Stable aqueous antibody formulation

Abstract

To provide, in the field of pharmaceutical formulations of antibodies, a stable liquid antibody formulation and its pharmaceutical preparation and use.SOLUTION: This invention is exemplified by an aqueous formulation of an anti-Tissue Factor Pathway Inhibitor (TFPI) antibody.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 934,781, filed November 13, 2019, and No. 63 / 081,409, filed September 22, 2020, which are incorporated by reference in their entireties.

[0002] Sequence Listing Reference This application has been filed electronically via EFS-Web and includes an electronically transmitted sequence listing in .txt format. The .txt file contains a sequence listing with the filename "PC72541A_Seq_Listing_ST25.txt" that is 42,550 bytes in size and was created on October 13, 2020. The sequence listing contained in this .txt file is a part of the specification and is incorporated herein by reference in its entirety.

[0003] The present invention relates to the field of pharmaceutical formulations of antibodies. In particular, the present invention relates to stable liquid antibody formulations and their pharmaceutical preparation and use. [Background technology]

[0004] Antibody formulations intended for therapeutic or prophylactic use require stabilizers to prevent loss of protein activity or structural integrity due to the effects of denaturation, deamidation, oxidation or aggregation over a period of time during storage and transport prior to use. These problems are exacerbated at the high concentrations of antibodies often desired for therapeutic administration. Although numerous formulation options are available, no one approach or system is suitable for all proteins (see, e.g., Wang et al., J. Pharm Sci. 96:1-26 (2007)).

[0005] The main goal in the development of antibody formulations is to maintain the antibody, solubility, stability, and efficacy of its antigen binding. For the antibody to remain biologically active, the formulation must maintain the conformational integrity of at least the core of the antigen-binding amino acids of the antibody. It is also particularly desirable to avoid aggregates and particulates in the solution that require sterile filtration before use for intravenous or subcutaneous injection and limit the route of administration. Tonicity agents such as salts, detergents, pH, and sugars can be used to overcome aggregation or denaturation problems. Formulating antibody preparations requires careful selection of these factors, among others, to avoid protein denaturation and loss of antigen-binding activity. Thus, there is a need for stable aqueous antibody formulations that stably support high concentrations of bioactive antibody in solution and are suitable for parenteral administration, including intravenous, intraocular, intravitreal, intraarterial, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, intraosseous, intraperitoneal, intradermal, or subcutaneous injection.

[0006] Tissue factor pathway inhibitor (TFPI) antibodies have been shown to be useful in the treatment and prevention of blood clotting disorders or bleeding disorders, such as hemophilia A and B (see, e.g., US2017 / 0073428). While liquid antibody formulations are known in the art (see, e.g., WO2006 / 096491, WO2010 / 032220, WO2013 / 186719, US2009 / 0110681, US2017 / 0360929, and US2018 / 0000933), there is a need to provide stable aqueous formulations of TFPI antibodies to meet the medical needs of patients suffering from blood clotting disorders or bleeding disorders.

[0007] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, and patent application was specifically and individually indicated to be incorporated by reference herein. In the event that one or more of the incorporated literature and similar materials differs or conflicts with this application in any respect, including, but not limited to, defined terms, term usage, techniques discussed, etc., this application will control. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] US2017 / 0073428 [Patent Document 2] WO2006 / 096491 [Patent Document 3] WO2010 / 032220 [Patent Document 4] WO2013 / 186719 [Patent Document 5] US2009 / 0110681 [Patent Document 6] US2017 / 0360929 [Patent Document 7] US2018 / 0000933 [Patent Document 8] U.S. Patent No. 4,816,567 [Patent Document 9] WO99 / 58572 [Patent Document 10] U.S. Patent No. 5,807,715 [Patent Document 11] U.S. Patent No. 5,866,692 [Patent Document 12] U.S. Patent No. 6,331,415 [Patent Document 13] U.S. Patent No. 5,530,101 [Patent Document 14] U.S. Patent No. 5,693,761 [Patent Document 15] U.S. Patent No. 5,693,762 [Patent Document 16] U.S. Patent No. 5,585,089 [Patent Document 17] U.S. Patent No. 6,180,370 [Patent Document 18] European Patent Publication No. 0519596 [Patent Document 19] PCT Publication No. WO99 / 58572 [Patent Document 20] UK Patent Application No. 9809951.8 [Patent Document 21] U.S. Patent No. 6,180,377 [Patent Document 22] U.S. Patent No. 6,054,297 [Patent Document 23] U.S. Patent No. 5,997,867 [Patent Document 24] U.S. Patent No. 5,866,692 [Patent Document 25] U.S. Patent No. 6,210,671 [Patent Document 26] U.S. Patent No. 6,350,861 [Patent Document 27] PCT Publication No. WO01 / 27160 [Patent Document 28] PCT / IB2019 / 058597 [Patent Document 29] US62 / 802,401 [Patent Document 30] US62 / 744,481 [Patent Document 31] WO2017 / 029583 [Patent Document 32] WO2010 / 017196 [Patent Document 33] WO2011 / 109452 [Patent Document 34] WO2014 / 144577 [Patent Document 35] WO2010 / 072687 [Patent Document 36] WO2012 / 001087 [Patent Document 37] WO2014 / 140240 [Patent Document 38] WO2015 / 007880 [Non-patent literature]

[0009] [Non-Patent Document 1] Wang et al., J. Pharm. Sci. 96: 1 - 26 (2007)

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[0010] Provided is a stable aqueous pharmaceutical formulation with extended shelf life that includes a tissue factor pathway inhibitor (TFPI) antibody (also known as an anti-TFPI antibody). It is demonstrated that the formulations of the invention having high TFPI antibody concentrations are stable (e.g., have low levels of % HMMS (high molecular weight species), % LMMS (low molecular weight species), % fragments, and oxidation) and thus are suitable for parenteral administration.

[0011] Disclosed and exemplified herein are preparations of antibodies (including antigen-binding fragments thereof) that bind to tissue factor pathway inhibitor (TFPI).Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.Such equivalents are intended to be encompassed by embodiment (E) below.

