatment are screened by subjecting the protein variants to defined temperatures (or other analytical conditions) and measuring the amount of protein activity that is retained after the treatment or other analytical conditions. . DNA containing the polynucleotide encoding the Factor VIII polypeptide is then isolated from the host cell, sequenced to identify nucleotide sequence changes, if any, and used to express the protein in different or the same host cell. Measuring protein activity of a library of expressions can be performed using any suitable method known in the art (such as provided in the Examples).

For engineered polypeptides of known sequence, the polynucleotide encoding the polypeptide can be prepared by standard solid phase methods according to known synthetic methods. In some embodiments, polynucleotide fragments can be synthesized individually and subsequently ligated (eg, by enzymatic or chemical ligation methods or polymerase-mediated methods) to form any desired contiguous sequence. For example, the polynucleotides and oligonucleotides disclosed herein can be synthesized by using classical aminophosphite methods (see, e.g., Beaucage et al., Tetra. Lett., 1981, 22:1859-69; and Matthes et al., EMBO J., 1984, 3:801-05) are prepared by chemical synthesis, as is typically performed by automated synthesis methods.

Accordingly, in some embodiments, a method for preparing a recombinant Factor VIII polypeptide may comprise: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from Tables 4.1 , 4.2 , The amino acid sequence of any variant provided in 5.1 , 5.2 , 6.1 , 6.2 , 7.1 , 7.2 , 8.1 , 8.2 , 9.1 , 9.2 , 10.1 , 10.2 , 11.1 and 15.1 , and SEQ ID NOs: 2-2346 and 2350 -2870, (b) represents a recombinant Factor VIII polypeptide encoded by a polynucleotide. In some embodiments of this method, the amino acid sequence encoded by the polynucleotide may optionally have one or several (eg, up to 3, 4, 5, or up to 10) amino acid residues deleted, Insertion and/or replacement. In some embodiments, the amino acid sequence optionally has 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45 or 1-50 amine groups Acid residue deletions, insertions and/or substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45 or 50 amino acid residues are deleted, inserted and/or substituted. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24 or 25 amino acid residues are deleted, inserted and/or substituted. In some embodiments, substitutions include conservative or non-conservative substitutions.

Expressed recombinant Factor VIII polypeptides can be evaluated for any desired improved properties (e.g., activity/potency, selection) using any suitable assay known in the art, including, but not limited to, the assays and conditions described herein. properties, stability, serum/plasma stability, alkaline pH tolerance, protease sensitivity, etc.).

In some embodiments, any recombinant Factor VIII polypeptide expressed in the host cell is recovered from the cell and/or culture medium using any one or more known protein purification techniques, including, inter alia, lysozyme or detergent treatment, sonication , filtration, salting out, ultracentrifugation and chromatography.

Chromatography techniques for isolating recombinant Factor VIII polypeptides include, inter alia, reverse phase chromatography, high performance liquid chromatography, ion exchange chromatography, hydrophobic interaction chromatography, gel electrophoresis and affinity chromatography. The conditions used to purify a particular polypeptide depend in part on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc. and will be apparent to those skilled in the art. In some embodiments, affinity technology can be used to isolate modified variant Factor VIII polypeptides. In some embodiments utilizing affinity chromatography for purification, any antibody that specifically binds a variant Factor VIII polypeptide may be used. In some embodiments utilizing affinity chromatography for purification, proteins that bind to glycans covalently linked to Factor VIII may be used. In still other embodiments utilizing affinity chromatography for purification, any small molecule that binds to the Factor VIII active site may be used. For the generation of antibodies, various host animals including, but not limited to, rabbits, mice, rats, etc. are immunized by injecting Factor VIII polypeptides (eg, Factor VIII variants) or fragments thereof. In some embodiments, a Factor VIII polypeptide or fragment is attached to a suitable carrier, such as BSA, via a side chain functionality or a linker attached to a side chain functionality.

In some embodiments, after the recombinant Factor VIII polypeptide is recovered or isolated from the recombinant host cell or cell culture medium, it is further purified by any suitable method known in the art. In some other embodiments, purified Factor VIII polypeptides are combined with other ingredients and compounds to provide compositions and formulations (eg, pharmaceutical compositions) including recombinant Factor VIII polypeptides, if desired for different applications and uses.

In some other embodiments, the purified recombinant Factor VIII polypeptide or the formulated recombinant Factor VIII polypeptide is lyophilized. In some embodiments, recombinant Factor VIII polypeptides are produced directly in the body (ie, cells in the body, such as humans or another animal) without purification. However, in some alternative embodiments, the recombinant Factor VIII polypeptide is produced within the body (ie, cells within the body, such as a human or another animal) and collected from the body using methods known in the art. In some other embodiments, the collected recombinant Factor VIII polypeptides are purified. pharmaceutical composition

In another aspect, the present disclosure provides various compositions and forms comprising recombinant Factor VIII polypeptides, including (but not limited to) those described below. In some embodiments, the present disclosure provides recombinant polypeptides or recombinant polynucleotides suitable for use in pharmaceutical compositions and other compositions.

Depending on the mode of administration, compositions comprising a therapeutically effective amount of recombinant Factor VIII described herein are in solid, semi-solid, or liquid form. In some embodiments, the compositions include other pharmaceutically acceptable components such as diluents, buffers, excipients, salts, emulsifiers, preservatives, stabilizers, fillers, and other ingredients. Technical details regarding formulation and administration are well known in the art and described in the literature. In some embodiments, these compositions are produced directly in humans after being introduced as gene therapy.

In some embodiments, the present disclosure provides a pharmaceutical composition comprising a recombinant Factor VIII polypeptide described herein. In some embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable carrier or excipient. Any suitable format for delivering recombinant Factor VIII polypeptides may be used in the present disclosure, including, but not limited to, pills, lozenges, gel lozenges, capsules, lozenges, dragees, powders, soft gels, sol-gels. Gums, gels, lotions, implants, patches, sprays, ointments, liniments, creams, pastes, jelly, paints, aerosols, gums, emollients, sticks, solutions, suspensions (including But not limited to oil-based suspensions, oil-in-water emulsions, etc.), slurries, syrups, controlled release formulations, etc.

In some embodiments, the recombinant Factor VIII polypeptide is provided in a form suitable for injection or infusion (i.e., in an injectable formulation). In some embodiments, the polynucleotide sequence of the recombinant Factor VIII polypeptide is provided in a form suitable for injection or infusion.

In some embodiments, recombinant Factor VIII polypeptides are provided in a biocompatible matrix such as a sol-gel, including silica-based (eg, oxysilane) sol-gels. In some embodiments, the recombinant Factor VIII polypeptide is encapsulated. In some alternative embodiments, the recombinant Factor VIII polypeptide is encapsulated in nanostructures (eg, nanotubes, nanotubules, nanocapsules or microcapsules, microspheres, liposomes, etc.).

In some embodiments, compositions comprising recombinant Factor VIII polypeptides of the present disclosure may include one or more commonly used carrier compounds, including, but not limited to, sugars (e.g., lactose, sucrose, mannitol, and/or sorbitol), Starch, cellulose (such as methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose), gum (such as acacia, tragacanth, guar gum, etc.) and/or protein (such as gelatin, collagen protein, etc.).

In some embodiments, recombinant Factor VIII polypeptides are chemically modified by glycosylation, chemical cross-linking reagents, pegylation (i.e., modified with polyethylene glycol (PEG) or activated PEG, etc.), or other compounds (see, e.g., Ikeda, Amino Acids, 2005, 29:283-287; U.S. Patent Nos. 7,531,341, 7,534,595, and 7,560,263; U.S. Patent Publication Nos. 2013/0039898, 2012/0177722, etc.).

It is contemplated that the recombinant Factor VIII polypeptide is administered by any suitable means known in the art, including, but not limited to, parenterally, orally, topically, transdermally, intranasally, intraocularly, intrathecally, via an implant wait. The present invention is not intended to be limited to any particular delivery method and/or mechanism.

In some embodiments, pharmaceutical compositions comprise recombinant polynucleotides encoding recombinant Factor VIII polynucleotides described herein, such as for use in gene therapy. In some embodiments, a gene therapy composition comprises a recombinant polynucleotide encoding any of the recombinant Factor VIII polypeptides described herein. In some embodiments, the gene therapy composition comprises a viral vector comprising a recombinant polynucleotide encoding a recombinant Factor VIII polypeptide. In some embodiments, viral delivery vectors include, but are not limited to, adenovirus (AV), adeno-associated virus (AAV), lentivirus (LV), retrovirus, or herpesvirus vectors. In some embodiments, gene therapy compositions comprise non-viral delivery compositions in which the recombinant polynucleotide is formulated with liposomes, cationic polymers, dendrimers or conjugated to cell-penetrating peptides. In some embodiments, recombinant Factor VIII polypeptides are provided for delivery to cells or tissues via mRNA therapy, such as by formulating polyribonucleotides (eg, mRNA) in an encapsulated delivery vehicle (eg, liposomes).

In some other embodiments, recombinant Factor VIII polypeptides are provided for delivery to cells or tissues via cell therapy, wherein a polynucleotide sequence encoding a recombinant Factor VIII polypeptide is introduced into an exogenous cell and the cell (or cells) Upon introduction into a recipient or individual (eg, a patient exhibiting Factor VIII deficiency). In some embodiments, the cells are stem cells, particularly human stem cells. Usage and method

In another aspect, the recombinant Factor VIII polypeptides disclosed herein are used for therapeutic purposes. In some embodiments, recombinant Factor VIII polypeptides and/or recombinant polynucleotides encoding recombinant Factor VIII polypeptides are used to treat conditions or diseases associated with Factor VIII deficiency.

In some embodiments, methods for treating and/or preventing symptoms of Factor VIII deficiency in an individual comprise administering to an individual suffering from Factor VIII deficiency an effective amount of a recombinant Factor VIII polypeptide described herein, or a pharmaceutical composition thereof things. In some embodiments, administration of recombinant Factor VIII is provided as enzyme replacement therapy.

In some embodiments, methods for treating and/or preventing symptoms of Factor VIII deficiency in an individual comprise providing to an individual suffering from Factor VIII deficiency a recombinant polynucleotide encoding a recombinant Factor VIII polypeptide, such as comprising the above Recombinant peptide gene therapy compositions. In some embodiments, the recombinant polynucleotide is provided in a viral vector. In some embodiments, the viral vector is an expression vector that provides expression of an encoded recombinant Factor VIII polypeptide when the vector is administered to a patient or inserted into a mammalian cell. In some embodiments, the viral vector system is based on adenovirus, adeno-associated virus, lentivirus, retrovirus or herpesvirus vectors. In some embodiments, recombinant polynucleotides are formulated in non-viral delivery compositions as described above. In some embodiments, recombinant polynucleotides are provided as gene replacement therapies, eg, using CRISPR technology.

In some embodiments, the condition or disease associated with Factor VIII deficiency and treated with a recombinant Factor VIII polypeptide or a polynucleotide encoding a recombinant Factor VIII polypeptide is hemophilia A. In some embodiments, the hemophilia is acquired hemophilia A. In some embodiments, hemophilia A results from a genetic defect in the gene encoding Factor VIII. In some embodiments, symptoms of Factor VIII deficiency improve with treatment. Example

The following examples, including experiments and results achieved, are provided for illustrative purposes only and are not to be construed as limiting the invention.

In the following experimental disclosures, the following abbreviations shall be used where applicable: ppm (parts per million); M (molar); mM (millimole), uM and µM (micromolar); nM (nanomolar) ); mol (mol); gm and g (gram); mg (milligram); ug and µg (microgram); L and l (liter); ml and mL (millilitre); cm (centimeter); mm (millimetre) ;um and µm (micron); sec. (second); min (minute); h and hr (hour); U (unit); MW (molecular weight); rpm (rotation/minute); ℃ (degrees Celsius); CDS ( Coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); FIOPC (fold improvement compared to positive control). Example 1 Obtaining FVIII gene and constructing expression vector

The synthetic gene (SEQ ID NO: 1) encoding the WT human FVIII sequence with a deletion of the B region (SEQ ID NO: 2) was constructed as previously described (see, eg, US Patent Publication No. 2017/0360900). The oligonucleotides BamHI-pcDNA-FVIII-F and XhoI-pcDNA-FVIII-R were used to amplify fragments of the coding sequence of FVIII variants. The PCR products were ligated into the BamHI/XhoI linearized mammalian expression vector pcDNA3.1(+) (Invitrogen) or a vector containing the CMV promoter and BGH-pA (bovine growth hormone polyadenylation) sequence. Directed evolution techniques, generally known to those skilled in the art, were used to generate genetic variants derived from SEQ ID NO: 2 in this plastid construct (see, eg, US Patent No. 8,383,346 and International Patent Publication WO2010/144103).

Codon-optimized secretory expression of human FVIII in mammalian cells is achieved using the native FVIII signal peptide. The pure line mainly expresses the pDH.1(+) vector in HEK293T cells, Expi293F™ (ThermoFisher Scientific) cells and HepG2 cells. This method uses plasma from hemophilia A patients to provide supernatants with measurable activity in chromogenic assays (see, e.g., Aniara Diagnostica BIOPHEN™ FVIII:C) and activated partial thromboplastin time (APTT) assays.

In some embodiments, combinatorial libraries are constructed to generate FVIII variants derived from SEQ ID NO: 2 in order to identify mutational diversity with similar stability and improved blood coagulation function compared to SEQ ID NO: 2. Screening above equivalent volumes in unchallenged conditions (unincubated, pH 7.4) or after incubation in human plasma (physiological pH 7.1-8.2) for one to seventeen hours at 37°C to 55°C Clear liquid. FVIII variant systems with improved activity due to increased FVIII performance or FVIII specific activity are primarily identified by their fold improvement compared to the parent FVIII. FVIII mutant systems with increased stability are identified by dividing the fold improvement observed under challenged conditions by the fold improvement observed under unchallenged conditions. This approach reduces the preference for selected variants based on increased performance but does not alter stability. The combined activity score (the product of fold improvements across all three conditions) and stability (the product of stability scores) were used to rank mutations in improved variants for inclusion in subsequent FVIII libraries. In additional embodiments, additional methods and sequences described in the Examples are used. Example 2 High-Throughput Growth High-Throughput (HTP) Growth of FVIII and FVIII Variants

Lipofectamine transfection was performed using ExpiFectamine™ 293 Transfection Kit (ThermoFisher Scientific) or LIPOFECTAMINE ^® 3000 Reagent (ThermoFisher Scientific), using a CMV promoter and a vector encoding wild-type FVIII or a variant with a deletion of the B region. BGH-pA pcDNA 3.1(+) was transfected into Expi293F™ (ThermoFisher Scientific) cells or HepG2 cells. Expi293F™ (ThermoFisher Scientific) cells were cultured in growth medium (Expi293™ Expression Medium, ThermoFisher Scientific). Immediately before transfection, cells were transferred to NUNC™ 96-well DeepWell™ plates (ThermoFisher Scientific) at a density of 10 ⁸ cells/well/400 µL of growth medium and incubated in an incubator at 37°C, 8% CO ₂ times to cultivate. HepG2 cells were cultured in growth medium (EMEM with 10% fetal calf serum, both from Corning). 24 hours before transfection, cells were seeded into NUNC ^® Edge 2.0 96-well culture plates (ThermoFisher Scientific) at a density of 10 ⁴ cells/well/250 µL of growth medium and incubated in an incubator at 37°C, 5 Cultivated under % CO ₂ . Cells are incubated for 24-96 hours under specific conditions to allow expression and secretion of FVIII variants. Conditioned medium (50-100 µL) from transfection was transferred to Corning 96-well solid black culture plates (Corning) for activity and/or stability analysis. Example 3 In vitro characterization of variants in HTP HTP analysis of supernatant (chromogenic analysis)

FVIII variant activity was determined by measuring the release of p-nitroaniline (pNa) from synthetic peptide substrates. For unchallenged analysis, 25 µL of Expi293F or HepG2 conditioned medium generated as described above was warmed to 37°C and mixed with 25 µL of "Reagent 1" (BIOPHEN™ FVIII:C, Aniara) and 25 µL of "Reagent 2" ( BIOPHEN™ FVIII:C, Aniara) was mixed in a 96-well black opaque bottom microtiter plate. The reaction was mixed briefly and incubated at 37°C for 5 minutes. 25 µL of "Reagent 3" (BIOPHEN™ FVIII:C, Aniara) was then added and the reaction was mixed briefly and incubated at 37°C for 5 minutes before being quenched with 25 µL pf 2% citric acid. The release of pNA was analyzed using an EnVision® 2105 multi-mode disk reader (PerkinElmer) monitoring absorbance at 405 nm. "Reagent 1", "Reagent 2" and "Reagent 3" are components of the chromogenic assay (BIOPHEN™ FVIII:C) supplied by Aniara and were used as recommended by the supplier. HTP analysis of supernatant (coagulation analysis)

FVIII variant activity was determined by measuring the coagulation rate of hemophilia A patient plasma (George King Bio-Medical, Inc.) using activated partial thromboplastin time analysis. For unchallenged analysis, 25 µL of Expi293F or HepG2 conditioned media generated as described above was warmed to 37°C and mixed with 30 µL of hemophilia A patient plasma (George King Bio-Medical, Inc.) and 50 µL of aPTT -XL reagent (Pacific Hemostasis) was mixed in a 96-well black opaque bottom microtiter plate. The reaction was mixed briefly and incubated at 37°C for 3 minutes. Then 10 µL of 200 µM calcium chloride (Sigma Aldrich) was added and the reaction was mixed briefly. Coagulation was analyzed using an EnVision® 2105 multimode disk reader (PerkinElmer) monitoring absorbance at 405 nm over a period of 5-10 minutes. HTP analysis of supernatants pretreated with plasma

FVIII variants were challenged with hemophilia A patient plasma (George King Bio-Medical, Inc.) to simulate conditions that the variants may encounter within the bloodstream. First, 50 µL of Expi293F or HepG2 conditioned medium and 50 µL of hemophilia A patient plasma (George King Bio-Medical, Inc.) generated as described above were added to a 96-well round-bottom microtiter plate. The plates were sealed with a ^PlateLoc® Thermal Microplate Sealer (Agilent) and incubated at 37-55°C for 1-24 hours. The sample is then analyzed by chromogenic or coagulation assays as described above. Example 4 FVIII variants derived from SEQ ID NO: 2

In this example, experiments performed to determine the activity of FVIII variants are described. These variants were tested using chromogenic analysis as described in Example 3. The results using the variants expressed in Expi293F cells are shown in Table 4.1 and Table 4.2 . Table 4.1 : Activity of FVIII variants relative to SEQ ID NO: 2 SEQ ID NO: (nt/aa) Amino acid differences ( relative to SEQ ID NO: 2) Unchallenged chromogenic FIOPC relative to SEQ ID NO: 2 1/2 + 3/4 T1148N + 5/6 P85Q/I708T/T1148N ++ 7/8 K798T + 9/10 L706G + 11/12 L706A + 13/14 L171Q + 15/16 Q335E + 17/18 V63R ++ 19/20 K344A ++ 21/22 S428F ++ 23/24 E879Q ++ 25/26 F59L + 27/28 Q429S + 29/30 K205R ++ 31/32 I1264W + 33/34 M797P + 35/36 L1248S + 37/38 I1264R + 39/40 Y873T + 41/42 T792Y + 43/44 I708K + 45/46 K344L ++ 47/48 F57V + 49/50 Q96L + 51/52 A362W + 53/54 V63G ++ 55/56 Y873L + 57/58 M1292Y ++ 59/60 T1148Q ++ 61/62 D346V ++ 63/64 M797G + 65/66 L706Y + 67/68 L1248R ++ 69/70 N433A ++ 71/72 Y65A + 73/74 F59W + 75/76 Y873Q + 77/78 V795C + 79/80 E684K/K798R ++ 81/82 F57L + 83/84 Q353S + 85/86 S332Q + 87/88 I708A + 89/90 G1259T + 91/92 V63M + 93/94 M356L ++ 95/96 L1248G ++ 97/98 L1248P ++ 99/100 Q436G + 101/102 F57A + 103/104 F59S ++ 105/106 T1245R + 107/108 F59R + 109/110 Q96W + 111/112 M797Y + 113/114 K1145R + 115/116 Q436D + 117/118 F716L + 119/120 S56E + 121/122 R773H + 123/124 T734I ++ 125/126 N358S ++ 127/128 Q335P + 129/130 F59Q ++ 131/132 S729T + 133/134 F716K ++ 135/136 T665G + 137/138 A362T + 139/140 F57T + 141/142 N359G + 143/144 T61S ++ 145/146 V1250A + 147/148 A362V + 149/150 N359E + 151/152 F716W + 153/154 S332W ++ 155/156 I708V + 157/158 V63H ++ 159/160 M797C + 161/162 T61Q + 163/164 M356N ++ 165/166 V1250S + 167/168 K798C + 169/170 N433G ++ 171/172 V795N ++ 173/174 Q96I + 175/176 S56P + 177/178 V63N + 179/180 T792W + 181/182 I1264L + 183/184 V1250P + 185/186 S332P + 187/188 F59V + 189/190 K726L ++ All activities are determined relative to the reference polypeptide of SEQ ID NO:2. The level of increased activity is defined as follows: "+" 0.9 to 1.1, "++"> 1.1 Table 4.2 : Activity of FVIII variants relative to SEQ ID NO: 2 SEQ ID NO: (nt/aa) Amino acid difference ( relative to SEQ ID NO: 2) Unchallenged chromogenic FIOPC relative to SEQ ID NO: 2 1/2 + 191/192 Q37H/Q96R/L171P/Q429L/I708T/K799R/T1148N ++ 193/194 L171P ++ 195/196 L171P/Q429L/K732R/T1148N ++ 197/198 Q96R/L171P ++ 199/200 L171P/E919G/G1151S +++ 201/202 L171P/T792Y/M797Q/K799R/T1148N ++ 203/204 L171P/P569T/T1148N ++ 205/206 Q96R/L171P/Q429L/T792Y/M797T/K799R ++ 207/208 L171P/Q429L/I708T/L1422I +++ 209/210 L171P/Q429L ++ 211/212 D321Y/A362P/K732E/M797T/K799R ++ 213/214 L171P/L1100I ++ 215/216 I708T/T1148N/A1186E +++++ 217/218 Q429L/L724I/K732E/T1148N ++ 219/220 L171P/A362E/V795T/M797Q/K799M ++ 221/222 L171P/Q429L/K732E ++ 223/224 Q96R/L171P/S643N/M797T/K799R ++ 225/226 L171P/T792Y/M797T/K799M ++ 227/228 R503S ++ 229/230 L171P/K732E ++ 231/232 T1148N/F1193L ++ 233/234 Q96R/L171P/A362E/Q429L/K732R/V795T/M797Q ++ 235/236 K726A + 237/238 F59M + 239/240 T734R ++ 241/242 A362N + 243/244 H334G ++ 245/246 K732G ++ 247/248 V63Q ++ 249/250 E351S ++ 251/252 L1377D +++++ 253/254 V795S ++ 255/256 N358K ++ 257/258 Q999R ++ 259/260 V1250T ++ 261/262 E306G ++ 263/264 P349G ++ 265/266 L1377G +++ 267/268 S332F ++ 269/270 L1377E +++++ 271/272 V257I ++ 273/274 L646I ++ 275/276 D346R ++ 277/278 P435Q ++ 279/280 Q96V ++ 281/282 L1377R ++ 283/284 L1377S +++ 285/286 F59G ++ 287/288 L171M ++ 289/290 T792H + 291/292 L1248D ++ 293/294 E306S ++ 295/296 L706M + 297/298 L171H ++ 299/300 L1377A +++ 301/302 L1377V ++ 303/304 T734S ++ 305/306 T1148R ++ 307/308 F57S ++ 309/310 F59D ++ 311/312 K344E + 313/314 A362G ++ 315/316 P58K ++ All activities are determined relative to the reference polypeptide of SEQ ID NO:2. The levels of increased activity are defined as follows: "+" 0.9 to 1.1, "++"> 1.1, "+++"> 2, "++++"> 3.5, "+++++"> 5 Example 5 FVIII variants derived from SEQ ID NO: 252

In this example, experiments performed to determine the activity of FVIII variants are described. These variants were tested using chromogenic analysis as described in Example 3. The results using the variants expressed in Expi293F cells are shown in Table 5.1 and Table 5.2 . Table 5.1 : Activity of FVIII variants relative to SEQ ID NO: 252 SEQ ID NO: (nt/aa) Amino acid differences ( relative to SEQ ID NO: 252) Amino acid difference ( relative to SEQ ID NO: 2) Unchallenged chromogenic FIOPC relative to SEQ ID NO: 252 251/252 L1377D + 317/318 M1380W L1377D/ M1380W +++ 319/320 I388A I388A/L1377D + 321/322 F1043E F1043E/L1377D + 323/324 F1043R F1043R/L1377D + 325/326 I610R I610R/L1377D + 327/328 I1274N I1274N/L1377D + 329/330 S1220T S1220T/L1377D + 331/332 I1442T L1377D/I1442T + 333/334 F1193L F1193L/L1377D + 335/336 Q621T Q621T/L1377D + 337/338 L1054C L1054C/L1377D + 339/340 A755Q A755Q/L1377D + 341/342 Y1041G Y1041G/L1377D + 343/344 Y748S Y748S/L1377D + 345/346 Y1049G Y1049G/L1377D + 347/348 L1221V L1221V/L1377D + 349/350 M1051E M1051E/L1377D + 351/352 R612G R612G/L1377D + 353/354 F1037H F1037H/L1377D + 355/356 N616E N616E/L1377D + 357/358 Y1041H Y1041H/L1377D + 359/360 F387S F387S/L1377D + 361/362 S489R S489R/L1377D + 363/364 Q621M Q621M/L1377D + 365/366 S489Q S489Q/L1377D + 367/368 L750G L750G/L1377D ++ 369/370 S1224P S1224P/L1377D + 371/372 A618C A618C/L1377D + 373/374 N616G N616G/L1377D + 375/376 R612T R612T/L1377D + 377/378 I1050Q I1050Q/L1377D + 379/380 T1053A T1053A/L1377D + 381/382 N616W N616W/L1377D + 383/384 I896V I896V/L1377D + 385/386 S489F S489F/L1377D + 387/388 I1050P I1050P/L1377D + 389/390 M897G M897G/L1377D + 391/392 L749T L749T/L1377D + 393/394 I1274V I1274V/L1377D + 395/396 S1220L S1220L/L1377D + 397/398 S260E S260E/L1377D + 399/400 L481V L481V/L1377D +++ 401/402 F613S F613S/L1377D + 403/404 F387G F387G/L1377D + 405/406 Q621R Q621R/L1377D + 407/408 I756R I756R/L1377D + 409/410 V63R V63R/L1377D + All activities were determined relative to the reference polypeptide of SEQ ID NO: 252. The level of increased activity is defined as follows: "+" 0.9 to 1.1, "++"> 1.1, "+++"> 2 Table 5.2 : Activity of FVIII variants relative to SEQ ID NO: 252 SEQ ID NO: (nt/aa) Amino acid differences ( relative to SEQ ID NO: 252) Amino acid difference ( relative to SEQ ID NO: 2) (Expi293F) Unchallenged chromogenic FIOPC relative to SEQ ID NO: 252 251/252 L1377D + 411/412 I265S I265S/L1377D + 413/414 L1221T L1221T/L1377D + 415/416 M1380G L1377D/M1380G ++ 417/418 K752G K752G/L1377D + 419/420 Y748K Y748K/L1377D ++ 421/422 A755L A755L/L1377D ++ 423/424 F1218A F1218A/L1377D ++ 425/426 Y748N Y748N/L1377D + 427/428 T1378E L1377D/T1378E ++ 429/430 L614I L614I/L1377D + 431/432 L749F L749F/L1377D ++ 433/434 F387V F387V/L1377D + 435/436 L750T L750T/L1377D + 437/438 F1193G F1193G/L1377D ++ 439/440 L749V L749V/L1377D ++ 441/442 T1378A T1378A/L1377D + 443/444 L750V L750V/L1377D ++ 445/446 F387T F387T/L1377D ++ 447/448 F387Q F387Q/L1377D ++ 449/450 L750A L750A/L1377D ++ 451/452 F1037N F1037N/L1377D ++ 453/454 H1440A H1440A/L1377D ++ 455/456 M1051S M1051S/L1377D +++ 457/458 L1221G L1221G/L1377D ++ 459/460 T1378D L1377D/T1378D ++ 461/462 H1440S L1377D/H1440S +++ 463/464 F387R F387R/L1377D + 465/466 F1193A F1193A/L1377D ++ 467/468 F387I F387I/L1377D ++ 469/470 F1218M F1218M/L1377D ++ 471/472 I265W I265W/L1377D ++ 473/474 H1440E L1377D/H1440E ++ 475/476 V1439L L1377D/V1439L + 477/478 H1440T L1377D/H1440T +++ 479/480 R612S R612S/L1377D + 481/482 Y1049W Y1049W/L1377D + 483/484 L1221S L1221S/L1377D +++ 485/486 S1220D S1220D/L1377D ++ 487/488 L749G L749G/L1377D ++ 489/490 L749S L749S/L1377D + 491/492 F1218G F1218G/L1377D ++ 493/494 I1205L I1205L/L1377D ++ 495/496 Y1222F Y1222F/L1377D ++ 497/498 F1193E F1193E/L1377D ++ 499/500 I1050S I1050S/L1377D ++ 501/502 S1389W L1377D/S1389W + 503/504 V1439E L1377D/V1439E +++ 505/506 S260T S260T/L1377D ++ 507/508 F1037P F1037P/L1377D + 509/510 M1051K M1051K/L1377D +++ 511/512 I1269F I1269F/L1377D + 513/514 I265R I265R/L1377D ++ 515/516 M1051R M1051R/L1377D ++ 517/518 F1218L F1218L/L1377D ++ 519/520 M897R M897R/L1377D ++ 521/522 I388P I388P/L1377D + 523/524 Y748G Y748G/L1377D ++ 525/526 I756P I756P/L1377D ++ 527/528 Y748F Y748F/L1377D + 529/530 I390A I390A/L1377D ++ 531/532 I610L I610L/L1377D + 533/534 A755G A755G/L1377D ++ 535/536 L749R L749R/L1377D ++ 537/538 Y748V Y748V/L1377D + 539/540 M1380A L1377D/M1380A + 541/542 H1440C L1377D/H1440C + 543/544 H397N/F1218M H397N/F1218M/L1377D ++ 545/546 F1037M F1037M/L1377D + 547/548 F1218P F1218P/L1377D + 549/550 E757K E757K/L1377D ++ 551/552 V1373S V1373S/L1377D ++ 553/554 H1440G L1377D/H1440G ++ 555/556 F1193I F1193I/L1377D ++ 557/558 V1439T L1377D/V1439T ++ 559/560 L750D L750D/L1377D ++ 561/562 I390G I390G/L1377D ++ 563/564 I756G I756G/L1377D + 565/566 S1220E S1220E/L1377D ++ 567/568 T1378C L1377D/T1378C + 569/570 A618G A618G/L1377D + 571/572 D346R D346R/L1377D ++ 573/574 V63Q/L1248D V63Q/L1248D/L1377D ++ 575/576 P349G P349G/L1377D ++ 577/578 E306G/R355C/L1422I E306G/R355C/ L1377D/L1422I ++ 579/580 L1422I L1377D/L1422I ++ 581/582 E306G/D346R/I708T E306G/D346R/I708T/L1377D ++ 583/584 T1148N T1148N/L1377D +++ 585/586 L171P/D346R/L1248D L171P/D346R/L1248D/L1377D +++ 587/588 V63Q/M797T/K799R/L1248D/L1422I V63Q/M797T/K799R/L1248D/L1377D/L1422I ++ 589/590 L171P/K799R L171P/K799R/L1377D ++ 591/592 A362P A362P/L1377D ++ 593/594 L1248D L1248D/L1377D ++ 595/596 V63Q V63Q/L1377D ++ 597/598 L171P/E306G/D346R/Q999R/L1248D L171P/E306G/D346R/Q999R/L1248D/L1377D +++ 599/600 L171P/T1148N L171P/T1148N/L1377D +++ 601/602 T734S/K799R/L1248D/L1422I T734S/K799R/L1248D/L1377D/L1422I ++ 603/604 V63Q/K732E V63Q/K732E/L1377D +++ 605/606 V63Q/L171P/E306G/D346R/K732G V63Q/L171P/E306G/D346R/K732G/L1377D ++ 607/608 L171P L171P/L1377D ++ 609/610 L171P/L1422I L171P/L1377D/L1422I ++ 611/612 V63Q/A362P/K732G/K799R/L1248D V63Q/A362P/K732G/K799R/L1248D/L1377D ++ 613/614 Q999R/T1148N/L1422I Q999R/T1148N/L1377D/L1422I + 615/616 D346R/P349G D346R/P349G/L1377D +++ 617/618 E306G/P349G E306G/P349G/L1377D ++ 619/620 V63Q/L171P/T1148N/G1151S V63Q/L171P/T1148N/L1377D/G1151S ++ 621/622 E306G E306G/L1377D + 623/624 K344E/P349G/L354R/V1250T K344E/P349G/L354R/V1250T/L1377D ++ 625/626 V63G V63G/L1377D + 627/628 V1250T V1250T/L1377D + 629/630 V63R/E306G/K344E/P349G/L354R/R503S/L1248R V63R/E306G/K344E/P349G/L354R/R503S/L1248R/L1377D +++ 631/632 F59D/V63S/S332P/P349G/R503S/L706M/I708K F59D/V63S/S332P/P349G/R503S/L706M/I708K/L1377D ++ 633/634 V63S/E306G/K344A/P349G/L354R/L706M/L1248R V63S/E306G/K344A/P349G/L354R/L706M/L1248R/L1377D ++ 635/636 L1248R L1248R/L1377D ++ 637/638 F59D/V63G F59D/V63G/L1377D ++ 639/640 S332P/L706M/I708K/L1248R/V1250T S332P/L706M/I708K/L1248R/V1250T/L1377D ++ 641/642 R503S R503S/L1377D ++ 643/644 F59D/V63R/P349G/L354R F59D/V63R/P349G/L354R/L1377D +++ 645/646 F59D/P349G/I708K F59D/P349G/I708K/L1377D +++ 647/648 F59D/E306G/K344E/L1248R F59D/E306G/K344E/L1248R/L1377D +++ 649/650 K344E/L1248R/V1250T K344E/L1248R/V1250T/L1377D ++ 651/652 I708K I708K/L1377D ++ 653/654 V63R/K344E/P349G V63R/K344E/P349G/L1377D ++ 655/656 F59D F59D/L1377D + 657/658 V63S/K344A/P349G/L354R V63S/K344A/P349G/L354R/L1377D ++ 659/660 L706M L706M/L1377D + 661/662 F59D/E306G/R770W F59D/E306G/R770W/L1377D + 663/664 V63G/S347N/L354R/L1248R V63G/S347N/L354R/L1248R/L1377D ++ 665/666 L354R/R503S L354R/R503S/L1377D +++ All activities were determined relative to the reference polypeptide of SEQ ID NO: 252. The level of increased activity is defined as follows: "+" 0.9 to 1.1, "++"> 1.1, "+++"> 2 Example 6 FVIII variants derived from SEQ ID NO: 504