[0012] E1. A formulation comprising about 15 mg / mL to about 250 mg / mL of an antibody that specifically binds tissue factor pathway inhibitor (TFPI), a buffer, a polyol, a surfactant, and a chelating agent, the formulation having a pH of about 5.0 to about 6.0.

[0013] E2. A formulation comprising about 15 mg / mL to about 250 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), a buffer, a polyol, a surfactant, and a chelating agent, wherein the formulation has a pH of about 5.0 to about 6.0, and the epitope comprises residues Ile105, Arg107, and Leu131 based on the numbering of SEQ ID NO:2.

[0014] E3. The formulation of any one of E1-E2, wherein the buffer is selected from the group consisting of acetate, succinate, gluconate, citrate, histidine, acetic acid, phosphate, phosphoric acid, ascorbate, tartaric acid, maleic acid, glycine, lactate, lactic acid, ascorbic acid, imidazole, bicarbonate and carbonic acid, succinic acid, sodium benzoate, benzoic acid, gluconate, edetate, acetate, malate, imidazole, Tris, phosphate, and mixtures thereof.

[0015] E4. The formulation of E3, wherein the buffer is histidine. E5. The formulation of E3, wherein the buffer is succinate. E6. The aqueous formulation according to any one of E1 to E5, wherein the concentration of the buffer is from about 0.1 mM to about 100 mM.

[0016] E7. The formulation of E6, wherein the concentration of the buffer is from about 1 mM to about 40 mM or from about 10 mM to about 40 mM. E8. The formulation of E7, wherein the concentration of the buffer is about 10 mM, about 20 mM, or about 40 mM.

[0017] E9. The formulation of E8, wherein the concentration of the buffer is about 20 mM. E10. The formulation of any one of E1-E9, wherein the polyol is selected from the group consisting of mannitol, trehalose, sorbitol, erythritol, isomalt, lactitol, maltitol, xylitol, glycerol, lactitol, propylene glycol, polyethylene glycol, inositol, fructose, glucose, mannose, sucrose, sorbose, xylose, lactose, maltose, sucrose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch, water soluble glucan, or mixtures thereof.

[0018] E11. The formulation of E10, wherein the polyol is sucrose or trehalose. E12. The formulation of any one of E1-E11, wherein the concentration of the polyol is from about 1 mg / mL to about 300 mg / mL.

[0019] E13. The formulation of E12, wherein the concentration of polyol is from about 1 mg / mL to about 120 mg / mL, from about 50 mg / mL to about 120 mg / mL, or from about 60 mg / mL to about 110 mg / mL.

[0020] E14. The formulation of E13, wherein the concentration of polyol is about 64 mg / mL, about 85 mg / mL, or about 106 mg / mL. E15. The formulation of E1-E14, wherein the concentration of polyol is about 85 mg / mL.

[0021] E16. The surfactant is polysorbate, poloxamer, triton, sodium dodecyl sulfate, sodium lauryl sulfate, sodium octyl glucoside, lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine, stearyl sulfobetaine, lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, stearyl sarcosine, linoleyl betaine, myristyl betaine, cetyl betaine, lauroamidopropyl betaine, cocamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmidopropyl betaine, isostearamidopropyl betaine The formulation of any one of E1-E15, wherein the isopropyl tert-butyl ether is selected from the group consisting of butyl ether, myristamidopropyl-dimethylamine, palmidopropyl-dimethylamine, isostearamidopropyl-dimethylamine, sodium methyl cocoyl-taurate, disodium methyl oleyl-taurate, dihydroxypropyl PEG5 linoleate ammonium chloride, polyethylene glycol, polypropylene glycol, polysorbate 20, polysorbate 21, polysorbate 40, polysorbate 60, polysorbate 61, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, PEG3350, and mixtures thereof.

[0022] E17. The formulation of E16, wherein the surfactant is polysorbate 80. E18. The formulation of any one of E1-E17, wherein the concentration of the surfactant is from about 0.01 mg / mL to about 10 mg / mL, from about 0.05 mg / mL to about 5 mg / mL, from about 0.1 mg / mL to about 1 mg / mL, or from about 0.1 mg / ml to about 0.5 mg / mL.

[0023] E19. The formulation of E18, wherein the concentration of the surfactant is about 0.1 mg / ml, about 0.2 mg / mL, or about 0.3 mg / mL. E20. The formulation of E19, wherein the concentration of the surfactant is about 0.2 mg / mL.

[0024] E21. The formulation of any one of E1-E20, wherein the formulation does not contain a surfactant (e.g., polysorbate 80). E22. Chelating agents include ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid 5 (DTPA), nitrilotriacetic acid (NTA), N-2-acetamido-2-iminodiacetic acid (ADA), bis(aminoethyl) glycol ether, N,N,N',N'-tetraacetic acid (EGTA), trans-diaminocyclohexanetetraacetic acid (DCTA), glutamic acid, and aspartic acid, N-hydroxyethyliminodiacetic acid (HIMDA), N,N-bis-hydroxyethylglycine (bicine) and N-(trishydroxymethyl)glycine (tricine), glycylglycine, The formulation of any one of E1-E21, wherein the amino acid is selected from the group consisting of sodium deoxycholate, ethylenediamine, propylenediamine, diethylenetriamine, triethylenetetraamine (triene), disodium edetate dihydrate (or disodium EDTA dihydrate or disodium salt of EDTA), calcium EDTA, oxalic acid, malic acid, citric acid, citric acid monohydrate, and trisodium citrate dihydrate, 8-hydroxyquinolate, amino acids, histidine, cysteine, methionine, peptides, polypeptides, and proteins, and mixtures thereof.

[0025] E23. The formulation of E22, wherein the chelating agent is edetate disodium dihydrate. E24. The formulation of any one of E1-E23, wherein the concentration of the chelating agent is from about 0.01 mg / mL to about 50 mg / mL, from about 0.01 mg / mL to about 10 mg / mL, from about 0.01 mg / mL to about 1 mg / mL, or from about 0.02 mg / mL to about 0.08 mg / mL.