In this example, experiments performed to determine the activity of FVIII variants are described. These variants were tested using chromogenic and coagulation assays as described in Example 3. Results using variants expressed in Expi293F cells or HepG2 cells are shown in Table 6.1 and Table 6.2 . Table 6.1 : Activity of FVIII variants relative to SEQ ID NO: 504 SEQ ID NO: (nt/aa) Amino acid differences ( relative to SEQ ID NO: 504) Amino acid differences ( relative to SEQ ID NO: 2) (Expi293F) Unchallenged chromogenic FIOPC relative to SEQ ID NO: 504 503/504 L1377D/V1439E + 667/668 L750D L750D/L1377D/V1439E ++ 669/670 M1051K M1051K/L1377D/V1439E ++ 671/672 M1051S M1051S/L1377D/V1439E ++ 673/674 S260T S260T/L1377D/V1439E ++ 675/676 I756P I756P/L1377D/V1439E + 677/678 F1193A F1193A/L1377D/V1439E ++ 679/680 S332P S332P/L1377D/V1439E + 681/682 M897R M897R/L1377D/V1439E ++ 683/684 F387Q F387Q/L1377D/V1439E + 685/686 F1037N F1037N/L1377D/V1439E + 687/688 F387R F387R/L1377D/V1439E ++ 689/690 E306G/L749F E306G/L749F/L1377D/V1439E ++ 691/692 I1270V I1270V/L1377D/V1439E + 693/694 S489E S489E/L1377D/V1439E ++ 695/696 Q487N Q487N/L1377D/V1439E ++ 697/698 A488G A488G/L1377D/V1439E ++ 699/700 Q903E Q903E/L1377D/V1439E + All activities were determined relative to the reference polypeptide of SEQ ID NO: 504. The level of increased activity is defined as follows: "+" 0.9 to 1.1, "++"> 1.1 Table 6.2 : Activity of FVIII variants relative to SEQ ID NO: 504 SEQ ID NO: (nt/aa) Amino acid differences ( relative to SEQ ID NO: 504) Amino acid differences ( relative to SEQ ID NO: 2) (Expi293F) Unchallenged chromogenic FIOPC relative to SEQ ID NO: 504 (HepG2) Unchallenged chromogenic FIOPC relative to SEQ ID NO: 504 (Expi293F) Unchallenged coagulation active FIOPC relative to SEQ ID NO: 504 503/504 L1377D/V1439E + + + 701/702 L171P/L1248D/H1440T L171P/L1248D/ L1377D/V1439E/H1440T +++ +++ ++ 703/704 S260T/T1148N/T1378D S260T/T1148N/L1377D/T1378D/V1439E +++ + + 705/706 L171P/S260T/F387I/L750D/L1248D L171P/S260T/F387I/L750D/L1248D/L1377D/V1439E +++ +++ +++ 707/708 L171P/S260T L171P/S260T/L1377D/V1439E +++ +++ ++ 709/710 L171P/S260T/F387I/L750D/M1051S/T1148N L171P/S260T/F387I/L750D/M1051S/T1148N/L1377D/V1439E +++ +++ +++ 711/712 L171P L171P/L1377D/V1439E +++ +++ ++ 713/714 L750D/M1051S/T1148N L750D/M1051S/T1148N/L1377D/V1439E +++ ++ ++ 715/716 L171P/S260T/I756P L171P/S260T/I756P/L1377D/V1439E +++ +++ ++ 717/718 L171P/L1248D L171P/L1248D/L1377D/V1439E +++ +++ ++ 719/720 L171P/T1148N L171P/T1148N/L1377D/V1439E ++ +++ ++ 721/722 F387I F387I/L1377D/V1439E ++ + + 723/724 F59D F59D/L1377D/V1439E ++ + ++ 725/726 H1440S L1377D/V1439E/H1440S ++ + + 727/728 S332P/L750D/L1248D S332P/L750D/L1248D/L1377D/V1439E +++ ++ +++ 729/730 I756P/L1248D I756P/L1248D/L1377D/V1439E ++ + ++ 731/732 L1248D L1248D/L1377D/V1439E ++ ++ 733/734 L171P/F387I/L1248D/H1440S L171P/F387I/L1248D/L1377D/H1440S/V1439E +++ ++ ++ 735/736 L750D/H1440S L750D/L1377D/V1439E/H1440S ++ ++ ++ 737/738 F59D/L171P/L750D F59D/L171P/L750D/L1377D/V1439E +++ +++ +++ 739/740 L171P/F1193A/L1248D L171P/F1193A/L1248D/L1377D/V1439E +++ +++ ++ 741/742 L750D/T1148N/F1193A/H1440S L750D/T1148N/F1193A/L1377D/V1439E/H1440S +++ ++ ++ 743/744 T1148N T1148N/L1377D/V1439E ++ ++ ++ 745/746 L171P/F387I/M1051K L171P/F387I/M1051K/L1377D/V1439E ++ +++ ++ 747/748 Y748G Y748G/L1377D/V1439E ++ + + 749/750 E306G E306G/L1377D/V1439E ++ 751/752 E306G/F387R/I390A/L749F/S1220D E306G/F387R/I390A/L749F/S1220D/L1377D/V1439E +++ +++ ++ 753/754 I390A I390A/L1377D/V1439E ++ +++ ++ 755/756 E306G/I390A/F1037N E306G/I390A/F1037N/L1377D/V1439E ++ ++ ++ 757/758 E306G/R612S/L749F E306G/R612S/L749F/L1377D/V1439E ++ + ++ 759/760 F387V/I390A/L749F F387V/I390A/L749F/L1377D/V1439E +++ ++ ++ 761/762 V63S/I390A/R503S/L749F V63S/I390A/R503S/L749F/L1377D/V1439E ++ ++ ++ 763/764 F387R/I390A/L749R F387R/I390A/L749R/L1377D/V1439E ++ ++ ++ 765/766 S1220D S1220D/L1377D/V1439E ++ 767/768 V63S/E306G V63S/E306G/L1377D/V1439E ++ ++ ++ 769/770 L749F L749F/L1377D/V1439E ++ ++ 771/772 E306G/I390A/L749F/F1037N E306G/I390A/L749F/F1037N/L1377D/V1439E ++ ++ ++ 773/774 F387V F387V/L1377D/V1439E ++ ++ + 775/776 E306G/L749F/S1220D E306G/L749F/S1220D/L1377D/V1439E ++ ++ + 777/778 E306G/F387V/I390A/R612S/L749F/F1037N E306G/F387V/I390A/R612S/L749F/F1037N/L1377D/V1439E ++ ++ ++ 779/780 F328S F328S/L1377D/V1439E ++ ++ + All activities were determined relative to the reference polypeptide of SEQ ID NO: 504. The level of increased activity is defined as follows: "+" 0.9 to 1.1, "++"> 1.1, "+++"> 2 Example 7 FVIII variants derived from SEQ ID NO: 706

In this example, experiments performed to determine the activity of FVIII variants are described. These variants were tested using chromogenic and coagulation assays as described in Example 3. Results using variants expressed in Expi293F cells or HepG2 cells are shown in Table 7.1 and Table 7.2 . Table 7.1 : Activity of FVIII variants relative to SEQ ID NO: 706 SEQ ID NO: (nt/aa) Amino acid differences ( relative to SEQ ID NO: 706) Amino acid differences ( relative to SEQ ID NO: 2) (Expi293F) Unchallenged chromogenic FIOPC relative to SEQ ID NO: 706 705/706 L171P/S260T/F387I/L750D/L1248D/L1377D/V1439E + 781/782 M1051K L171P/S260T/F387I/L750D/M1051K/L1248D/L1377D/V1439E ++ 783/784 L1221G L171P/S260T/F387I/L750D/L1221G/L1248D/L1377D/V1439E + 785/786 F1218G L171P/S260T/F387I/L750D/F1218G/L1248D/L1377D/V1439E + 787/788 I387R L171P/S260T/F387R/L750D/L1248D/L1377D/V1439E + 789/790 M1051R L171P/S260T/F387I/L750D/M1051R/L1248D/L1377D/V1439E ++ 791/792 L749F/F1218G L171P/S260T/F387I/L749F/L750D/F1218G/L1248D/L1377D/V1439E ++ 793/794 I387T L171P/S260T/F387T/L750D/L1248D/L1377D/V1439E + 795/796 S332P L171P/S260T/S332P/F387I/L750D/L1248D/L1377D/V1439E + 797/798 M897K L171P/S260T/F387I/L750D/M897K/L1248D/L1377D/V1439E + 799/800 T696E L171P/S260T/F387I/T696E/L750D/L1248D/L1377D/V1439E + 801/802 F816W L171P/S260T/F387I/L750D/F816W/L1248D/L1377D/V1439E + 803/804 F824W L171P/S260T/F387I/L750D/F824W/L1248D/L1377D/V1439E + 805/806 A826G L171P/S260T/F387I/L750D/A826G/L1248D/L1377D/V1439E + 807/808 V393W L171P/S260T/F387I/V393W/L750D/L1248D/L1377D/V1439E + 809/810 I825L L171P/S260T/F387I/L750D/I825L/L1248D/L1377D/V1439E + 811/812 M897R L171P/S260T/F387I/L750D/M897R/L1248D/L1377D/V1439E + 813/814 F816V L171P/S260T/F387I/L750D/F816V/L1248D/L1377D/V1439E ++ 815/816 F698W L171P/S260T/F387I/F698W/L750D/L1248D/L1377D/V1439E ++ 817/818 V393Q L171P/S260T/F387I/V393Q/L750D/L1248D/L1377D/V1439E + All activities were determined relative to the reference polypeptide of SEQ ID NO: 706. The level of increased activity is defined as follows: "+" 0.9 to 1.1, "++"> 1.1 Table 7.2 : Activity of FVIII variants relative to SEQ ID NO: 706 SEQ ID NO: (nt/aa) Amino acid differences ( relative to SEQ ID NO: 706) Amino acid differences ( relative to SEQ ID NO: 2) (Expi293F) Unchallenged chromogenic FIOPC relative to SEQ ID NO: 706 (HepG2) Unchallenged chromogenic FIOPC relative to SEQ ID NO: 706 (Expi293F) Unchallenged coagulation active FIOPC relative to SEQ ID NO: 706 705/706 L171P/S260T/F387I/L750D/L1248D/L1377D/V1439E + + + 819/820 S332P/I387R/I390A/T1148N L171P/S260T/S332P/F387R/I390A/L750D/T1148N/L1248D/L1377D/V1439E +++ +++ +++ 821/822 F328S/I387R/L749F L171P/S260T/F328S/F387R/L749F/L750D/L1248D/L1377D/V1439E + + ++ 823/824 L749F L171P/S260T/F387I/L749F/L750D/L1248D/L1377D/V1439E + + + 825/826 E306G/F328S/S332P/I390A/M1051R/F1193A/F1218G L171P/S260T/E306G/F328S/S332P/F387I/I390A/L750D/M1051R/F1193A/F1218G/L1248D/L1377D/V1439E +++ +++ + 827/828 V63Q/E306G/S332P/M1051R/T1148N V63Q/L171P/S260T/E306G/S332P/F387I/L750D/M1051R/T1148N/L1248D/L1377D/V1439E +++ +++ +++ 829/830 V63Q/E306G/S332P/I387T/M1051K V63Q/L171P/S260T/E306G/S332P/F387T/L750D/M1051K/L1248D/L1377D/V1439E ++ +++ ++ 831/832 I387T/I390A/M1051R L171P/S260T/F387T/I390A/L750D/M1051R/L1248D/L1377D/V1439E ++ +++ ++ 833/834 D186A/E306G/F328S/S332P/I390A/L749F/M1051K/T1148N/F1193A L171P/D186A/S260T/E306G/F328S/S332P/F387I/I390A/L749F/L750D/M1051K/T1148N/F1193A/L1248D/L1377D/V1439E +++ ++ +++ 835/836 I387T/I390A/I756P/M1051R L171P/S260T/F387T/I390A/L750D/I756P/M1051R/L1248D/L1377D/V1439E ++ ++ ++ 837/838 E306G/I390A/M1051R L171P/S260T/E306G/F387I/I390A//L750D/M1051R/L1248D/L1377D/V1439E +++ ++ +++ 839/840 E306G/S332P/I390A/L749F L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E +++ +++ +++ 841/842 E306G/I387R/I390A/T1148N L171P/S260T/E306G/F387R/I390A/L750D/T1148N/L1248D/L1377D/V1439E ++ ++ +++ 843/844 E306G/F328S/S332P/I387T/I390A/L749F/M1051R/T1148N/F1193A L171P/S260T/E306G/F328S/S332P/F387T/I390A/L749F/L750D/M1051R/T1148N/F1193A/L1248D/L1377D/V1439E +++ +++ +++ 845/846 S332P/I387R/I390A/L749F/T1148N L171P/S260T/S332P/F387R/I390A/L749F/L750D/T1148N/L1248D/L1377D/V1439E +++ ++ +++ 847/848 I387R/I390A/L749F L171P/S260T/F387R/I390A/L749F/L750D/L1248D/L1377D/V1439E ++ ++ ++ 849/850 E306G/I387T/L749F/M1051K/T1148N L171P/S260T/E306G/F387T/L749F/L750D/M1051K/T1148N/L1248D/L1377D/V1439E ++ ++ ++ 851/852 I387R/L749F L171P/S260T/F387R/L749F/L750D/L1248D/L1377D/V1439E ++ ++ ++ 853/854 E306G/Q945K/M1051R/F1193A/L1221G L171P/S260T/E306G/F387I/L750D/Q945K/M1051R/F1193A/L1221G/L1248D/L1377D/V1439E ++ + + 855/856 E306G/I390A/L749F/M1051K L171P/S260T/E306G/F387I/I390A/L749F/L750D/M1051K/L1248D/L1377D/V1439E ++ + ++ 857/858 V63Q/E306G/S332P/I390A V63Q/L171P/S260T/E306G/S332P/F387I/I390A/L750D/L1248D/L1377D/V1439E ++ ++ +++ 859/860 E306G/I387T/I390A/L749F/T1148N/F1193A/L1221G L171P/S260T/E306G/F387T/I390A/L749F/L750D/T1148N/F1193A/L1221G/L1248D/L1377D/V1439E +++ ++ + 861/862 E306G/S332P/I387R/I390A/L749F/F1218M L171P/S260T/E306G/S332P/F387R//I390A/L749F/L750D/F1218M/L1248D/L1377D/V1439E +++ +++ ++ 863/864 V63Q/E306G/S332P/I390A/M1051R/T1148N/F1193A/F1218G V63Q/L171P/S260T/E306G/S332P/F387I/I390A/L750D/M1051R/T1148N/F1193A/F1218G/L1248D/L1377D/V1439E +++ +++ + 865/866 G924I L171P/S260T/F387I/L750D/G924I/L1248D/L1377D/V1439E + ++ + 867/868 A755S L171P/S260T/F387I/L750D/A755S/L1248D/L1377D/V1439E + + + 869/870 K752F L171P/S260T/F387I/L750D/K752F/L1248D/L1377D/V1439E + + ++ 871/872 S751Y L171P/S260T/F387I/L750D/S751Y/L1248D/L1377D/V1439E ++ ++ ++ 873/874 Q923M L171P/S260T/F387I/L750D/Q923M/L1248D/L1377D/V1439E + + + 875/876 M797Y L171P/S260T/F387I/L750D/M797Y/L1248D/L1377D/V1439E + + + 877/878 N754W L171P/S260T/F387I/L750D/N754W/L1248D/L1377D/V1439E + + + 879/880 I793G L171P/S260T/F387I/L750D/I793G/L1248D/L1377D/V1439E + + + 881/882 N753D L171P/S260T/F387I/L750D/N753D/L1248D/L1377D/V1439E ++ + +++ 883/884 R922W L171P/S260T/F387I/L750D/R922W/L1248D/L1377D/V1439E + + + 885/886 I896L L171P/S260T/F387I/L750D/I896L/L1248D/L1377D/V1439E + + + 887/888 K938R L171P/S260T/F387I/L750D/K938R/L1248D/L1377D/V1439E + + + 889/890 I775G L171P/S260T/F387I/L750D/I775G/L1248D/L1377D/V1439E ++ ++ ++ 891/892 S751E L171P/S260T/F387I/L750D/S751E/L1248D/L1377D/V1439E + + + 893/894 K732W L171P/S260T/F387I/L750D/K732W/L1248D/L1377D/V1439E + + ++ 895/896 K752G L171P/S260T/F387I/L750D/K752G/L1248D/L1377D/V1439E + + ++ 897/898 N810V L171P/S260T/F387I/L750D/N810V/L1248D/L1377D/V1439E + + + 899/900 K938V L171P/S260T/F387I/L750D/K938V/L1248D/L1377D/V1439E + + + 901/902 A747D L171P/S260T/F387I/L750D/A747D/L1248D/L1377D/V1439E ++ + ++ 903/904 A755G L171P/S260T/F387I/L750D/A755G/L1248D/L1377D/V1439E + + + 905/906 V393G/A407V L171P/S260T/F387I/V393G/A407V/L750D/L1248D/L1377D/V1439E ++ ++ + 907/908 M797W L171P/S260T/F387I/L750D/M797W/L1248D/L1377D/V1439E + + ++ 909/910 K819V L171P/S260T/F387I/L750D/K819V/L1248D/L1377D/V1439E + + + 911/912 V393G L171P/S260T/F387I/V393G/L750D/L1248D/L1377D/V1439E ++ +++ + 913/914 I787W L171P/S260T/F387I/L750D/I787W/L1248D/L1377D/V1439E + ++ ++ 915/916 V393R L171P/S260T/F387I/V393R/L750D/L1248D/L1377D/V1439E ++ +++ + 917/918 F802V L171P/S260T/F387I/L750D/F802V/L1248D/L1377D/V1439E + ++ + 919/920 I775E L171P/S260T/F387I/L750D/I775E/L1248D/L1377D/V1439E ++ + + 921/922 G924P L171P/S260T/F387I/L750D/G924P/L1248D/L1377D/V1439E + + + 923/924 K732G L171P/S260T/F387I/K732G/L750D/L1248D/L1377D/V1439E + + + 925/926 Q811F L171P/S260T/F387I/L750D/Q811F/L1248D/L1377D/V1439E + + ++ 927/928 F802S L171P/S260T/F387I/L750D/F802S/L1248D/L1377D/V1439E + ++ + 929/930 K752L L171P/S260T/F387I/L750D/K752L/L1248D/L1377D/V1439E + + +++ 931/932 S392A L171P/S260T/F387I/S392A/L750D/L1248D/L1377D/V1439E + + + 933/934 H397G L171P/S260T/F387I/H397G/L750D/L1248D/L1377D/V1439E + ++ + All activities were determined relative to the reference polypeptide of SEQ ID NO: 706. The level of increased activity is defined as follows: "+" 0.9 to 1.1, "++"> 1.1, "+++"> 2 Example 8 FVIII variants derived from SEQ ID NO: 840

In this example, experiments performed to determine the activity of FVIII variants by analyzing protein activity after challenge are described. These variants were tested for FVIII activity after no incubation and after incubation in hemophilia A patient plasma (George King Bio-Medical, Inc.), as described in Example 3. Variants were tested using chromogenic and coagulation assays as described in Example 3. Results using variants expressed in Expi293F cells or HepG2 cells are shown in Table 8.1 and Table 8.2 . Table 8.1 : Activity of FVIII variants relative to SEQ ID NO: 840 SEQ ID NO: (nt/aa) Amino acid differences ( relative to SEQ ID NO: 840) Amino acid differences ( relative to SEQ ID NO: 2) (Expi293F) Unchallenged chromogenic FIOPC relative to SEQ ID NO: 840 (Expi293F) Chromogenic activity of FIOPC relative to SEQ ID NO: 840 after plasma challenge 839/840 L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E + + 935/936 V620N L171P/S260T/E306G/S332P/F387I/I390A/V620N/L749F/L750D/L1248D/L1377D/V1439E + + 937/938 P491E L171P/S260T/E306G/S332P/F387I/I390A/P491E/L749F/L750D/L1248D/L1377D/V1439E ++ + 939/940 M1229D L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/M1229D/L1248D/L1377D/V1439E + + 941/942 P615N L171P/S260T/E306G/S332P/F387I/I390A/P615N/L749F/L750D/L1248D/L1377D/V1439E ++ 943/944 I1283S L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/I1283S/L1377D/V1439E ++ 945/946 H822S L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/H822S/L1248D/L1377D/V1439E ++ 947/948 F328G L171P/S260T/E306G/F328G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E ++ + 949/950 H212L L171P/H212L/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E +++ ++ 951/952 K818W L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/K818W/L1248D/L1377D/V1439E ++ + 953/954 I1283L L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/I1283L/L1377D/V1439E + ++ 955/956 F555G/E684G L171P/S260T/E306G/S332P/F387I/I390A/F555G/E684G/L749F/L750D/L1248D/L1377D/V1439E ++ + 957/958 V828K L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/V828K/L1248D/L1377D/V1439E ++ ++ 959/960 R550G L171P/S260T/E306G/S332P/F387I/I390A/R550G/L749F/L750D/L1248D/L1377D/V1439E + + 961/962 R830M L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/R830M/L1248D/L1377D/V1439E ++ + 963/964 S839A L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/S839A/L1248D/L1377D/V1439E + + 965/966 H822T L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/H822T/L1248D/L1377D/V1439E + + 967/968 L13Y L13Y/L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E ++ ++ 969/970 L14G L14G/L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E + + 971/972 C17V C17V/L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E ++ 973/974 M836V L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/M836V/L1248D/L1377D/V1439E + ++ 975/976 S839P L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/S839P/L1248D/L1377D/V1439E + + 977/978 K1210W L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/K1210W/L1248D/L1377D/V1439E + + 979/980 Y1281R L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/Y1281R/L1377D/V1439E ++ 981/982 V64D V64D/L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E ++ 983/984 H212G L171P/H212G/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E ++ ++ 985/986 V682R L171P/S260T/E306G/S332P/F387I/I390A/V682R/L749F/L750D/L1248D/L1377D/V1439E ++ 987/988 L14M L14M/L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E ++ 989/990 L318C L171P/S260T/E306G/L318C/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E ++ 991/992 C17S C17S/L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E ++ ++ 993/994 A20L A20L/L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E ++ ++ 995/996 K818G L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/K818G/L1248D/L1377D/V1439E + + 997/998 M836G L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/M836G/L1248D/L1377D/V1439E + + 999/1000 T21M T21M/L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E ++ ++ 1001/1002 M836R L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/M836R/L1248D/L1377D/V1439E + ++ 1003/1004 F325Y L171P/S260T/E306G/F325Y/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E ++ ++ 1005/1006 T533V L171P/S260T/E306G/S332P/F387I/I390A/T533V/L749F/L750D/L1248D/L1377D/V1439E ++ 1007/1008 S19G S19G/L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E ++ 1009/1010 L14V L14V/L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E ++ 1011/1012 Q817D L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/Q817D/L1248D/L1377D/V1439E + + 1013/1014 L831W L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L831W/L1248D/L1377D/V1439E + + 1015/1016 K225R L171P/K225R/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E ++ ++ 1017/1018 H1156R L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/H1156R/L1248D/L1377D/V1439E + ++ 1019/1020 R830V L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/R830V/L1248D/L1377D/V1439E ++ ++ 1021/1022 V393G/A747D L171P/S260T/E306G/S332P/F387I/I390A/V393G/A747D/L749F/L750D/L1248D/L1377D/V1439E ++ ++ 1023/1024 N753D L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/N753D/L1248D/L1377D/V1439E ++ 1025/1026 V393G/K732W/S751Y L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/S751Y/L1248D/L1377D/V1439E ++ ++ 1027/1028 V63Q/M797W V63Q/L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/M797W/L1248D/L1377D/V1439E ++ + All activities were determined relative to the reference polypeptide of SEQ ID NO: 840. The level of increased activity is defined as follows: "+" 0.9 to 1.1, "++"> 1.1, "+++"> 2 Table 8.2 : Activity of FVIII variants relative to SEQ ID NO: 840 SEQ ID NO: (nt/aa) Amino acid differences ( relative to SEQ ID NO: 840) Amino acid difference ( relative to SEQ ID NO: 2) (Expi293F) Unchallenged chromogenic FIOPC relative to SEQ ID NO: 840 (Expi293F) Chromogenic activity of FIOPC relative to SEQ ID NO: 840 after plasma challenge (HepG2) Unchallenged chromogenic FIOPC relative to SEQ ID NO: 840 (Expi293F) Unchallenged coagulation active FIOPC relative to SEQ ID NO: 840 839/840 L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E + + + + 1029/1030 L622D L171P/S260T/E306G/S332P/F387I/I390A/L622D/L749F/L750D/L1248D/L1377D/V1439E ++ + + + 1031/1032 P617E L171P/S260T/E306G/S332P/F387I/I390A/P617E/L749F/L750D/L1248D/L1377D/V1439E ++ ++ + ++ 1033/1034 P617N L171P/S260T/E306G/S332P/F387I/I390A/P617N/L749F/L750D/L1248D/L1377D/V1439E ++ + + 1035/1036 P907D L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/P907D/L1248D/L1377D/V1439E ++ + + 1037/1038 N1040E L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/N1040E/L1248D/L1377D/V1439E ++ +++ ++ 1039/1040 P617D L171P/S260T/E306G/S332P/F387I/I390A/P617D/L749F/L750D/L1248D/L1377D/V1439E ++ ++ + + 1041/1042 Q611E L171P/S260T/E306G/S332P/F387I/I390A/Q611E/L749F/L750D/L1248D/L1377D/V1439E ++ ++ ++ + 1043/1044 V620P L171P/S260T/E306G/S332P/F387I/I390A/V620P/L749F/L750D/L1248D/L1377D/V1439E ++ ++ + 1045/1046 P617G L171P/S260T/E306G/S332P/F387I/I390A/P617G/L749F/L750D/L1248D/L1377D/V1439E ++ ++ + ++ 1047/1048 A488G L171P/S260T/E306G/S332P/F387I/I390A/A488G/L749F/L750D/L1248D/L1377D/V1439E + + 1049/1050 P1192D L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/P1192D/L1248D/L1377D/V1439E ++ ++ + 1051/1052 S1194P L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/S1194P/L1248D/L1377D/V1439E ++ ++ + 1053/1054 G619E L171P/S260T/E306G/S332P/F387I/I390A/G619E/L749F/L750D/L1248D/L1377D/V1439E ++ ++ ++ + 1055/1056 P615D L171P/S260T/E306G/S332P/F387I/I390A/P615D/L749F/L750D/L1248D/L1377D/V1439E + + + + 1057/1058 F16A F16A/L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E ++ ++ ++ + 1059/1060 F802S L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/F802S/L1248D/L1377D/V1439E ++ + ++ + 1061/1062 K818V L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/K818V/L1248D/L1377D/V1439E + ++ + + 1063/1064 L211K L171P/L211K/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E + ++ + ++ 1065/1066 M836W L171P/S260T/E306G/S332P/F387I/I390A/L749F/M836W/L750D/L1248D/L1377D/V1439E ++ ++ ++ 1067/1068 F555R L171P/S260T/E306G/S332P/F387I/I390A/F555R/L749F/L750D/L1248D/L1377D/V1439E ++ ++ ++ 1069/1070 V536A L171P/S260T/E306G/S332P/F387I/I390A/V536A/L749F/L750D/L1248D/L1377D/V1439E + ++ + + 1071/1072 I527A L171P/S260T/E306G/S332P/F387I/I390A/I527A/L749F/L750D/L1248D/L1377D/V1439E + + 1073/1074 F555E L171P/S260T/E306G/S332P/F387I/I390A/F555E/L749F/L750D/L1248D/L1377D/V1439E ++ ++ + ++ 1075/1076 H841W L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/H841W/L1248D/L1377D/V1439E + ++ + 1077/1078 M320Q L171P/S260T/E306G/M320Q/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E + ++ + + 1079/1080 S837G L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/S837G/L1248D/L1377D/V1439E ++ ++ + 1081/1082 M558R L171P/S260T/E306G/S332P/F387I/I390A/M558R/L749F/L750D/L1248D/L1377D/V1439E + + 1083/1084 F555G L171P/S260T/E306G/S332P/F387I/I390A/F555G/L749F/L750D/L1248D/L1377D/V1439E + ++ + + 1085/1086 L211R L171P/L211R/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E + + + + 1087/1088 V64G V64G/L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E ++ + + + 1089/1090 M320R L171P/S260T/E306G/M320R/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E ++ +++ ++ ++ 1091/1092 T533Y L171P/S260T/E306G/S332P/F387I/I390A/T533Y/L749F/L750D/L1248D/L1377D/V1439E + ++ ++ 1093/1094 L914V L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L914V/L1248D/L1377D/V1439E ++ + ++ ++ 1095/1096 F16V F16V/L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E ++ + ++ ++ 1097/1098 I1283E L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/I1283E/L1377D/V1439E ++ ++ + 1099/1100 V536M L171P/S260T/E306G/S332P/F387I/I390A/V536M/L749F/L750D/L1248D/L1377D/V1439E + ++ + 1101/1102 F312L L171P/S260T/E306G/F312L/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E ++ ++ ++ ++ 1103/1104 M681N L171P/S260T/E306G/S332P/F387I/I390A/M681N/L749F/L750D/L1248D/L1377D/V1439E ++ +++ + ++ 1105/1106 S838T L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/S838T/L1248D/L1377D/V1439E ++ + ++ + 1107/1108 V536R L171P/S260T/E306G/S332P/F387I/I390A/V536R/L749F/L750D/L1248D/L1377D/V1439E + + ++ + 1109/1110 R15G R15G/L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E ++ + ++ + 1111/1112 V536P L171P/S260T/E306G/S332P/F387I/I390A/V536P/L749F/L750D/L1248D/L1377D/V1439E ++ ++ ++ 1113/1114 M558W L171P/S260T/E306G/S332P/F387I/I390A/M558W/L749F/L750D/L1248D/L1377D/V1439E ++ ++ ++ + 1115/1116 M558E L171P/S260T/E306G/S332P/F387I/I390A/M558E/L749F/L750D/L1248D/L1377D/V1439E ++ ++ + 1117/1118 L1427Y L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/L1427Y/V1439E ++ ++ ++ ++ 1119/1120 S839G L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/S839G/L1248D/L1377D/V1439E ++ + 1121/1122 F18L F18L/L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E + ++ + + 1123/1124 L13G L13G/L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E ++ ++ + + 1125/1126 K1210M L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/K1210M/L1248D/L1377D/V1439E + ++ + 1127/1128 H1156G L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/H1156G/L1248D/L1377D/V1439E + ++ ++ + 1129/1130 M558L L171P/S260T/E306G/S332P/F387I/I390A/M558L/L749F/L750D/L1248D/L1377D/V1439E + ++ + 1131/1132 S839K L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/S839K/L1248D/L1377D/V1439E + ++ ++ ++ 1133/1134 L211P L171P/L211P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E + + + 1135/1136 L211G L171P/L211G/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E + + + 1137/1138 L211Q L171P/L211Q/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E ++ ++ + ++ 1139/1140 F16D F16D/L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E ++ + + ++ 1141/1142 M320G L171P/S260T/E306G/M320G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E + + 1143/1144 Y683W L171P/S260T/E306G/S332P/F387I/I390A/Y683W/L749F/L750D/L1248D/L1377D/V1439E ++ ++ + 1145/1146 Y24V Y24V/L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E ++ ++ + 1147/1148 V534I L171P/S260T/E306G/S332P/F387I/I390A/V534I/L749F/L750D/L1248D/L1377D/V1439E ++ ++ + ++ 1149/1150 M681A L171P/S260T/E306G/S332P/F387I/I390A/M681A/L749F/L750D/L1248D/L1377D/V1439E ++ ++ + + 1151/1152 P840G L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/P840G/L1248D/L1377D/V1439E + + + 1153/1154 F1160R L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/F1160R/L1248D/L1377D/V1439E ++ + + 1155/1156 M681C L171P/S260T/E306G/S332P/F387I/I390A/M681C/L749F/L750D/L1248D/L1377D/V1439E + ++ ++ + 1157/1158 F328Q L171P/S260T/E306G/F328Q/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E + + 1159/1160 R15S R15S/L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E + + + + 1161/1162 V536K L171P/S260T/E306G/S332P/F387I/I390A/V536K/L749F/L750D/L1248D/L1377D/V1439E + + + + 1163/1164 V536G L171P/S260T/E306G/S332P/F387I/I390A/V536G/L749F/L750D/L1248D/L1377D/V1439E ++ ++ + 1165/1166 T533R L171P/S260T/E306G/S332P/F387I/I390A/T533R/L749F/L750D/L1248D/L1377D/V1439E + ++ + ++ 1167/1168 M681E L171P/S260T/E306G/S332P/F387I/I390A/M681E/L749F/L750D/L1248D/L1377D/V1439E ++ ++ ++ 1169/1170 V536W L171P/S260T/E306G/S332P/F387I/I390A/V536W/L749F/L750D/L1248D/L1377D/V1439E ++ ++ ++ 1171/1172 H1156N L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/H1156N/L1248D/L1377D/V1439E + + ++ ++ 1173/1174 F16P F16P/L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E ++ + + + 1175/1176 Q817A L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/Q817A/L1248D/L1377D/V1439E ++ ++ ++ 1177/1178 F18R F18R/L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E ++ ++ + 1179/1180 Y551W L171P/S260T/E306G/S332P/F387I/I390A/Y551W/L749F/L750D/L1248D/L1377D/V1439E ++ + + + 1181/1182 F555T L171P/S260T/E306G/S332P/F387I/I390A/F555T/L749F/L750D/L1248D/L1377D/V1439E + ++ + 1183/1184 M681G L171P/S260T/E306G/S332P/F387I/I390A/M681G/L749F/L750D/L1248D/L1377D/V1439E ++ +++ ++ + 1185/1186 H212A L171P/H212A/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E + ++ + + 1187/1188 F16G F16G/L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E ++ + ++ + 1189/1190 S838G L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/S838G/L1248D/L1377D/V1439E + + + 1191/1192 M681R L171P/S260T/E306G/S332P/F387I/I390A/M681R/L749F/L750D/L1248D/L1377D/V1439E + + + + 1193/1194 V393G L171P/S260T/E306G/S332P/F387I/I390A/V393G/L749F/L750D/L1248D/L1377D/V1439E ++ ++ ++ 1195/1196 V393G/A747D/K752G/Q811F/K819V/Q923M/T1148N L171P/S260T/E306G/S332P/F387I/I390A/V393G/A747D/L749F/L750D/K752G/Q811F/K819V/Q923M/T1148N L1248D/L1377D/V1439E ++ ++ ++ ++ 1197/1198 V393G/S751Y/I775G L171P/S260T/E306G/S332P/F387I/I390A/V393G/L749F/L750D/S751Y/I775G/L1248D/L1377D/V1439E ++ +++ + + 1199/1200 V63Q/V393G/K732W/I775G/T1148N V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ +++ ++ ++ 1201/1202 V393G/K732W/T1148N L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/T1148N/L1248D/L1377D/V1439E ++ ++ ++ ++ 1203/1204 I775G/T1148N L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ +++ ++ ++ 1205/1206 V393G/K752L/N753D/I775G/I793G/M797W L171P/S260T/E306G/S332P/F387I/I390A/V393G/L749F/L750D/K752L/N753D/I775G/I793G/M797W/L1248D/L1377D/V1439E ++ +++ + ++ 1207/1208 V393G/Q923M L171P/S260T/E306G/S332P/F387I/I390A/V393G/L749F/L750D/Q923M/L1248D/L1377D/V1439E ++ ++ ++ + 1209/1210 V393G/Q923M/T1148N L171P/S260T/E306G/S332P/F387I/I390A/V393G/L749F/L750D/Q923M/T1148N/L1248D/L1377D/V1439E ++ +++ ++ + 1211/1212 V393G/A747D/S751Y L171P/S260T/E306G/S332P/F387I/I390A/V393G/A747D/L749F/L750D/S751Y/L1248D/L1377D/V1439E ++ ++ ++ ++ 1213/1214 V393G/A747D/N753D/I775G/T1148N L171P/S260T/E306G/S332P/F387I/I390A/V393G/A747D/L749F/L750D/N753D/I775G/T1148N/L1248D/L1377D/V1439E ++ +++ ++ ++ 1215/1216 K752L/Q923M/M1051R L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/K752L/Q923M/M1051R/L1248D/L1377D/V1439E ++ ++ ++ + 1217/1218 V63Q/I775G V63Q/L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/I775G/L1248D/L1377D/V1439E ++ +++ ++ ++ 1219/1220 V63Q/V393G/K752L/I775G/I793G/M1051R/T1053R V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/L749F/L750D/K752L/I775G/I793G/M1051R/T1053R L1248D/L1377D/V1439E ++ ++ + + 1221/1222 K752G/N753D/I775G/I793G/K819V L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/K752G/N753D/I775G/I793G/K819V/L1248D/L1377D/V1439E ++ ++ ++ ++ 1223/1224 V63Q/M1051R V63Q/L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/M1051R/L1248D/L1377D/V1439E ++ +++ ++ ++ 1225/1226 V63Q/A747D/K752G/N753D/I775G/Q811F/K819V/Q923M/M1051R V63Q/L171P/S260T/E306G/S332P/F387I/I390A/A747D/L749F/L750D/K752G/N753D/I775G/Q811F/K819V/Q923M/M1051R L1248D/L1377D/V1439E ++ +++ ++ ++ 1227/1228 V393G/K752G/I787W L171P/S260T/E306G/S332P/F387I/I390A/V393G/L749F/L750D/K752G/I787W/L1248D/L1377D/V1439E + ++ ++ ++ 1229/1230 S751Y L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/S751Y/L1248D/L1377D/V1439E + + + ++ 1231/1232 V63Q/T1148N V63Q/L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/T1148N/L1248D/L1377D/V1439E ++ ++ ++ ++ 1233/1234 V63H/A747D/K752G/N753D/K819V/Q923M/T1148N V63H/L171P/S260T/E306G/S332P/F387I/I390A/A747D/L749F/L750D/K752G/N753D/K819V/Q923M/T1148N L1248D/L1377D/V1439E ++ + ++ ++ 1235/1236 T1148N L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/T1148N/L1248D/L1377D/V1439E + ++ + ++ 1237/1238 A747D/K752L/N753D/I793G/Q811F L171P/S260T/E306G/S332P/F387I/I390A/A747D/L749F/L750D/K752L/N753D/I793G/Q811F/L1248D/L1377D/V1439E ++ ++ + ++ 1239/1240 V63Q V63Q/L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/L1248D/L1377D/V1439E ++ +++ ++ ++ 1241/1242 M797W/T1148N L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/M797W/T1148N/L1248D/L1377D/V1439E + + ++ + 1243/1244 S751Y/K752L/N753D/M1051R L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/S751Y/K752L/N753D/M1051R/L1248D/L1377D/V1439E ++ +++ ++ ++ 1245/1246 V63Q/K732W/M1051R V63Q/L171P/S260T/E306G/S332P/F387I/I390A/K732W/L749F/L750D/M1051R/L1248D/L1377D/V1439E ++ ++ ++ + 1247/1248 K752G/M797W/S815I/Q923M/M1051R/T1148N L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/K752G/M797W/S815I/Q923M/M1051R/T1148N L1248D/L1377D/V1439E ++ +++ ++ ++ 1249/1250 A747D/N753D L171P/S260T/E306G/S332P/F387I/I390A/A747D/L749F/L750D/N753D/L1248D/L1377D/V1439E ++ ++ ++ ++ All activities were determined relative to the reference polypeptide of SEQ ID NO: 840. The level of increased activity is defined as follows: "+" 0.9 to 1.1, "++"> 1.1, "+++"> 2 Example 9 FVIII variants derived from SEQ ID NO: 1200

In this example, experiments performed to determine the activity of FVIII variants by analyzing protein activity after challenge are described. These variants were tested for FVIII activity after no incubation and after incubation in hemophilia A patient plasma (George King Bio-Medical, Inc.), as described in Example 3. Variants were tested using chromogenic and coagulation assays as described in Example 3. Results using variants expressed in Expi293F cells or HepG2 cells are shown in Table 9.1 and Table 9.2 . Table 9.1 : Activity of FVIII variants relative to SEQ ID NO: 1200 SEQ ID NO: (nt/aa) Amino acid differences ( relative to SEQ ID NO: 1200) Amino acid differences ( relative to SEQ ID NO: 2) (Expi293F) Unchallenged chromogenic FIOPC relative to SEQ ID NO: 1200 (Expi293F) Chromogenic activity of FIOPC relative to SEQ ID NO: 1200 after plasma challenge 1199/1200 V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E + + 1251/1252 M681A V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/M681A/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E + +++ 1253/1254 L622D V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/L622D/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E + + 1255/1256 P617G V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/P617G/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ + 1257/1258 M558L V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/M558L/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E + +++ 1259/1260 V536K V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/V536K/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E + +++ 1261/1262 M681G V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E + +++ 1263/1264 L211Q V63Q/L171P/L211Q/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E + + 1265/1266 M320R V63Q/L171P/S260T/E306G/M320R/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E +++ +++ 1267/1268 M558W V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/M558W/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E + +++ 1269/1270 S839K V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/S839K/T1148N/L1248D/L1377D/V1439E +++ +++ 1271/1272 L211K V63Q/L171P/L211K/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ + 1273/1274 M797W V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/M797W/T1148N/L1248D/L1377D/V1439E + +++ 1275/1276 T533R V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/T533R/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E + ++ 1277/1278 S751A V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/S751A/I775G/T1148N/L1248D/L1377D/V1439E + ++ 1279/1280 A303T V63Q/L171P/S260T/A303T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E +++ + 1281/1282 T951V V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/T951V/I775G/T1148N/L1248D/L1377D/V1439E ++ + 1283/1284 G164E V63Q/G164E/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ +++ 1285/1286 K818G V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/K818G/T1148N/L1248D/L1377D/V1439E + ++ 1287/1288 E807L V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/E807L/T1148N/L1248D/L1377D/V1439E +++ + 1289/1290 K799A V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/K799A/T1148N/L1248D/L1377D/V1439E ++ + 1291/1292 G735S V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/G735S/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ ++ 1293/1294 S794R V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/S794R/T1148N/L1248D/L1377D/V1439E + ++ 1295/1296 E926V V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/E926V/T1148N/L1248D/L1377D/V1439E + ++ 1297/1298 A755P V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/A755P/I775G/T1148N/L1248D/L1377D/V1439E + +++ 1299/1300 Q921W V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/Q921W/T1148N/L1248D/L1377D/V1439E +++ + 1301/1302 D801A V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/D801A/T1148N/L1248D/L1377D/V1439E + ++ 1303/1304 E1094S V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/E1094S/T1148N/L1248D/L1377D/V1439E + ++ 1305/1306 L968A V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/L968A/T1148N/L1248D/L1377D/V1439E ++ + 1307/1308 K929Q V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/K929Q/T1148N/L1248D/L1377D/V1439E ++ ++ 1309/1310 G924W V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/G924W/T1148N/L1248D/L1377D/V1439E ++ +++ 1311/1312 E807P V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/E807P/T1148N/L1248D/L1377D/V1439E + ++ 1313/1314 F698D V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/F698D/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E + +++ 1315/1316 S168G V63Q/S168G/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E + ++ 1317/1318 K929V V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/K929V/T1148N/L1248D/L1377D/V1439E ++ + 1319/1320 K818A V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/K818A/T1148N/L1248D/L1377D/V1439E + + 1321/1322 K818S V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/K818S/T1148N/L1248D/L1377D/V1439E + + 1323/1324 R581K V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/R581K/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E + + 1325/1326 K818P V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/K818P/T1148N/L1248D/L1377D/V1439E + +++ 1327/1328 S916K V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/S916K/T1148N/L1248D/L1377D/V1439E + + 1329/1330 F698S V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/F698S/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E + + 1331/1332 A303G V63Q/L171P/S260T/A303G/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E +++ + 1333/1334 S916C V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/S916C/T1148N/L1248D/L1377D/V1439E + + 1335/1336 T1428G V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/T1428G/V1439E + ++ 1337/1338 Y805L V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/Y805L/T1148N/L1248D/L1377D/V1439E +++ ++ 1339/1340 G924M V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/G924M/T1148N/L1248D/L1377D/V1439E ++ + 1341/1342 L1114M V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/L1114M/T1148N/L1248D/L1377D/V1439E ++ + 1343/1344 T311G V63Q/L171P/S260T/E306G/T311G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E +++ + 1345/1346 Y737N V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/Y737N/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E + + 1347/1348 Y805V V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/Y805V/T1148N/L1248D/L1377D/V1439E ++ ++ 1349/1350 D801I V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/D801I/T1148N/L1248D/L1377D/V1439E + ++ 1351/1352 S751D V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/S751D/I775G/T1148N/L1248D/L1377D/V1439E + + 1353/1354 S693T V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/S693T/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ + 1355/1356 K818E V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/K818E/T1148N/L1248D/L1377D/V1439E + + 1357/1358 K929A V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/K929A/T1148N/L1248D/L1377D/V1439E ++ + 1359/1360 T311E V63Q/L171P/S260T/E306G/T311E/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E +++ + 1361/1362 Y737L V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/Y737L/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E + ++ 1363/1364 E809W V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/E809W/T1148N/L1248D/L1377D/V1439E +++ + 1365/1366 E757A V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/E757A/I775G/T1148N/L1248D/L1377D/V1439E +++ + 1367/1368 A1099G V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/A1099G/T1148N/L1248D/L1377D/V1439E + + 1369/1370 K799G V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/K799G/T1148N/L1248D/L1377D/V1439E + ++ 1371/1372 S916I V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/S916I/T1148N/L1248D/L1377D/V1439E + + 1373/1374 Y805G V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/Y805G/T1148N/L1248D/L1377D/V1439E +++ + 1375/1376 Y737F V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/Y737F/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E + ++ 1377/1378 Y737M V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/Y737M/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ ++ 1379/1380 K799P V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/K799P/T1148N/L1248D/L1377D/V1439E ++ + 1381/1382 E807C V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/E807C/T1148N/L1248D/L1377D/V1439E ++ + 1383/1384 S916E V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/S916E/T1148N/L1248D/L1377D/V1439E ++ + 1385/1386 T676Q V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/T676Q/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ + 1387/1388 R581V V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/R581V/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ ++ 1389/1390 L968P V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/L968P/T1148N/L1248D/L1377D/V1439E ++ +++ 1391/1392 A755Y V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/A755Y/I775G/T1148N/L1248D/L1377D/V1439E ++ ++ 1393/1394 S916Q V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/S916Q/T1148N/L1248D/L1377D/V1439E +++ +++ 1395/1396 G735D V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/G735D/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E +++ + 1397/1398 D740G V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/D740G/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E +++ +++ 1399/1400 F698Y V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/F698Y/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E + + 1401/1402 E757G V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/E757G/I775G/T1148N/L1248D/L1377D/V1439E ++ ++ 1403/1404 D791T V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/D791T/T1148N/L1248D/L1377D/V1439E +++ + 1405/1406 L1114A V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/L1114A/T1148N/L1248D/L1377D/V1439E + ++ 1407/1408 G735V V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/G735V/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E + ++ All activities were determined relative to the reference polypeptide of SEQ ID NO: 1200. The level of increased activity is defined as follows: "+" 0.9 to 1.1, "++"> 1.1, "+++"> 2 Table 9.2 : Activity of FVIII variants relative to SEQ ID NO: 1200 SEQ ID NO: (nt/aa) Amino acid differences ( relative to SEQ ID NO: 1200) Amino acid difference ( relative to SEQ ID NO: 2) (Expi293F) Unchallenged chromogenic FIOPC relative to SEQ ID NO: 1200 (Expi293F) Chromogenic activity of FIOPC relative to SEQ ID NO: 1200 after plasma challenge (HepG2) Unchallenged chromogenic FIOPC relative to SEQ ID NO: 1200 (HepG2) Chromogenic activity FIOPC relative to SEQ ID NO: 1200 after plasma challenge (Expi293F) Unchallenged coagulation active FIOPC relative to SEQ ID NO: 1200 1199/1200 V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E + + + + + 1409/1410 V536P V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/V536P/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ + ++ ++ + 1411/1412 L211R/V536K/L622D/M681A V63Q/L171P/L211R/S260T/E306G/S332P/F387I/I390A/V393G/V536K/L622D/M681A/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ ++ + ++ + 1413/1414 V536K/M558L V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/V536K/M558L/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ + ++ + + 1415/1416 L211R/V536R/F555G/P617G/W732K V63Q/L171P/L211R/S260T/E306G/S332P/F387I/I390A/V393G/V536R/F555G/P617G/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ ++ ++ ++ + 1417/1418 L211R/M681R/W732K V63Q/L171P/L211R/S260T/E306G/S332P/F387I/I390A/V393G/M681R/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ ++ + + 1419/1420 L211R/V536K/F555E V63Q/L171P/L211R/S260T/E306G/S332P/F387I/I390A/V393G/V536K/F555E/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ ++ + + + 1421/1422 V536R V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/V536R/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ ++ + + + 1423/1424 F555E V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/F555E/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ ++ ++ ++ ++ 1425/1426 L211R/V536P V63Q/L171P/L211R/S260T/E306G/S332P/F387I/I390A/V393G/V536P/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ ++++ ++ ++ + 1427/1428 L211R/H212P V63Q/L171P/L211R/H212P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ + ++ ++ + 1429/1430 L211R/V536K V63Q/L171P/L211R/S260T/E306G/S332P/F387I/I390A/V393G/V536K/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E + + + ++ + 1431/1432 L211R/V536K/P617G/M681A/W732K V63Q/L171P/L211R/S260T/E306G/S332P/F387I/I390A/V393G/ V536K/P617G/M681A/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ + ++ ++ + 1433/1434 L211R V63Q/L171P/L211R/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ ++ ++ ++ ++ 1435/1436 L211R/V536P/M558L/P617G/M681A V63Q/L171P/L211R/S260T/E306G/S332P/F387I/I390A/V393G/V536P/M558L/P617G/M681A/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1 439E ++ + ++ ++ ++ 1437/1438 F555G/W732K V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/F555G/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ + ++ ++ + 1439/1440 V536P/F555E/M558L/P617G/W732K V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/V536P/F555E/M558L/P617G/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ +++ ++ ++ + 1441/1442 V536K/P617G/M681A/W732K V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/V536K/P617G/M681A/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ ++ + ++ + 1443/1444 V536K/F555G V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/V536K/F555G/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ + ++ ++ + 1445/1446 V536P/L622D/M681A V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/V536P/L622D/M681A/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ ++ + + 1447/1448 W732K V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ ++ ++ + 1449/1450 H212A/M320Q/T533R/P617E/M681G V63Q/L171P/H212A/S260T/E306G/M320Q/S332P/F387I/I390A/V393G/T533R/P617E/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1 439E ++ ++ ++ ++ + 1451/1452 L211K/F312L/T533R/V534I/P617D/G619E/M681N/S839K V63Q/L171P/L211K/S260T/E306G/F312L/S332P/F387I/I390A/V393G/T533R/V534I/P617D/G619E/M681N/K732W/L749F/L750D/I775G/S839K/T1148 N/L1248D/L1377D/V1439E ++ + ++ ++ ++ 1453/1454 H212A V63Q/L171P/H212A/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E + + + + + 1455/1456 L211K/H212A/M320Q/G619E/M681G/S839K V63Q/L171P/L211K/H212A/S260T/E306G/M320Q/S332P/F387I/I390A/V393G/G619E/M681G/K732W/L749F/L750D/I775G/S839K/T1148N/L1248D/L13 77D/V1439E +++ ++ ++ ++ ++ 1457/1458 M797W/S839K V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/M797W/S839K/T1148N/L1248D/L1377D/V1439E + ++ + + + 1459/1460 H212A/F312L/M320Q/T533R/V534I/P617E/M681G V63Q/L171P/H212A/S260T/E306G/F312L/M320Q/S332P/F387I/I390A/V393G/T533R/V534I/P617E/M681G/K732W/L749F/L750D/I775G/T1148N/L124 8D/L1377D/V1439E ++ +++ ++ ++ + 1461/1462 M558W/G619E/M681G V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/M558W/G619E/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ ++ ++ +++ ++ 1463/1464 M320Q/M558W/M681G V63Q/L171P/S260T/E306G/M320Q/S332P/F387I/I390A/V393G/ M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ ++ ++ ++ + 1465/1466 H212A/M320Q/V534I/G619E/M681G/M797W/S839K V63Q/L171P/H212A/S260T/E306G/M320Q/S332P/F387I/I390A/V393G/V534I/G619E/M681G/K732W/L749F/L750D/I775G/M797W/S839K/T1148N/L124 8D/L1377D/V1439E ++ ++ ++ ++ + 1467/1468 M320R/M558W/G619E/W732K/S839K V63Q/L171P/S260T/E306G/M320R/S332P/F387I/I390A/V393G/M558W/G619E/L749F/L750D/I775G/S839K/T1148N/L1248D/L1377D/V1439E ++ ++++ + ++ + 1469/1470 L211K/H212A/F312L/V534I/M558W/G619E/M681N/W732K/S839K V63Q/L171P/L211K/H212A/S260T/E306G/F312L/S332P/F387I/I390A/V393G/V534I/M558W/G619E/M681N/L749F/L750D/I775G/ S839K/T1148N/L124 8D/L1377D/V1439E +++ ++++ ++ ++ + 1471/1472 M320R/V534I/M558R/G619E/W732K/M797W V63Q/L171P/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558R/G619E/L749F/L750D/I775G/M797W/T1148N/L1248D/L1377D/V1439E +++ ++ ++ ++ ++ 1473/1474 M558W/P617D/G619E/M681N V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/M558W/P617D/G619E/M681N/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ ++ ++ ++ ++ 1475/1476 L211Q/V534I/M558W/Q611E/M681N V63Q/L171P/L211Q/S260T/E306G/S332P/F387I/I390A/V393G/V534I/M558W/Q611E/M681N/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1 439E ++ ++ ++ ++ ++ 1477/1478 L211K/M320R/P617E/G619E/M681G V63Q/L171P/L211K/S260T/E306G/M320R/S332P/F387I/I390A/V393G/P617E/G619E/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1 439E +++ ++ ++ +++ ++ 1479/1480 M558W/P617D/M681G/M797W V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/M558W/P617D/M681G/K732W/L749F/L750D/I775G/M797W/T1148N/L1248D/L1377D/V1439E ++ ++ ++ ++ ++ 1481/1482 M320R/V534I/G619E/W732K V63Q/L171P/S260T/E306G/M320R/S332P/F387I/I390A/V393G/ V534I/G619E//L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E +++ +++ ++ ++ ++ 1483/1484 L211K/G619E/M681N V63Q/L171P/L211K/S260T/E306G/S332P/F387I/I390A/V393G/ G619E/M681N/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E +++ ++ ++ ++ ++ 1485/1486 L211K/H212A/V534I/M558W/G619E/W732K/S839K V63Q/L171P/L211K/H212A/S260T/E306G/S332P/F387I/I390A/V393G/V534I/M558W/G619E/L749F/L750D/I775G/S839K/T1148N/L1248D/L1377D/V1 439E ++ +++ ++ ++ + 1487/1488 H212A/M320R/G619E/M681N/M797W/S839K V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/G619E/M681N/K732W/L749F/L750D/I775G/M797W/S839K/T1148N/L1248D/L13 77D/V1439E +++ +++++ +++ ++ ++ 1489/1490 H212A/M320R/Q611E/S839K V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/Q611E/K732W/L749F/L750D/I775G/S839K/T1148N/L1248D/L1377D/V1439E ++ +++ ++ ++ + 1491/1492 M320Q/M558W/G619E/M681N V63Q/L171P/S260T/E306G/M320Q/S332P/F387I/I390A/V393G/M558W/G619E/M681N/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E +++ +++ +++ +++ ++ 1493/1494 F312L/T533R/P617D/M681N/M797W/S839K V63Q/L171P/S260T/E306G/F312L/S332P/F387I/I390A/V393G/T533R/P617D/M681N/K732W/L749F/L750D/I775G/M797W/S839K/T1148N/L1248D/L13 77D/V1439E ++ +++ ++ ++ ++ 1495/1496 M558W/Q611E/M681N V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/M558W/Q611E/M681N/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ +++ + ++ ++ 1497/1498 F312L V63Q/L171P/S260T/E306G/F312L/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ ++ ++ ++ + 1499/1500 L211Q/M320R/M558W/G619E/M681G/W732K/M797W/S839K V63Q/L171P/L211Q/S260T/E306G/M320R/S332P/F387I/I390A/V393G/M558W/G619E/M681G/L749F/L750D/I775G/M797W/S839K/T1148N/L1248D/L13 77D/V1439E ++ ++ ++ ++ + 1501/1502 M320Q/M558W/W732K/M797W V63Q/L171P/S260T/E306G/M320Q/S332P/F387I/I390A/V393G/M558W/L749F/L750D/I775G/M797W/T1148N/L1248D/L1377D/V1439E ++ + ++ ++ + 1503/1504 T533R/M558W/Q611E/G619E/F716L/W732K/S839K V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/T533R/M558W/Q611E/G619E/F716L//L749F/L750D/I775G/S839K/T1148N/L1248D/L1377D/V 1439E ++ + ++ ++ + 1505/1506 F312L/M681G V63Q/L171P/S260T/E306G/F312L/S332P/F387I/I390A/V393G/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E +++ ++ ++ +++ ++ 1507/1508 M320Q/V534I/G619E/M681N V63Q/L171P/S260T/E306G/M320Q/S332P/F387I/I390A/V393G/V534I/G619E/M681N/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ +++ ++ ++ ++ 1509/1510 L211K/M320Q/T533R/V536L/G619E V63Q/L171P/L211K/S260T/E306G/M320Q/S332P/F387I/I390A/V393G/T533R/V536L/G619E/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1 439E + ++ + + + 1511/1512 M558W/P617E/M681N/S839K V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/M558W/P617E/M681N/K732W/L749F/L750D/I775G/S839K/T1148N/L1248D/L1377D/V1439E ++ ++ ++ ++ ++ 1513/1514 H212A/M320R/V534I/M558W/M681G V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1 439E +++ ++++ ++ ++ + 1515/1516 T533R/V534I/M681N/S839K V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/T533R/V534I/M681N/K732W/L749F/L750D/I775G/S839K/T1148N/L1248D/L1377D/V1439E ++ ++ + ++ + 1517/1518 H212A/V534I/G619E/M681G V63Q/L171P/H212A/S260T/E306G/S332P/F387I/I390A/V393G/V534I/G619E/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E +++ +++ ++ +++ ++ 1519/1520 P617E/G619E/M681G V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/P617E/G619E/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ ++ ++ ++ ++ 1521/1522 L211Q/H212A/M320R/M558W/P617E/M681N V63Q/L171P/L211Q/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/M558W/P617E/M681N/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E +++ +++ ++ ++ ++ 1523/1524 H212A/V534I/M558W/M681N/M797W V63Q/L171P/H212A/S260T/E306G/S332P/F387I/I390A/V393G/V534I/M558W/M681N/K732W/L749F/L750D/I775G/M797W/T1148N/L1248D/L1377D/V1 439E ++ ++ ++ ++ ++ 1525/1526 H212A/M320R/V534I V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E +++ +++ ++ ++ ++ 1527/1528 L211K/F312L/G619E V63Q/L171P/L211K/S260T/E306G/F312L/S332P/F387I/I390A/V393G/G619E/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ ++ + + ++ 1529/1530 H212A/F312L/G619E/S839K V63Q/L171P/H212A/S260T/E306G/F312L/S332P/F387I/I390A/V393G/G619E/K732W/L749F/L750D/I775G/S839K/T1148N/L1248D/L1377D/V1439E ++ ++ ++ ++ + 1531/1532 T210P/H212A/M320R/M558W/M681G V63Q/L171P/T210P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1 439E ++ +++ ++ ++ + 1533/1534 H212A/F312L/V534I/M681N/M797W/S839K V63Q/L171P/H212A/S260T/E306G/F312L/S332P/F387I/I390A/V393G/V534I/M681N/K732W/L749F/L750D/I775G/M797W/S839K/T1148N/L1248D/L13 77D/V1439E +++ ++ ++ +++ ++ 1535/1536 L211K/M320R/M558W/S839K V63Q/L171P/L211K/S260T/E306G/M320R/S332P/F387I/I390A/V393G/M558W/K732W/L749F/L750D/I775G/S839K/T1148N/L1248D/L1377D/V1439E ++ ++ ++ ++ ++ 1537/1538 G924Q V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/G924Q/T1148N/L1248D/L1377D/V1439E + + ++ ++ + 1539/1540 S916L V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/S916L/T1148N/L1248D/L1377D/V1439E + + + + ++ 1541/1542 E757T V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/E757T/I775G/T1148N/L1248D/L1377D/V1439E + ++ ++ + 1543/1544 Y737K V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/Y737K/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ ++ ++ ++ 1545/1546 K818V V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/K818V/T1148N/L1248D/L1377D/V1439E ++ ++ ++ + 1547/1548 D801G V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/D801G/T1148N/L1248D/L1377D/V1439E ++ ++ ++ ++ ++ 1549/1550 T294G V63Q/L171P/S260T/T294G/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ ++ + + 1551/1552 K799S V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/K799S/T1148N/L1248D/L1377D/V1439E ++ ++ ++ ++ 1553/1554 K799E V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/K799E/T1148N/L1248D/L1377D/V1439E + + + ++ 1555/1556 E809G V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/E809G/T1148N/L1248D/L1377D/V1439E + + + ++ + 1557/1558 D791M V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/D791M/T1148N/L1248D/L1377D/V1439E + ++ ++ + + 1559/1560 Y737V V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/Y737V/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ ++ ++ ++ + 1561/1562 Y805Q V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/Y805Q/T1148N/L1248D/L1377D/V1439E ++ +++ ++ ++ + 1563/1564 K799Q V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/K799Q/T1148N/L1248D/L1377D/V1439E + + + ++ + 1565/1566 Y737S V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/Y737S/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E + ++ + + + 1567/1568 S916M V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/S916M/T1148N/L1248D/L1377D/V1439E ++ + + + ++ 1569/1570 R759S V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/R759S/I775G/T1148N/L1248D/L1377D/V1439E + + ++ 1571/1572 K799V V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/K799V/T1148N/L1248D/L1377D/V1439E ++ + ++ ++ ++ 1573/1574 D538V V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/D538V/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ + + ++ + 1575/1576 E1109G V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/E1109G/T1148N/L1248D/L1377D/V1439E ++ ++ + + ++ 1577/1578 S168R V63Q/S168R/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E + ++ + ++ 1579/1580 Q921R V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/Q921R/T1148N/L1248D/L1377D/V1439E ++ + ++ ++ + 1581/1582 E926T V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/E926T/T1148N/L1248D/L1377D/V1439E ++ ++ + ++ 1583/1584 S916T V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/S916T/T1148N/L1248D/L1377D/V1439E ++ +++ + + + 1585/1586 Y737G V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/Y737G/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E ++ + ++ + + 1587/1588 D801R V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/L749F/L750D/I775G/D801R/T1148N/L1248D/L1377D/V1439E + ++ + + + All activities were determined relative to the reference polypeptide of SEQ ID NO: 1200. The levels of increased activity are defined as follows: "+" 0.9 to 1.1, "++"> 1.1, "+++"> 2, "++++"> 3.5, "+++++"> 5 Example 10 FVIII variants derived from SEQ ID NO: 1514