[0026] E25. The formulation of E24, wherein the concentration of the chelating agent is about 0.038 mg / mL, about 0.05 mg / mL, or about 0.063 mg / mL. E26. The formulation of E25, wherein the concentration of the chelating agent is about 0.05 mg / mL.

[0027] E27. The formulation of any one of E1-E26, wherein the formulation has a pH of about 5.0 to 6.6 or about 5.2 to 6.4. E28. The formulation of E27, wherein the formulation has a pH of about 5.2, 5.8 or 6.4.

[0028] E29. The formulation of E27, wherein the formulation has a pH of about 5.5 or 5.8. E30. The formulation of any one of E1-E29, wherein the antibody does not bind to Kunitz domain 1 (K1) of TFPI.

[0029] E31. The formulation of any one of E2-E30, wherein the epitope further comprises residues Cys106, Gly108, Cys130, Leu131, and Gly132, based on the numbering of SEQ ID NO:2.

[0030] E32. The formulation of any one of E2-E31, wherein the epitope further includes Asp102, Arg112, Tyr127, Gly129, Met134, and Glu138, based on the numbering of SEQ ID NO:2.

[0031] E33. The formulation of any one of E2-E32, wherein the epitope does not include E100, E101, P103, Y109, T111, Y113, F114, N116, Q118, Q121, C122, E123, R124, F125, K126, and L140, based on the numbering of SEQ ID NO: 2.

[0032] E34. The formulation of any one of E2-E32, wherein the epitope does not include D31, D32, P34, C35, K36, E100, E101, P103, Y109, K126, and G128, based on the numbering of SEQ ID NO:2.

[0033] E35. The formulation of any one of E1-E34, wherein the antibody comprises a heavy chain variable region (VH) comprising: (a) VH complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 13; (b) a VH complementarity determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 14; and (c) VH complementarity determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO:15.

[0034] E36. The formulation of any one of E1-E35, wherein the antibody comprises a VH comprising an amino acid sequence at least 90%, at least 95%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 18, and 20.

[0035] The formulation of any one of E1-E36, wherein the antibody comprises a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 18, and 20. E38. The formulation of any one of E1 to E37, wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:16.

[0036] E39. The formulation of any one of E1-E37, wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:18. E40. The formulation of any one of E1-E37, wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:20.

[0037] E41. The formulation of any one of E1-E40, wherein the antibody comprises a light chain variable region (VL) comprising: (a) VL complementarity determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO:8; (b) a VL complementarity determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO:9; and (c) VL complementarity determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO:10.

[0038] E42. The formulation of any one of E1-E41, wherein the antibody comprises a VL comprising an amino acid sequence at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:11.

[0039] E43. The formulation of any one of E1-E42, wherein the antibody comprises a VL comprising the amino acid sequence of SEQ ID NO:11. E44. The formulation of any one of E1-E43, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:17.

[0040] E45. The formulation of any one of E1-E43, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:19. E46. The formulation of any one of E1-E43, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:21.

[0041] E47. The formulation of any one of E1-E46, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:12. E48. The formulation of any one of E1-E47, wherein the antibody comprises: (i) a heavy chain variable region (VH) comprising: (a) a VH complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 13; (b) a VH complementarity determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 14; and (c) a VH complementarity determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 15; and (ii) a light chain variable region (VL) comprising: (a) a VL complementarity determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 8; (b) a VL complementarity determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 9; and (c) a VL complementarity determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 10.

[0042] E49. The formulation of E48, wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 18, and a VL comprising the amino acid sequence of SEQ ID NO: 11. E50. The formulation of any one of E1-E49, wherein the antibody comprises a VH sequence encoded by an insert present in the plasmid deposited under ATCC Accession No. PTA-122329.

[0043] E51. The formulation of any one of E1-E50, wherein the antibody comprises a VL sequence encoded by an insert present in the plasmid deposited under ATCC Accession No. PTA-122328.

[0044] E52. The formulation of any one of E1-E51, wherein the antibody comprises a sequence encoded by an insert present in a plasmid deposited under ATCC Accession No. PTA-122329 and a sequence encoded by an insert present in a plasmid deposited under ATCC Accession No. PTA-122328.

[0045] E53. The formulation of any one of E1 to E52, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 12. E54. The formulation of E48, wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 16, and a VL comprising the amino acid sequence of SEQ ID NO: 11.

[0046] E55. The formulation of E54, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a light chain comprising the amino acid sequence of SEQ ID NO: 12. E56. The formulation of E48, wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 20, and a VL comprising the amino acid sequence of SEQ ID NO: 11.

[0047] E57. The formulation of E56, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 21, and a light chain comprising the amino acid sequence of SEQ ID NO: 12. E58. The formulation of any one of E1, E3 to E29, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 23 and a light chain comprising the amino acid sequence of SEQ ID NO: 22.

[0048] E59. The formulation of any one of E1, E3 to E29, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ ID NO: 24. E60. The formulation of any one of E1-E59, wherein the antibody has a serum half-life of at least 25 hours, at least 29 hours, at least 30 hours, at least 35 hours, at least 40 hours, at least 50 hours, at least 55 hours, at least 60 hours, at least 65 hours, at least 70 hours, at least 75 hours, at least 80 hours, at least 85 hours, at least 90 hours, at least 95 hours, at least 100 hours, at least 105 hours, at least 110 hours, at least 115 hours, at least 120 hours, or at least 125 hours.

[0049] E61. The antibody is about 5 × 10 -7 M to approx. 5 x 10 -11 Binding affinity of M (K D The formulation according to any one of E1 to E60, comprising: E62. The formulation of any one of E1-E61, wherein the antibody has a subcutaneous (SC) bioavailability of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95, or at least 99% compared to the intravenous bioavailability.

[0050] E63. The formulation of any one of E1-E62, wherein the concentration of the antibody is about 20 mg / mL, 25 mg / mL, 50 mg / mL, 75 mg / mL, 100 mg / ml, 125 mg / ml, 150 mg / mL, 175 mg / mL, 200 mg / ml, 225 mg / ml, or 250 mg / mL.

[0051] E64. An aqueous formulation comprising about 150 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), about 20 mM succinate or histidine buffer; about 85 mg / mL sucrose or trehalose; about 0.2 mg / mL polysorbate 80 or polysorbate 20; about 0.05 mg / mL edetate disodium dihydrate or ethylenediaminetetraacetic acid (EDTA), wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 18, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11, and the formulation has a pH of about 5.5 or 5.8.

[0052] E65. A pharmaceutical formulation comprising 150 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 20 mM histidine buffer, 85 mg / mL sucrose, 0.2 mg / mL polysorbate 80, 0.05 mg / mL edetate disodium dihydrate, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:18 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:11, and the formulation has a pH of 5.8.

[0053] E66. A pharmaceutical formulation comprising 150 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 20 mM histidine buffer, 85 mg / mL sucrose, 0.2 mg / mL polysorbate 80, 0.05 mg / mL edetate disodium dihydrate, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:19 and a light chain comprising the amino acid sequence of SEQ ID NO:12, and the formulation has a pH of 5.8.

[0054] E67. A pharmaceutical formulation comprising about 50 mg / mL to about 250 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 20 mM histidine buffer, 85 mg / mL sucrose, 0.2 mg / mL polysorbate 80, 0.05 mg / mL edetate disodium dihydrate, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:23 and a light chain comprising the amino acid sequence of SEQ ID NO:22, and the formulation has a pH of 5.8.

[0055] E68. A pharmaceutical formulation comprising about 50 mg / mL to about 250 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 20 mM histidine buffer, 85 mg / mL sucrose, 0.2 mg / mL polysorbate 80, 0.05 mg / mL edetate disodium dihydrate, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:25 and a light chain comprising the amino acid sequence of SEQ ID NO:24, and the formulation has a pH of 5.8.

[0056] E69. A pharmaceutical formulation comprising 150 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 10 mM histidine buffer, 64 mg / mL sucrose, 0.1 mg / mL polysorbate 80, 0.038 mg / mL edetate disodium dihydrate, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:18 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:11, and the formulation has a pH of 6.4.

[0057] E70. A pharmaceutical formulation comprising 150 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 40 mM histidine buffer, 106 mg / mL sucrose, 0.3 mg / mL polysorbate 80, 0.063 mg / mL edetate disodium dihydrate, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:18 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:11, and the formulation has a pH of 5.2.

[0058] E71. A pharmaceutical formulation comprising 150 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 10 mM histidine buffer, 64 mg / mL sucrose, 0.1 mg / mL polysorbate 80, 0.038 mg / mL edetate disodium dihydrate, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:18 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:11, and the formulation has a pH of 5.2.

[0059] E72. A pharmaceutical formulation comprising 150 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 20 mM histidine buffer, 85 mg / mL sucrose, 0.05 mg / mL of edetate disodium dihydrate, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:18 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:11, and the formulation has a pH of 5.8.

[0060] E73. The pharmaceutical formulation of E72, wherein the formulation does not contain a surfactant (e.g., polysorbate 80). E74. The formulation of any one of E1-E73, wherein the formulation has a shelf life (e.g., at about -20°C, about 5°C, about 25°C, or about 40°C) of at least about 1 month, about 3 months, about 4 months, about 6 months, about 12 months, about 24 months, about 36 months, about 48 months, or about 60 months.

[0061] E75. The formulation of any one of E1-E74, wherein the formulation is an aqueous formulation. E76. The formulation of any one of E1-E75, wherein the formulation is a pharmaceutical formulation. E77. A method of reducing bleeding time comprising administering to a subject in need thereof a therapeutically effective amount of a formulation according to any one of E1-E76.

[0062] E78. A method for treating or preventing a disorder in blood clotting or a bleeding disorder, comprising the step of administering to a subject in need thereof a therapeutically effective amount of a formulation according to any one of E1-E76.

[0063] E79. A method of treating or preventing hemophilia A, B, or C, comprising the step of administering to a subject in need thereof a therapeutically effective amount of a formulation of any one of E1-E76.

[0064] E80. A method of treating or preventing von Willebrand's disease (vWD), comprising administering to a subject in need thereof a therapeutically effective amount of a formulation of any one of E1-E76.

[0065] E81. A method for reducing the activity of TFPI, comprising the step of administering to a subject in need thereof a therapeutically effective amount of a formulation according to any one of E1-E76. E82. The method of any one of E77-E81, wherein the subject is afflicted with or susceptible to a disorder in blood clotting or a bleeding disorder.

[0066] The method of any one of E77-E82, wherein the subject has or is susceptible to hemophilia A, B, or C. The method of any one of E77-E83, wherein the subject has or is susceptible to hemophilia A or B.

[0067] E85. The method of any one of E77-E82, wherein the subject is affected by or susceptible to von Willebrand's disease (vWD). E86. The method of any one of E77-E82, wherein the subject is suffering from or susceptible to a platelet disorder.

[0068] E87. The method of any one of E77-82, wherein the subject is suffering from or susceptible to a factor VII deficiency. E88. The method of any one of E77-E82, wherein the subject is affected by or susceptible to a factor XI deficiency.

[0069] E89. The method of any one of E77-E88, further comprising administering a coagulant to the subject. E90. The method of E89, wherein the coagulation agent is selected from the group consisting of factor VIIa, factor VIII, factor IX, tranexamic acid and a bypassing agent (e.g., FEIBA).

[0070] E91. Use of a formulation according to any one of E1 to E76 in a method according to any one of E77 to E90. E92. A formulation according to any one of E1 to E76 for use in a method according to any one of E77 to E90.

[0071] E93. A formulation for use in the method of any of E77 to E90, comprising 150 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 20 mM histidine buffer, 85 mg / mL sucrose, 0.2 mg / mL polysorbate 80, 0.05 mg / mL edetate disodium dihydrate, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 12, and the formulation has a pH of 5.8.