In this example, experiments performed to determine the activity of FVIII variants by analyzing protein activity after challenge are described. These variants were tested for FVIII activity after no incubation and after incubation in hemophilia A patient plasma (George King Bio-Medical, Inc.), as described in Example 3. Variants were tested using chromogenic and coagulation assays as described in Example 3. surface 10.1and surface 10.2Results using variants expressed in Expi293F cells or HepG2 cells are shown in . Table 10.1 : Activity of FVIII variants relative to SEQ ID NO: 1514 SEQ ID NO: (nt/aa) Amino acid differences ( relative to SEQ ID NO: 1514) Amino acid differences ( relative to SEQ ID NO: 2) (Expi293F) Unchallenged chromogenic FIOPC relative to SEQ ID NO: 1514 (Expi293F) Chromogenic activity of FIOPC relative to SEQ ID NO: 1514 after plasma challenge 1513/1514 V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1 439E + + 1589/1590 R1042E V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/R1042E/T1148N/L1248D/L1 377D/V1439E + + 1591/1592 Y1041N V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/Y1041N/T1148N/L1248D/L1 377D/V1439E + + 1593/1594 M1051H V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/M1051H/T1148N/L1248D/L1 377D/V1439E + + 1595/1596 I1283H V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/I1283H/L1 377D/V1439E + 1597/1598 I1283T V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/I1283T/L1 377D/V1439E + 1599/1600 Y1281Q V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/Y1281Q/L1 377D/V1439E + 1601/1602 Y1281P V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/Y1281P/L1 377D/V1439E + + 1603/1604 S916E V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/S916E/T1148N/L1248D/L13 77D/V1439E + + 1605/1606 M1051W V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/M1051W/T1148N/L1248D/L1 377D/V1439E ++ ++ 1607/1608 L187C V63Q/L171P/L187C/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + 1609/1610 I215E V63Q/L171P/H212A/I215E/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + 1611/1612 I215P V63Q/L171P/H212A/I215P/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + 1613/1614 I215S V63Q/L171P/H212A/I215S/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + 1615/1616 L178C V63Q/L171P/L178C/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ ++ 1617/1618 I1050T V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/I1050T/T1148N/L1248D/L1 377D/V1439E ++ + 1619/1620 K1210T V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/K1210T/L1248D/L1 377D/V1439E ++ + 1621/1622 Y1281K V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/Y1281K/L1 377D/V1439E ++ + 1623/1624 R1042A V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/R1042A/T1148N/L1248D/L1 377D/V1439E + + 1625/1626 V104S V63Q/V104S/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + 1627/1628 P615D V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/P615D/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + 1629/1630 I610S V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/I610S/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + ++ 1631/1632 I610K V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/I610K/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + 1633/1634 I610T V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/I610T/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + ++ 1635/1636 V536P V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/V536P/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + ++ 1637/1638 A618Y V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/A618Y/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ + 1639/1640 Q621W V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/Q621W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + 1641/1642 A618P V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/A618P/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ ++ 1643/1644 L614F V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/L614F/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + 1645/1646 M110H V63Q/M110H/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + 1647/1648 R301K V63Q/L171P/H212A/S260T/R301K/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + All activities were determined relative to the reference polypeptide of SEQ ID NO: 1514. The level of increased activity is defined as follows: "+" 0.9 to 1.1, "++"> 1.1 Table 10.2 : Activity of FVIII variants relative to SEQ ID NO: 1514 SEQ ID NO: (nt/aa) Amino acid differences ( relative to SEQ ID NO: 1514) Amino acid differences ( relative to SEQ ID NO: 2) (Expi293F) Unattacked chromogenic FIOPC (Expi293F) Unchallenged coagulation-active FIOPC (Expi293F) Chromogenic FIOPC after plasma challenge (Expi293F) Coagulation activity of FIOPC after plasma challenge (HepG2) Unchallenged chromogenic FIOPC (HepG2) Unchallenged coagulantly active FIOPC Chromogenic FIOPC after (HepG2) plasma challenge (HepG2) coagulation activity FIOPC after plasma challenge 1513/1514 V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1 439E + + + + + + + + 1649/1650 I1078S V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/I1078S/T1148N/L1248D/L1 377D/V1439E ++ + ++ ++ + + 1651/1652 S812G V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/S812G/T1148N/L1248D/L13 77D/V1439E ++ + ++ ++ + 1653/1654 E786C V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/E786C/T1148N/L1248D/L13 77D/V1439E ++ + + ++ ++ 1655/1656 A127V V63Q/A127V/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + ++ ++ + + + + 1657/1658 Y175G V63Q/L171P/Y175G/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + 1659/1660 K1093W V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/K1093W/T1148N/L1248D/L1 377D/V1439E ++ ++ ++ ++ + ++ + ++ 1661/1662 F1088G V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/F1088G/T1148N/L1248D/L1 377D/V1439E ++ + ++ ++ ++ + 1663/1664 K1093T V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/K1093T/T1148N/L1248D/L1 377D/V1439E ++ ++ + ++ ++ ++ + + 1665/1666 L650M V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/L650M/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + ++ ++ ++ ++ 1667/1668 R301M V63Q/L171P/H212A/S260T/R301M/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + + + 1669/1670 T535N V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/T535N/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ + ++ ++ + 1671/1672 D806R V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/D806R/T1148N/L1248D/L13 77D/V1439E ++ + ++ ++ ++ ++ + 1673/1674 Y738E V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ + + + ++ ++ ++ 1675/1676 S700E V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/S700E/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ ++ + ++ ++ ++ ++ 1677/1678 E132R V63Q/E132R/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + ++ + ++ 1679/1680 E1076G V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/E1076G/T1148N/L1248D/L1 377D/V1439E + + ++ ++ ++ + + 1681/1682 S1084L V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/S1084L/T1148N/L1248D/L1 377D/V1439E + ++ + 1683/1684 G498A V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/G498A/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + ++ + 1685/1686 E786L V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/E786L/T1148N/L1248D/L13 77D/V1439E + + ++ ++ + + + 1687/1688 Y738F V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738F/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + + ++ + 1689/1690 T535G V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/T535G/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ ++ + ++ ++ + ++ 1691/1692 D806E V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/D806E/T1148N/L1248D/L13 77D/V1439E + + + + + 1693/1694 I756G V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I756G/I775G/T1148N/L1248D/L13 77D/V1439E ++ + ++ ++ + + 1695/1696 F1108S V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/F1108S/T1148N/L1248D/L1 377D/V1439E ++ + ++ ++ ++ + + + 1697/1698 K1093V V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/K1093V/T1148N/L1248D/L1 377D/V1439E ++ ++ + ++ ++ + 1699/1700 S746P V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/S746P/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ + + 1701/1702 L160E V63Q/L160E/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ + + + + + + + 1703/1704 L160T V63Q/L160T/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + + + + + + 1705/1706 L178V V63Q/L171P/L178V/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + + ++ +++ + + + 1707/1708 S543L V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/S543L/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + ++ +++ + + 1709/1710 I310G V63Q/L171P/H212A/S260T/E306G/I310G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ ++ ++ ++ + 1711/1712 S700Q V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/S700Q/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ + ++ + ++ ++ ++ 1713/1714 K1093A V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/K1093A/T1148N/L1248D/L1 377D/V1439E ++ ++ +++ ++ + ++ ++ 1715/1716 Q584A V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/Q584A/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + 1717/1718 E918S V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/E918S/T1148N/L1248D/L13 77D/V1439E ++ ++ +++ ++ ++ ++ 1719/1720 E1452L V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1 439E/E1452L + + + + + + + 1721/1722 Y738P V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738P/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + 1723/1724 D790W V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/D790W/T1148N/L1248D/L13 77D/V1439E + + ++ ++ + 1725/1726 D790I V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/D790I/T1148N/L1248D/L13 77D/V1439E ++ + ++ + + + + 1727/1728 G292D V63Q/L171P/H212A/S260T/G292D/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + ++ ++ + + 1729/1730 E800M V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/E800M/T1148N/L1248D/L13 77D/V1439E + + ++ + + + 1731/1732 F1108M V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/F1108M/T1148N/L1248D/L1 377D/V1439E + + ++ ++ ++ + ++ + 1733/1734 F1088S V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/F1088S/T1148N/L1248D/L1 377D/V1439E + + + 1735/1736 A167G V63Q/A167G/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + + + ++ + + + 1737/1738 V892T V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/V892T/T1148N/L1248D/L13 77D/V1439E + + + + + + 1739/1740 D790P V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/D790P/T1148N/L1248D/L13 77D/V1439E ++ + ++ + + 1741/1742 E800R V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/E800R/T1148N/L1248D/L13 77D/V1439E + + ++ + + + 1743/1744 Y738K V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738K/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ + + +++ + + + 1745/1746 S304G V63Q/L171P/H212A/S260T/S304G/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + + ++ 1747/1748 S1116G V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/S1116G/T1148N/L1248D/L1 377D/V1439E + + + ++ ++ + 1749/1750 L178Q V63Q/L171P/L178Q/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ + + ++++ ++ ++ ++ 1751/1752 S1116A V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/S1116A/T1148N/L1248D/L1 377D/V1439E + + + + + 1753/1754 K752G V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/K752G/I775G/T1148N/L1248D/L13 77D/V1439E + ++ ++ ++ ++ + + 1755/1756 F1108W V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/F1108W/T1148N/L1248D/L1 377D/V1439E + + + ++ + 1757/1758 Y738D V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738D/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ + ++ + + + ++ 1759/1760 T281A V63Q/L171P/H212A/S260T/T281A/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + ++ + + + + 1761/1762 Y917G V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/Y917G/T1148N/L1248D/L13 77D/V1439E + + ++ + ++ + + + 1763/1764 H712M V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/H712M/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + + ++ ++ + ++ 1765/1766 S1080V V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/S1080V/T1148N/L1248D/L1 377D/V1439E ++ + + ++ + + + 1767/1768 D806G/K1352E V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/D806G/T1148N/L1248D/K13 52E/L1377D/V1439E ++ + ++ ++ + + 1769/1770 E1095G V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/E1095G/T1148N/L1248D/L1 377D/V1439E + + + ++ + 1771/1772 K1093G V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/K1093G/T1148N/L1248D/L1 377D/V1439E ++ ++ ++ ++ ++ ++ ++ ++ 1773/1774 D744A V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/D744A/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ + 1775/1776 S543G V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/S543G/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ + + ++ ++ ++ 1777/1778 M1098G V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/M1098G/T1148N/L1248D/L1 377D/V1439E + + + + + + 1779/1780 T281S V63Q/L171P/H212A/S260T/T281S/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ + + + + + 1781/1782 I756T V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I756T/I775G/T1148N/L1248D/L13 77D/V1439E ++ + ++ ++ + + ++ 1783/1784 M948S V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/M948S/T1148N/L1248D/L13 77D/V1439E + + ++ + + 1785/1786 A119V V63Q/A119V/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + 1787/1788 S1116L V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/S1116L/T1148N/L1248D/L1 377D/V1439E ++ + + + + + + 1789/1790 E785V V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/E785V/T1148N/L1248D/L13 77D/V1439E ++ + + + + + + + 1791/1792 M1113F V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/M1113F/T1148N/L1248D/L1 377D/V1439E + + + + + 1793/1794 S700A V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/S700A/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ + + + + + 1795/1796 E786D V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/E786D/T1148N/L1248D/L13 77D/V1439E + + + ++ + + 1797/1798 Y175A V63Q/L171P/Y175A/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + + 1799/1800 D790M V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/D790M/T1148N/L1248D/L13 77D/V1439E ++ + ++ + + + + 1801/1802 F667I V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/F667I/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + + + + + + + 1803/1804 Q158G V63Q/Q158G/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + ++ + 1805/1806 Q580S V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/Q580S/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ + + ++ + + + + 1807/1808 W707H V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/W707H/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ + + + + 1809/1810 D731I V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/D731I/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + + 1811/1812 E918M V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/E918M/T1148N/L1248D/L13 77D/V1439E ++ + + + + 1813/1814 L1100S V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/L1100S/T1148N/L1248D/L1 377D/V1439E ++ + + + + + + 1815/1816 T1089S V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1089S/T1148N/L1248D/L1 377D/V1439E + + + + + + 1817/1818 H712Y V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/H712Y/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + + + + + + + 1819/1820 Y175H V63Q/L171P/Y175H/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + + + + 1821/1822 K1092G V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/K1092G/T1148N/L1248D/L1 377D/V1439E + ++ + + 1823/1824 T939E V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T939E/T1148N/L1248D/L13 77D/V1439E + 1825/1826 Y1104E V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/Y1104E/T1148N/L1248D/L1 377D/V1439E + + + + 1827/1828 E800G V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/E800G/T1148N/L1248D/L13 77D/V1439E ++ + ++ ++ + + + + 1829/1830 D736F V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/D736F/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + ++ + 1831/1832 S543W V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/S543W/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + ++ + + 1833/1834 N1297A V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/N1297A/L1 377D/V1439E ++ + + ++ ++ + ++ 1835/1836 H712L V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/H712L/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + + + + + + 1837/1838 I1127V V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/I1127V/T1148N/L1248D/L1 377D/V1439E ++ + ++ + + 1839/1840 T939S V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T939S/T1148N/L1248D/L13 77D/V1439E + + + + + + + + 1841/1842 L178M V63Q/L171P/L178M/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + ++ + ++ ++ 1843/1844 D731R V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/D731R/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ + + + + 1845/1846 T686W V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/T686W/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + + ++ + 1847/1848 S543P V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/S543P/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ + + ++ +++ ++ ++ ++ 1849/1850 D736G V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/D736G/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + 1851/1852 E1129P V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/E1129P/L1248D/L1 377D/V1439E + + ++ ++ + + + + 1853/1854 L1138Q V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/L1138Q/T1148N/L1248D/L1 377D/V1439E + + + + + + 1855/1856 S812V V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/S812V/T1148N/L1248D/L13 77D/V1439E ++ + ++ + + ++ 1857/1858 E786D/R821Q V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/E786D/R821Q/T1148N/L124 8D/L1377D/V1439E + + + + + 1859/1860 T535C V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/T535C/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + + 1861/1862 K680R V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/K680R/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + + ++ + + 1863/1864 S1116I V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/S1116I/T1148N/L1248D/L1 377D/V1439E + + + + ++ ++ ++ 1865/1866 T1089F V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1089F/T1148N/L1248D/L1 377D/V1439E + + + + + + 1867/1868 S304E V63Q/L171P/H212A/S260T/S304E/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ + ++ + + + + 1869/1870 L178W V63Q/L171P/L178W/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + 1871/1872 Y1104C V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/Y1104C/T1148N/L1248D/L1 377D/V1439E + + 1873/1874 D744R V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/D744R/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + ++ + 1875/1876 Y738R V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738R/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + ++ 1877/1878 K1093S V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/K1093S/T1148N/L1248D/L1 377D/V1439E ++ ++ ++ ++ + ++ ++ + 1879/1880 H712S V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/H712S/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ + ++ ++ + + + 1881/1882 L187G V63Q/L171P/L187G/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + ++ + 1883/1884 E132P V63Q/L171P/H212A/S260T/E306G/E132P/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + ++ 1885/1886 S812I V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/S812I/T1148N/L1248D/L13 77D/V1439E + + ++ 1887/1888 S1080G V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/S1080G/T1148N/L1248D/L1 377D/V1439E ++ + ++ + + + 1889/1890 E800C V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/E800C/T1148N/L1248D/L13 77D/V1439E + + ++ + + 1891/1892 Y1101G V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/Y1101G/T1148N/L1248D/L1 377D/V1439E ++ ++ ++ + ++ + 1893/1894 D808K V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/D808K/T1148N/L1248D/L13 77D/V1439E + ++ + ++ + + ++ 1895/1896 D808S V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/D808S/T1148N/L1248D/L13 77D/V1439E ++ + ++ + + + + + 1897/1898 D790A V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/D790A/T1148N/L1248D/L13 77D/V1439E ++ + + + + 1899/1900 E800V V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/E800V/T1148N/L1248D/L13 77D/V1439E ++ + ++ +++ ++ ++ ++ + 1901/1902 K680L V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/K680L/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + + + ++ 1903/1904 A167R V63Q/A167R/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ + + ++ + + + + 1905/1906 K752E V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/K752E/I775G/T1148N/L1248D/L13 77D/V1439E ++ ++ ++ ++ + ++ ++ 1907/1908 K1093N V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/K1093N/T1148N/L1248D/L1 377D/V1439E + ++ + ++ ++ + + 1909/1910 A119G V63Q/A119G/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ + ++ ++ ++ + 1911/1912 E739G V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/E739G/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ + ++ ++ ++++ + ++ ++ 1913/1914 S1116R V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/S1116R/T1148N/L1248D/L1 377D/V1439E + + ++ + + + + + 1915/1916 Q580Y V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/Q580Y/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + ++ ++ + + 1917/1918 S304A V63Q/L171P/H212A/S260T/S304A/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + + ++ ++ + 1919/1920 H712Q V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/H712Q/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ + + ++ ++ ++ 1921/1922 D806S V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/D806S/T1148N/L1248D/L13 77D/V1439E + + ++ ++ + + 1923/1924 H712G V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/H712G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + + + + ++ + 1925/1926 D790G V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/D790G/T1148N/L1248D/L13 77D/V1439E + + + + + 1927/1928 F1108V V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/F1108V/T1148N/L1248D/L1 377D/V1439E + + + ++ + + ++ 1929/1930 D731L V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/D731L/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + + ++ ++ + + + 1931/1932 S1084N V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/S1084N/T1148N/L1248D/L1 377D/V1439E ++ + ++ + + + 1933/1934 I1127L V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/I1127L/T1148N/L1248D/L1 377D/V1439E + + ++ ++ + + 1935/1936 S308P V63Q/L171P/H212A/S260T/E306G/S308P/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + + + + + + 1937/1938 E1076S V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/E1076S/T1148N/L1248D/L1 377D/V1439E ++ + ++ ++ ++ ++ ++ ++ 1939/1940 K1093P V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/K1093P/T1148N/L1248D/L1 377D/V1439E + ++ ++ + + + 1941/1942 K1093Y V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/K1093Y/T1148N/L1248D/L1 377D/V1439E + ++ + ++ + ++ + ++ 1943/1944 E739R V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/E739R/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + 1945/1946 E739P V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/E739P/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + + + + + + + 1947/1948 I310V V63Q/L171P/H212A/S260T/E306G/I310V/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ + ++ ++ + + 1949/1950 H712T V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/H712T/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + 1951/1952 A167H V63Q/A167H/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + + + + + + 1953/1954 K1093R V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/K1093R/T1148N/L1248D/L1 377D/V1439E + + + ++ + + + 1955/1956 V536P/F555E/G619E/W732K/K799S/Y805Q V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/V536P/F555E/M558W/G619E/M681G/L749F/L750D/I775G/K799S/Y805Q /T1148N/L1248D/L1377D/V1439E ++ + ++++ + + ++ 1957/1958 F555E/G619E/D801G V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/F555E/M558W/G619E/M681G/K732W/L749F/L750D/I775G/D801G/T1148 N/L1248D/L1377D/V1439E ++ ++ ++ ++ + ++ + 1959/1960 F555E/G619E/K799S V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/F555E/M558W/G619E/M681G/K732W/L749F/L750D/I775G/K799S/T1148 N/L1248D/L1377D/V1439E ++ ++ ++ ++ ++ ++ ++ + 1961/1962 G619E/K799S/D801G/Y805Q V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/G619E/M681G/K732W/L749F/L750D/I775G/K799S/D801G/Y805Q /T1148N/L1248D/L1377D/V1439E ++ ++ ++ ++ ++ + ++ 1963/1964 V536P/F555E/G619E/W732K/K799S V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/V536P/F555E/M558W/G619E/M681G/L749F/L750D/I775G/K799S/T1148 N/L1248D/L1377D/V1439E ++ + ++ ++ + + + 1965/1966 K799S/Y805Q V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/K799S/Y805Q/T1148N/L124 8D/L1377D/V1439E ++ + ++ + ++ + + 1967/1968 D801G V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/D801G/T1148N/L1248D/L13 77D/V1439E ++ + ++ + + + 1969/1970 A246D/G619E/K799S/D801G V63Q/L171P/H212A/A246D/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/G619E/M681G/K732W/L749F/L750D/I775G/K799S/D801G /T1148N/L1248D/L1377D/V1439E ++ ++ ++ ++ + + 1971/1972 V536P/F555E/G619E V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/V536P/F555E/M558W/G619E/M681G/K732W/L749F/L750D/I775G/T1148 N/L1248D/L1377D/V1439E ++ ++ ++ ++ ++ + + + 1973/1974 G619E/S839K V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/G619E/M681G/K732W/L749F/L750D/I775G/S839K/T1148N/L124 8D/L1377D/V1439E ++ + ++ + ++ + 1975/1976 G619E/K799S/Y805Q V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/G619E/M681G/K732W/L749F/L750D/I775G/K799S/Y805Q/T1148 N/L1248D/L1377D/V1439E ++ ++ ++ ++ + + 1977/1978 V536P/D801G/Y805Q V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/V536P/M558W/M681G/K732W/L749F/L750D/I775G/D801G/Y805Q/T1148 N/L1248D/L1377D/V1439E ++ + ++ ++ + + + + 1979/1980 F555E/G619E/K799S/Y805Q V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/F555E/M558W/G619E/M681G/K732W/L749F/L750D/I775G/K799S/Y805Q /T1148N/L1248D/L1377D/V1439E ++ ++ ++ ++ + + + + 1981/1982 G619E/D801G/Y805Q V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/G619E/M681G/K732W/L749F/L750D/I775G/D801G/Y805Q/T1148 N/L1248D/L1377D/V1439E ++ ++ ++ ++ + + + 1983/1984 F555E/G619E V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/F555E/M558W/G619E/M681G/K732W/L749F/L750D/I775G/T1148N/L124 8D/L1377D/V1439E ++ + + ++ + + + 1985/1986 V536P/G619E/D801G V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/V536P/M558W/G619E/M681G/K732W/L749F/L750D/I775G/D801G/T1148 N/L1248D/L1377D/V1439E ++ ++ ++ ++ + + + + 1987/1988 D801G/Y805Q V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/D801G/Y805Q/T1148N/L124 8D/L1377D/V1439E + + ++ + ++ + + + 1989/1990 V536P/G619E/K799S/Y805Q V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/V536P/M558W/G619E/M681G/K732W/L749F/L750D/I775G/K799S/Y805Q /T1148N/L1248D/L1377D/V1439E ++ ++ ++ ++ + + + 1991/1992 F555E/G619E/W732K/K799S/D801G/Y805Q V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/F555E/M558W/G619E/M681G/L749F/L750D/I775G/ K799S/D801G/Y805Q /T1148N/L1248D/L1377D/V1439E ++ + ++ + ++ + + 1993/1994 Y1281H V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/Y1281H/L1 377D/V1439E + + + 1995/1996 F749L V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L750D/I775G/T1148N/L1248D/L1377D/V1439E + ++ + ++ 1997/1998 L614H V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/L614H/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + 1999/2000 N616D V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/N616D/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + + + + + + + 2001/2002 Q621K V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/Q621K/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + + ++ ++ + 2003/2004 I1050E V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/I1050E/T1148N/L1248D/L1 377D/V1439E ++ + + + + + 2005/2006 I610H V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/I610H/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + + 2007/2008 Q611D V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/Q611D/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + + + + 2009/2010 R1042T V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/R1042T/T1148N/L1248D/L1 377D/V1439E + + + 2011/2012 N616Q V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/N616Q/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ + + ++ + 2013/2014 L614N V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/L614N/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + ++ + + + 2015/2016 T370A/L614A V63Q/L171P/H212A/S260T/E306G/M320R/S332P/T370A/F387I/I390A/V393G/V534I/M558W/L614A/M681G/K732W/L749F/L750D/I775G/T1148N/L124 8D/L1377D/V1439E + + ++ ++ + + 2017/2018 H1156D V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/H1156D/L1248D/L1 377D/V1439E + + + + + + 2019/2020 K1210V V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/K1210V/L1248D/L1 377D/V1439E + + + + ++ + + 2021/2022 L614T V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/L614T/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ + + + + + 2023/2024 R1158N V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/R1158N/L1248D/L1 377D/V1439E + + + 2025/2026 Y1281S V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/Y1281S/L1 377D/V1439E + + + + + + + 2027/2028 A618E V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/A618E/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ + + + + + + + 2029/2030 F555E/G681N/Y737K/K799Q V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/F555E/M558W/M681N/K732W/Y737K/L749F/L750D/I775G/K799Q/T1148 N/L1248D/L1377D/V1439E + + ++ + + 2031/2032 V536P/G619E/E809G/E1109G V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/V536P/M558W/G619E/M681G/K732W/L749F/L750D/I775G/E809G/E1109 G/T1148N/L1248D/L1377D/V1439E ++ ++ ++ ++ ++ ++ + 2033/2034 V536K/F555G/G619E/Y737K/K799Q/E809G/S839K/S916T/Q921R V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/V536K/F555G/M558W/G619E/M681G/K732W/Y737K/L749F/L750D/I775G /K799Q/E809G/S839K/S916T/ Q921R/T1148N/L1248D/L1377D/V1439E ++ ++ 2035/2036 S916T/E1109G V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/S916T/E1109G/T1148N/L12 48D/L1377D/V1439E + + ++ ++ + + + 2037/2038 V536R/G619E/G681N/K799Q/S916T/E1109G V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/V536R/M558W/G619E/M681N/K732W/L749F/L750D/I775G/K799Q/S916T /E1109G/T1148N/L1248D/L1377D/ V1439E ++ + ++ ++ + 2039/2040 F555G/G619E/G681N/E807R/E1109G V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/F555G/M558W/G619E/M681N/K732W/L749F/L750D/I775G/E807R/E1109 G/T1148N/L1248D/L1377D/V1439E ++ ++ ++ ++ + ++ + ++ 2041/2042 F555E/G619E/G681N/Y737K/K799Q V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/F555E/M558W/G619E/M681N/L749F/L750D/I775G/K799Q/T1148N/L124 8D/L1377D/V1439E ++ ++ + + 2043/2044 G619E/G681N/K799S/S916T/E1109G V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/G619E/M681N/K732W/L749F/L750D/I775G/K799S/S916T/E1109 G/T1148N/L1248D/L1377D/V1439E ++ ++ ++ ++ + ++ + ++ 2045/2046 G619E/G681N V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/G619E/M681N/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ ++ ++ +++ + ++ + ++ 2047/2048 F555G/Q921R V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/F555G/M558W/M681G/K732W/L749F/L750D/I775G/Q921R/T1148N/L124 8D/L1377D/V1439E + + ++ + + + 2049/2050 G619E/G681N/S916T V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/G619E/M681N/K732W/L749F/L750D/I775G/S916T/T1148N/L124 8D/L1377D/V1439E ++ ++ ++ ++ + ++ + 2051/2052 F555G/G619E/G681N/K799S V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/F555G/M558W/G619E/M681N/K732W/L749F/L750D/I775G/K799S/T1148 N/L1248D/L1377D/V1439E + ++ ++ ++ + ++ + + 2053/2054 F555E V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/F555E/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + + + 2055/2056 W732K V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1439E + + + 2057/2058 G619E/K799S V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/G619E/M681G/K732W/L749F/L750D/I775G/K799S/T1148N/L124 8D/L1377D/V1439E ++ ++ ++ ++ + + + 2059/2060 G619E V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/G619E/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E + + + ++ ++ 2061/2062 G681N V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681N/K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/V1 439E + ++ ++ ++ + + + 2063/2064 F555G/S916T V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/F555G/M558W/M681G/K732W/L749F/L750D/I775G/S916T/T1148N/L124 8D/L1377D/V1439E + + ++ + + + 2065/2066 S839K V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/S839K/T1148N/L1248D/L13 77D/V1439E + + + ++ + 2067/2068 K799Q V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/K799Q/T1148N/L1248D/L13 77D/V1439E + + + ++ + + + All activities were determined relative to the reference polypeptide of SEQ ID NO: 1514. The levels of increased activity are defined as follows: "+" 0.9 to 1.1, "++"> 1.1, "+++"> 2, "++++"> 2.5 Example 11 Derived from SEQ ID NO: 2072 Of FVIII variant

In this example, experiments performed to determine the activity of FVIII variants by analyzing protein activity after challenge are described. These variants were tested for FVIII activity after no incubation and after incubation in hemophilia A patient plasma (George King Bio-Medical, Inc.), as described in Example 3. Variants were tested using chromogenic and coagulation assays as described in Example 3. Results using variants expressed in Expi293F cells or HepG2 cells are shown in Table 11.1 . Table 11.1 : Activity of FVIII variants relative to SEQ ID NO: 2072 SEQ ID NO: (nt/aa) Amino acid differences ( relative to SEQ ID NO: 2072) Amino acid differences ( relative to SEQ ID NO: 2) Relative to the unattacked Biophen of FIOP of SEQ ID NO: 2072 (Expi) Relative to the unattacked APTT of FIOP inv of SEQ ID NO: 2072 (Expi) Relative to the FIOP of SEQ ID NO: 2072 attacked Biophen (Expi) Relative to the unattacked Biophen (HepG2) of FIOP of SEQ ID NO: 2072 Relative to the unattacked APTT of FIOP inv of SEQ ID NO: 2072 (HepG2) Relative to the FIOP of SEQ ID NO: 2072 attacked Biophen (HepG2) 2073/2074 A167G V63Q/A167G/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/I775G/T1148 N/L1248D/L1377D/V1439E +++ ++ + + + 2075/2076 A167G/E738Y/E1076S/K1093G V63Q/A167G/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/E1076S/K109 3G/T1148N/L1248D/L1377D/V1439E ++ ++ + + + ++ 2077/2078 A167G/E738Y/K752E/E800V/Y805Q/D808S/E809G/S1116I V63Q/A167G/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/K752E/I775G/E800V /Y805Q/D808S/E809G/S1116I/ T1148N/L1248D/L1377D/V1439E ++ ++ + + + ++ 2079/2080 A167G/E800V/E809G/E1076S V63Q/A167G/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/I775G/E800V /E809G/E1076S/T1148N/L1248D/ L1377D/V1439E ++ ++ + + + + 2081/2082 A167G/G318L V63Q/A167G/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/I775G/T1148N/L124 8D/L1377D/V1439E +++ + ++ ++ ++ ++ 2083/2084 A167G/G318L/E800V/Y805Q/D808S/E809G V63Q/A167G/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/I775G/E800V/Y805Q /D808S/E809G/T1148N/L1248D/ L1377D/V1439E +++ ++ + ++ ++ ++ 2085/2086 A167G/G318L/G681N V63Q/A167G/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681N/K732W/Y738E/L749F/L750D/I775G/T1148N/L124 8D/L1377D/V1439E ++ + + ++ ++ ++ 2087/2088 A167G/G318L/G681N/S700E/Y805Q/D808S/E809G/E1076S/S1116I V63Q/A167G/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681N/S700E/K732W/Y738E/L749F/L750D/I775G/Y805Q /D808S/E809G/E1076S/S1116I/ T1148N/L1248D/L1377D/V1439E ++ ++ + ++ ++ ++ 2089/2090 A167G/G318L/S700E V63Q/A167G/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/S700E/K732W/Y738E/L749F/L750D/I775G/T1148 N/L1248D/L1377D/V1439E ++ ++ + ++ ++ ++ 2091/2092 A167G/G318L/S700E/E1076S V63Q/A167G/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/S700E/K732W/Y738E/L749F/L750D/I775G/E1076 S/T1148N/L1248D/L1377D/V1439E ++ + ++ ++ ++ ++ 2093/2094 A167G/G318L/S700E/E738Y/E739G/E1076S V63Q/A167G/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/S700E/K732W/E739G/L749F/L750D/I775G/E1076 S/T1148N/L1248D/L1377D/V1439E ++ + + ++ ++ ++ 2095/2096 A167G/G681N/E738Y/E800V/D808S/E809G V63Q/A167G/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681N/K732W/L749F/L750D/I775G/E800V/D808S /E809G/T1148N/L1248D/L1377D/ V1439E ++ ++ + + + ++ 2097/2098 A167G/L178Q/S700E/K752E/K1093G V63Q/A167G/L171P/L178Q/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/S700E/K732W/Y738E/L749F/L750D /K752E/I775G/K1093G/T1148N/ L1248D/L1377D/V1439E ++ ++ + ++ ++ ++ 2099/2100 A167G/S700E V63Q/A167G/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/S700E/K732W/Y738E/L749F/L750D/I775G /T1148N/L1248D/L1377D/V1439E +++ + + + + ++ 2101/2102 A167G/S700E/E738Y/E739G/K1093G V63Q/A167G/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/S700E/K732W/E739G/L749F/L750D/I775G /K1093G/T1148N/L1248D/L1377D/ V1439E + ++ + + + ++ 2103/2104 A167G/S700E/E738Y/K752E/K1093G V63Q/A167G/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/S700E/K732W/L749F/L750D/K752E/I775G /K1093G/T1148N/L1248D/L1377D/ V1439E + ++ + + ++ +++ 2105/2106 A167G/S700E/E738Y/K752E/Y805Q/D808S/E809G/K1093A/S1116I V63Q/A167G/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/S700E/K732W/L749F/L750D/K752E/I775G /Y805Q/D808S/E809G/K1093A/ S1116I/T1148N/L1248D/L1377D/V1439E + ++ + + + ++ 2107/2108 D808S/E809G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/I775G/D808S/E809G /T1148N/L1248D/L1377D/V1439E ++ ++ ++ + + ++ 2109/2110 E1076S V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/I775G/E1076S/T114 8N/L1248D/L1377D/V1439E + + + + + + 2111/2112 E738Y V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ ++ + ++ + ++ 2113/2114 E738Y/D808S/E809G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/D808S/E809G/T1148 N/L1248D/L1377D/V1439E ++ ++ + ++ + ++ 2115/2116 E738Y/E1076S V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/E1076S/T1148N/L12 48D/L1377D/V1439E ++ + + + + ++ 2117/2118 E738Y/E739G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/E739G/L749F/L750D/I775G/T1148N/L124 8D/L1377D/V1439E ++ + + ++ + + 2119/2120 E738Y/E739G/E809G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/E739G/L749F/L750D/I775G/E809G/T1148 N/L1248D/L1377D/V1439E ++ + + ++ ++ + 2121/2122 E738Y/E739G/E809G/K1093G/S1116I V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/E739G/L749F/L750D/I775G/E809G/K1093 G/S1116I/T1148N/L1248D/L1377D/ V1439E + + + ++ ++ ++ 2123/2124 E738Y/E739G/K1093G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/E739G/L749F/L750D/I775G/K1093G/T114 8N/L1248D/L1377D/V1439E +++ ++ + ++ + ++ 2125/2126 E738Y/E739G/K752E/K1093G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/E739G/L749F/L750D/K752E/I775G/K1093 G/T1148N/L1248D/L1377D/V1439E ++ ++ ++ ++ ++ ++ 2127/2128 E738Y/E739G/K799S/E800V/K1093G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/E739G/L749F/L750D/I775G/K799S/E800V /K1093G/T1148N/L1248D/L1377D/ V1439E ++ + + ++ ++ 2129/2130 E738Y/E739G/S1116I V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/E739G/L749F/L750D/I775G/S1116I/T114 8N/L1248D/L1377D/V1439E ++ + + + + 2131/2132 E738Y/E800V V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/E800V/T1148N/L124 8D/L1377D/V1439E ++ + + + + 2133/2134 E738Y/K1093G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/K1093G/T1148N/L12 48D/L1377D/V1439E ++ ++ + ++ ++ 2135/2136 E738Y/K752E V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/K752E/I775G/T1148N/L124 8D/L1377D/V1439E + ++ + ++ ++ ++ 2137/2138 E738Y/K752E/K1093G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/K752E/I775G/K1093G/T114 8N/L1248D/L1377D/V1439E +++ ++ + ++ ++ 2139/2140 E738Y/K752E/K799S V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/K752E/I775G/K799S/T1148 N/L1248D/L1377D/V1439E ++ + + + + 2141/2142 E738Y/K799S/E809G/K1093G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/K799S/E809G/K1093 G/T1148N/L1248D/L1377D/V1439E + + + ++ ++ ++ 2143/2144 E738Y/K799S/K1093G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/L749F/L750D/I775G/K799S/K1093G/T114 8N/L1248D/L1377D/V1439E ++ + ++ + ++ ++ 2145/2146 E739G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/E739G/L749F/L750D/I775G/T1148 N/L1248D/L1377D/V1439E ++ + + + + ++ 2147/2148 E739G/E800V/E809G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/E739G/L749F/L750D/I775G/E800V /E809G/T1148N/L1248D/L1377D/ V1439E ++ + + + + + 2149/2150 E800V V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/I775G/E800V/T1148 N/L1248D/L1377D/V1439E ++ + + + + 2151/2152 E800V/Y805Q/E809G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/I775G/E800V/Y805Q /E809G/T1148N/L1248D/L1377D/ V1439E ++ ++ + + ++ ++ 2153/2154 E809G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/I775G/E809G/T1148 N/L1248D/L1377D/V1439E ++++ + + ++ + ++ 2155/2156 E809G/K1093G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/I775G/E809G/K1093 G/T1148N/L1248D/L1377D/V1439E ++ + ++ ++ + 2157/2158 E809G/K1093G/S1116I V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/I775G/E809G/K1093 G/S1116I/T1148N/L1248D/L1377D/ V1439E ++ + ++ + + ++ 2159/2160 E809G/S1116I V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/I775G/E809G/S1116 I/T1148N/L1248D/L1377D/V1439E ++ + + ++ + ++ 2161/2162 G318L V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E +++ + ++ ++ ++ ++ 2163/2164 G318L/E1076S V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/I775G/E1076S/T1148N/L12 48D/L1377D/V1439E +++ ++ + ++ ++ ++ 2165/2166 G318L/E1076S/S1116I V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/I775G/E1076S/S1116I/T11 48N/L1248D/L1377D/V1439E ++ + + ++ ++ ++ 2167/2168 G318L/E739G/Y805Q/D808S/E809G/E1076S V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/E739G/L749F/L750D/I775G/Y805Q/D808S /E809G/E1076S/T1148N/L1248D/ L1377D/V1439E ++ ++ ++ ++ ++ ++ 2169/2170 G318L/I499L/S700E/E738Y/E809G V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/I499L/V534I/M558W/M681G/S700E/K732W/L749F/L750D/I775G/E809G/T1148 N/L1248D/L1377D/V1439E ++++ ++ + ++ ++ +++ 2171/2172 G318L/K1093A V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/I775G/K1093A/T1148N/L12 48D/L1377D/V1439E ++ ++ ++ ++ ++ ++ 2173/2174 G318L/K752E/K1093A V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/K752E/I775G/K1093A/T114 8N/L1248D/L1377D/V1439E ++ ++ ++ ++ ++ +++ 2175/2176 G318L/S1116I V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/I775G/S1116I/T1148N/L12 48D/L1377D/V1439E ++ ++ + ++ ++ ++ 2177/2178 G318L/S543P/E739G V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/S543P/M558W/M681G/K732W/Y738E/E739G/L749F/L750D/I775G/T1148 N/L1248D/L1377D/V1439E ++ + + ++ ++ ++ 2179/2180 G318L/S543P/G681N/K752E/S1116I V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/S543P/M558W/M681N/K732W/Y738E/L749F/L750D/K752E/I775G/S1116 I/T1148N/L1248D/L1377D/V1439E ++ ++ + ++ ++ +++ 2181/2182 G318L/S543P/K1093A V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/S543P/M558W/M681G/K732W/Y738E/L749F/L750D/I775G/K1093A/T114 8N/L1248D/L1377D/V1439E ++ + + ++ ++ ++ 2183/2184 G318L/S700E/E738Y/K1093A/S1414N V63Q/L171P/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/S700E/K732W/L749F/L750D/I775G/K1093A/T1148N/L12 48D/L1377D/S1414N/V1439E +++ ++ + ++ ++ ++++ 2185/2186 G681N V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681N/K732W/Y738E/L749F/L750D/I775G/T1148N/L124 8D/L1377D/V1439E ++ ++ + ++ + ++ 2187/2188 G681N/E738Y V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681N/K732W/L749F/L750D/I775G/T1148N/L1248D/L13 77D/V1439E ++ ++ + + + ++ 2189/2190 G681N/E738Y/E739G/K1093G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681N/K732W/E739G/L749F/L750D/I775G/K1093G/T114 8N/L1248D/L1377D/V1439E ++ + ++ + ++ ++ 2191/2192 G681N/E738Y/E739G/K752E/K799S/E800V/E809G/S1116I/G1339C V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681N/K732W/E739G/L749F/L750D/K752E/I775G/K799S /E800V/E809G/S1116I/T1148N/ L1248D/G1339C/L1377D/V1439E ++ ++ + + + 2193/2194 G681N/E738Y/E739G/S1116I V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681N/K732W/E739G/L749F/L750D/I775G/S1116I/T114 8N/L1248D/L1377D/V1439E +++ ++ + ++ + ++ 2195/2196 G681N/E738Y/E800V V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681N/K732W/L749F/L750D/I775G/E800V/T1148N/L124 8D/L1377D/V1439E ++ ++ + ++ + 2197/2198 G681N/E800V/Y805Q/E809G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681N/K732W/Y738E/L749F/L750D/I775G/E800V/Y805Q /E809G/T1148N/L1248D/L1377D/ V1439E ++++ ++ + ++ ++ ++ 2199/2200 G681N/E809G/K1093G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681N/K732W/Y738E/L749F/L750D/I775G/E809G/K1093 G/T1148N/L1248D/L1377D/V1439E ++ ++ ++ + ++ 2201/2202 G681N/K752E/K1093A V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681N/K732W/Y738E/L749F/L750D/K752E/I775G/K1093 A/T1148N/L1248D/L1377D/V1439E ++ ++ + + ++ +++ 2203/2204 G681N/K752E/K1093G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681N/K732W/Y738E/L749F/L750D/K752E/I775G/K1093 G/T1148N/L1248D/L1377D/V1439E ++ ++ + ++ ++ +++ 2205/2206 G681N/S700E V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681N/S700E/K732W/Y738E/L749F/L750D/I775G/T1148 N/L1248D/L1377D/V1439E ++ ++ + ++ ++ ++ 2207/2208 G681N/S700E/E738Y/E739G/K1093G/S1116I V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681N/S700E/K732W/E739G/L749F/L750D/I775G/K1093 G/S1116I/T1148N/L1248D/L1377D/ V1439E ++ ++ + ++ + ++ 2209/2210 G681N/S700E/E809G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681N/S700E/K732W/Y738E/L749F/L750D/I775G/E809G /T1148N/L1248D/L1377D/V1439E + + + ++ + ++ 2211/2212 G681N/S700E/K1093G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681N/S700E/K732W/Y738E/L749F/L750D/I775G/K1093 G/T1148N/L1248D/L1377D/V1439E ++ ++ + ++ ++ ++ 2213/2214 G681N/S700E/K752E/D808S/E809G/K1093G/S1116I V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681N/S700E/K732W/Y738E/L749F/L750D/K752E/I775G /D808S/E809G/K1093G/S1116I/ T1148N/L1248D/L1377D/V1439E + ++ ++ + ++ 2215/2216 G681N/S700E/K752E/K1093G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681N/S700E/K732W/Y738E/L749F/L750D/K752E/I775G /K1093G/T1148N/L1248D/L1377D/ V1439E +++ ++ ++ ++ ++ 2217/2218 G681N/S700E/R844H V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681N/S700E/K732W/Y738E/L749F/L750D/I775G/R844H /T1148N/L1248D/L1377D/V1439E + + + + + ++ 2219/2220 G681N/S700E/Y805Q/E809G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681N/S700E/K732W/Y738E/L749F/L750D/I775G/Y805Q /E809G/T1148N/L1248D/L1377D/ V1439E ++ ++ ++ ++ + ++ 2221/2222 K1093A V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/I775G/K1093A/T114 8N/L1248D/L1377D/V1439E ++ ++ + + ++ 2223/2224 K1093G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/I775G/K1093G/T114 8N/L1248D/L1377D/V1439E ++ + + ++ + + 2225/2226 K1093G/S1116I V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/I775G/K1093G/S111 6I/T1148N/L1248D/L1377D/V1439E ++ ++ + ++ ++ ++ 2227/2228 K752E V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/K752E/I775G/T1148 N/L1248D/L1377D/V1439E ++ + ++ ++ + 2229/2230 K752E/E1076S V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/K752E/I775G/E1076 S/T1148N/L1248D/L1377D/V1439E ++ ++ + + ++ ++ 2231/2232 K752E/E800V/E809G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/K752E/I775G/E800V /E809G/T1148N/L1248D/L1377D/ V1439E ++ ++ + + + ++ 2233/2234 K752E/E800V/K1093G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/K752E/I775G/E800V /K1093G/T1148N/L1248D/L1377D/ V1439E ++ ++ + ++ ++ ++ 2235/2236 K752E/E809G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/K752E/I775G/E809G /T1148N/L1248D/L1377D/V1439E + + + + + ++ 2237/2238 K752E/G866A V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/K752E/I775G/G866A /T1148N/L1248D/L1377D/V1439E ++ ++ + + + 2239/2240 K752E/K1093G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/K752E/I775G/K1093 G/T1148N/L1248D/L1377D/V1439E + ++ ++ ++ ++ ++ 2241/2242 K752E/S1116I V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/K752E/I775G/S1116 I/T1148N/L1248D/L1377D/V1439E + + + + + ++ 2243/2244 L178Q/G318L/E739G/K752E/E809G/E1076S/S1116I V63Q/L171P/L178Q/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/E739G/L749F/L750D/K752E/I775G /E809G/E1076S/S1116I/T1148N/ L1248D/L1377D/V1439E ++ ++ + ++ ++ ++ 2245/2246 L178Q/G318L/E800V/E809G V63Q/L171P/L178Q/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/I775G/E800V/E809G /T1148N/L1248D/L1377D/V1439E ++ + + ++ ++ ++ 2247/2248 L178Q/G318L/G681N/E800V/D808S/E809G/E1076S V63Q/L171P/L178Q/H212A/S260T/E306G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681N/K732W/Y738E/L749F/L750D/I775G/E800V/D808S /E809G/E1076S/T1148N/L1248D/ L1377D/V1439E ++ ++ + ++ ++ ++ 2249/2250 L178Q/G681N V63Q/L171P/L178Q/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681N/K732W/Y738E/L749F/L750D/I775G/T1148 N/L1248D/L1377D/V1439E ++ ++ + ++ ++ ++ 2251/2252 L178Q/G681N/E738Y/S1116I V63Q/L171P/L178Q/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681N/K732W/L749F/L750D/I775G/S1116I/T114 8N/L1248D/L1377D/V1439E ++ ++ + ++ ++ ++ 2253/2254 L178Q/K752E/E809G V63Q/L171P/L178Q/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/K752E/I775G /E809G/T1148N/L1248D/L1377D/ V1439E + ++ + ++ + ++ 2255/2256 L178Q/S700E/E800V/Y805Q/D808S/E809G V63Q/L171P/L178Q/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/S700E/K732W/Y738E/L749F/L750D/I775G /E800V/Y805Q/D808S/E809G/ T1148N/L1248D/L1377D/V1439E ++ ++ + ++ + ++ 2257/2258 L178Q/Y805Q/E809G/G866A V63Q/L171P/L178Q/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/I775G/Y805Q /E809G/G866A/T1148N/L1248D/ L1377D/V1439E ++ + + ++ ++ + 2259/2260 S1116I V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/I775G/S1116I/T114 8N/L1248D/L1377D/V1439E ++ + + + + 2261/2262 S543P V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/S543P/M558W/M681G/K732W/Y738E/L749F/L750D/I775G/T1148 N/L1248D/L1377D/V1439E ++ + + + + + 2263/2264 S543P/E738Y/E739G/E809G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/S543P/M558W/M681G/K732W/E739G/L749F/L750D/I775G/E809G /T1148N/L1248D/L1377D/V1439E ++ ++ + ++ + 2265/2266 S543P/E738Y/E809G/K1093G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/S543P/M558W/M681G/K732W/L749F/L750D/I775G/E809G/K1093 G/T1148N/L1248D/L1377D/V1439E ++ + + + + ++ 2267/2268 S543P/E739G/K1093A V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/S543P/M558W/M681G/K732W/Y738E/E739G/L749F/L750D/I775G /K1093A/T1148N/L1248D/L1377D/ V1439E ++ + + + ++ + 2269/2270 S543P/E800V/K1093G/S1116I V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/S543P/M558W/M681G/K732W/Y738E/L749F/L750D/I775G/E800V /K1093G/S1116I/T1148N/L1248D/ L1377D/V1439E ++ ++ + + + 2271/2272 S543P/E809G/K1093G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/S543P/M558W/M681G/K732W/Y738E/L749F/L750D/I775G/E809G /K1093G/T1148N/L1248D/L1377D/ V1439E + + + ++ ++ ++ 2273/2274 S543P/E809G/K1093G/S1116I V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/S543P/M558W/M681G/K732W/Y738E/L749F/L750D/I775G/E809G /K1093G/S1116I/T1148N/L1248D/ L1377D/V1439E + ++ + ++ + 2275/2276 S543P/G681N/E738Y/E739G/K752E/E809G/K1093G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/S543P/M558W/M681N/K732W/E739G/L749F/L750D/K752E/I775G /E809G/K1093G/T1148N/L1248D/ L1377D/V1439E + ++ + ++ + ++ 2277/2278 S543P/G681N/E738Y/E739G/K752E/K1093G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/S543P/M558W/M681N/K732W/E739G/L749F/L750D/K752E/I775G /K1093G/T1148N/L1248D/L1377D/ V1439E ++ ++ + ++ ++ 2279/2280 S543P/G681N/E739G/K752E/E800V/Y805Q/E809G/K1093G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/S543P/M558W/M681N/K732W/Y738E/E739G/L749F/L750D/K752E /I775G/E800V/Y805Q/E809G/ K1093G/T1148N/L1248D/L1377D/V1439E +++ ++ + ++ ++ 2281/2282 S543P/G681N/S700E/E738Y/E739G/E800V/K1093G/S1116I V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/S543P/M558W/M681N/S700E/K732W/E739G/L749F/L750D/I775G /E800V/K1093G/S1116I/T1148N/ L1248D/L1377D/V1439E + ++ + + + + 2283/2284 S543P/G681N/S700E/E738Y/E739G/Y805Q/D808S/E809G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/S543P/M558W/M681N/S700E/K732W/E739G/L749F/L750D/I775G /Y805Q/D808S/E809G/T1148N/ L1248D/L1377D/V1439E +++ ++ + + + + 2285/2286 S543P/G681N/S700E/K752E V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/S543P/M558W/M681N/S700E/K732W/Y738E/L749F/L750D/K752E /I775G/T1148N/L1248D/L1377D/ V1439E + ++ + + + ++ 2287/2288 S543P/G681N/S700E/K752E/E809G/K1093G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/S543P/M558W/M681N/S700E/K732W/Y738E/L749F/L750D/K752E /I775G/E809G/K1093G/T1148N/ L1248D/L1377D/V1439E + + + + + ++ 2289/2290 S543P/K1093G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/S543P/M558W/M681G/K732W/Y738E/L749F/L750D/I775G/K1093 G/T1148N/L1248D/L1377D/V1439E ++ + + ++ ++ ++ 2291/2292 S543P/S700E V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/S543P/M558W/M681G/S700E/K732W/Y738E/L749F/L750D/I775G /T1148N/L1248D/L1377D/V1439E +++ ++ + ++ + 2293/2294 S543P/S700E/E738Y/E739G/K752E/E800V/K1093G/S1116I V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/S543P/M558W/M681G/S700E/K732W/E739G/L749F/L750D/K752E /I775G/E800V/K1093G/S1116I/ T1148N/L1248D/L1377D/V1439E ++ ++ + + + ++ 2295/2296 S543P/S700E/E809G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/S543P/M558W/M681G/S700E/K732W/Y738E/L749F/L750D/I775G /E809G/T1148N/L1248D/L1377D/ V1439E + ++ + ++ ++ 2297/2298 S543P/S700E/K1093G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/S543P/M558W/M681G/S700E/K732W/Y738E/L749F/L750D/I775G /K1093G/T1148N/L1248D/L1377D/ V1439E + ++ + ++ + ++ 2299/2300 S543P/S700E/K752E V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/S543P/M558W/M681G/S700E/K732W/Y738E/L749F/L750D/K752E /I775G/T1148N/L1248D/L1377D/ V1439E + ++ + + + ++ 2301/2302 S543P/S700E/K752E/E800V/K1093G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/S543P/M558W/M681G/S700E/K732W/Y738E/L749F/L750D/K752E /I775G/E800V/K1093G/T1148N/ L1248D/L1377D/V1439E + ++ + + + ++ 2303/2304 S543P/S700E/K752E/E809G/S1116I V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/S543P/M558W/M681G/S700E/K732W/Y738E/L749F/L750D/K752E /I775G/E809G/S1116I/T1148N/ L1248D/L1377D/V1439E ++ ++ + + + ++ 2305/2306 S543P/S700E/K752E/K1093G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/S543P/M558W/M681G/S700E/K732W/Y738E/L749F/L750D/K752E /I775G/K1093G/T1148N/L1248D/ L1377D/V1439E + ++ + ++ + +++ 2307/2308 S700E V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/S700E/K732W/Y738E/L749F/L750D/I775G/T1148 N/L1248D/L1377D/V1439E +++ + + + + 2309/2310 S700E/E738Y/E739G/E800V/K1093G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/S700E/K732W/E739G/L749F/L750D/I775G/E800V /K1093G/T1148N/L1248D/L1377D/ V1439E +++ ++ + ++ ++ 2311/2312 S700E/E738Y/E739G/K799S/K1093G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/S700E/K732W/E739G/L749F/L750D/I775G/K799S /K1093G/T1148N/L1248D/L1377D/ V1439E +++ + + + + 2313/2314 S700E/E738Y/E800V/K1093G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/S700E/K732W/L749F/L750D/I775G/E800V/K1093 G/T1148N/L1248D/L1377D/V1439E +++ ++ + ++ ++ +++ 2315/2316 S700E/E738Y/S1116I V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/S700E/K732W/L749F/L750D/I775G/S1116I/T114 8N/L1248D/L1377D/V1439E + + + + + ++ 2317/2318 S700E/E809G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/S700E/K732W/Y738E/L749F/L750D/I775G/E809G /T1148N/L1248D/L1377D/V1439E ++ ++ + ++ ++ ++ 2319/2320 S700E/E809G/K1093G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/S700E/K732W/Y738E/L749F/L750D/I775G/E809G /K1093G/T1148N/L1248D/L1377D/ V1439E ++ ++ + ++ ++ ++ 2321/2322 S700E/K1093G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/S700E/K732W/Y738E/L749F/L750D/I775G/K1093 G/T1148N/L1248D/L1377D/V1439E ++ ++ + ++ ++ ++ 2323/2324 S700E/K1093G/S1116I V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/S700E/K732W/Y738E/L749F/L750D/I775G/K1093 G/S1116I/T1148N/L1248D/L1377D/ V1439E ++ + + ++ ++ ++ 2325/2326 S700E/K752E V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/S700E/K732W/Y738E/L749F/L750D/K752E/I775G /T1148N/L1248D/L1377D/V1439E ++ ++ + + + 2327/2328 S700E/K752E/D808S/E809G/K1093G/S1116I V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/S700E/K732W/Y738E/L749F/L750D/K752E/I775G /D808S/E809G/K1093G/S1116I/ T1148N/L1248D/L1377D/V1439E ++ ++ + ++ + ++ 2329/2330 S700E/K752E/E809G/K1093G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/S700E/K732W/Y738E/L749F/L750D/K752E/I775G /E809G/K1093G/T1148N/L1248D/ L1377D/V1439E ++ ++ ++ + + 2331/2332 S700E/K752E/K799S/S1116I V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/S700E/K732W/Y738E/L749F/L750D/K752E/I775G /K799S/S1116I/T1148N/L1248D/ L1377D/V1439E + ++ + + + ++ 2333/2334 S700E/K752E/Y805Q/E809G/K1093A V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/S700E/K732W/Y738E/L749F/L750D/K752E/I775G /Y805Q/E809G/K1093A/T1148N/ L1248D/L1377D/V1439E + ++ ++ + + ++ 2335/2336 S700E/K799S/E800V V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/S700E/K732W/Y738E/L749F/L750D/I775G/K799S /E800V/T1148N/L1248D/L1377D/ V1439E + ++ + ++ + ++ 2337/2338 S700E/S1116I V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/S700E/K732W/Y738E/L749F/L750D/I775G/S1116 I/T1148N/L1248D/L1377D/V1439E ++ + + + + 2339/2340 S700E/Y805Q/D808S/E809G/E1076S V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/S700E/K732W/Y738E/L749F/L750D/I775G/Y805Q /D808S/E809G/E1076S/T1148N/ L1248D/L1377D/V1439E ++ ++ ++ ++ + ++ 2341/2342 V514L/S543P/G681N/Y805Q/D808S/E809G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V514L/V534I/S543P/M558W/M681N/K732W/Y738E/L749F/L750D/I775G /Y805Q/D808S/E809G/T1148N/ L1248D/L1377D/V1439E + + + ++ + ++ 2343/2344 Y805Q/D808S/E809G V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/I775G/Y805Q/D808S /E809G/T1148N/L1248D/L1377D/ V1439E ++ + + + + + 2345/2346 Y805Q/E809G/S1116I V63Q/L171P/H212A/S260T/E306G/L318G/M320R/S332P/F387I/I390A/V393G/V534I/M558W/M681G/K732W/Y738E/L749F/L750D/I775G/Y805Q/E809G /S1116I/T1148N/L1248D/L1377D/ V1439E + ++ + ++ + All activities were determined relative to the reference polypeptide of SEQ ID NO: 2072. The levels of increased activity are defined as follows: "+" 0.9 to 1.1, "++"> 1.1, "+++"> 2, "++++"> 2.5 Example 12 Identification of active FVIII variants with reduced immunogenicity