[0072] E94. A pharmaceutical formulation consisting of 150 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 20 mM histidine buffer, 85 mg / mL sucrose, 0.2 mg / mL polysorbate 80, 0.05 mg / mL edetate disodium dihydrate, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:19 and a light chain comprising the amino acid sequence of SEQ ID NO:12, and the formulation has a pH of 5.8. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0073] Disclosed herein is a stable aqueous pharmaceutical formulation having an extended shelf life that includes a tissue factor pathway inhibitor (TFPI) antibody. The application is based on the discovery that a formulation that includes a histidine buffer and has a pH of 5.8 stably supports high concentrations of TFPI antibody.

[0074] In particular, anti-TFPI antibodies stored in histidine-buffered aqueous formulations at pH 5.8 were demonstrated to have low levels of degradation as assessed by % high molecular weight species (%HMMS) using size-exclusion high performance liquid chromatography (SE-HPLC) (see Table 1). For example, at the intended storage conditions of 5±3°C, anti-TFPI antibodies were stable for up to 24 months with little to no degradation observed (%HMMS ranged from 0.5 to 0.9). At heat-stress storage conditions of 40°C / 75%RH, some degradation was observed (%HMMS ranged from 0.9 to 1.2 at 1 month (i.e., 4 weeks) and 1.5 to 2.4 at 3 months (i.e., 12 weeks)), but this degradation was less than that observed with other IgG1 antibodies, such as anti-VEGF antibodies (see Table 9; data for anti-VEGF antibodies taken from Table 11 of US2018 / 0000933). In particular, the amount of high molecular weight species (% HMMS) of anti-VEGF antibody stored in a histidine buffer formulation at pH 5.8 for one month (i.e., 4 weeks) at 40° C. was 3.1 (compared to 0.9 to 1.2 for anti-TFPI antibody) and 5.6 (compared to 1.5 to 2.4 for anti-TFPI antibody) for three months (i.e., 12 weeks). Thus, the histidine buffer formulation at pH 5.8 slows degradation, reduces aggregate formation, and improves the stability of anti-TFPI antibody compared to anti-VEGF antibody. This result was surprising and unexpected.

[0075] Thus, in one aspect, provided is a formulation comprising about 15 mg / mL to about 250 mg / mL of a tissue factor pathway inhibitor (TFPI) antibody, a buffer, a polyol, a surfactant, and a chelating agent, the formulation having a pH of about 5.0 to about 6.0. For example, in some embodiments, provided is a formulation comprising about 15 mg / mL to about 250 mg / mL of a TFPI antibody (e.g., an anti-TFPI antibody), about 1 mM to about 40 mM of a buffer (e.g., a histidine buffer), about 1 mg / mL to about 300 mg / mL of a polyol (e.g., sucrose), about 0.01 mg / mL to about 10 mg / mL of a surfactant (e.g., polysorbate 80), about 0.01 mg / mL to about 50.0 mg / mL of a chelating agent (e.g., edetate disodium dihydrate), the formulation having a pH of about 5.0 to about 6.0. In some embodiments, the antibody concentration is about 15 mg / mL to about 250 mg / mL. In other embodiments, the antibody concentration is about 100 mg / mL, about 115 mg / mL, about 150 mg / mL or about 158 ​​mg / mL.

[0076] general technique The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are described in Molecular Cloning: A Laboratory Manual, 2nd ed. (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (ed. M. J. Gait, 1984) Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (ed. J. E. Elis, 1998) Academic Press; Animal Cell Culture (ed. R. I. Freshney, 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Oberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (eds. A. Doyle, J. B. Griffiths, and D. G. Newell, 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (eds. D. M. Weir and C. C. Blackwell); Gene Transfer Vectors for Mammalian Cells (eds. JM Miller and MP Calos, 1987); Current Protocols in Molecular Biology (eds. FMA Usubel et al., 1987); PCR: The Polymerase Chain Reaction, (eds. Mullis et al., 1994); Current Protocols in Immunology (eds. JE Coligan et al., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P.Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (edited by D. Catty, IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (edited by P. Shepherd and C. Dean, Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (edited by M. Zanetti and JD Capra, Harwood Academic Publishers, 1995).

[0077] definition The following terms shall be understood to have the following meanings unless otherwise indicated: the term "isolated molecule" or "purified molecule" (where the molecule is, for example, a polypeptide, polynucleotide, or antibody) is a molecule that, by its origin or source of derivation, (1) is not associated with naturally associated components that accompany it in its natural state, (2) is substantially free of other molecules from the same species, (3) is expressed by cells of a different species, or (4) does not occur in nature. Thus, a molecule that is chemically synthesized or expressed in a cellular system different from the cell from which it is naturally derived, will be "isolated" from its naturally associated components. A molecule can also be rendered substantially free of naturally associated components by isolation using purification techniques well known in the art. The purity or homogeneity of a molecule can be assayed by many means well known in the art. For example, the purity of a polypeptide sample can be assayed using polyacrylamide gel electrophoresis and staining of the gel to visualize the polypeptide using techniques well known in the art. For certain purposes, higher resolution can be provided by using HPLC or other means well known in the art for purification.

[0078] As used herein, the term "formulation" in reference to an antibody is intended to describe an antibody preparation in a form that allows the biological activity of the antibody to be effective.

[0079] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation of an antibody in combination with a pharma- ceutical acceptable carrier / excipient. As used herein, an "aqueous formulation" refers to a formulation that contains water as a component.

[0080] A "pharmaceutical acceptable carrier / excipient" (vehicle, additive) is one that can be safely administered to a subject to provide an effective amount of the active ingredient used. The term "excipient" or "carrier" as used herein refers to an inert substance that is commonly used as a diluent, vehicle, preservative, binder or stabilizer for drugs. Compositions containing such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th Edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy, 20th Edition, Mack Publishing, 2000). As used herein, the term "diluent" refers to a pharmaceutical acceptable (safe and non-toxic for administration to humans) solvent that is useful in the preparation of the formulations described herein. Exemplary diluents include, but are not limited to, sterile water and bacteriostatic water for injection (BWFI).

[0081] An "antibody" is an immunoglobulin molecule capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid, or polypeptide, through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses complete polyclonal or monoclonal antibodies, as well as antigen-binding fragments / portions thereof that compete with the complete antibody for specific binding, unless otherwise specified, fusion proteins containing antigen-binding portions, and other modified configurations of immunoglobulin molecules that contain antigen recognition sites. Antigen-binding portions include, for example, Fab, Fab', F(ab')2, Fd, Fv, domain antibodies (dAbs, e.g., shark and camel antibodies), fragments containing complementarity determining regions (CDRs), single chain variable fragment antibodies (scFv), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR, and bis-scFv, and polypeptides that contain at least a portion of an immunoglobulin sufficient to confer specific antigen binding to a polypeptide. Antibodies include antibodies of any class, such as IgG, IgA, or IgM (or subclasses thereof), and need not be of a particular class. Immunoglobulins can be assigned to different classes depending on the antibody amino acid sequence of the constant region of their heavy chains. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0082] The "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, either alone or in combination. As known in the art, the variable regions of the heavy and light chains each consist of four framework regions (FRs) linked by three complementarity determining regions (CDRs), also known as hypervariable regions, which contribute to the formation of the antigen-binding site of the antibody. When a variant of a subject variable region is desired, particularly involving the substitution of amino acid residues outside the CDRs (i.e., within the framework regions), suitable amino acid substitutions, preferably conservative amino acid substitutions, can be identified by comparing the subject variable region with the variable regions of other antibodies that contain the CDR1 and CDR2 sequences of the same canonical class as the subject variable region (Chothia and Lesk, J Mol Biol 196(4):901-917, 1987).

[0083] In certain embodiments, the delineation of CDRs and the identification of the residues that comprise the binding site of an antibody are achieved by solving the structure of an antibody and / or solving the structure of an antibody-ligand complex. In certain embodiments, it can be achieved by any of a variety of techniques known in the art, such as X-ray crystallography. In certain embodiments, various analytical methods can be used to identify or estimate CDR regions. In certain embodiments, various analytical methods can be used to identify or estimate CDR regions. Examples of such methods include, but are not limited to, Kabat definition, Chothia definition, AbM definition, contact definition, and conformational definition.

[0084] The Kabat definition is a standard for numbering residues in an antibody and is typically used to identify CDR regions. See, e.g., Johnson & Wu, 2000, Nucleic Acids Res., 28:214-8. The Chothia definition is similar to the Kabat definition, but takes into account the location of certain structural loop regions. See, e.g., Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83. The AbM definition uses an integrated suite of computer programs created by the Oxford Molecular Group to model antibody structures. See, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbM TMSee, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," Oxford Molecular, Ltd., Oxford, UK. The AbM definition uses a combination of knowledge databases and ab initio methods, such as those described in Samudrala et al., 1999, PROTEINS, Structure, Function and Genetics Suppl., 3:194-198, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," to model the tertiary structure of an antibody from the primary sequence. The contact definition is based on analysis of available complex crystal structures. See, e.g., MacCallum et al., 1996, J. Mol. Biol., 5:732-45. In another approach, referred to herein as the "conformational definition" of the CDRs, the positions of the CDRs may be identified as residues that contribute enthalpic-wise to antigen binding. See, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166. It should be noted that other CDR boundary definitions may not strictly follow one of the above approaches and may be shortened or extended in light of predictions or experimental results that a particular residue or group of residues does not significantly affect antigen binding, but will nevertheless overlap with at least a portion of the Kabat CDRs. As used herein, CDRs may refer to CDRs defined by any approach known in the art, including combinations of approaches. The methods used herein may utilize CDRs defined according to any of these approaches. For any given embodiment that includes multiple CDRs, the CDRs may be defined according to any of the Kabat, Chothia, extended, AbM, contact, and / or conformational definitions.

[0085] As known in the art, a "constant region" of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, alone or in combination. As used herein, "monoclonal antibody" refers to an antibody obtained from a population of antibodies that are substantially homogeneous, i.e., the individual antibodies constituting the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which usually contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody obtained from a substantially homogeneous antibody population and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or by recombinant DNA methods such as those described in U.S. Pat. No. 4,816,567. The monoclonal antibodies may also be isolated from phage libraries generated using the techniques described in McCafferty et al., 1990, Nature 348:552-554, for example.

[0086] A "human antibody" is one that has an amino acid sequence that corresponds to that of an antibody produced by a human and / or that has been made using any of the techniques for making human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. As used herein, the term "human antibody" is intended to include antibodies that have variable and constant regions derived from human germline immunoglobulin sequences. This definition of a human antibody includes antibodies that contain at least one human heavy chain polypeptide or at least one human light chain polypeptide. The human antibodies of the invention may contain amino acid residues (e.g., mutations introduced by random or site-specific mutagenesis in vitro or somatic mutation in vivo) that are not encoded by human germline immunoglobulin sequences, for example in the CDRs, particularly CDR3. However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0087] The term "chimeric antibody" is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as antibodies in which the variable region sequences are derived from a murine antibody and the constant region sequences are derived from a human antibody.

[0088] As used herein, a "humanized" antibody refers to a form of a non-human (e.g., murine) antibody that is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequence of an antibody) that contains minimal sequence derived from the non-human immunoglobulin. Preferably, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. Generally, a humanized antibody comprises substantially all of at least one, typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are of a human immunoglobulin consensus sequence. A humanized antibody also optimally comprises at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Preferred are antibodies with Fc regions modified as described in WO99 / 58572. Other forms of humanized antibodies have one or more CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, or CDR H3) that are altered compared to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs of the original antibody.

[0089] There are four general steps to humanize a monoclonal antibody. These are: (1) determining the nucleotide and predicted amino acid sequences of the light and heavy chain variable domains of the starting antibody, (2) designing the humanized antibody, i.e., determining the antibody framework regions to be used during the humanization process, (3) the actual humanization method / technology, and (4) transfection and expression of the humanized antibody. See, for example, U.S. Patent Nos. 4,816,567, 5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761; 5,693,762; 5,585,089; and 6,180,370.