Putative T cell epitopes in the B-region deleted form of wild-type human FVIII were determined using tools as known in the art as the Immune Epitope Database (IEDB; Immune Epitope Database and Analysis Resource Website) and proprietary statistics. Analysis tools to identify (see, for example, iedb.org and Vita et al., Nucl. Acids Res., 2010, 38 (Database Issue): D854-62, electronic version November 11, 2009). The B-region deleted form of wild-type human FVIII (SEQ ID NO: 2) was resolved into all possible 15-mer analytical frames, where each frame overlapped the last one by 14 amino acids. Using the method recommended on the IEDB website, by matching eight common class II HLA-DR allele genes (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*0801, DRB1*1101, DRB1*1301 and DRB1*1501) were combined to perform scoring to evaluate the immunogenic potential of the 15-mer analysis framework. Potential T cell epitope clusters contained within the polypeptide (ie, subregions contained within human FVIII that have an unusually high potential for immunogenicity) were identified using statistical analysis tools, as are known in the art. The identified T cell epitope clusters were screened against the IEDB database of known epitopes.

The predicted immunogenicity potential of the active FVIII variants identified in the screen in Example 3 was identified by evaluating their binding to eight common class II HLA-DR alleles. The total immunogenicity score and the immunogenicity hit count were calculated for each variant. The total immunogenicity score (TIS) reflects the overall predicted immunogenicity of a variant (ie, a higher score indicates a higher predicted immunogenicity level). The immunogenic "hit count" (IHC) indicates the number of 15-mer assay frames with unusually high immunogenic potential (i.e., higher scores indicate higher immunogenic potential). Mutations that cause a decrease in the overall immunogenicity score and/or a decrease in the immunogenicity hit count compared to the reference sequence are considered potential "deimmunizing mutations" and are shown in surface 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8as well as 15.2middle. The collection of mostly deimmunizing mutations was recombined to generate multiple active variants and predicted to be significantly less immunogenic than the B-region deleted form of WT human FVIII (SEQ ID NO: 2). Table 12.1 : Reduction in Total Immunogenicity Score (TIS) and Immunogenic Hit Count (IHC) of FVIII variants relative to SEQ ID NO: 2 SEQ ID NO: (nt/aa) Amino acid differences ( relative to SEQ ID NO: 2) Reduced TIS relative to SEQ ID NO: 2 Reduced IHC relative to SEQ ID NO: 2 1/2 191/192 Q37H/Q96R/L171P/Q429L/I708T/K799R/T1148N ++ + 193/194 L171P + + 195/196 L171P/Q429L/K732R/T1148N + + 197/198 Q96R/L171P ++ + 199/200 L171P/E919G/G1151S + 201/202 L171P/T792Y/M797Q/K799R/T1148N +++ + 203/204 L171P/P569T/T1148N +++ + 205/206 Q96R/L171P/Q429L/T792Y/M797T/K799R + 207/208 L171P/Q429L/I708T/L1422I ++ + 209/210 L171P/Q429L + 3/4 T1148N + + 211/212 D321Y/A362P/K732E/M797T/K799R + 213/214 L171P/L1100I ++ + 215/216 I708T/T1148N/A1186E +++ + 217/218 Q429L/L724I/K732E/T1148N + 219/220 L171P/A362E/V795T/M797Q/K799M ++ + 221/222 L171P/Q429L/K732E + 223/224 Q96R/L171P/S643N/M797T/K799R ++ + 225/226 L171P/T792Y/M797T/K799M + + 227/228 R503S ++ + 229/230 L171P/K732E + + 231/232 T1148N/F1193L + + 5/6 P85Q/I708T/T1148N ++ + 233/234 Q96R/L171P/A362E/Q429L/K732R/V795T/M797Q + + 7/8 K798T + 9/10 L706G ++ ++ 11/12 L706A + + 13/14 L171Q + 15/16 Q335E + 17/18 V63R ++ ++ 235/236 K726A + 19/20 K344A + 237/238 F59M + 241/242 A362N + 243/244 H334G + 25/26 F59L + 247/248 V63Q +++ ++ 251/252 L1377D +++ + 27/28 Q429S + 31/32 I1264W + 259/260 V1250T ++ + 261/262 E306G + 265/266 L1377G ++ + 269/270 L1377E +++ + 273/274 L646I + 33/34 M797P + 35/36 L1248S + + 37/38 I1264R + 39/40 Y873T + 41/42 T792Y + 43/44 I708K + + 45/46 K344L + 47/48 F57V + 49/50 Q96L + 277/278 P435Q + 51/52 A362W ++ 53/54 V63G +++ ++ 55/56 Y873L + 59/60 T1148Q + 63/64 M797G + 65/66 L706Y ++ ++ 67/68 L1248R ++ + 71/72 Y65A + + 73/74 F59W + 75/76 Y873Q + 77/78 V795C + 279/280 Q96V + 81/82 F57L + 83/84 Q353S + 85/86 S332Q + + 87/88 I708A + 89/90 G1259T + + 91/92 V63M + 93/94 M356L ++ 95/96 L1248G + + 97/98 L1248P ++ + 99/100 Q436G + 101/102 F57A + 103/104 F59S + + 105/106 T1245R + 107/108 F59R + 109/110 Q96W + 111/112 M797Y + 113/114 K1145R + 281/282 L1377R + 115/116 Q436D + 117/118 F716L + 119/120 S56E + 283/284 L1377S + + 285/286 F59G + 287/288 L171M + 121/122 R773H + 289/290 T792H + 129/130 F59Q + + 131/132 S729T + 133/134 F716K + 135/136 T665G + + 137/138 A362T ++ 139/140 F57T + 141/142 N359G + 291/292 L1248D + + 145/146 V1250A ++ + 147/148 A362V + 149/150 N359E ++ 151/152 F716W + 153/154 S332W + ++ 155/156 I708V + 157/158 V63H ++ ++ 159/160 M797C + 295/296 L706M + ++ 161/162 T61Q + + 163/164 M356N ++ 297/298 L171H + 165/166 V1250S + + 167/168 K798C + 169/170 N433G + 299/300 L1377A ++ 301/302 L1377V + 171/172 V795N + 173/174 Q96I + 175/176 S56P + 177/178 V63N ++ ++ 179/180 T792W ++ 181/182 I1264L + 183/184 V1250P ++ + 185/186 S332P + + 187/188 F59V + 309/310 F59D + + 189/190 K726L + 311/312 K344E + TIS reduction is measured as a reduction in counts compared to the reference polypeptide of SEQ ID NO: 2 and is defined as follows: "+" 1-10, "++"> 10, "+++"> 20. IHC reduction is measured as a reduction in counts compared to the reference polypeptide of SEQ ID NO: 2 and is defined as follows: "+" 1-3, "++"> 3, "+++"> 5. Table 12.2 : Reduction in Total Immunogenicity Score (TIS) and Immunogenic Hit Count (IHC) of FVIII variants relative to SEQ ID NO: 252 SEQ ID NO: (nt/aa) Amino acid differences ( relative to SEQ ID NO: 252) Reduced TIS relative to SEQ ID NO: 252 Reduced IHC relative to SEQ ID NO: 252 251/252 411/412 I265S ++ + 413/414 L1221T + ++ 415/416 M1380G + 417/418 K752G ++ + 419/420 Y748K +++ +++ 423/424 F1218A ++ ++ 425/426 Y748N +++ +++ 427/428 T1378E + 429/430 L614I + + 431/432 L749F + + 433/434 F387V ++ + 435/436 L750T + + 437/438 F1193G ++ + 441/442 T1378A + 443/444 L750V + 445/446 F387T +++ +++ 447/448 F387Q +++ +++ 451/452 F1037N ++ + 453/454 H1440A + 455/456 M1051S + + 457/458 L1221G ++ ++ 459/460 T1378D + 461/462 H1440S + + 463/464 F387R ++ +++ 465/466 F1193A + + 467/468 F387I + + 469/470 F1218M ++ ++ 319/320 I388A ++ ++ 471/472 I265W +++ + 473/474 H1440E +++ +++ 475/476 V1439L + + 477/478 H1440T ++ +++ 479/480 R612S + 481/482 Y1049W + ++ 321/322 F1043E +++ +++ 323/324 F1043R ++ ++ 483/484 L1221S + ++ 325/326 I610R ++ + 327/328 I1274N +++ +++ 329/330 S1220T + + 331/332 I1442T + + 485/486 S1220D ++ ++ 333/334 F1193L + 335/336 Q621T + + 337/338 L1054C + + 339/340 A755Q + + 487/488 L749G +++ +++ 341/342 Y1041G ++ ++ 343/344 Y748S +++ +++ 345/346 Y1049G +++ +++ 347/348 L1221V + ++ 489/490 L749S ++ ++ 491/492 F1218G ++ ++ 497/498 F1193E ++ + 499/500 I1050S ++ ++ 501/502 S1389W + 349/350 M1051E +++ +++ 503/504 V1439E +++ +++ 505/506 S260T + + 507/508 F1037P ++ + 351/352 R612G + + 353/354 F1037H ++ + 355/356 N616E ++ + 509/510 M1051K ++ ++ 511/512 I1269F + + 513/514 I265R + + 357/358 Y1041H ++ +++ 515/516 M1051R ++ ++ 517/518 F1218L + ++ 359/360 F387S +++ +++ 361/362 S489R ++ + 363/364 Q621M + + 365/366 S489Q ++ + 367/368 L750G + + 519/520 M897R + + 521/522 I388P +++ +++ 369/370 S1224P + + 523/524 Y748G +++ +++ 371/372 A618C + + 525/526 I756P ++ +++ 373/374 N616G ++ + 375/376 R612T + 529/530 I390A ++ + 377/378 I1050Q ++ ++ 531/532 I610L + 533/534 A755G + + 535/536 L749R + + 379/380 T1053A + + 381/382 N616W + + 537/538 Y748V + + 383/384 I896V + + 539/540 M1380A + 541/542 H1440C ++ +++ 385/386 S489F + + 543/544 H397N/F1218M ++ ++ 387/388 I1050P ++ ++ 389/390 M897G ++ + 545/546 F1037M + + 547/548 F1218P ++ ++ 549/550 E757K + + 391/392 L749T ++ + 551/552 V1373S + 553/554 H1440G ++ +++ 555/556 F1193I + + 557/558 V1439T ++ +++ 393/394 I1274V + + 395/396 S1220L + + 559/560 L750D +++ +++ 397/398 S260E ++ + 561/562 I390G +++ + 563/564 I756G ++ ++ 399/400 L481V + 565/566 S1220E ++ ++ 401/402 F613S + + 403/404 F387G +++ +++ 405/406 Q621R + + 567/568 T1378C + 407/408 I756R ++ + 569/570 A618G ++ + 573/574 V63Q/L1248D +++ +++ 577/578 E306G/R355C/L1422I +++ 579/580 L1422I + 581/582 E306G/D346R/I708T ++ 583/584 T1148N + + 585/586 L171P/D346R/L1248D ++ + 587/588 V63Q/M797T/K799R/L1248D/L1422I +++ +++ 589/590 L171P/K799R + + 591/592 A362P + 593/594 L1248D + + 595/596 V63Q +++ ++ 597/598 L171P/E306G/D346R/Q999R/L1248D ++ + 599/600 L171P/T1148N ++ + 601/602 T734S/K799R/L1248D/L1422I ++ + 603/604 V63Q/K732E +++ ++ 605/606 V63Q/L171P/E306G/D346R/K732G +++ +++ 607/608 L171P + + 609/610 L171P/L1422I ++ + 611/612 V63Q/A362P/K732G/K799R/L1248D +++ +++ 613/614 Q999R/T1148N/L1422I ++ + 617/618 E306G/P349G + 619/620 V63Q/L171P/T1148N/G1151S +++ +++ 621/622 E306G + 623/624 K344E/P349G/L354R/V1250T +++ + 625/626 V63G +++ ++ 627/628 V1250T ++ + 629/630 V63R/E306G/K344E/P349G/L354R/R503S/L1248R ++++ ++++ 631/632 F59D/V63S/S332P/P349G/R503S/L706M/I708K ++++ ++++ 633/634 V63S/E306G/K344A/P349G/L354R/L706M/L1248R ++++ ++++ 635/636 L1248R ++ + 637/638 F59D/V63G +++ ++ 639/640 S332P/L706M/I708K/L1248R/V1250T +++ ++++ 641/642 R503S ++ + 643/644 F59D/V63R/P349G/L354R +++ ++ 645/646 F59D/P349G/I708K ++ + 647/648 F59D/E306G/K344E/L1248R +++ + 409/410 V63R ++ ++ 649/650 K344E/L1248R/V1250T +++ + 651/652 I708K + + 653/654 V63R/K344E/P349G +++ ++ 655/656 F59D + + 657/658 V63S/K344A/P349G/L354R +++ ++ 659/660 L706M + ++ 661/662 F59D/E306G/R770W ++ + 663/664 V63G/S347N/L354R/L1248R ++++ +++ 665/666 L354R/R503S +++ + TIS reduction is measured as a reduction in counts compared to the reference polypeptide of SEQ ID NO: 252 and is defined as follows: "+" 1-10, "++"> 10, "+++"> 20, "+++ +"> 50, "+++++". IHC reduction is measured as a reduction in counts compared to the reference polypeptide of SEQ ID NO: 252 and is defined as follows: "+" 1-3, "++"> 3, "+++"> 5, "+++ +》＞8. Table 12.3 : Reduction in Total Immunogenicity Score (TIS) and Immunogenic Hit Count (IHC) of FVIII variants relative to SEQ ID NO: 504 SEQ ID NO: (nt/aa) Amino acid differences ( relative to SEQ ID NO: 504) Reduced TIS relative to SEQ ID NO: 504 Reduced IHC relative to SEQ ID NO: 504 503/504 701/702 L171P/L1248D/H1440T ++ + 703/704 S260T/T1148N/T1378D ++ ++ 705/706 L171P/S260T/F387I/L750D/L1248D ++++ ++++ 707/708 L171P/S260T ++ ++ 709/710 L171P/S260T/F387I/L750D/M1051S/T1148N ++++ +++++ 711/712 L171P + + 667/668 L750D +++ +++ 713/714 L750D/M1051S/T1148N +++ ++++ 715/716 L171P/S260T/I756P +++ ++++ 669/670 M1051K ++ ++ 717/718 L171P/L1248D ++ + 671/672 M1051S + + 719/720 L171P/T1148N ++ + 721/722 F387I + + 723/724 F59D + + 727/728 S332P/L750D/L1248D +++ ++++ 729/730 I756P/L1248D +++ +++ 673/674 S260T + + 731/732 L1248D + + 675/676 I756P ++ +++ 733/734 L171P/F387I/L1248D/H1440S +++ + 677/678 F1193A + + 735/736 L750D/H1440S +++ +++ 737/738 F59D/L171P/L750D +++ ++++ 739/740 L171P/F1193A/L1248D ++ ++ 679/680 S332P + + 741/742 L750D/T1148N/F1193A/H1440S +++ ++++ 743/744 T1148N + + 745/746 L171P/F387I/M1051K +++ +++ 747/748 Y748G +++ +++ 681/682 M897R + + 683/684 F387Q +++ +++ 749/750 E306G + 751/752 E306G/F387R/I390A/L749F/S1220D ++++ ++++ 685/686 F1037N ++ + 753/754 I390A ++ + 755/756 E306G/I390A/F1037N +++ + 757/758 E306G/R612S/L749F + + 759/760 F387V/I390A/L749F +++ +++ 761/762 V63S/I390A/R503S/L749F ++++ ++++ 763/764 F387R/I390A/L749R +++ +++ 765/766 S1220D ++ ++ 687/688 F387R ++ +++ 767/768 V63S/E306G +++ ++ 689/690 E306G/L749F + + 769/770 L749F + + 771/772 E306G/I390A/L749F/F1037N +++ + 773/774 F387V ++ + 775/776 E306G/L749F/S1220D +++ ++ 777/778 E306G/F387V/I390A/R612S/L749F/F1037N ++++ ++++ 691/692 I1270V + + 693/694 S489E +++ + 779/780 F328S ++ +++ 695/696 Q487N ++ + 697/698 A488G ++ + 699/700 Q903E ++ +++ TIS reduction is measured as a reduction in counts compared to the reference polypeptide of SEQ ID NO: 504 and is defined as follows: "+" 1-10, "++"> 10, "+++"> 20, "+++ +"> 50, "+++++"> 75. IHC reduction is measured as a reduction in counts compared to the reference polypeptide of SEQ ID NO: 504 and is defined as follows: "+" 1-3, "++"> 3, "+++"> 5, "+++ +"> 8, "+++++"> 15. Table 12.4 : Reduction in Total Immunogenicity Score (TIS) and Immunogenic Hit Count (IHC) of FVIII variants relative to SEQ ID NO: 706 SEQ ID NO: (nt/aa) Amino acid differences ( relative to SEQ ID NO: 706) Reduced TIS relative to SEQ ID NO: 706 Reduced IHC relative to SEQ ID NO: 706 705/706 819/820 S332P/I387R/I390A/T1148N +++ +++ 821/822 F328S/I387R/L749F +++ ++++ 781/782 M1051K ++ ++ 823/824 L749F + 783/784 L1221G ++ ++ 825/826 E306G/F328S/S332P/I390A/M1051R/F1193A/F1218G ++++ ++++ 827/828 V63Q/E306G/S332P/M1051R/T1148N ++++ ++++ 829/830 V63Q/E306G/S332P/I387T/M1051K ++++ +++++ 831/832 I387T/I390A/M1051R +++ ++++ 833/834 D186A/E306G/F328S/S332P/I390A/L749F/M1051K/T1148N/F1193A ++++ ++++ 835/836 I387T/I390A/I756P/M1051R ++++ ++++ 837/838 E306G/I390A/M1051R +++ +++ 839/840 E306G/S332P/I390A/L749F +++ ++ 785/786 F1218G ++ ++ 841/842 E306G/I387R/I390A/T1148N +++ +++ 843/844 E306G/F328S/S332P/I387T/I390A/L749F/M1051R/T1148N/F1193A +++++ ++++ 845/846 S332P/I387R/I390A/L749F/T1148N +++ +++ 847/848 I387R/I390A/L749F +++ ++ 787/788 I387R ++ ++ 849/850 E306G/I387T/L749F/M1051K/T1148N +++ ++++ 789/790 M1051R ++ ++ 851/852 I387R/L749F ++ ++ 853/854 E306G/Q945K/M1051R/F1193A/L1221G +++ ++ 855/856 E306G/I390A/L749F/M1051K +++ +++ 857/858 V63Q/E306G/S332P/I390A +++ ++++ 791/792 L749F/F1218G ++ ++ 859/860 E306G/I387T/I390A/L749F/T1148N/F1193A/L1221G ++++ ++++ 861/862 E306G/S332P/I387R/I390A/L749F/F1218M ++++ ++++ 793/794 I387T ++ ++ 863/864 V63Q/E306G/S332P/I390A/M1051R/T1148N/F1193A/F1218G +++++ +++++ 795/796 S332P + + 867/868 A755S + 875/876 M797Y + 797/798 M897K + + 877/878 N754W + 879/880 I793G + 881/882 N753D + 883/884 R922W + 885/886 I896L + 799/800 T696E + + 889/890 I775G + 801/802 F816W + + 803/804 F824W ++ +++ 805/806 A826G ++ +++ 891/892 S751E + 807/808 V393W ++ + 809/810 I825L + + 811/812 M897R + + 895/896 K752G + 899/900 K938V + 903/904 A755G + 905/906 V393G/A407V +++ ++ 813/814 F816V + + 907/908 M797W + 909/910 K819V + + 911/912 V393G +++ ++ 913/914 I787W + 921/922 G924P + 815/816 F698W + ++ 931/932 S392A + + 933/934 H397G + 817/818 V393Q + + TIS reduction is measured as a reduction in counts compared to the reference polypeptide of SEQ ID NO: 706 and is defined as follows: "+" 1-10, "++"> 10, "+++"> 20, "+++ +"> 50, "+++++"> 75. IHC reduction is measured as a reduction in counts compared to the reference polypeptide of SEQ ID NO: 706 and is defined as follows: "+" 1-3, "++"> 3, "+++"> 5, "+++ +"> 8, "+++++"> 15. Table 12.5 : Reduction in Total Immunogenicity Score (TIS) and Immunogenic Hit Count (IHC) of FVIII variants relative to SEQ ID NO: 840 SEQ ID NO: (nt/aa) Amino acid differences ( relative to SEQ ID NO: 840) Reduced TIS relative to SEQ ID NO: 840 Reduced IHC relative to SEQ ID NO: 840 839/840 1029/1030 L622D + + 1031/1032 P617E + + 1033/1034 P617N + + 935/936 V620N + + 1035/1036 P907D + + 1037/1038 N1040E + + 1039/1040 P617D + + 1041/1042 Q611E + + 937/938 P491E + + 1043/1044 V620P + + 1045/1046 P617G + + 1047/1048 A488G ++ + 1049/1050 P1192D + + 1051/1052 S1194P + + 1053/1054 G619E + + 939/940 M1229D +++ ++ 1055/1056 P615D ++ + 941/942 P615N + + 1057/1058 F16A + + 943/944 I1283S +++ ++ 945/946 H822S + 1063/1064 L211K ++ + 1065/1066 M836W + + 947/948 F328G +++ ++ 1067/1068 F555R + + 951/952 K818W + + 1069/1070 V536A + + 955/956 F555G/E684G ++ + 1071/1072 I527A + + 1073/1074 F555E ++ ++ 957/958 V828K + 1075/1076 H841W + 959/960 R550G + + 1077/1078 M320Q + + 1079/1080 S837G ++ + 1081/1082 M558R ++ ++ 1083/1084 F555G ++ ++ 1085/1086 L211R ++ + 961/962 R830M + 963/964 S839A + + 965/966 H822T + + 1087/1088 V64G + ++ 1089/1090 M320R + 1091/1092 T533Y ++ ++ 1095/1096 F16V + 1097/1098 I1283E +++ +++ 1099/1100 V536M + + 969/970 L14G ++ ++ 1101/1102 F312L + 1103/1104 M681N +++ ++ 1107/1108 V536R + + 1109/1110 R15G + + 1111/1112 V536P ++ ++ 973/974 M836V + + 1113/1114 M558W ++ ++ 1115/1116 M558E +++ ++ 975/976 S839P + + 977/978 K1210W +++ ++ 979/980 Y1281R ++ ++ 981/982 V64D +++ ++ 985/986 V682R + + 987/988 L14M + 989/990 L318C +++ +++ 1119/1120 S839G ++ + 1123/1124 L13G ++ + 1125/1126 K1210M + + 1127/1128 H1156G + + 1129/1130 M558L + + 1131/1132 S839K ++ + 1133/1134 L211P ++ + 1135/1136 L211G ++ + 1137/1138 L211Q ++ + 1139/1140 F16D +++ ++++ 995/996 K818G + + 1141/1142 M320G ++ + 997/998 M836G ++ + 1143/1144 Y683W + + 1145/1146 Y24V + 1147/1148 V534I + + 1149/1150 M681A +++ ++ 1001/1002 M836R + + 1151/1152 P840G + 1153/1154 F1160R +++ + 1155/1156 M681C +++ ++ 1157/1158 F328Q ++ ++ 1159/1160 R15S ++ + 1005/1006 T533V + + 1007/1008 S19G ++ +++ 1161/1162 V536K + + 1163/1164 V536G + ++ 1165/1166 T533R + ++ 1167/1168 M681E +++ ++ 1011/1012 Q817D ++ + 1013/1014 L831W + +++ 1169/1170 V536W ++ ++ 1171/1172 H1156N + + 1173/1174 F16P ++ +++ 1175/1176 Q817A + + 1177/1178 F18R + + 1179/1180 Y551W + 1181/1182 F555T ++ ++ 1017/1018 H1156R + 1183/1184 M681G +++ ++ 1185/1186 H212A + + 1187/1188 F16G ++ ++ 1189/1190 S838G + + 1019/1020 R830V ++ 1191/1192 M681R + + 1021/1022 V393G/A747D ++ + 1193/1194 V393G ++ + 1195/1196 V393G/A747D/K752G/Q811F/K819V/Q923M/T1148N +++ +++ 1197/1198 V393G/S751Y/I775G ++ + 1023/1024 N753D ++ 1199/1200 V63Q/V393G/K732W/I775G/T1148N +++ ++++ 1201/1202 V393G/K732W/T1148N +++ ++ 1203/1204 I775G/T1148N + + 1205/1206 V393G/K752L/N753D/I775G/I793G/M797W +++ + 1207/1208 V393G/Q923M ++ + 1209/1210 V393G/Q923M/T1148N +++ ++ 1211/1212 V393G/A747D/S751Y ++ + 1025/1026 V393G/K732W/S751Y ++ + 1213/1214 V393G/A747D/N753D/I775G/T1148N +++ ++ 1215/1216 K752L/Q923M/M1051R ++ ++ 1217/1218 V63Q/I775G +++ ++ 1219/1220 V63Q/V393G/K752L/I775G/I793G/M1051R/T1053R ++++ ++++ 1221/1222 K752G/N753D/I775G/I793G/K819V +++ + 1027/1028 V63Q/M797W +++ ++ 1223/1224 V63Q/M1051R +++ ++++ 1225/1226 V63Q/A747D/K752G/N753D/I775G/Q811F/K819V/Q923M/M1051R ++++ ++++ 1227/1228 V393G/K752G/I787W +++ + 1231/1232 V63Q/T1148N +++ +++ 1233/1234 V63H/A747D/K752G/N753D/K819V/Q923M/T1148N +++ ++++ 1235/1236 T1148N + + 1237/1238 A747D/K752L/N753D/I793G/Q811F ++ 1239/1240 V63Q +++ ++ 1241/1242 M797W/T1148N ++ + 1243/1244 S751Y/K752L/N753D/M1051R +++ ++ 1245/1246 V63Q/K732W/M1051R +++ ++++ 1247/1248 K752G/M797W/S815I/Q923M/M1051R/T1148N +++ ++ 1249/1250 A747D/N753D ++ TIS reduction is measured as a reduction in counts compared to the reference polypeptide of SEQ ID NO: 840 and is defined as follows: "+" 1-10, "++"> 10, "+++"> 20, "+++ +》＞50. IHC reduction is measured as a reduction in counts compared to the reference polypeptide of SEQ ID NO: 840 and is defined as follows: "+" 1-3, "++"> 3, "+++"> 5, "+++ +》＞8. Table 12.6 : Reduction in Total Immunogenicity Score (TIS) and Immunogenic Hit Count (IHC) of FVIII variants relative to SEQ ID NO: 1200 SEQ ID NO: (nt/aa) Amino acid differences ( relative to SEQ ID NO: 1200) Reduced TIS relative to SEQ ID NO: 1200 Reduced IHC relative to SEQ ID NO: 1200 1199/1200 1409/1410 V536P ++ ++ 1411/1412 L211R/V536K/L622D/M681A +++ ++++ 1413/1414 V536K/M558L ++ ++ 1415/1416 L211R/V536R/F555G/P617G/W732K +++ ++++ 1417/1418 L211R/M681R/W732K +++ ++ 1419/1420 L211R/V536K/F555E +++ +++ 1421/1422 V536R + + 1423/1424 F555E ++ ++ 1425/1426 L211R/V536P +++ ++ 1427/1428 L211R/H212P ++ + 1251/1252 M681A +++ ++ 1429/1430 L211R/V536K +++ ++ 1431/1432 L211R/V536K/P617G/M681A/W732K +++ ++++ 1433/1434 L211R ++ + 1435/1436 L211R/V536P/M558L/P617G/M681A ++++ ++++ 1437/1438 F555G/W732K ++ ++ 1439/1440 V536P/F555E/M558L/P617G/W732K +++ ++++ 1253/1254 L622D + + 1255/1256 P617G + + 1441/1442 V536K/P617G/M681A/W732K +++ +++ 1443/1444 V536K/F555G +++ +++ 1257/1258 M558L + + 1445/1446 V536P/L622D/M681A +++ ++++ 1447/1448 W732K + 1259/1260 V536K + + 1449/1450 H212A/M320Q/T533R/P617E/M681G +++ ++++ 1451/1452 L211K/F312L/T533R/V534I/P617D/G619E/M681N/S839K ++++ +++++ 1453/1454 H212A + + 1455/1456 L211K/H212A/M320Q/G619E/M681G/S839K +++ ++++ 1457/1458 M797W/S839K ++ + 1459/1460 H212A/F312L/M320Q/T533R/V534I/P617E/M681G +++ +++++ 1461/1462 M558W/G619E/M681G +++ ++++ 1261/1262 M681G +++ ++ 1463/1464 M320Q/M558W/M681G +++ ++++ 1465/1466 H212A/M320Q/V534I/G619E/M681G/M797W/S839K ++++ ++++ 1467/1468 M320R/M558W/G619E/W732K/S839K +++ ++++ 1469/1470 L211K/H212A/F312L/V534I/M558W/G619E/M681N/W732K/S839K ++++ ++++ 1471/1472 M320R/V534I/M558R/G619E/W732K/M797W +++ +++ 1473/1474 M558W/P617D/G619E/M681N +++ ++++ 1475/1476 L211Q/V534I/M558W/Q611E/M681N ++++ ++++ 1477/1478 L211K/M320R/P617E/G619E/M681G +++ ++++ 1479/1480 M558W/P617D/M681G/M797W +++ ++++ 1481/1482 M320R/V534I/G619E/W732K ++ ++ 1483/1484 L211K/G619E/M681N +++ +++ 1263/1264 L211Q ++ + 1485/1486 L211K/H212A/V534I/M558W/G619E/W732K/S839K ++++ ++++ 1487/1488 H212A/M320R/G619E/M681N/M797W/S839K +++ ++++ 1489/1490 H212A/M320R/Q611E/S839K ++ ++++ 1265/1266 M320R + 1491/1492 M320Q/M558W/G619E/M681N +++ ++++ 1493/1494 F312L/T533R/P617D/M681N/M797W/S839K +++ ++++ 1495/1496 M558W/Q611E/M681N +++ ++++ 1497/1498 F312L + 1499/1500 L211Q/M320R/M558W/G619E/M681G/W732K/M797W/S839K ++++ ++++ 1501/1502 M320Q/M558W/W732K/M797W +++ ++ 1503/1504 T533R/M558W/Q611E/G619E/F716L/W732K/S839K +++ ++++ 1505/1506 F312L/M681G ++ ++ 1267/1268 M558W ++ ++ 1507/1508 M320Q/V534I/G619E/M681N +++ +++ 1509/1510 L211K/M320Q/T533R/V536L/G619E +++ +++ 1269/1270 S839K ++ + 1511/1512 M558W/P617E/M681N/S839K ++++ ++++ 1271/1272 L211K ++ + 1513/1514 H212A/M320R/V534I/M558W/M681G +++ ++++ 1515/1516 T533R/V534I/M681N/S839K +++ ++++ 1517/1518 H212A/V534I/G619E/M681G +++ +++ 1519/1520 P617E/G619E/M681G +++ +++ 1521/1522 L211Q/H212A/M320R/M558W/P617E/M681N ++++ ++++ 1523/1524 H212A/V534I/M558W/M681N/M797W +++ ++++ 1525/1526 H212A/M320R/V534I + ++ 1527/1528 L211K/F312L/G619E ++ + 1529/1530 H212A/F312L/G619E/S839K ++ +++ 1531/1532 T210P/H212A/M320R/M558W/M681G ++++ ++++ 1273/1274 M797W + 1275/1276 T533R + ++ 1533/1534 H212A/F312L/V534I/M681N/M797W/S839K +++ ++++ 1535/1536 L211K/M320R/M558W/S839K +++ ++++ 1277/1278 S751A + 1279/1280 A303T + + 1539/1540 S916L + 1549/1550 T294G + + 1285/1286 K818G + + 1551/1552 K799S + 1553/1554 K799E + 1299/1300 Q921W + 1563/1564 K799Q + 1567/1568 S916M + 1305/1306 L968A + 1309/1310 G924W + 1569/1570 R759S + 1313/1314 F698D +++ ++ 1315/1316 S168G + 1319/1320 K818A + + 1321/1322 K818S + + 1323/1324 R581K + 1325/1326 K818P + + 1327/1328 S916K + + 1329/1330 F698S +++ ++ 1331/1332 A303G + + 1333/1334 S916C ++ ++ 1335/1336 T1428G + + 1343/1344 T311G + 1351/1352 S751D + 1355/1356 K818E ++ + 1367/1368 A1099G + 1369/1370 K799G + 1371/1372 S916I + + 1379/1380 K799P + 1383/1384 S916E +++ ++ 1583/1584 S916T + 1385/1386 T676Q + + 1387/1388 R581V + 1389/1390 L968P ++ 1391/1392 A755Y + 1393/1394 S916Q ++ + 1399/1400 F698Y + ++ 1405/1406 L1114A + TIS reduction is measured as a reduction in counts compared to the reference polypeptide of SEQ ID NO: 1200, and is defined as follows: "+" 1-10, "++"> 10, "+++"> 20, "+++ +》＞50. IHC reduction is measured as a reduction in counts compared to the reference peptide of SEQ ID NO: 1200 and is defined as follows: "+" 1-3, "++"> 3, "+++"> 5, "+++ +"> 8, "+++++"> 15. Table 12.7 : Reduction in Total Immunogenicity Score (TIS) and Immunogenic Hit Count (IHC) of FVIII variants relative to SEQ ID NO: 1514 SEQ ID NO: (nt/aa) Amino acid differences ( relative to SEQ ID NO: 1514) Reduced TIS relative to SEQ ID NO: 1514 Reduced IHC relative to SEQ ID NO: 1514 1513/1514 1609/1610 I215E ++++ + 2069/2070 L318G ++++ + 1611/1612 I215P ++++ + 2033/2034 V536K/F555G/G619E/Y737K/K799Q/E809G/S839K/S916T/Q921R +++ + 2003/2004 I1050E +++ + 1593/1594 M1051H +++ + 1595/1596 I1283H +++ + 1597/1598 I1283T +++ + 1613/1614 I215S +++ + 1599/1600 Y1281Q +++ + 1601/1602 Y1281P +++ + 1603/1604 S916E +++ + 1857/1858 E786D/R821Q +++ + 1589/1590 R1042E +++ + 1615/1616 L178C +++ + 1869/1870 L178W +++ + 1955/1956 V536P/F555E/G619E/W732K/K799S/Y805Q +++ + 1963/1964 V536P/F555E/G619E/W732K/K799S +++ + 1617/1618 I1050T ++ + 1619/1620 K1210T ++ + 1999/2000 N616D ++ + 2005/2006 I610H ++ + 2025/2026 Y1281S ++ + 1591/1592 Y1041N ++ + 1657/1658 Y175G ++ + 1971/1972 V536P/F555E/G619E ++ + 2007/2008 Q611D ++ + 2027/2028 A618E ++ + 1605/1606 M1051W ++ + 1627/1628 P615D ++ + 1825/1826 Y1104E ++ 1989/1990 V536P/G619E/K799S/Y805Q ++ + 1621/1622 Y1281K ++ + 1853/1854 L1138Q ++ 1973/1974 G619E/S839K ++ + 1985/1986 V536P/G619E/D801G ++ + 1993/1994 Y1281H ++ + 1623/1624 R1042A ++ + 1749/1750 L178Q ++ + 1797/1798 Y175A ++ + 1939/1940 K1093P ++ 1629/1630 I610S ++ + 1659/1660 K1093W ++ 1661/1662 F1088G ++ 1663/1664 K1093T ++ 2009/2010 R1042T ++ + 1607/1608 L187C ++ + 1631/1632 I610K ++ + 1633/1634 I610T ++ + 1675/1676 S700E ++ + 1697/1698 K1093V ++ 1907/1908 K1093N ++ 2023/2024 R1158N ++ + 1625/1626 V104S ++ + 1739/1740 D790P ++ 1881/1882 L187G ++ + 1635/1636 V536P ++ + 1637/1638 A618Y ++ + 1645/1646 M110H ++ + 1733/1734 F1088S ++ 1871/1872 Y1104C ++ 1925/1926 D790G ++ 1977/1978 V536P/D801G/Y805Q ++ + 2015/2016 T370A/L614A ++ + 2019/2020 K1210V ++ + 2021/2022 L614T ++ + 2065/2066 S839K ++ + 1639/1640 Q621W + + 1819/1820 Y175H + + 1877/1878 K1093S + 1991/1992 F555E/G619E/W732K/K799S/D801G/Y805Q + + 2031/2032 V536P/G619E/E809G/E1109G + + 2037/2038 V536R/G619E/G681N/K799Q/S916T/E1109G + + 2049/2050 G619E/G681N/S916T + + 1693/1694 I756G + 1777/1778 M1098G + 2001/2002 Q621K + + 1669/1670 T535N + + 1723/1724 D790W + 1771/1772 K1093G + 1781/1782 I756T + 1959/1960 F555E/G619E/K799S + + 1969/1970 A246D/G619E/K799S/D801G + + 1979/1980 F555E/G619E/K799S/Y805Q + + 2011/2012 N616Q + + 2041/2042 F555E/G619E/G681N/Y737K/K799Q + + 2051/2052 F555G/G619E/G681N/K799S + + 1653/1654 E786C + 1805/1806 Q580S + 1909/1910 A119G + 1919/1920 H712Q + 1923/1924 H712G + 1957/1958 F555E/G619E/D801G + + 1983/1984 F555E/G619E + + 2063/2064 F555G/S916T + 1705/1706 L178V + + 1713/1714 K1093A + 1719/1720 E1452L + 1745/1746 S304G + + 1761/1762 Y917G + 1793/1794 S700A + + 1961/1962 G619E/K799S/D801G/Y805Q + + 1975/1976 G619E/K799S/Y805Q + + 1997/1998 L614H + + 2017/2018 H1156D + + 2057/2058 G619E/K799S + + 1641/1642 A618P + + 1689/1690 T535G + + 1753/1754 K752G + 1755/1756 F1108W + 1783/1784 M948S + 1785/1786 A119V + 1791/1792 M1113F + 1795/1796 E786D + 1931/1932 S1084N + 1981/1982 G619E/D801G/Y805Q + + 2043/2044 G619E/G681N/K799S/S916T/E1109G + + 2045/2046 G619E/G681N + + 2059/2060 G619E + + 1643/1644 L614F + + 1727/1728 G292D + + 1747/1748 S1116G + 1787/1788 S1116L + 1813/1814 L1100S + 1905/1906 K752E + 2013/2014 L614N + + 1647/1648 R301K + + 1867/1868 S304E + + 1941/1942 K1093Y + 2029/2030 F555E/G681N/Y737K/K799Q + 1709/1710 I310G + 1711/1712 S700Q + + 1759/1760 T281A + 1809/1810 D731I + 1879/1880 H712S + 1929/1930 D731L + 1949/1950 H712T + 2053/2054 F555E + 2055/2056 W732K + 1649/1650 I1078S + 1665/1666 L650M + 1725/1726 D790I + 1731/1732 F1108M + 1751/1752 S1116A + 1845/1846 T686W + 1859/1860 T535C + + 1897/1898 D790A + 1953/1954 K1093R + 1965/1966 K799S/Y805Q + 2039/2040 F555G/G619E/G681N/E807R/E1109G + + 2067/2068 K799Q + TIS reduction is measured as a reduction in counts compared to the reference polypeptide of SEQ ID NO: 1514 and is defined as follows: "+" 1-10, "++"> 10, "+++"> 20, "+++ +》＞30. IHC reduction is measured as a reduction in counts compared to the reference polypeptide of SEQ ID NO: 1514 and is defined as follows: "+" 1-3, "++"> 3, "+++"> 5. Table 12.8 : Reduction in Total Immunogenicity Score (TIS) and Immunogenic Hit Count (IHC) of FVIII variants relative to SEQ ID NO: 2072 SEQ ID NO: (nt/aa) Amino acid differences ( relative to SEQ ID NO: 2072) TIS decreased ( relative to SEQ ID NO 2072 = 1998) Decreased IHC ( relative to SEQ ID NO 2072) 2097/2098 A167G/L178Q/S700E/K752E/K1093G +++ +++ 2101/2102 A167G/S700E/E738Y/E739G/K1093G ++ ++ 2103/2104 A167G/S700E/E738Y/K752E/K1093G +++ ++ 2105/2106 A167G/S700E/E738Y/K752E/Y805Q/D808S/E809G/K1093A/S1116I +++ ++ 2121/2122 E738Y/E739G/E809G/K1093G/S1116I ++ 2137/2138 E738Y/K752E/K1093G ++ 2141/2142 E738Y/K799S/E809G/K1093G ++ 2189/2190 G681N/E738Y/E739G/K1093G ++ 2203/2204 G681N/K752E/K1093G ++ 2205/2206 G681N/S700E ++ ++ 2209/2210 G681N/S700E/E809G ++ ++ 2211/2212 G681N/S700E/K1093G +++ ++ 2213/2214 G681N/S700E/K752E/D808S/E809G/K1093G/S1116I +++ ++ 2215/2216 G681N/S700E/K752E/K1093G +++ ++ 2219/2220 G681N/S700E/Y805Q/E809G ++ ++ 2225/2226 K1093G/S1116I ++ 2227/2228 K752E + 2233/2234 K752E/E800V/K1093G + 2235/2236 K752E/E809G + 2241/2242 K752E/S1116I + 2249/2250 L178Q/G681N ++ + 2251/2252 L178Q/G681N/E738Y/S1116I ++ + 2253/2254 L178Q/K752E/E809G ++ + 2265/2266 S543P/E738Y/E809G/K1093G ++ 2267/2268 S543P/E739G/K1093A ++ 2271/2272 S543P/E809G/K1093G ++ 2275/2276 S543P/G681N/E738Y/E739G/K752E/E809G/K1093G ++ 2277/2278 S543P/G681N/E738Y/E739G/K752E/K1093G ++ 2279/2280 S543P/G681N/E739G/K752E/E800V/Y805Q/E809G/K1093G + 2281/2282 S543P/G681N/S700E/E738Y/E739G/E800V/K1093G/S1116I ++ ++ 2283/2284 S543P/G681N/S700E/E738Y/E739G/Y805Q/D808S/E809G ++ ++ 2287/2288 S543P/G681N/S700E/K752E/E809G/K1093G +++ ++ 2289/2290 S543P/K1093G ++ 2301/2302 S543P/S700E/K752E/E800V/K1093G ++ ++ 2305/2306 S543P/S700E/K752E/K1093G +++ ++ 2307/2308 S700E ++ ++ 2309/2310 S700E/E738Y/E739G/E800V/K1093G ++ ++ 2313/2314 S700E/E738Y/E800V/K1093G ++ ++ 2315/2316 S700E/E738Y/S1116I ++ ++ 2317/2318 S700E/E809G ++ ++ 2319/2320 S700E/E809G/K1093G +++ ++ 2321/2322 S700E/K1093G +++ ++ 2323/2324 S700E/K1093G/S1116I +++ ++ 2325/2326 S700E/K752E ++ ++ 2329/2330 S700E/K752E/E809G/K1093G +++ ++ 2333/2334 S700E/K752E/Y805Q/E809G/K1093A +++ ++ 2335/2336 S700E/K799S/E800V ++ ++ 2337/2338 S700E/S1116I ++ ++ 2339/2340 S700E/Y805Q/D808S/E809G/E1076S ++ + TIS reduction is measured as a reduction in counts compared to the reference polypeptide of SEQ ID NO: 2072 and is defined as follows: "+" 1-4, "++" 5-20, "+++"> 20. IHC reduction is measured as a reduction in counts compared to the reference polypeptide of SEQ ID NO: 2072 and is defined as follows: "+" 4, "++" 5, "+++"> 5. Example 13 peptide T cell analysis