[0090] Many "humanized" antibody molecules have been described that contain antigen-binding sites derived from non-human immunoglobulins, including chimeric antibodies with rodent V regions or modified rodent V regions and their associated complementarity determining regions (CDRs) fused to human constant domains.See, for example, Winter et al., Nature 349:293-299 (1991); Lobuglio et al., Proc. Nat. Acad. Sci. USA 86:4220-4224 (1989); Shaw et al., J Immunol. 138:4534-4538 (1987); and Brown et al., Cancer Res. 47:3577-3583 (1987). Other references describe rodent CDRs grafted into human supporting framework regions (FRs) before fusion with the appropriate human antibody constant domains. See, e.g., Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988); and Jones et al., Nature 321:522-525 (1986). Other references describe rodent CDRs supported by recombinantly modified rodent framework regions. See, e.g., European Patent Publication No. 0519596. These "humanized" molecules are designed to minimize unwanted immune responses against these moieties that would limit the duration and effectiveness of therapeutic applications of rodent anti-human antibody molecules in human recipients. For example, antibody constant regions can be modified to be immunologically inert (e.g., not trigger complement lysis). See, e.g., PCT Publication No. WO99 / 58572; UK Patent Application No. 9809951.8. Other methods for antibody humanization that may be utilized are disclosed in Daugherty et al., Nucl. Acids Res. 19:2471-2476 (1991) and U.S. Patent Nos. 6,180,377; 6,054,297; 5,997,867; 5,866,692; 6,210,671; and 6,350,861; and PCT Publication No. WO 01 / 27160.

[0091] As used herein, the term "recombinant antibody" is intended to include any antibody prepared, expressed, created or isolated by recombinant methods, e.g., antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant combinatorial human antibody library, antibodies isolated from an animal (e.g., a mouse) transgenic for human immunoglobulin genes, or antibodies prepared where a recombinant human antibody can be subjected to in vitro mutagenesis.

[0092] The term "epitope" refers to a portion of a molecule that can be recognized and bound by an antibody at one or more antigen-binding regions of the antibody. Epitopes often consist of surface groupings of molecules, such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics and specific charge characteristics. In some embodiments, an epitope can be a protein epitope. A protein epitope can be linear or conformational. In a linear epitope, all of the points of interaction between the protein and an interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. A "non-linear epitope" or "conformational epitope" includes non-contiguous polypeptides (or amino acids) within an antigenic protein to which an epitope-specific antibody binds. The term "antigenic epitope" as used herein is defined as a portion of an antigen to which an antibody can specifically bind, as determined by any method known in the art, e.g., conventional immunoassays. Once the desired epitope on the antigen is determined, it is possible to generate antibodies against that epitope, for example, using the techniques described herein. Alternatively, during the discovery process, the generation and characterization of antibodies may reveal information about the desired epitope. From this information, it is then possible to competitively screen antibodies for binding to the same epitope. An approach to achieve this is to perform competitive and cross-competitive studies to find antibodies that compete or cross-compete with each other, e.g., antibodies compete for binding to antigens, for binding to TFPI.

[0093] The term "antagonist antibody" refers to an antibody that binds to a target and prevents or reduces the biological effect of the target.In some embodiments, this term can refer to an antibody that reduces the target that the antibody binds to, for example, TFPI, from exerting biological function.

[0094] An antibody that "preferentially binds" or "specifically binds" (used interchangeably herein) to an epitope is a term well understood in the art, and methods for determining such specific or preferential binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates more frequently, more rapidly, for a longer duration, and / or with a higher affinity to a particular cell or substance than to alternative cells or substances. An antibody "specifically binds" or "preferentially binds" to a target if it binds with higher affinity, avidity, more readily, and / or for a longer duration than it binds to other substances. For example, an antibody that specifically or preferentially binds to a TFPI epitope is an antibody that binds to this epitope sequence with higher affinity, avidity, more readily, and / or for a longer duration than it binds to other sequences. It is also understood by reading this definition that, for example, an antibody (or moiety or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. Generally, but not necessarily, reference to binding refers to preferential binding.

[0095] A variety of assay formats can be used to select antibodies or peptides that specifically bind to a molecule of interest. For example, solid-phase ELISA immunoassays, immunoprecipitation, Biacore™ (GE Healthcare, Piscataway, NJ), KinExA, fluorescence-activated cell sorting (FACS), Octet™ (ForteBio, Inc., Menlo Park, CA) and Western blot analysis are some of the many assays that can be used to identify antibodies that specifically react with an antigen or receptor, or its ligand-binding portion that specifically binds to its cognate ligand or binding partner. Typically, a specific or selective reaction will be at least twice the background signal or noise, more typically greater than 10 times the background, even more typically greater than 50 times the background, even more typically greater than 100 times the background, even more typically greater than 500 times the background, even more typically greater than 1000 times the background, and even more typically greater than 10,000 times the background. Also, the equilibrium dissociation constant (K D ) is 7 nM or less, an antibody is said to "specifically bind" to an antigen.

[0096] The term "binding affinity" is used herein as a measure of the strength of a non-covalent interaction between two molecules, such as an antibody or fragment thereof and an antigen. The term "binding affinity" is used to describe a monovalent interaction (intrinsic activity).

[0097] The binding affinity between two molecules, e.g., an antibody or fragment thereof and an antigen, via a monovalent interaction is determined by the dissociation constant (K D ) can be quantified by determining K D can be determined by measuring the kinetics of complex formation and dissociation, for example, using surface plasmon resonance (SPR) techniques (Biacore). The rate constants corresponding to the association and dissociation of monovalent complexes are the binding rate constant k, respectively. a (or k on ) and the dissociation rate constant k d (or koff ) is called. D is the formula K D =k d / k a By k a and k d The value of the dissociation constant can be determined directly by well-known methods and can be calculated even for complex mixtures, for example, by methods such as those described in Caceci et al. (1984, Byte 9:340-362). For example, K D can be established using a double filter nitrocellulose filter binding assay such as that disclosed by Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90:5428-5432). Other standard assays for evaluating the binding ability of a ligand, such as an antibody, to a target antigen are known in the art, including, for example, ELISA, Western blot, RIA, and flow cytometry analysis, and other assays exemplified elsewhere herein. The binding kinetics and binding affinity of an antibody can also be evaluated by standard assays known in the art, such as surface plasmon resonance (SPR), for example by using the Biacore™ system or KinExA.