In this example, the peptide was designed based on wild-type human Factor VIII with a deletion of the B region, with or without mutations thought to alter the immunogenicity of the protein. Specifically, two polypeptides (SEQ ID NO: 2347 and SEQ ID NO: 2348) were prepared by Abzena Ltd. (Cambridge, United Kingdom). The sequence of the peptide of SEQ ID NO: 2347 has no amino acid changes compared with the sequence of SEQ ID NO: 2, while the sequence of the peptide of SEQ ID NO: 2348 contains L→G at amino acid position 12 of the peptide. Substitution, which is equivalent to the L318G substitution relative to SEQ ID NO: 2. Peripheral blood mononuclear cells (PBMC) from 50 healthy human donors were analyzed for the presence of CD4 ⁺ T cell epitopes of these peptides using EpiScreen ^™ T cell epitope matching technology. PBMC were adjusted to a cell density of 2.5-3.0E6 cells/ml using AIM V ^© medium (Invitrogen, Paisley, UK). Add 5 µM peptide or control medium to PBMC in flat-bottom 96-well plates. For each peptide donor pair, 6 reactions were performed. Cultures were grown for 6 days before 0.75 µCi of tritium-labeled thymidine (Perkin Elmer ^© , Beaconsfield, UK) was added to each well. Cultures were incubated for a further 18 hours before cells were harvested onto filter pads using a TomTec Mach III cell harvester (Receptor Tech. Ltd., Warwick, UK). Counts per minute (CPM) for each well were determined by Meltilex™ (Perkin Elmer®, Beaconsfield, UK) scintillation counting in parallel, low background counting mode on a microplate beta counter (Perkin Elmer®, Beaconsfield, UK). Student's t test compares the response of wells receiving peptide to wells receiving medium to determine the significance of the response (p<0.05). A positive reaction is a reaction in which the average CPM value of the individual peptide-donor pairs divided by the average CPM value of the medium control is greater than 1.90. The results of this experiment are shown in Figure 1 .

Similar to Example 1, directed evolution techniques were used to generate genes containing mutations tested using the Abzena, Ltd. Peptide T Cell Assay (SEQ ID NO: 2069 (nucleotide sequence); SEQ ID NO: 2070 (amino acid sequence)) Variant (SEQ ID NO: 1514 derived from the same plasmid construct) (see, eg, US Patent No. 8,383,346 and WO2010/144103). This variant (SEQ ID NO: 2070) was generated using the method described in Example 2 and tested for FVIII activity after no incubation and after incubation in hemophilia A patient plasma (George King Bio-Medical, Inc.) , as described in Example 3. Variants were tested using chromogenic analysis as described in Example 3. The results are shown in Figure 2 . Example 14 Activity of Truncated FVIII Variants Derived from SEQ ID NO: 2

Similar to Example 1, directed evolution techniques commonly known to those skilled in the art were used to generate genetic variants derived from SEQ ID NO: 2 containing a truncated form of Factor VIII within the same plastid construct (see, e.g., U.S. Patent No. 8,383,346 and WO2010 /144103). These variants were generated using the method described in Example 2 and tested using chromogenic analysis, as described in Example 3. The results are shown in Figure 3 . Example 15 Truncated FVIII variants derived from SEQ ID NO: 2

In this example, experiments performed to determine the activity of FVIII variants by analyzing protein activity are described. These variants were tested for FVIII activity using a chromogenic assay as described in Example 3. Results using variants expressed in Expi293F cells are shown in Table 15.1 . Table 15.1 : Activity of FVIII variants relative to SEQ ID NO: 2 SEQ ID NO: (nt/aa) Amino acid differences ( relative to SEQ ID NO: 2) Expi293F Biophen FIOP ( Level 1 ) relative to SEQ ID NO: 2 3/4 T1148N + 13/14 L171Q +++ 17/18 V63R ++ 35/36 L1248S ++ 67/68 L1248R ++ 85/86 S332Q ++ 95/96 L1248G +++ 153/154 S332W + 157/158 V63H ++ 185/186 S332P ++ 193/194 L171P +++ 245/246 K732G ++ 251/252 L1377D ++++ 261/262 E306G +++ 265/266 L1377G +++ 267/268 S332F + 269/270 L1377E ++++ 281/282 L1377R +++ 283/284 L1377S ++++ 287/288 L171M +++ 291/292 L1248D ++ 293/294 E306S + 297/298 L171H +++ 299/300 L1377A +++ 301/302 L1377V +++ 305/306 T1148R ++ 2359/2360 E306T + 2361/2362 E809L + 2363/2364 E800L + 2365/2366 F387A + 2367/2368 Y738A + 2369/2370 I499C + 2371/2372 M681R + 2373/2374 K1093E + 2375/2376 T1148A + 2377/2378 V63W + 2379/2380 K752Q + 2381/2382 I390E + 2383/2384 V534M + 2385/2386 L749P + 2387/2388 K1093M + 2389/2390 V1439S + 2391/2392 S260K + 2393/2394 M558V + 2395/2396 S332N + 2397/2398 I390T + 2399/2400 Y738D + 2401/2402 S700V + 2403/2404 S1414P + 2405/2406 K1093P + 2407/2408 L749D + 2409/2410 H212Y + 2411/2412 M681C + 2413/2414 Y805R + 2415/2416 T1148S + 2417/2418 E800R + 2419/2420 S332V + 2421/2422 L171K + 2423/2424 E809Q + 2425/2426 M681L + 2427/2428 E800G + 2429/2430 Y738T + 2431/2432 M681E + 2433/2434 K732C + 2435/2436 L749C + 2437/2438 S1414T + 2439/2440 V63C + 2441/2442 L171F + 2443/2444 L318M + 2445/2446 M681T + 2447/2448 V534F + 2449/2450 L750K + 2451/2452 Y738N + 2453/2454 K1093V + 2455/2456 L749Q + 2457/2458 H212F + 2459/2460 H212M + 2461/2462 I775S + 2463/2464 F387W + 2465/2466 I499L + 2467/2468 F387R + 2469/2470 T1148L + 2471/2472 V1439T + 2473/2474 E800M + 2475/2476 L750R + 2477/2478 V1439A + 2479/2480 V63I + 2481/2482 V393H + 2483/2484 M558R + 2485/2486 V63S + 2487/2488 M558T + 2489/2490 S332A + 2491/2492 Y805C + 2493/2494 L749F + 2495/2496 H212A + 2497/2498 L749A + 2499/2500 K752L + 2501/2502 L1248I + 2503/2504 M681A + 2505/2506 L171R + 2507/2508 I775N + 2509/2510 F387L + 2511/2512 E809A + 2513/2514 Y805G + 2515/2516 K752R + 2517/2518 M320I + 2519/2520 V63E + 2521/2522 Y805H + 2523/2524 L749Y + 2525/2526 S332G + 2527/2528 Y805K + 2529/2530 M558S + 2531/2532 E800V + 2533/2534 L1377M + 2535/2536 E800W + 2537/2538 E809C + 2539/2540 Y738E + 2541/2542 V1439H + 2543/2544 L318V + 2545/2546 E800Y + 2547/2548 H212V + 2549/2550 H212N + 2551/2552 E800Q + 2553/2554 H212K + 2555/2556 L749M + 2557/2558 S1414H + 2559/2560 L1248C ++ 2561/2562 L749K ++ 2563/2564 M681Q ++ 2565/2566 V393Y ++ 2567/2568 Y738H ++ 2569/2570 L750S ++ 2571/2572 K732L ++ 2573/2574 K752F ++ 2575/2576 K1093S ++ 2577/2578 L750T ++ 2579/2580 E800I ++ 2581/2582 E809R ++ 2583/2584 S1414W ++ 2585/2586 M558L ++ 2587/2588 L318Q ++ 2589/2590 V393Q ++ 2591/2592 T1148E ++ 2593/2594 L1248F ++ 2595/2596 H212R ++ 2597/2598 H212P ++ 2599/2600 M681N ++ 2601/2602 L749T ++ 2603/2604 L171N ++ 2605/2606 E800C ++ 2607/2608 E809K ++ 2609/2610 S260T ++ 2611/2612 S332R ++ 2613/2614 L750N ++ 2615/2616 V534L ++ 2617/2618 I390P ++ 2619/2620 Y805S ++ 2621/2622 M320Q ++ 2623/2624 K732T ++ 2625/2626 Y738S ++ 2627/2628 V63K ++ 2629/2630 E800N ++ 2631/2632 L1248M ++ 2633/2634 Y805L ++ 2635/2636 E306A ++ 2637/2638 S1414C ++ 2639/2640 Y805P ++ 2641/2642 H212L ++ 2643/2644 L750G ++ 2645/2646 K752M ++ 2647/2648 S700E ++ 2649/2650 S332Y ++ 2651/2652 I390V ++ 2653/2654 M681G ++ 2655/2656 M558Q ++ 2657/2658 V63F ++ 2659/2660 E809W ++ 2661/2662 M558E ++ 2663/2664 M558G ++ 2665/2666 L749I ++ 2667/2668 K732Q ++ 2669/2670 Y805M ++ 2671/2672 I775K ++ 2673/2674 F387T ++ 2675/2676 L750Q ++ 2677/2678 I390L ++ 2679/2680 K732V ++ 2681/2682 K752D ++ 2683/2684 K752S ++ 2685/2686 K732D ++ 2687/2688 T1148C ++ 2689/2690 M558P ++ 2691/2692 E809I ++ 2693/2694 L750W ++ 2695/2696 S332H ++ 2697/2698 E800P ++ 2699/2700 K732R ++ 2701/2702 L1248W ++ 2703/2704 K752E ++ 2705/2706 V63Y ++ 2707/2708 V63T ++ 2709/2710 L749G ++ 2711/2712 I775G ++ 2713/2714 K1093T ++ 2715/2716 E306P ++ 2717/2718 V393R ++ 2719/2720 E809N ++ 2721/2722 E809S ++ 2723/2724 M681F ++ 2725/2726 K732S ++ 2727/2728 L750V ++ 2729/2730 S1414A ++ 2731/2732 E809T ++ 2733/2734 K1093Q ++ 2735/2736 L1248E ++ 2737/2738 L171W ++ 2739/2740 K732Y ++ 2741/2742 L171T ++ 2743/2744 Y805W ++ 2745/2746 E809G ++ 2747/2748 F387V ++ 2749/2750 H212S ++ 2751/2752 T1148M ++ 2753/2754 I775P ++ 2755/2756 K732M ++ 2757/2758 V63L ++ 2759/2760 L750P ++ 2761/2762 V393F ++ 2763/2764 M320P ++ 2765/2766 V393G ++ 2767/2768 S700T ++ 2769/2770 K752V ++ 2771/2772 E809V ++ 2773/2774 K1093A ++ 2775/2776 M320G ++ 2777/2778 I390M ++ 2779/2780 L1377I ++ 2781/2782 M681W ++ 2783/2784 S1414G ++ 2785/2786 L750M ++ 2787/2788 E800T ++ 2789/2790 L318T ++ 2791/2792 L171Y ++ 2793/2794 M558W ++ 2795/2796 Y805Q ++ 2797/2798 M558A ++ 2799/2800 M320S ++ 2801/2802 L750D ++ 2803/2804 I775V ++ 2805/2806 S700A ++ 2807/2808 L1377Y ++ 2809/2810 M320L +++ 2811/2812 L318R +++ 2813/2814 V393A +++ 2815/2816 L171V +++ 2817/2818 V1439E +++ 2819/2820 L1248Y +++ 2821/2822 S332T +++ 2823/2824 L1248Q +++ 2825/2826 L1377C +++ 2827/2828 L1248H +++ 2829/2830 M681S +++ 2831/2832 I390G +++ 2833/2834 L171A +++ 2835/2836 L1248T +++ 2837/2838 V393M +++ 2839/2840 L171S +++ 2841/2842 E306D +++ 2843/2844 L1248A +++ 2845/2846 S1414E +++ 2847/2848 S1414D +++ 2849/2850 L1248K +++ 2851/2852 I390S +++ 2853/2854 L318K +++ 2855/2856 M320T +++ 2857/2858 L171D +++ 2859/2860 L171E +++ 2861/2862 M320V +++ 2863/2864 L1377P +++ 2865/2866 M320R +++ 2867/2868 L1377T +++ 2869/2870 L1377H +++ All activities are determined relative to the reference polypeptide of SEQ ID NO:2. The levels of increased activity are defined as follows: "+" 0.9 to 1.1, "++"> 1.1, "+++"> 1.5, "++++"> 3.5.

The predicted immunogenicity potential of the active FVIII variants identified in the screen of this example was assessed by evaluating their binding to eight common class II HLA-DR alleles, as in Example 12. The total immunogenicity score and the immunogenicity hit count were calculated for each variant. The total immunogenicity score (TIS) reflects the overall predicted immunogenicity of a variant (ie, a higher score indicates a higher predicted immunogenicity level). The immunogenic "hit count" (IHC) indicates the number of 15-mer assay frames that have an unusually high immunogenic potential (i.e., higher scores indicate higher immunogenic potential). Mutations that cause a decrease in the overall immunogenicity score and/or a decrease in the immunogenicity hit count compared to the reference sequence are considered potential "deimmunizing mutations" and are shown in Table 15.2 . Table 15.2 : Reduction in Total Immunogenicity Score (TIS) and Immunogenic Hit Count (IHC) of FVIII variants relative to SEQ ID NO: 2 SEQ ID NO: (nt/aa) Amino acid difference ( relative to SEQ ID NO: 2) Reduced TIS relative to SEQ ID NO: 2 Reduced IHC relative to SEQ ID NO: 2 2365/2366 F387A +++ + 2369/2370 I499C ++ 2371/2372 M681R + + 2373/2374 K1093E ++ 2379/2380 K752Q ++ + 2381/2382 I390E +++ + 2385/2386 L749P ++ + 2389/2390 V1439S ++ + 2391/2392 S260K + + 2393/2394 M558V ++ + 2395/2396 S332N + + 2397/2398 I390T ++ + 2401/2402 S700V + 2405/2406 K1093P ++ 2407/2408 L749D +++ + 2411/2412 M681C +++ + 2419/2420 S332V + 2421/2422 L171K + 2431/2432 M681E +++ + 2435/2436 L749C +++ + 2439/2440 V63C +++ + 2445/2446 M681T +++ + 2447/2448 V534F + + 2449/2450 L750K + + 2453/2454 K1093V ++ 2455/2456 L749Q ++ + 2459/2460 H212M + 2463/2464 F387W + + 2465/2466 I499L + 2467/2468 F387R ++ + 2471/2472 V1439T ++ + 2475/2476 L750R + 2477/2478 V1439A + + 2483/2484 M558R ++ + 2485/2486 V63S +++ + 2487/2488 M558T +++ + 2493/2494 L749F + + 2495/2496 H212A + + 2497/2498 L749A + + 2499/2500 K752L + + 2503/2504 M681A +++ + 2505/2506 L171R + 2509/2510 F387L + + 2517/2518 M320I + + 2519/2520 V63E +++ + 2525/2526 S332G + + 2529/2530 M558S ++ + 2533/2534 L1377M + + 2541/2542 V1439H + + 2543/2544 L318V ++ + 2553/2554 H212K + 2555/2556 L749M + 2559/2560 L1248C + + 2561/2562 L749K + + 2563/2564 M681Q +++ + 2569/2570 L750S ++ + 2573/2574 K752F + + 2575/2576 K1093S + 2577/2578 L750T + + 2585/2586 M558L + + 2587/2588 L318Q +++ + 2589/2590 V393Q + 2591/2592 T1148E + + 2593/2594 L1248F + + 2597/2598 H212P + + 2599/2600 M681N +++ + 2601/2602 L749T ++ + 2603/2604 L171N ++ + 2609/2610 S260T + + 2613/2614 L750N ++ + 2617/2618 I390P +++ + 2621/2622 M320Q + + 2627/2628 V63K ++ + 2631/2632 L1248M + + 2637/2638 S1414C + 2643/2644 L750G + + 2645/2646 K752M + 2647/2648 S700E ++ + 2649/2650 S332Y + + 2651/2652 I390V + + 2653/2654 M681G +++ + 2655/2656 M558Q +++ + 2661/2662 M558E +++ + 2663/2664 M558G +++ + 2671/2672 I775K + 2673/2674 F387T +++ + 2675/2676 L750Q + + 2681/2682 K752D ++ + 2683/2684 K752S + + 2687/2688 T1148C + + 2689/2690 M558P +++ + 2693/2694 L750W + + 2701/2702 L1248W + 2703/2704 K752E ++ + 2707/2708 V63T +++ + 2709/2710 L749G +++ + 2711/2712 I775G + 2713/2714 K1093T ++ 2727/2728 L750V + 2733/2734 K1093Q ++ 2735/2736 L1248E + + 2737/2738 L171W + 2741/2742 L171T + 2747/2748 F387V ++ + 2749/2750 H212S + + 2753/2754 I775P + 2759/2760 L750P ++ + 2761/2762 V393F + 2763/2764 M320P ++ + 2765/2766 V393G +++ + 2767/2768 S700T + + 2769/2770 K752V ++ + 2773/2774 K1093A + 2775/2776 M320G ++ + 2777/2778 I390M + 2779/2780 L1377I + + 2781/2782 M681W + + 2783/2784 S1414G + 2789/2790 L318T ++ 2793/2794 M558W ++ + 2797/2798 M558A +++ + 2799/2800 M320S + 2801/2802 L750D +++ + 2803/2804 I775V + 2805/2806 S700A + + 2807/2808 L1377Y ++ + 2809/2810 M320L + + 2817/2818 V1439E +++ + 2819/2820 L1248Y + + 2823/2824 L1248Q + + 2825/2826 L1377C ++ + 2827/2828 L1248H + + 2829/2830 M681S +++ + 2831/2832 I390G +++ + 2833/2834 L171A + + 2835/2836 L1248T + + 2839/2840 L171S + + 2841/2842 E306D + 2843/2844 L1248A + + 2847/2848 S1414D + 2849/2850 L1248K ++ + 2851/2852 I390S + + 2853/2854 L318K ++ + 2855/2856 M320T ++ + 2857/2858 L171D ++ + 2859/2860 L171E ++ + 2861/2862 M320V + + 2863/2864 L1377P ++ + 2865/2866 M320R + 2867/2868 L1377T ++ + 2869/2870 L1377H ++ + TIS reduction is measured as a reduction in counts compared to the reference polypeptide of SEQ ID NO: 2 and is defined as follows: "+" 1-10, "++"> 10, "+++"> 20. IHC reduction is measured as a reduction in counts compared to the reference polypeptide of SEQ ID NO: 2 and is defined as follows: "+" 1-3, "++"> 3, "+++"> 5. Example 16 In vitro characterization of FVIII variants

In this example, experiments performed to characterize FVIII variants generated as indicated herein are described. Production of Factor VIII in Expi293F™ cells or HepG2 cells

Lipofectamine transfection was performed using the ExpiFectamine™ 293 Transfection Kit (ThermoFisher Scientific) or LIPOFECTAMINE ^® 3000 reagent (ThermoFisher Scientific) using the pcDNA 3.1(+) or CMV promoter and a protein encoding wild-type FVIII with a deletion of the B region. or variant vector BGH-pA to transfect Expi293F™ (ThermoFisher Scientific) cells or HepG2 cells. Expi293F™ (ThermoFisher Scientific) cells were cultured in growth medium (Expi293™ Expression Medium; ThermoFisher Scientific). HepG2 cells were cultured in growth medium (EMEM with 10% fetal calf serum, both from Corning). Expi293F™ cells or HepG2 cells are transfected with plastid DNA and grown in suspension for 3-7 days. The conditioned medium was then collected, clarified by centrifugation and filtered, and stored at -80°C until characterization. Analysis of expression of FVIII variants from Expi293F™ cells or HepG2 cells (ELISA)

The expression of FVIII variants in conditioned media was analyzed using human factor VIII ELISA kit (Ab272771, Abcam) according to the company's standard protocol. The colorimetric assay measures total human FVIII in the range of 0.0002 IU/ml to 0.84 IU/ml. FVIII variants greater than 0.84 IU/ml were diluted in tris-BSA buffer (A221406-RUO, Aniara Diagnostica) in 96-well microtiter plates. The absorbance of all wells was measured at 450 nm using an EnVision multi-mode disk reader. Results from this analysis are presented in Figures 4 and 5 , plotting the mean ± standard deviation of triplicates. Analysis of FVIII variant activity (chromogenic analysis)

FVIII variant activity was determined by measuring the release of p-nitroaniline (pNa) from synthetic peptide substrates. For unchallenged assays, 25 µL of diluted or undiluted Expi293F or HepG2 conditioned medium generated as described above was warmed to 37°C and mixed with 25 µL of "Reagent 1" (BIOPHEN™ FVIII:C, Aniara) and 25 µL of "Reagent 2" (BIOPHEN™ FVIII:C, Aniara) was mixed in a 96-well black opaque bottom microtiter plate. The reaction was mixed briefly and incubated at 37°C for 5 minutes. 25 µL of "Reagent 3" (BIOPHEN™ FVIII:C, Aniara) was then added and the reaction was mixed briefly and incubated at 37°C for 5 minutes before being quenched with 25 µL pf 2% citric acid. The release of pNA was analyzed using an EnVision® 2105 multi-mode disk reader (PerkinElmer) monitoring absorbance at 405 nm. "Reagent 1", "Reagent 2" and "Reagent 3" are components of the chromogenic assay (BIOPHEN™ FVIII:C) supplied by Aniara and were used as recommended by the supplier. Results from this analysis are presented in Figures 6 and 7 ( before normalization) and in Figures 8 and 9 (after normalization using protein concentrations determined by the ELISA described above), plotting the mean ± standard deviation of triplicates . Analysis of FVIII variant activity (coagulation analysis)

FVIII variant activity was determined by measuring the coagulation rate of hemophilia A patient plasma (George King Bio-Medical, Inc.) using activated partial thromboplastin time analysis. For unchallenged analysis, 25 µL of diluted or undiluted Expi293F or HepG2 conditioned media generated as described above was warmed to 37°C and mixed with 30 µL of hemophilia A patient plasma (George King Bio-Medical, Inc.) and 50 µL aPTT-XL reagent (Pacific Hemostasis) were mixed in a 96-well black opaque bottom microtiter plate. The reaction was mixed briefly and incubated at 37°C for 3 minutes. Then 10 µL of 200 µM calcium chloride (Sigma Aldrich) was added and the reaction was mixed briefly. Coagulation was analyzed using an EnVision® 2105 multimode disk reader (PerkinElmer) monitoring absorbance at 405 nm over a period of 5-10 minutes. The analysis was performed using R scripts to extrapolate the clotting onset time from the collected data points. Results from this analysis are presented in Figure 10 , plotting the mean ± standard deviation of triplicates. Analysis of the stability of FVIII variants in plasma of patients with hemophilia A

FVIII variants were challenged with hemophilia A patient plasma (George King Bio-Medical, Inc.) to simulate conditions that the variants may encounter within the bloodstream. First, 50 µL of Expi293F or HepG2 conditioned medium and 50 µL of hemophilia A patient plasma (George King Bio-Medical, Inc.) generated as described above were added to a 96-well round-bottom microtiter plate. The plates were sealed with a ^PlateLoc® Thermal Microplate Sealer (Agilent) and incubated at 37-55°C for 1-96 hours. The samples were then analyzed by chromogenic analysis as described above. Results from this analysis are presented in Figure 11 , plotting the mean ± standard deviation of triplicates.

Although the invention has been described with reference to specific embodiments, various changes may be made and equivalents may be substituted to adapt the particular situation, materials, compositions of matter, method, method step or steps without departing from what is claimed. The benefits of the present invention are achieved within the scope of the content.

Each publication and patent document cited in this disclosure is hereby incorporated by reference for all purposes as if each such publication or document was specifically and individually indicated to be incorporated by reference. Average. Citation of publications and patent documents is intended to be an indication that any such document is not relevant prior art and does not constitute an admission of the contents or date of the document.

(without)

Figure 1 shows the percentage of 50 donors whose peripheral blood mononuclear cells (PBMC) generated a CD4 T cell response when incubated with peptides of recombinant Factor VIII in a T cell assay used to examine immunogenicity.

Figure 2 shows the results of studies in which recombinant Factor VIII polypeptides were expressed using Expi293F or HepG2 cultures and before or after incubation with hemophilia A patient plasma (George King Bio-Medical, Inc.) using a chromogenic assay (BIOPHEN ™ FVIII:C, Aniara) to assess activity. Activity is shown relative to SEQ ID NO: 1514.

Figure 3 shows the results of studies in which truncated recombinant Factor VIII polypeptides were expressed using Expi293F cultures and activity assessed using a chromogenic assay (BIOPHEN™ FVIII:C, Aniara). Activity is shown on the left axis relative to SEQ ID NO:2. The number of amino acids removed relative to SEQ ID NO: 2 is shown on the right axis.

Figure 4 shows the performance of recombinant Factor VIII polypeptide variants in Expi293F cells as measured by ELISA.

Figure 5 shows the performance of recombinant Factor VIII polypeptide variants in HepG2 cells as measured by ELISA.

Figure 6 shows the activity of recombinant Factor VIII polypeptide variants expressed in Expi293F cells as measured by chromogenic analysis of conditioned media before normalization with protein concentrations determined by ELISA.

Figure 7 shows the activity of recombinant Factor VIII polypeptide variants expressed in HepG2 cells as measured by chromogenic analysis of conditioned media before normalization with protein concentrations determined by ELISA.

Figure 8 shows the activity of recombinant Factor VIII polypeptide variants expressed in Expi293F cells as measured by chromogenic analysis of conditioned media after normalization with protein concentrations determined by ELISA.

Figure 9 shows the activity of recombinant Factor VIII polypeptide variants expressed in HepG2 cells as measured by chromogenic analysis of conditioned media after normalization with protein concentrations determined by ELISA.

Figure 10 shows coagulation activity measured by activated partial thromboplastin time analysis using conditioned media of Expi293F cells expressing recombinant Factor VIII polypeptide variants.

Figure 11 shows residual activity as measured using a chromogenic assay of recombinant Factor VIII polypeptide variants after exposure to plasma obtained from hemophilia A patients for various periods of time.

TW202334195A_112100265_SEQL.xml

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Claims (82)

A recombinant Factor VIII polypeptide, or a biologically active fragment thereof, comprising a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072 or corresponding to One of the reference sequences of SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072 has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity of a monoamine group Acid sequence, wherein the amino acid sequence comprises the reference sequence relative to residues 20-1457 corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072 or relative to residues 20-1457 corresponding to SEQ ID NO: One or more substitutions of the reference sequence of NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072. The recombinant Factor VIII polypeptide of claim 1, which comprises a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2 or at least 70%, 75%, or 80% of the reference sequence corresponding to SEQ ID NO: 2 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, An amino acid sequence with 97%, 98%, or 99% or more sequence identity, wherein the amino acid sequence comprises or is relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2 One or more substitutions of the reference sequence corresponding to SEQ ID NO: 2. The recombinant Factor VIII polypeptide of claim 1, comprising a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, 504, 706, 840, 1200, 1514 or 2072 or corresponding to SEQ ID NO: 2 , 252, 504, 706, 840, 1200, 1514 or 2072, the reference sequence has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to an amino acid sequence in which the amine The amino acid sequence contains one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2. The recombinant Factor VIII polypeptide of claim 1, comprising a reference sequence corresponding to residues 20-1457 of one of the even-numbered SEQ ID NO.s of SEQ ID NO. 4-2346 and 2360-2870 or corresponding to SEQ ID NO. One of the even-numbered SEQ ID NO. reference sequences in ID NO. 4-2346 and 2350-2870 has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity of a monoamine group An acid sequence, wherein the amino acid sequence comprises one or more substitutions relative to a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2 or relative to a reference sequence corresponding to SEQ ID NO: 2. The recombinant Factor VIII polypeptide of any one of claims 2 to 4, wherein the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution at the following amino acid positions: 13, 14, 15, 16, 17 ,18,19,20,21,24,37,56,57,58,59,61,63,64,65,85,96,104,110,119,127,132,158,160,164,167 ,168,171,175,178,186,187,205,210,211,212,215,225,246,257,260,265,281,292,294,301,303,304,306,308,310 ,311,312,318,320,321,325,328,332,334,335,344,346,347,349,351,353,354,355,356,358,359,362,370,387,388 ,390,392,393,397,407,428,429,433,435,436,481,487,488,489,491,498,499,503,514,527,533,534,535,536,538 ,543,550,551,555,558,569,580,581,584,610,611,612,613,614,615,616,617,618,619,620,621,622,643,646,650 ,665,667,676,680,681,682,683,684,686,693,696,698,700,706,707,708,712,716,724,726,729,731,732,734,735 ,736,737,738,739,740,744,746,747,748,749,750,751,752,753,754,755,756,757,759,770,773,775,785,786,787 ,790,791,792,793,794,795,797,798,799,800,801,802,805,806,807,808,809,810,811,812,815,816,817,818,819 ,821,822,824,825,826,828,830,831,836,837,838,839,840,841,844,866,873,879,892,896,897,903,907,914,916 ,917,918,919,921,922,923,924,926,929,938,939,945,948,951,968,999,1037,1040,1041,1042,1043,1049,1050,1051,1053 ,1054,1076,1078,1080,1084,1088,1089,1092,1093,1094,1095,1098,1099,1100,1101,1104,1108,1109,1113,1114,1116,1127,1129,1138,11 45 ,1148,1151,1156,1158,1160,1186,1192,1193,1194,1205,1210,1218,1220,1221,1222,1224,1229,1245,1248,1250,1259,1264,1269,1270,12 74 , 1281, 1283, 1292, 1297, 1339, 1352, 1373, 1377, 1378, 1380, 1389, 1414, 1422, 1427, 1428, 1439, 1440, 1442 or 1452 or combinations thereof, wherein the amino acid positions are Relative to the reference sequence corresponding to SEQ ID NO: 2. The recombinant Factor VIII polypeptide of any one of claims 2 to 5, wherein the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or amino acid residue 13G/Y, 14G/M/V, 15G/ S, 16A/D/G/P/V, 17S/V, 18L/R, 19G, 20L, 21M, 24V, 37H, 56E/P, 57A/L/S/T/V, 58K, 59D/G/ L/M/Q/R/S/V/W, 61Q/S, 63C/E/F/G/H/I/K/L/M/N/Q/R/S/T/W/Y, 64D/G, 65A, 85Q, 96I/L/R/V/W, 104S, 110H, 119G/V, 127V, 132P/R, 158G, 160E/T, 164E, 167G/H/R, 168G/R, 171A/D/E/F/H/K/N/M/P/Q/R/S/T/V/W/Y, 175A/G/H, 178C/M/Q/V/W, 186A, 187C/G, 205R, 210P, 211G/K/P/Q/R, 212A/F/G/K/L/M/N/P/R/S/V/Y, 215E/P/S, 225R, 246D, 257I, 260E/K/T, 265R/S/W, 281A/S, 292D, 294G, 301K/M, 303G/T, 304A/E/G, 306A/D/G/P/S/T, 308P, 310G/V, 311E/G, 312L, 318C/G/K/M/Q/R/T/V, 320G/I/L/P/Q/R/S/T/V, 321Y, 325Y, 328G/Q/S, 332A/F/G/H/N/P/Q/R/T/V/W/Y, 334G, 335E/P, 344A/E/L, 346R/V, 347N, 349G, 351S, 353S, 354R, 355C, 356L/N, 358K/S, 359E/G, 362E/G/N/P/T/V/W, 370A, 387A/G/I/L/Q/R/S/ T/V/W, 388A/P, 390A/E/G/L/M/P/S/T/V, 392A, 393A/F/G/H/M/Q/R/W/Y, 397G/ N, 407V, 428F, 429L/S, 433A/G, 435Q, 436D/G, 481V, 487N, 488G, 489E/F/Q/R, 491E, 498A, 499C/L, 503S, 514L, 527A, 533R/ V/Y, 534F/I/L/M, 535C/G/N, 536A/G/K/L/M/P/R/W, 538V, 543G/L/P/W, 550G, 551W, 555E/ G/R/T, 558A/E/G/L/P/Q/R/S/T/V/W, 569T, 580S/Y, 581K/V, 584A, 610H/K/L/R/S/ T, 611D/E, 612G/S/T, 613S, 614A/F/H/I/N/T, 615D/N, 616D/E/G/Q/W, 617D/E/G/N, 618C/ E/G/P/Y, 619E, 620N/P, 621K/M/R/T/W, 622D, 643N, 646I, 650M, 665G, 667I, 676Q, 680L/R, 681A/C/E/F/ G/L/N/R/Q/S/T/W, 682R, 683W, 684G/K, 686W, 693T, 696E, 698D/S/W/Y, 700A/E/Q/T/V, 706A/ G/M/Y, 707H, 708A/K/T/V, 712G/L/M/Q/S/T/Y, 716K/L/W, 724I, 726A/L, 729T, 731I/L/R, 732C/D/E/G/L/R/W/K/M/Q/S/T/V/Y, 734I/R/S, 735D/S/V, 736F/G, 737F/G/K/ L/M/N/S/V, 738A/D/E/F/H/K/N/P/R/S/T, 739G/P/R, 740G, 744A/R, 746P, 747D, 748F/ G/K/N/S/V, 749A/C/D/F/G/I/K/L/M/P/Q/R/S/T/V/Y, 750A/D/G/K/ M/N/P/Q/R/S/T/V/W, 751A/D/E/Y, 752D/E/F/G/L/M/Q/R/S/V, 753D, 754W, 755G/L/P/Q/S/Y, 756G/P/R/T, 757A/G/K/T, 759S, 770W, 773H, 775E/G/K/N/P/S/V, 785V, 786C/D/L, 787W, 790A/G/I/M/P/W, 791M/T, 792H/W/Y, 793G, 794R, 795C/N/S/T, 797C/G/P/Q/ T/W/Y, 798C/R/T, 799A/E/G/M/P/Q/R/S/V, 800C/G/I/L/M/N/P/Q/R/T/ V/W/Y, 801A/G/I/R, 802S/V, 805C/G/H/K/L/M/P/Q/R/S/V/W, 806E/G/R/S, 807C/L/P/R, 808K/S, 809A/C/G/I/K/L/N/Q/R/S/T/V/W, 810V, 811F, 812G/I/V, 815I, 816V/W, 817A/D, 818A/E/G/P/S/V/W, 819V, 821Q, 822S/T, 824W, 825L, 826G, 828K, 830M/V, 831W, 836G/R/V/ W, 837G, 838G/T, 839A/G/K/P, 840G, 841W, 844H, 866A, 873L/Q/T, 879Q, 892T, 896L/V, 897G/K/R, 903E, 907D, 914V, 916C/E/I/K/L/M/Q/T, 917G, 918M/S, 919G, 921R/W, 922W, 923M, 924I/M/P/Q/W, 926T/V, 929A/Q/ V, 938R/V, 939E/S, 945K, 948S, 951V, 968A/P, 999R, 1037H/M/N/P, 1040E, 1041G/H/N, 1042A/E/T, 1043E/R, 1049G/ W, 1050E/P/Q/S/T, 1051E/H/K/R/S/W, 1053A/R, 1054C, 1076G/S, 1078S, 1080G/V, 1084L/N, 1088G/S, 1089F/ S, 1092G, 1093A/E/G/M/N/P/Q/R/S/T/V/W/Y, 1094S, 1095G, 1098G, 1099G, 1100I/S, 1101G, 1104C/E, 1108M/ S/V/W, 1109G, 1113F, 1114A/M, 1116A/G/I/L/R, 1127L/V, 1129P, 1138Q, 1145R, 1148A/C/E/L/M/N/Q/R/ S, 1151S, 1156D/G/N/R, 1158N, 1160R, 1186E, 1192D, 1193A/E/G/I/L, 1194P, 1205L, 1210M/T/V/W, 1218A/G/L/M/ P, 1220D/E/L/T, 1221G/S/T/V, 1222F, 1224P, 1229D, 1245R, 1248A/C/D/E/F/G/H/I/K/M/P/Q/ R/S/T/W/Y, 1250A/P/S/T, 1259T, 1264L/R/W, 1269F, 1270V, 1274N/V, 1281H/K/P/Q/R/S, 1283E/H/ L/S/T, 1292Y, 1297A, 1339C, 1352E, 1373S, 1377A/C/D/E/G/H/I/M/P/R/S/V/T/Y, 1378A/C/D/ E, 1380A/G/W, 1389W, 1414A/C/D/E/G/H/N/P/T/W, 1422I, 1427Y, 1428G, 1439A/E/H/L/S/T, 1440A/ C/E/G/S/T, 1442T or 1452L or a combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2. The recombinant Factor VIII polypeptide of any one of claims 2 to 6, wherein the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution at the following amino acid positions: 63, 171, 212, 260, 306 , 317, 320, 332, 387, 390, 393, 534, 558, 681, 732, 738, 749, 750, 775, 1148, 1248, 1377 or 1439 or combinations thereof, wherein the amino acid positions are relative to This reference sequence corresponds to SEQ ID NO: 2. The recombinant Factor VIII polypeptide of claim 7, wherein the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or amino acid residue 63C/E/F/G/H/I/K/L/M/ N/Q/R/S/T/W/Y, 171A/D/E/F/H/K/N/M/P/Q/R/S/T/V/W/Y, 212A/F/ G/L/K/M/N/P/R/S/V/Y, 260E/K/T, 306A/D/G/P/S/T, 318C/G/K/L/M/Q/ R/T/V, 320G/I/L/P/Q/R/S/T/V, 332A/F/G/H/N/P/Q/R/T/V/W/Y, 387A/ G/I/L/Q/R/S/T/V/W, 390A/E/G/L/M/P/S/T/V, 393A/F/G/H/M/Q/R/ W/Y, 499C/L, 534I/F/L/M, 558A/E/G/L/P/Q/R/S/T/V/W, 681A/C/E/F/G/L/ N/R/Q/S/T/W, 700A/E/Q/T/V, 732C/D/E/G/L/R/W/K/M/Q/S/T/V/Y, 738A/D/E/F/H/K/N/P/R/S/T/Y、749A/C/D/F/G/I/K/L/M/P/Q/R/S/ T/V/Y, 750A/D/G/K/M/N/P/Q/R/S/T/V/W, 752D/E/F/G/L/M/Q/R/S/ V, 775E/G/K/N/P/S/V, 800C/G/I/L/M/N/P/Q/R/T/V/W/Y, 805C/G/H/K/ L/M/P/Q/R/S/V/W, 809A/C/G/I/K/L/N/Q/R/S/T/V/W, 1093A/E/G/M/ N/P/Q/R/S/T/V/W/Y, 1148A/C/E/L/M/N/Q/R/S, 1248A/C/D/E/F/G/H/ I/K/M/P/Q/R/S/T/W/Y, 1377A/C/D/E/G/H/I/M/P/R/S/V/T/Y, 1414A/ C/D/E/G/H/N/P/T/W or 1439A/E/H/L/S/T or a combination thereof, wherein the amino acid positions are relative to those corresponding to SEQ ID NO: 2 the reference sequence. The recombinant Factor VIII polypeptide of claim 7, wherein the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution 63Q, 171P, 212A, 260T, 306G, 317G, 320R, 332P, 387I, 390A, 393G, 534I, 558W, 681G, 732W, 7338E, 749F, 750D, 775G, 1148N, 1248D, 1377D or 1439E or a combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2. The recombinant Factor VIII polypeptide of any one of claims 2 to 6, wherein the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or set of substitutions at the following amino acid positions: 1148, 85/708/ 1148, 798, 706, 171, 335, 63, 344, 428, 879, 59, 429, 205, 1264, 797, 1248, 873, 792, 708, 57, 96, 362, 1292, 346, 433, 65, 795, 684/798, 353, 332, 1259, 356, 436, 1245, 1145, 716, 56, 773, 734, 358, 729, 665, 359, 61, 1250, 726, 37/96/171/429/ 708/799/1148, 171/429/732/1148, 96/171, 171/919/1151, 171/792/797/799/1148, 171/569/1148, 96/171/429/792/797/ 799, 171/429/708/1422, 171/429, 321/362/732/797/799, 171/1100, 708/1148/1186, 429/724/732/1148, 171/362/795/797/ 799, 171/429/732, 96/171/643/797/799, 171/792/797/799, 503, 171/732, 1148/1193, 96/171/362/429/732/795/797, 334, 732, 351, 1377, 999, 306, 349, 257, 646, 435 or 58, wherein these positions are relative to the reference sequence corresponding to SEQ ID NO: 2. The recombinant Factor VIII polypeptide of any one of claims 2 to 6 or 10, wherein the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or substitution set 1148N, 85Q/708T/1148N, 798T, 706G, 706A , 171Q, 335E, 63R, 344A, 428F, 879Q, 59L, 429S, 205R, 1264W, 797P, 1248S, 1264R, 873T, 792Y, 708K, 344L, 57V, 96L, 362W, 63G, 873L, 1292Y, 1148Q, 34 6V , 797G, 706Y, 1248R, 433A, 65A, 59W, 873Q, 795C, 684K/798R, 57L, 353S, 332Q, 708A, 1259T, 63M, 356L, 1248G, 1248P, 436G, 57A, 59S, 1245R, 59R, 96W , 797Y, 1145R, 436D, 716L, 56E, 773H, 734I, 358S, 335P, 59Q, 729T, 716K, 665G, 362T, 57T, 359G, 61S, 1250A, 362V, 359E, 716W, 332W, 708V, 63H, 797C , 61Q, 356N, 1250S, 798C, 433G, 795N, 96I, 56P, 63N, 792W, 1264L, 1250P, 332P, 59V, 726L, 37H/96R/171P/429L/708T/799R/1148N, 171P, 171P/429L /732R/1148N、96R/171P、171P/919G/1151S、171P/792Y/797Q/799R/1148N、171P/569T/1148N、96R/171P/429L/792Y/797T/799R、171P/429L/708T/1 422I , 171P/429L, 321Y/362P/732E/797T/799R, 171P/1100I, 708T/1148N/1186E, 429L/724I/732E/1148N, 171P/362E/795T/797Q/799M, 171P/429L/732 E.96R /171P/643N/797T/799R、171P/792Y/797T/799M、503S、171P/732E、1148N/1193L、96R/171P/362E/429L/732R/795T/797Q、726A、59M、734R、362N、334 G , 732G, 63Q, 351S, 1377D, 795S, 358K, 999R, 1250T, 306G, 349G, 1377G, 332F, 1377E, 257I, 646I, 346R, 435Q, 96V, 1377R, 1377S, 59G, 171M, 792H, 1248 D、306S , 706M, 171H, 1377A, 1377V, 734S, 1148R, 57S, 59D, 344E, 362G or 58K, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2. The recombinant Factor VIII polypeptide of claim 1, comprising a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, 504, 706, 840, 1200, 1514 or 2072 or corresponding to SEQ ID NO: 252 , 504, 706, 840, 1200, 1514 or 2072 reference sequences have at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, An amino acid sequence with 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity. The recombinant Factor VIII polypeptide of claim 1, comprising a reference sequence corresponding to residues 20-1457 of one of the even-numbered SEQ ID NO.s of SEQ ID NO. 4-2346 and 2360-2870 or corresponding to SEQ ID NO. The reference sequence of one of the even-numbered SEQ ID NO.s in NO. 4-2346 and 2350-2870 has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, An amino acid sequence with 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity . The recombinant Factor VIII polypeptide of claim 1, comprising a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, 504, 706, 840, 1200, 1514 or 2072 or corresponding to SEQ ID NO: 252 , 504, 706, 840, 1200, 1514 or 2072 reference sequences have at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, An amino acid sequence with 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity, wherein the amino acid The sequence includes this reference sequence relative to residues 20-1457 corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514 or 2072 or relative to residues 20-1457 corresponding to SEQ ID NO: 252, 504, 706, 840, One or more substitutions of 1200, 1514 or 2072 of the reference sequence. The recombinant Factor VIII polypeptide of claim 14, wherein the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution at the following amino acid positions: 13, 14, 15, 16, 17, 18, 19, 20 ,21,24,37,56,57,58,59,61,63,64,65,85,96,104,110,119,127,132,158,160,164,167,168,171,175 ,178,186,187,205,210,211,212,215,225,246,257,260,265,281,292,294,301,303,304,306,308,310,311,312,318 ,320,321,325,328,332,334,335,344,346,347,349,351,353,354,355,356,358,359,362,370,387,388,390,392,393 ,397,407,428,429,433,435,436,481,487,488,489,491,498,499,503,514,527,533,534,535,536,538,543,550,551 ,555,558,569,580,581,584,610,611,612,613,614,615,616,617,618,619,620,621,622,643,646,650,665,667,676 , 680, 681, 682, 683, 684, 686, 693, 696, 698, 700, 706, 707, 708, 712, 716, 724, 726, 729, 731, 732, 734, 735, 736, 737, 738 ,739,740,744,746,747,748,749,750,751,752,753,754,755,756,757,759,770,773,775,785,786,787,790,791,792 ,793,794,795,797,798,799,800,801,802,805,806,807,808,809,810,811,812,815,816,817,818,819,821,822,824 ,825,826,828,830,831,836,837,838,839,840,841,844,866,873,879,892,896,897,903,907,914,916,917,918,919 ,921,922,923,924,926,929,938,939,945,948,951,968,999,1037,1040,1041,1042,1043,1049,1050,1051,1053,1054,1076,1078 ,1080,1084,1088,1089,1092,1093,1094,1095,1098,1099,1100,1101,1104,1108,1109,1113,1114,1116,1127,1129,1138,1145,1148,1151,11 56 ,1158,1160,1186,1192,1193,1194,1205,1210,1218,1220,1221,1222,1224,1229,1245,1248,1250,1259,1264,1269,1270,1274,1281,1283,12 92 , 1297, 1339, 1352, 1373, 1377, 1378, 1380, 1389, 1414, 1422, 1427, 1428, 1439, 1440, 1442 or 1452 or a combination thereof, wherein the amino acid positions are relative to those corresponding to SEQ ID The reference sequence of NO: 252, 504, 706, 840, 1200, 1514 or 2072. The recombinant Factor VIII polypeptide of claim 14 or 15, wherein the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or amino acid residue 13G/Y, 14G/M/V, 15G/S, 16A/ D/G/P/V, 17S/V, 18L/R, 19G, 20L, 21M, 24V, 37H, 56E/P, 57A/L/S/T/V, 58K, 59D/G/L/M/ Q/R/S/V/W, 61Q/S, 63C/E/F/G/H/I/K/L/M/N/Q/R/S/T/W/Y, 64D/G, 65A, 85Q, 96I/L/R/V/W, 104S, 110H, 119G/V, 127V, 132P/R, 158G, 160E/T, 164E, 167G/H/R, 168G/R, 171A/D/ E/F/H/K/N/M/P/Q/R/S/T/V/W/Y, 175A/G/H, 178C/M/Q/V/W, 186A, 187C/G, 205R, 210P, 211G/K/P/Q/R, 212A/F/G/L/K/M/N/P/R/S/V/Y, 215E/P/S, 225R, 246D, 257I, 260E/K/T, 265R/S/W, 281A/S, 292D, 294G, 301K/M, 303G/T, 304A/E/G, 306A/D/G/P/S/T, 308P, 310G/ V, 311E/G, 312L, 318C/G/K/L/M/Q/R/T/V, 320G/I/L/P/Q/R/S/T/V, 321Y, 325Y, 328G/ Q/S, 332A/F/G/H/N/P/Q/R/T/V/W/Y, 334G, 335E/P, 344A/E/L, 346R/V, 347N, 349G, 351S, 353S, 354R, 355C, 356L/N, 358K/S, 359E/G, 362E/G/N/P/T/V/W, 370A, 387A/G/I/L/Q/R/S/T/ V/W, 388A/P, 390A/E/G/L/M/P/S/T/V, 392A, 393A/F/G/H/M/Q/R/W/Y, 397G/N, 407V, 428F, 429L/S, 433A/G, 435Q, 436D/G, 481V, 487N, 488G, 489E/F/Q/R, 491E, 498A, 499C/L, 503S, 514L, 527A, 533R/V/ Y, 534I/F/L/M, 535C/G/N, 536A/G/K/L/M/P/R/W, 538V, 543G/L/P/W, 550G, 551W, 555E/G/ R/T, 558A/E/G/L/P/Q/R/S/T/V/W, 569T, 580S/Y, 581K/V, 584A, 610H/K/L/R/S/T, 611D/E, 612G/S/T, 613S, 614A/F/H/I/N/T, 615D/N, 616D/E/G/Q/W, 617D/E/G/N, 618C/E/ G/P/Y, 619E, 620N/P, 621K/M/R/T/W, 622D, 643N, 646I, 650M, 665G, 667I, 676Q, 680L/R, 681A/C/E/F/G/ L/N/R/Q/S/T/W, 682R, 683W, 684G/K, 686W, 693T, 696E, 698D/S/W/Y, 700A/E/Q/T/V, 706A/G/ M/Y, 707H, 708A/K/T/V, 712G/L/M/Q/S/T/Y, 716K/L/W, 724I, 726A/L, 729T, 731I/L/R, 732C/ D/E/G/L/R/W/K/M/Q/S/T/V/Y, 734I/R/S, 735D/S/V, 736F/G, 737F/G/K/L/ M/N/S/V, 738A/D/E/F/H/K/N/P/R/S/T/Y, 739G/P/R, 740G, 744A/R, 746P, 747D, 748F/ G/K/N/S/V, 749A/C/D/F/G/I/K/L/M/P/Q/R/S/T/V/Y, 750A/D/G/K/ M/N/P/Q/R/S/T/V/W, 751A/D/E/Y, 752D/E/F/G/L/M/Q/R/S/V, 753D, 754W, 755G/L/P/Q/S/Y, 756G/P/R/T, 757A/G/K/T, 759S, 770W, 773H, 775E/G/K/N/P/S/V, 785V, 786C/D/L, 787W, 790A/G/I/M/P/W, 791M/T, 792H/W/Y, 793G, 794R, 795C/N/S/T, 797C/G/P/Q/ T/W/Y, 798C/R/T, 799A/E/G/M/P/Q/R/S/V, 800C/G/I/L/M/N/P/Q/R/T/ V/W/Y, 801A/G/I/R, 802S/V, 805C/G/H/K/L/M/P/Q/R/S/V/W, 806E/G/R/S, 807C/L/P/R, 808K/S, 809A/C/G/I/K/L/N/Q/R/S/T/V/W, 810V, 811F, 812G/I/V, 815I, 816V/W, 817A/D, 818A/E/G/P/S/V/W, 819V, 821Q, 822S/T, 824W, 825L, 826G, 828K, 830M/V, 831W, 836G/R/V/ W, 837G, 838G/T, 839A/G/K/P, 840G, 841W, 844H, 866A, 873L/Q/T, 879Q, 892T, 896L/V, 897G/K/R, 903E, 907D, 914V, 916C/E/I/K/L/M/Q/T, 917G, 918M/S, 919G, 921R/W, 922W, 923M, 924I/M/P/Q/W, 926T/V, 929A/Q/ V, 938R/V, 939E/S, 945K, 948S, 951V, 968A/P, 999R, 1037H/M/N/P, 1040E, 1041G/H/N, 1042A/E/T, 1043E/R, 1049G/ W, 1050E/P/Q/S/T, 1051E/H/K/R/S/W, 1053A/R, 1054C, 1076G/S, 1078S, 1080G/V, 1084L/N, 1088G/S, 1089F/ S, 1092G, 1093A/E/G/M/N/P/Q/R/S/T/V/W/Y, 1094S, 1095G, 1098G, 1099G, 1100I/S, 1101G, 1104C/E, 1108M/ S/V/W, 1109G, 1113F, 1114A/M, 1116A/G/I/L/R, 1127L/V, 1129P, 1138Q, 1145R, 1148A/C/E/L/M/N/Q/R/ S, 1151S, 1156D/G/N/R, 1158N, 1160R, 1186E, 1192D, 1193A/E/G/I/L, 1194P, 1205L, 1210M/T/V/W, 1218A/G/L/M/ P, 1220D/E/L/T, 1221G/S/T/V, 1222F, 1224P, 1229D, 1245R, 1248A/C/D/E/F/G/H/I/K/M/P/Q/ R/S/T/W/Y, 1250A/P/S/T, 1259T, 1264L/R/W, 1269F, 1270V, 1274N/V, 1281H/K/P/Q/R/S, 1283E/H/ L/S/T, 1292Y, 1297A, 1339C, 1352E, 1373S, 1377A/C/D/E/G/H/I/M/P/R/S/V/T/Y, 1378A/C/D/ E, 1380A/G/W, 1389W, 1414A/C/D/E/G/H/N/P/T/W, 1422I, 1427Y, 1428G, 1439A/E/H/L/S/T, 1440A/ C/E/G/S/T, 1442T or 1452L or a combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514 or 2072 . The recombinant Factor VIII polypeptide of any one of claims 14 to 16, wherein the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution at the following amino acid positions: 63, 171, 212, 260, 306 , 317, 320, 332, 387, 390, 393, 534, 558, 681, 732, 738, 749, 750, 775, 1148, 1248, 1377 or 1439 or combinations thereof, wherein the amino acid positions are relative To the reference sequence corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514 or 2072. The recombinant Factor VIII polypeptide of claim 17, wherein the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or amino acid residue 63C/E/F/G/H/I/K/L/M/ N/Q/R/S/T/W/Y, 171A/D/E/F/H/K/N/M/P/Q/R/S/T/V/W/Y, 212A/F/ G/L/K/M/N/P/R/S/V/Y, 260E/K/T, 306A/D/G/P/S/T, 318C/G/K/L/M/Q/ R/T/V, 320G/I/L/P/Q/R/S/T/V, 332A/F/G/H/N/P/Q/R/T/V/W/Y, 387A/ G/I/L/Q/R/S/T/V/W, 390A/E/G/L/M/P/S/T/V, 393A/F/G/H/M/Q/R/ W/Y, 499C/L, 534I/F/L/M, 558A/E/G/L/P/Q/R/S/T/V/W, 681A/C/E/F/G/L/ N/R/Q/S/T/W, 700A/E/Q/T/V, 732C/D/E/G/L/R/W/K/M/Q/S/T/V/Y, 738A/D/E/F/H/K/N/P/R/S/T/Y、749A/C/D/F/G/I/K/L/M/P/Q/R/S/ T/V/Y, 750A/D/G/K/M/N/P/Q/R/S/T/V/W, 752D/E/F/G/L/M/Q/R/S/ V, 775E/G/K/N/P/S/V, 800C/G/I/L/M/N/P/Q/R/T/V/W/Y, 805C/G/H/K/ L/M/P/Q/R/S/V/W, 809A/C/G/I/K/L/N/Q/R/S/T/V/W, 1093A/E/G/M/ N/P/Q/R/S/T/V/W/Y, 1148A/C/E/L/M/N/Q/R/S, 1248A/C/D/E/F/G/H/ I/K/M/P/Q/R/S/T/W/Y, 1377A/C/D/E/G/H/I/M/P/R/S/V/T/Y, 1414A/ C/D/E/G/H/N/P/T/W or 1439A/E/H/L/S/T or a combination thereof, wherein the amino acid positions are relative to those corresponding to SEQ ID NO: 252 , 504, 706, 840, 1200, 1514 or 2072 of the reference sequence. The recombinant Factor VIII polypeptide of claim 17, wherein the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substituted or amino acid amino acid residue 63Q, 171P, 212A, 260T, 306G, 317G, 320R, 332P , 387I, 390A, 393G, 534I, 558W, 681G, 732W, 7338E, 749F, 750D, 775G, 1148N, 1248D, 1377D or 1439E or a combination thereof, wherein the amino acid positions are relative to those corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514 or 2072 of the reference sequence. The recombinant Factor VIII polypeptide of claim 14, which comprises a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252 or at least 70%, 75%, 80% with the reference sequence corresponding to SEQ ID NO: 252 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, An amino acid sequence with 97%, 98%, or 99% or more sequence identity, wherein the amino acid sequence comprises the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252 or is relative to One or more substitutions of the reference sequence corresponding to SEQ ID NO: 252. The recombinant Factor VIII polypeptide of claim 20, wherein the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or set of substitutions at the following amino acid positions: 1380, 388, 1043, 610, 1274, 1220, 1442, 1193, 621, 1054, 755, 1041, 748, 1049, 1221, 1051, 612, 1037, 616, 387, 489, 750, 1224, 618, 1050, 1053, 896, 897, 749, 260, 481, 613, 756, 63, 265, 752, 1218, 1378, 614, 1440, 1439, 1205, 1222, 1389, 1269, 390, 397/1218, 757, 1373, 346, 63/1248, 349, 306/355/ 1422, 1422, 306/346/708, 1148, 171/346/1248, 63/797/799/1248/1422, 171/799, 362, 1248, 171/306/346/999/1248, 171/1148, 734/799/1248/1422, 63/732, 63/171/306/346/732, 171, 171/1422, 63/362/732/799/1248, 999/1148/1422, 346/349, 306/ 349, 63/171/1148/1151, 306, 344/349/354/1250, 1250, 63/306/344/349/354/503/1248, 59/63/332/349/503/706/708, 63/306/344/349/354/706/1248, 59/63, 332/706/708/1248/1250, 503, 59/63/349/354, 59/349/708, 59/306/344/ 1248, 344/1248/1250, 708, 63/344/349, 59, 63/344/349/354, 706, 59/306/770, 63/347/354/1248 or 354/503, wherein these amines Acid positions are relative to the reference sequence corresponding to SEQ ID NO: 252. The recombinant Factor VIII polypeptide of claim 20 or 21, wherein the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or substitution set 1380W, 388A, 1043E, 1043R, 610R, 1274N, 1220T, 1442T, 1193L, 621T , 1054C, 755Q, 1041G, 748S, 1049G, 1221V, 1051E, 612G, 1037H, 616E, 1041H, 387S, 489R, 621M, 489Q, 750G, 1224P, 618C, 616G, 612T, 1050Q, 1053A, 61 6W, 896V, 489F , 1050P, 897G, 749T, 1274V, 1220L, 260E, 481V, 613S, 387G, 621R, 756R, 63R, 265S, 1221T, 1380G, 752G, 748K, 755L, 1218A, 748N, 1378E, 614I, 749F, 3 87V, 750T , 1193G, 749V, 1378A, 750V, 387T, 387Q, 750A, 1037N, 1440A, 1051S, 1221G, 1378D, 1440S, 387R, 1193A, 387I, 1218M, 265W, 1440E, 1439L, 1440T, 612S ,1049W,1221S,1220D , 749G, 749S, 1218G, 1205L, 1222F, 1193E, 1050S, 1389W, 1439E, 260T, 1037P, 1051K, 1269F, 265R, 1051R, 1218L, 897R, 388P, 748G, 756P, 748F, 390A, 610L, 755G, 749R , 748V, 1380A, 1440C, 397N/1218M, 1037M, 1218P, 757K, 1373S, 1440G, 1193I, 1439T, 750D, 390G, 756G, 1220E, 1378C, 618G, 346R, 63Q/1248D, 349G, 3 06G/355C/1422I , 1422I, 306G/346R/708T, 1148N, 171P/346R/1248D, 63Q/797T/799R/1248D/1422I, 171P/799R, 362P, 1248D, 63Q, 171P/306G/346R/999R/1248D, 17 1P/1148N , 734S/799R/1248D/1422I, 63Q/732E, 63Q/171P/306G/346R/732G, 171P, 171P/1422I, 63Q/362P/732G/799R/1248D, 999R/1148N/1422I, 346R/349G ,306G /349G、63Q/171P/1148N/1151S、306G、344E/349G/354R/1250T、63G、1250T、63R/306G/344E/349G/354R/503S/1248R、59D/63S/332P/349G/503S/706 M /708K、63S/306G/344A/349G/354R/706M/1248R、1248R、59D/63G、332P/706M/708K/1248R/1250T、503S、59D/63R/349G/354R、59D/349G/708K、59D /306g/344E/1248R, 344E/1248R/1250T, 708K, 63R/344E/349G, 59D, 63S/344A/354R, 706M, 59D/306G/770W, 63g/354R/1248R/503S , wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 252. The recombinant Factor VIII polypeptide of claim 14, which comprises a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 504 or at least 70%, 75%, 80% with the reference sequence corresponding to SEQ ID NO: 504 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, An amino acid sequence with 97%, 98%, or 99% or more sequence identity, wherein the amino acid sequence comprises the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 504 or is relative to One or more substitutions of the reference sequence corresponding to SEQ ID NO: 504. The recombinant Factor VIII polypeptide of claim 23, wherein the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or set of substitutions at the following amino acid positions: 750, 1051, 260, 756, 1193, 332, 897, 387, 1037, 306/749, 1270, 489, 487, 488, 903, 171/1248/1440, 260/1148/1378, 171/260/387/750/1248, 171/260, 171/260/ 387/750/1051/1148, 171, 750/1051/1148, 171/260/756, 171/1248, 171/1148, 59, 1440, 332/750/1248, 756/1248, 1248, 171/387/ 1248/1440, 750/1440, 59/171/750, 171/1193/1248, 750/1148/1193/1440, 1148, 171/387/1051, 748, 306, 306/387/390/749/1220, 390, 306/390/1037, 306/612/749, 387/390/749, 63/390/503/749, 1220, 63/306, 749, 306/390/749/1037, 306/749/1220, 306/387/390/612/749/1037 or 328, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 504. The recombinant Factor VIII polypeptide of claim 23 or 24, wherein the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or substitution set 750D, 1051K, 1051S, 260T, 756P, 1193A, 332P, 897R, 387Q, 1037N , 387R, 306G/749F, 1270V, 489E, 487N, 488G, 903E, 171P/1248D/1440T, 260T/1148N/1378D, 171P/260T/387I/750D/1248D, 171P/260T, 171P/260T/3 87I/750D /1051S/1148N, 171P, 750D/1051S/1148N, 171P/260T/756P, 171P/1248D, 171P/1148N, 387I, 59D, 1440S, 332P/750D/1248D, 756P/1248D, 1248D, 171P /387I/1248D /1440S, 750D/1440S, 59D/171P/750D, 171P/1193A/1248D, 750D/1148N/1193A/1440S, 1148N, 171P/387I/1051K, 748G, 306G, 306G/387R/390A/749F/ 1220D, 390A , 306G/390A/1037N, 306G/612S/749F, 387V/390A/749F, 63S/390A/503S/749F, 387R/390A/749R, 1220D, 63S/306G, 749F, 306G/390A/749F/1037N, 3 87V , 306G/749F/1220D, 306G/387V/390A/612S/749F/1037N or 328S, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 504. The recombinant Factor VIII polypeptide of claim 14, which comprises a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 706 or at least 70%, 75%, or 80% of the reference sequence corresponding to SEQ ID NO: 706 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, An amino acid sequence with 97%, 98%, or 99% or more sequence identity, wherein the amino acid sequence comprises the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 706 or is relative to One or more substitutions of the reference sequence corresponding to SEQ ID NO: 706. The recombinant Factor VIII polypeptide of claim 26, wherein the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or set of substitutions at the following amino acid positions: 1051, 1221, 1218, 387, 749/1218, 332, 897, 696, 816, 824, 826, 393, 825, 698, 332/387/390/1148, 328/387/749, 749, 306/328/332/390/1051/1193/1218, 63/ 306/332/1051/1148, 63/306/332/387/1051, 387/390/1051, 186/306/328/332/390/749/1051/1148/1193, 387/390/756/1051, 306/390/1051, 306/332/390/749, 306/387/390/1148, 306/328/332/387/390/749/1051/1148/1193, 332/387/390/749/1148, 387/390/749, 306/387/749/1051/1148, 387/749, 306/945/1051/1193/1221, 306/390/749/1051, 63/306/332/390, 306/387/ 390/749/1148/1193/1221, 306/332/387/390/749/1218, 63/306/332/390/1051/1148/1193/1218, 924, 755, 752, 751, 923, 797, 754, 793, 753, 922, 896, 938, 775, 732, 810, 747, 393/407, 819, 787, 802, 811, 392 or 397, wherein the amino acid positions are relative to those corresponding to SEQ ID The reference sequence of NO: 706. The recombinant Factor VIII polypeptide of claim 26 or 27, wherein the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or substitution set 1051K, 1221G, 1218G, 387R, 1051R, 749F/1218G, 387T, 332P, 897K , 696E, 816W, 824W, 826G, 393W, 825L, 897R, 816V, 698W, 393Q, 332P/387R/390A/1148N, 328S/387R/749F, 749F, 306G/328S/332P/390A/1051R/1193A /1218G , 63Q/306G/332P/1051R/1148N, 63Q/306G/332P/387T/1051K, 387T/390A/1051R, 186A/306G/328S/332P/390A/749F/1051K/1148N/1193A, 387T/39 0A/756P /1051R、306G/390A/1051R、306G/332P/390A/749F、306G/387R/390A/1148N、306G/328S/332P/387T/390A/749F/1051R/1148N/1193A、332P/387R/39 0A/749F /1148N、387R/390A/749F、306G/387T/749F/1051K/1148N、387R/749F、306G/945K/1051R/1193A/1221G、306G/390A/749F/1051K、63Q/306G/332P/39 0A, 306G 7 51Y、923M , 797Y, 754W, 793G, 753D, 922W, 896L, 938R, 775G, 751E, 732W, 752G, 810V, 938V, 747D, 755G, 393G/407V, 797W, 819V, 393G, 787W, 393R, 802V, 775E, 92 4P , 732G, 811F, 802S, 752L, 392A or 397G, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 706. The recombinant Factor VIII polypeptide of claim 14, which comprises a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 840 or at least 70%, 75%, 80% with the reference sequence corresponding to SEQ ID NO: 840 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, An amino acid sequence with 97%, 98%, or 99% or more sequence identity, wherein the amino acid sequence comprises the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 840 or is relative to One or more substitutions of the reference sequence corresponding to SEQ ID NO: 840. The recombinant Factor VIII polypeptide of claim 29, wherein the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or set of substitutions at the following amino acid positions: 620, 491, 1229, 615, 1283, 822, 328, 212, 818, 555/684, 828, 550, 830, 839, 13, 14, 17, 836, 1210, 1281, 64, 682, 318, 20, 21, 325, 533, 19, 817, 831, 225, 1156, 393/747, 753, 393/732/751, 63/797, 622, 617, 907, 1040, 611, 488, 1192, 1194, 619, 16, 802, 211, 555, 536, 527, 841, 320, 837, 558, 914, 312, 681, 838, 15, 1427, 18, 683, 24, 534, 840, 1160, 551, 393, 393/747/752/811/819/923/1148, 393/751/775, 63/393/732/775/1148, 393/732/1148, 775/1148, 393/752/753/775/793/797, 393/923, 393/923/1148, 393/ 747/751, 393/747/753/775/1148, 752/923/1051, 63/775, 63/393/752/775/793/1051/1053, 752/753/775/793/819, 63/ 1051, 63/747/752/753/775/811/819/923/1051, 393/752/787, 751, 63/1148, 63/747/752/753/819/923/1148, 1148, 747/ 752/753/793/811, 63, 797/1148, 751/752/753/1051, 63/732/1051, 752/797/815/923/1051/1148 or 747/753, wherein these amino acids Positions are relative to the reference sequence corresponding to SEQ ID NO: 840. The recombinant Factor VIII polypeptide of claim 29 or 30, wherein the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or substitution set 620N, 491E, 1229D, 615N, 1283S, 822S, 328G, 212L, 818W, 1283L , 555G/684G, 828K, 550G, 830M, 839A, 822T, 13Y, 14G, 17V, 836V, 839P, 1210W, 1281R, 64D, 212G, 682R, 14M, 318C, 17S, 20L, 818G, 836G, 21M, 836R , 325Y, 533V, 19G, 14V, 817D, 831W, 225R, 1156R, 830V, 393G/747D, 753D, 393G/732W/751Y, 63Q/797W, 622D, 617E, 617N, 907D, 1040E, 617D, 611E, 620 P , 617G, 488G, 1192D, 1194P, 619E, 615D, 16A, 802S, 818V, 211K, 836W, 555R, 536A, 527A, 555E, 841W, 320Q, 837G, 558R, 555G, 211R, 64G, 320R, 533Y, 91 4V , 16V, 1283E, 536M, 312L, 681N, 838T, 536R, 15G, 536P, 558W, 558E, 1427Y, 839G, 18L, 13G, 1210M, 1156G, 558L, 839K, 211P, 211G, 211Q, 16D, 320G, 683 W , 24V, 534I, 681A, 840G, 1160R, 681C, 328Q, 15S, 536K, 536G, 533R, 681E, 536W, 1156N, 16P, 817A, 18R, 551W, 555T, 681G, 212A, 16G, 838G, 681R, 393G , 393G/747D/752G/811F/819V/923M/1148N, 393G/751Y/775G, 63Q/393G/732W/775G/1148N, 393G/732W/1148N, 775G/1148N, 393G/752L/753D/775G /793G /797W、393G/923M、393G/923M/1148N、393G/747D/751Y、393G/747D/753D/775G/1148N、752L/923M/1051R、63Q/775G、63Q/393G/752L/775G/793G/10 51R /1053R、752G/753D/775G/793G/819V、63Q/1051R、63Q/747D/752G/753D/775G/811F/819V/923M/1051R、393G/752G/787W、751Y、63Q/1148N、63H/747 D /752G/753D/819V/923M/1148N, 1148N, 747D/752L/753D/793G/811F, 63Q, 797W/1148N, 751Y/752L/753D/1051R, 63Q/732W/1051R, 752G/797W/815I/ 923M /1051R/1148N or 747D/753D, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 840. The recombinant Factor VIII polypeptide of claim 14, which comprises a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 1200 or at least 70%, 75%, or 80% of the reference sequence corresponding to SEQ ID NO: 1200 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, An amino acid sequence with 97%, 98%, or 99% or more sequence identity, wherein the amino acid sequence comprises the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 1200 or is relative to One or more substitutions of the reference sequence corresponding to SEQ ID NO: 1200. The recombinant Factor VIII polypeptide of claim 32, wherein the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or set of substitutions at the following amino acid positions: 681, 622, 617, 558, 536, 211, 320, 839, 797, 533, 751, 303, 951, 164, 818, 807, 799, 735, 794, 926, 755, 921, 801, 1094, 968, 929, 924, 698, 168, 581, 916, 1428, 805, 1114, 311, 737, 693, 809, 757, 1099, 676, 740, 791, 211/536/622/681, 536/558, 211/536/555/617/732, 211/681/ 732, 211/536/555, 555, 211/536, 211/212, 211/536/617/681/732, 211/536/558/617/681, 555/732, 536/555/558/617/ 732, 536/617/681/732, 536/555, 536/622/681, 732, 212/320/533/617/681, 211/312/533/534/617/619/681/839, 212, 211/212/320/619/681/839, 797/839, 212/312/320/533/534/617/681, 558/619/681, 320/558/681, 212/320/534/619/ 681/797/839, 320/558/619/732/839, 211/212/312/534/558/619/681/732/839, 320/534/558/619/732/797, 558/617/ 619/681, 211/534/558/611/681, 211/320/617/619/681, 558/617/681/797, 320/534/619/732, 211/619/681, 211/212/ 534/558/619/732/839, 212/320/619/681/797/839, 212/320/611/839, 320/558/619/681, 312/533/617/681/797/839, 558/611/681, 312, 211/320/558/619/681/732/797/839, 320/558/732/797, 533/558/611/619/716/732/839, 312/681, 320/534/619/681, 211/320/533/536/619, 558/617/681/839, 212/320/534/558/681, 533/534/681/839, 212/534/619/ 681, 617/619/681, 211/212/320/558/617/681, 212/534/558/681/797, 212/320/534, 211/312/619, 212/312/619/839, 210/212/320/558/681, 212/312/534/681/797/839, 211/320/558/839, 294, 759, 538 or 1109, wherein the amino acid positions are relative to those corresponding to The reference sequence of SEQ ID NO: 1200. The recombinant Factor VIII polypeptide of claim 32 or 33, wherein the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or substitution set 681A, 622D, 617G, 558L, 536K, 681G, 211Q, 320R, 558W, 839K , 211K, 797W, 533R, 751A, 303T, 951V, 164E, 818G, 807L, 799A, 735S, 794R, 926V, 755P, 921W, 801A, 1094S, 968A, 929Q, 924W, 807P, 698D, 168G, 929V, 8 18A , 818S, 581K, 818P, 916K, 698S, 303G, 916C, 1428G, 805L, 924M, 1114M, 311G, 737N, 805V, 801I, 751D, 693T, 818E, 929A, 311E, 737L, 809W, 757A, 1099G ,799G , 916I, 805G, 737F, 737M, 799P, 807C, 916E, 676Q, 581V, 968P, 755Y, 916Q, 735D, 740G, 698Y, 757G, 791T, 1114A, 735V, 536P, 211R/536K/622D/681A, 5 36K /558L、211R/536R/555G/617G/732K、211R/681R/732K、211R/536K/555E、536R、555E、211R/536P、211R/212P、211R/536K、211R/536K/617G/681A/732 K , 211R, 211R/536P/558L/617G/681A, 555G/732K, 536P/555E/558L/617G/732K, 536K/617G/681A/732K, 536K/555G, 536P/622D/681A, 732K, 212A/32 0Q /533R/617E/681G, 211K/312L/533R/534I/617D/619E/681N/839K, 212A, 211K/212A/320Q/619E/681G/839K, 797W/S839K, 212A/312L/320Q/533R/53 4I /617E/681G、558W/619E/681G、320Q/558W/681G、212A/320Q/534I/619E/681G/797W/839K、320R/558W/619E/732K/839K、211K/212A/312L/534I/558 W /619E/681N/732K/839K、320R/534I/558R/619E/732K/797W、558W/617D/619E/681N、211Q/534I/558W/611E/681N、211K/320R/617E/619E/681G、558 W /617D/681G/797W, 320R/534I/619E/732K, 211K/619E/681N, 211K/212A/534I/558W/619E/732K/839K, 212A/320R/619E/681N/797W/839K, 212A/320 R /611E/839K、320Q/558W/619E/681N、312L/533R/617D/681N/797W/839K、558W/611E/681N、312L、211Q/320R/558W/619E/681G/732K/797W/839K、320 Q /558W/732K/797W、533R/558W/611E/619E/716L/732K/839K、312L/681G、320Q/534I/619E/681N、211K/320Q/533R/536L/619E、558W/617E/681N/839 K , 212A/320R/534I/558W/681G, 533R/534I/681N/839K, 212A/534I/619E/681G, 617E/619E/681G, 211Q/212A/320R/558W/617E/681N, 212A/534I/55 8W /681N/797W、212A/320R/534I、211K/312L/619E、212A/312L/619E/839K、210P/212A/320R/558W/681G、212A/312L/534I/681N/797W/839K、211K/320 R /558W/839K, 924Q, 916L, 757T, 737K, 818V, 801G, 294G, 799S, 799E, 809G, 791M, 737V, 805Q, 799Q, 737S, 916M, 759S, 799V, 538V, 1109G, 168R, 921R, 92 6T , 916T, 737G or 801R, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 1200. The recombinant Factor VIII polypeptide of claim 14, which comprises a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 1514 or at least 70%, 75%, 80% with the reference sequence corresponding to SEQ ID NO: 1514 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, An amino acid sequence with 97%, 98%, or 99% or more sequence identity, wherein the amino acid sequence comprises the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 1514 or is relative to One or more substitutions of the reference sequence corresponding to SEQ ID NO: 1514. The recombinant Factor VIII polypeptide of claim 35, wherein the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or set of substitutions at the following amino acid positions: 1042, 1041, 1051, 1283, 1281, 916, 187, 215, 178, 1050, 1210, 104, 615, 610, 536, 618, 621, 614, 110, 301, 1078, 812, 786, 127, 175, 1093, 1088, 650, 535, 806, 738, 700, 132, 1076, 1084, 498, 756, 1108, 746, 160, 543, 310, 584, 918, 1452, 790, 292, 800, 167, 892, 304, 1116, 752, 281, 917, 712, 1080, 806/1352, 1095, 744, 1098, 948, 119, 785, 1113, 667, 158, 580, 707, 731, 1100, 1089, 1092, 939, 1104, 736, 1297, 1127, 686, 1129, 1138, 786/821, 680, 1101, 808, 739, 308, 536/555/619/732/799/805, 555/619/801, 555/619/799, 619/799/801/805, 536/ 555/619/732/799, 799/805, 801, 246/619/799/801, 536/555/619, 619/839, 619/799/805, 536/801/805, 555/619/799/ 805, 619/801/805, 555/619, 536/619/801, 801/805, 536/619/799/805, 555/619/732/799/801/805, 749, 616, 611, 370/ 614, 1156, 1158, 555/681/737/799, 536/619/809/1109, 536/555/619/737/799/809/839/916/921, 916/1109, 536/619/681/ 799/916/1109, 555/619/681/807/1109, 555/619/681/737/799, 619/681/799/916/1109, 619/681, 555/921, 619/681/916, 555/619/681/799, 555, 732, 619/799, 619, 681, 555/916, 839 or 799, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 1514. The recombinant Factor VIII polypeptide of claim 35 or 36, wherein the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or substitution set 1042E, 1041N, 1051H, 1283H, 1283T, 1281Q, 1281P, 916E, 1051W, 187C , 215E, 215P, 215S, 178C, 1050T, 1210T, 1281K, 1042A, 104S, 615D, 610S, 610K, 610T, 536P, 618Y, 621W, 618P, 614F, 110H, 301K, 1078S, 812G, 786C, 12 7V, 175G , 1093W, 1088G, 1093T, 650M, 301M, 535N, 806R, 738E, 700E, 132R, 1076G, 1084L, 498A, 786L, 738F, 535G, 806E, 756G, 1108S, 1093V, 746P, 160E, 160T, 178V, 543L , 310G, 700Q, 1093A, 584A, 918S, 1452L, 738P, 790W, 790I, 292D, 800M, 1108M, 1088S, 167G, 892T, 790P, 800R, 738K, 304G, 1116G, 178Q, 1116A, 752G, 1 108W, 738D , 281A, 917G, 712M, 1080V, 806G/1352E, 1095G, 1093G, 744A, 543G, 1098G, 281S, 756T, 948S, 119V, 1116L, 785V, 1113F, 700A, 786D, 175A, 790M, 667I, 158G, 580S , 707H, 731I, 918M, 1100S, 1089S, 712Y, 175H, 1092G, 939E, 1104E, 800G, 736F, S543W, 1297A, 712L, 1127V, 939S, 178M, 731R, 686W, 543P, 736G, 1129P ,1138Q,812V , 786D/821Q, 535C, 680R, 1116I, 1089F, 304E, 178W, 1104C, 744R, 738R, 1093S, 712S, 187G, 132P, 812I, 1080G, 800C, 1101G, 808K, 808S, 790A, 800V, 6 80L, 167R , 752E, 1093N, 119G, 739G, 1116R, 580Y, 304A, 712Q, 806S, 712G, 790G, 1108V, 731L, 1084N, 1127L, 308P, 1076S, 1093P, 1093Y, 739R, 739P, 310V, 712T ,167H,1093R , 536P/555E/619E/732K/799S/805Q, 555E/619E/801G, 555E/619E/799S, 619E/799S/801G/805Q, 536P/555E/619E/732K/799S, 799S/805Q, 801G, 24 6D /619E/799S/801G、536P/555E/619E、619E/839K、619E/799S/805Q、536P/801G/805Q、555E/619E/799S/805Q、619E/801G/805Q、555E/619E、536P/619 E /801G, 801G/805Q, 536P/619E/799S/805Q, 555E/619E/732K/799S/801G/805Q, 1281H, 749L, 614H, 616D, 621K, 1050E, 610H, 611D, 1042T, 616Q, 614N, 370A /614A, 1156D, 1210V, 614T, 1158N, 1281S, 618E, 555E/681N/737K/799Q, 536P/619E/809G/1109G, 536K/555G/619E/737K/799Q/809G/839K/916T/921 R、916T /1109G、536R/619E/681N/799Q/916T/1109G、555G/619E/681N/807R/1109G、555E/619E/681N/737K/799Q、619E/681N/799S/916T/1109G、619E/681N ,555G /921R, 619E/681N/916T, 555G/619E/681N/799S, 555E, 732K, 619E/799S, 619E, 681N, 555G/916T, 839K or 799Q, where the amino acid positions are relative to those corresponding to SEQ. The reference sequence of ID NO: 1514. The recombinant Factor VIII polypeptide of claim 14, which comprises a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2072 or at least 70%, 75%, 80% with the reference sequence corresponding to SEQ ID NO: 2072 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, An amino acid sequence with 97%, 98%, or 99% or more sequence identity, wherein the amino acid sequence comprises the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2072 or is relative to One or more substitutions of the reference sequence corresponding to SEQ ID NO: 2072. The recombinant Factor VIII polypeptide of claim 38, wherein the amino acid sequence of the recombinant Factor VIII includes at least one substitution or set of substitutions at the following amino acid positions: 167, 167/738/1076/1093, 167/738 /752/800/805/808/809/1116, 167/800/809/1076, 167/318, 167/318/800/805/808/809, 167/318/681, 167/318/681/700 /805/808/809/1076/1116, 167/318/700, 167, 167/738/1076/1093, 167/738/752/800/805/808/809/1116, 167/800/809/1076 ,167/318, 167/318/800/805/808/809, 167/318/681, 167/318/681/700/805/808/809/1076/1116, 167/318/700, 167/318 /700/1076、167/318/700/738/739/1076、167/681/738/800/808/809、167/178/700/752/1093、167/700、167/700/738/739 /1093、167/700/738/752/1093、167/700/738/752/805/808/809/1093/1116、808/809、1076、738、738/808/809、738/1076、738 /739、738/739/809、738/739/809/1093/1116、738/739/1093、738/739/752/1093、738/739/799/800/1093、738/739/1116、738 /800、738/1093、738/752、738/752/1093、738/752/799、738/799/809/1093、738/799/1093、739、739/800/809、800、800/805 /809, 809, 809/1093, 809/1093/1116, 809/1116, 318, 318/1076, 318/1076/1116, 318/739/805/808/809/1076, 318/499/700/738 /809、318/1093、318/752/1093、318/1116、318/543/739、318/543/681/752/1116、318/543/1093、318/700/738/1093/1414、681 , 681/738, 681/738/739/1093, 681/738/739/752/799/800/809/1116/1339, 681/738/739/1116, 681/738/800, 681/800/805 /809, 681/809/1093, 681/752/1093, 681/752/1093, 681/700, 681/700/738/739/1093/1116, 681/700/809, 681/700/1093, 681 /700/752/808/809/1093/1116, 681/700/752/1093, 681/700/844, 681/700/805/809, 1093, 1093/1116, 752, 752/1076, 752/800 /809, 752/800/1093, 752/809, 752/866, 752/1093, 752/1116, 178/318/739/752/809/1076/1116, 178/318/800/809, 178/318 /681/800/808/809/1076, 178/681, 178/681/738/1116, 178/752/809, 178/700/800/805/808/809, 178/805/809/866, 1116 ,543, 543/738/739/809, 543/738/809/1093, 543/739/1093, 543/800/1093/1116, 543/809/1093, 543/809/1093/1116, 543/681 /738/739/752/809/1093, 543/681/738/739/752/1093, 543/681/739/752/800/805/809/1093, 543/681/700/738/739/800 /1093/1116、543/681/700/738/739/805/808/809、543/681/700/752、543/681/700/752/809/1093、543/1093、543/700、543 /700/738/739/752/800/1093/1116, 543/700/809, 543/700/1093, 543/700/752, 543/700/752/800/1093, 543/700/752/809 /1116, 543/700/752/1093, 700, 700/738/739/800/1093, 700/738/739/799/1093, 700/738/800/1093, 700/738/1116, 700/809 ,700/809/1093, 700/1093, 700/1093/1116, 700/752, 700/752/808/809/1093/1116, 700/752/809/1093, 700/752/799/1116, 700 /752/805/809/1093, 700/799/800, 700/1116, 700/805/808/809/1076, 514/543/681/805/808/809, 805/808/809 or 805/809 /1116, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2072. The recombinant Factor VIII polypeptide of claim 38 or 39, wherein the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one substitution or substitution set 167G, 167G/738Y/1076S/1093G, 167G/738Y/752E/800V/805Q /808S/809G/1116I, 167G/800V/809G/1076S, 167G/318L, 167G/318L/800V/805Q/808S/809G, 167G/318L/681N, 167G/318L/681N/700E/805Q/808S/8 09G /1076S/1116I, 167G/318L/700E, 167G/318L/700E/1076S, 167G/318L/700E/738Y/739G/1076S, 167G/681N/738Y/800V/808S/809G, 167G/178Q/700E /752E /1093G、167G/700E、167G/700E/738Y/739G/1093G、167G/700E/738Y/752E/1093G、167G/700E/738Y/752E/805Q/808S/809G/1093A/1116I、808S/809 G、1076S 738Y 8Y/739G /799S/800V/1093G、738Y/739G/1116I、738Y/800V、738Y/1093G、738Y/752E、738Y/752E/1093G、738Y/752E/799S、738Y/799S/809G/1093G、738Y/799 S/1093G , 739G, 739G/800V/809G, 800V, 800V/805Q/809G, 809G, 809G/1093G, 809G/1093G/1116I, 809G/1116I, 318L, 318L/1076S, 318L/1076S/1116I, 318L/ 739G/805Q /808S/809G/1076S, 318L/499L/700E/738Y/809G, 318L/1093A, 318L/752E/1093A, 318L/1116I, 318L/543P/739G, 318L/543P/681N/752E/1116I, 318 L/543P /1093A、318L/700E/738Y/1093A/1414N、681N、681N/738Y、681N/738Y/739G/1093G、681N/738Y/739G/752E/799S/800V/809G/1116I/1339C、681N/73 8Y/739G /1116I、681N/738Y/800V、681N/800V/805Q/809G、681N/809G/1093G、681N/752E/1093A、681N/752E/1093G、681N/700E、681N/700E/738Y/739G/1093 G/1116I , 681N/700E/809G, 681N/700E/1093G, 681N/700E/752E/808S/809G/1093G/1116I, 681N/700E/752E/1093G, 681N/700E/844H, 681N/700E/805Q/809 G、1093A , 1093G, 1093G/1116I, 752E, 752E/1076S, 752E/800V/809G, 752E/800V/1093G, 752E/809G, 752E/866A, 752E/1093G, 752E/1116I, 178Q/318L/739G/ 752E/809G /1076S/1116I, 178Q/318L/800V/809G, 178Q/318L/681N/800V/808S/809G/1076S, 178Q/681N, 178Q/681N/738Y/1116I, 178Q/752E/809G, 178Q/700E /800V /805Q/808S/809G, 178Q/805Q/809G/866A, 1116I, 543P, 543P/738Y/739G/809G, 543P/738Y/809G/1093G, 543P/739G/1093A, 543P/800V/1093G/1116 I.543P /809G/1093G、543P/809G/1093G/1116I、543P/681N/738Y/739G/752E/809G/1093G、543P/681N/738Y/739G/752E/1093G、543P/681N/739G/752E/800 V/805Q /809G/1093G、543P/681N/700E/738Y/739G/800V/1093G/1116I、543P/681N/700E/738Y/739G/805Q/808S/809G、543P/681N/700E/752E、543P/681N/ 700E /752E/809G/1093G, 543P/1093G, 543P/700E, 543P/700E/738Y/739G/752E/800V/1093G/1116I, 543P/700E/809G, 543P/700E/1093G, 543P/700E/752 E. 543P /700E/752E/800V/1093G, 543P/700E/752E/809G/1116I, 543P/700E/752E/1093G, 700E, 700E/738Y/739G/800V/1093G, 700E/738Y/739G/799S/1093 G、700E /738Y/800V/1093G, 700E/738Y/1116I, 700E/809G, 700E/809G/1093G, 700E/1093G, 700E/1093G/1116I, 700E/752E, 700E/752E/808S/809G/1093G/1 116I, 700E /752E/809G/1093G, 700E/752E/799S/1116I, 700E/752E/805Q/809G/1093A, 700E/799S/800V, 700E/1116I, 700E/805Q/808S/809G/1076S, 514L/543 P/681N /805Q/808S/809G, 805Q/808S/809G or 805Q/809G/1116I, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2072. Such as the recombinant Factor VIII polypeptide of claim 1, wherein the amino acid sequence of the recombinant Factor VIII polypeptide includes at least one of Tables 4.1 , 4.2 , 5.1 , 5.2 , 6.1 , 6.2 , 7.1 , 7.2 , 8.1 , 8.2 , 9.1 , 9.2 , A substitution or set of substitutions of one of the variants described in 10.1 , 10.2 , 11.1 and 15.1 , wherein the amino acid positions are relative to those corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072 of this reference sequence. For example, the recombinant Factor VIII polypeptide of claim 1 includes an amino acid sequence, and the amino acid sequence includes Table 4.1 , 4.2 , 5.1 , 5.2 , 6.1 , 6.2 , 7.1 , 7.2 , 8.1, 8.2 , 9.1 , 9.2 , 10.1 , Residues 20-1457 of one of the even-numbered SEQ ID NO. sequences set forth in 10.2 , 11.1 and 15.1 , or include Tables 4.1 , 4.2 , 5.1 , 5.2 , 6.1 , 6.2 , 7.1 , 7.2, 8.1 , 8.2 , 9.1 , The sequence of one of the even-numbered SEQ ID NO. described in 9.2 , 10.1 , 10.2 , 11.1 and 15.1 . The recombinant Factor VIII polypeptide of claim 1, wherein the amino acid sequence of the recombinant Factor VIII polypeptide includes residues 20-1457 of one of the even-numbered SEQ ID NO.s among SEQ ID NOs: 4-2346 and 2360-2870, Or comprise one of the even-numbered SEQ ID NOs: 4-2346 and 2350-2870, wherein the amino acid sequence optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 amino acid substitutions. The recombinant Factor VIII polypeptide of claim 1, wherein the amino acid sequence of the recombinant Factor VIII polypeptide includes residues 20-1457 of SEQ ID NO: 252, 504, 706, 840, 1200, 1514 or 2072, or includes SEQ ID NO: 252, 504, 706, 840, 1200, 1514 or 2072, wherein the amino acid sequence optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 amine groups acid substitution. The recombinant Factor VIII polypeptide of any one of claims 1 to 44, wherein the combination of the recombinant Factor VIII polypeptide and Factor IX promotes blood coagulation activity. The recombinant Factor VIII polypeptide of any one of claims 1 to 45, wherein the recombinant Factor VIII polypeptide has increased activity/potency compared to a reference Factor VIII polypeptide. The recombinant Factor VIII polypeptide of claim 46, wherein the activity is coagulation activity/potency. The recombinant Factor VIII polypeptide of any one of claims 1 to 47, wherein the recombinant Factor VIII polypeptide is more stable to serum or plasma exposure than a reference Factor VIII polypeptide. The recombinant Factor VIII polypeptide of any one of claims 1 to 48, wherein the recombinant Factor VIII polypeptide is more thermally stable than a reference Factor VIII polypeptide. The recombinant Factor VIII polypeptide of any one of claims 1 to 45, which is similar to a reference Factor VIII polypeptide having a sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072 Ratio, the recombinant Factor VIII polypeptide exhibits at least one improved property selected from the following: i) increased activity/potency; ii) increased coagulation activity/potency; iii) increased plasma or serum stability; iv) increased thermal stability sex; v) reduced immunogenicity; vi) increased expression and/or secretion from cells transfected with an expression vector; vii) increased transduction and/or secretion from cells transduced with a gene therapy vector Secretion, or a combination of any of i), ii), iii), iv), v), vi and/or vii). The recombinant Factor VIII polypeptide of any one of claims 46 to 50, wherein the reference Factor VIII polypeptide has a sequence corresponding to SEQ ID NO: 2. The recombinant Factor VIII polypeptide of any one of claims 1 to 51, which includes a deimmunized recombinant Factor VIII polypeptide. The recombinant Factor VIII polypeptide of claim 52, wherein the deimmunized recombinant Factor VIII polypeptide comprises residues 20-1457 or residue 1 corresponding to one of the sequences set forth in Tables 12-1 to 12-8 and 15-2 -1457 amino acid sequence. The recombinant Factor VIII polypeptide of any one of claims 1 to 53, wherein the recombinant Factor VIII polypeptide is purified. A biologically active fragment of the recombinant Factor VIII polypeptide of any one of claims 1 to 53. The biologically active fragment of claim 55, wherein the biological fragment is processed in a mammalian cell or in a human patient. The biologically active fragment of any one of claims 55 to 56, which has one or more cleavages between amino acid residues 760 to 773, and the polypeptide does not contain some corresponding to amino acid residues 760 to 773 or All amino acid sequences, where the positions are relative to the reference sequence of SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514 or 2072. The biologically active fragment of any one of claims 55 to 57, which has a cleavage between amino acid residues 391 and 392, wherein these positions are relative to SEQ ID NOs: 2, 252, 504, 706, 840, 1200, 1514 or 2072 of the reference sequence. A recombinant polynucleotide encoding at least one recombinant Factor VIII polypeptide as set forth in any one of claims 1 to 53 or a recombinant Factor VIII polypeptide as set forth in any one of claims 55 to 58. active fragment. The recombinant polynucleotide of claim 59, comprising a reference polynucleotide corresponding to nucleotide residues 58-4371 of one of the odd-numbered SEQ ID NO.s in SEQ ID NOs: 3-2345 and 2359-2869 The nucleotide sequence is at least 70%, 75%, 80%, 81%, 82% identical to a reference polynucleotide sequence corresponding to one of the odd-numbered SEQ ID NO.s in SEQ ID NOs: 3-2345 and 2349-2869. %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or A polynucleotide sequence with 99% or more sequence identity; wherein the recombinant polynucleotide encodes a recombinant Factor VIII polypeptide. The recombinant polynucleotide of claim 59, comprising a reference polynucleoside corresponding to nucleotide residues 58-4371 of SEQ ID NO: 1, 251, 503, 705, 839, 1199, 1513 or 2071 The acid sequence or the reference polynucleotide sequence has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or a polynucleotide sequence with greater sequence identity; wherein the recombinant polynucleotide encodes a recombinant Factor VIII polypeptide. The recombinant polynucleotide of any one of claims 59 to 61, comprising a polynucleotide sequence codon-optimized to express the encoded recombinant Factor VIII polypeptide. The recombinant polynucleotide of claim 59, wherein the polynucleotide sequence includes odd-numbered nucleotide residues 58-4371 of one of SEQ ID NOs: 3-2345 and 2359-2869, or Contains one of the odd-numbered SEQ ID NOs: 3-2345 and 2349-2869. The recombinant polynucleotide of claim 59, wherein the polynucleotide sequence includes nucleotide residues 58-4371 of SEQ ID NO: 1, 251, 503, 705, 839, 1199, 1513 or 2071, or includes SEQ ID NO: 1, 251, 503, 705, 839, 1199, 1513 or 2071. An expression vector comprising the recombinant polynucleotide of any one of claims 59 to 64. The expression vector of claim 65, wherein the recombinant polynucleotide sequence is operably linked to one or more control sequences. The expression vector of claim 66, wherein the control sequence includes a promoter. A host cell comprising the expression vector of any one of claims 65 to 67. The host cell of claim 68, wherein the host cell is a eukaryotic cell or a prokaryotic cell. The host cell of claim 68, wherein the host cell is a cultured human cell. A method of producing a Factor VIII polypeptide comprising culturing the host cell of any one of claims 68 to 70 under conditions such that the Factor VIII polypeptide encoded by the recombinant polynucleotide is produced. The method of claim 71, further comprising the step of recovering the Factor VIII polypeptide. The method of claim 71 or 72, further comprising the step of purifying the Factor VIII polypeptide. A pharmaceutical composition comprising the recombinant Factor VIII polypeptide according to any one of claims 1 to 54. Such as the pharmaceutical composition of claim 74, which further includes a pharmaceutically acceptable carrier and/or excipient. For example, the pharmaceutical composition of claim 74 or 75, wherein the composition is suitable for parenteral injection or infusion into a human being. A method for treating and/or preventing symptoms of factor VIII deficiency in an individual, comprising administering to an individual suffering from factor VIII deficiency an effective amount of a recombinant factor as claimed in any one of claims 1 to 54 VIII polypeptide or a pharmaceutical composition according to any one of claims 74 to 76. The method of claim 77, wherein the individual has hemophilia A. The method of any one of claims 77 to 78, wherein the symptoms of factor VIII deficiency are improved. Use of a recombinant Factor VIII polypeptide according to any one of claims 1 to 54 or a pharmaceutical composition according to any one of claims 74 to 76 for the treatment of a factor VIII deficiency in a subject. Use of a recombinant Factor VIII polypeptide according to any one of claims 1 to 54 for the preparation of a medicament for the treatment of Factor VIII deficiency in a subject. Such as the use of claim 80 or 81, wherein the factor VIII deficiency is hemophilia A.