[0098] An antibody that specifically binds to its target may bind to its target with high affinity, i.e., a low K D and may bind other non-target molecules with lower affinity. For example, an antibody may have a 1×10 -6 M or more, preferably 1×10 -5 M or more, preferably 1×10 -4 M or more, preferably 1×10 -3 M or more, and even more preferably 1×10 -2 K over M D Antibodies of the invention can bind to their target with an affinity that is preferably at least 2-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold, or 10,000-fold or more than their affinity for binding to another non-TFPI molecule.

[0099] As used herein, "immunospecific" binding of an antibody refers to an antigen-specific binding interaction that occurs between the antigen-binding site of an antibody and the specific antigen recognized by that antibody (i.e., the antibody reacts with a protein in an ELISA or other immunoassay and does not detectably react with unrelated proteins).

[0100] The term "compete" as used herein with respect to antibodies means that a first antibody or antigen-binding portion thereof binds to an epitope in a manner sufficiently similar to the binding of a second antibody or antigen-binding portion thereof such that the result of binding of the first antibody to its cognate epitope is detectably reduced in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. Alternatively, the binding of the second antibody to its epitope in the presence of the first antibody may, but need not, also be detectably reduced. That is, the first antibody may inhibit the binding of the second antibody to its epitope, but the second antibody does not inhibit the binding of the first antibody to its respective epitope. However, if each antibody detectably inhibits the binding of the other antibody to its cognate epitope or ligand, to the same extent or to a greater or lesser extent, the antibodies are said to "cross-compete" with each other for binding of their respective epitopes. Both competing and cross-competing antibodies are encompassed by the present invention. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational changes, or binding to a common epitope or portion thereof), one of skill in the art will recognize, based on the teachings provided herein, that such competing and / or cross-competing antibodies can be encompassed and useful in the methods disclosed herein.

[0101] As used herein, the term "tissue factor pathway inhibitor or TFPI" refers to any form of TFPI and variants thereof that retain at least some of the activity of TFPI. TFPI is a multivalent Kunitz domain containing protease inhibitor. Exemplary sequences of human, mouse, cynomolgus monkey, rabbit, and rat TFPI are provided in Table 20. Human TFPI is an extracellular glycoprotein with two major forms, TFPI-α and TFPI-β. TFPI alpha, a 276 amino acid glycosylated protein (molecular weight 43 kD), is the largest form of TFPI and consists of three Kunitz-like domains and a basic carboxy-terminal region. Alternative splicing generates TFPI-β, which contains Kunitz domain 1 (K1) and Kunitz domain 2 (K2) but lacks Kunitz domain 3 (K3) and an alternative C-terminal portion that lacks the basic region. TFPI-β is anchored to the plasma membrane by post-translational modification with a glycosylphosphatidylinositol (GPI) anchor.

[0102] The main targets of TFPI are the proteases factor Xa (FXa) and factor VIIa (FVIIa), which are key factors in the initiation step of the coagulation cascade. Biochemical analysis reveals that K2 is an inhibitor of FXa, while K1 inhibits the FVIIa-tissue factor complex. The role of K3 is unclear, as it does not appear to have direct protease inhibitory activity, but may function as a recognition site for the cofactor protein S. A unique C-terminal domain of TFPI-α may be involved in the recognition of prothrombinase on the platelet surface.

[0103] Kunitz domain 1 (K1) corresponds to amino acid residues 26-76 of SEQ ID NO: 2, and Kunitz domain 2 (K2) corresponds to residues 91-147 of SEQ ID NO: 2. K1 and K2 domains from other TFPI homologs, isoforms, variants, or fragments can be identified by sequence or structural alignment to SEQ ID NO: 2.

[0104] TFPI of the present disclosure includes any naturally occurring form of TFPI that may be derived from any suitable organism. For example, TFPI may be mammalian TFPI, such as human, mouse, rat, non-human primate, bovine, ovine, canine, feline, or porcine TFPI. In certain embodiments, TFPI is human TFPI. TFPI may be a mature form of TFPI (i.e., a TFPI protein that has undergone post-translational processing in a suitable cell). Such mature TFPI protein may, for example, be glycosylated.

[0105] TFPI of the present disclosure includes any functional fragment or variant derived from naturally occurring TFPI. A functional fragment of TFPI can be any element or portion of TFPI that retains the activity of TFPI, such as the ability to inhibit factor Xa (FXa), the ability to inhibit the activity of FVIIa-tissue factor complex, and / or the ability to function as a negative regulator of coagulation or hemostasis. For example, a functional fragment can include a Kunitz domain, such as the K1 domain, the K2 domain, or both the K1 and K2 domains of TFPI.

[0106] A functional variant may contain one or more mutations compared to naturally occurring TFPI, but still retain an activity of naturally occurring TFPI, such as the ability to inhibit factor Xa (FXa) or the activity of the FVIIa-tissue factor complex. For example, variants may have varying degrees of sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7, such as being at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.

[0107] The TFPI fragments, variants, isoforms and homologs of the invention should maintain important epitope residues as described herein (such as Ile105, Arg107, and Leu131 when TFPI-23 and TFPI-106 antibodies are used). Additionally, TFPI may contain 5 or more, 8 or more, 10 or more, 12 or more, or 15 or more surface accessible residues of the K2 domain of TFPI. Surface accessible residues are those residues with a relative accessibility of greater than 40%.

[0108] For example, for the K2 domain of TFPI (see, e.g., SEQ ID NO:2), the following amino acid residues have a relative accessibility of greater than 40%: 94-95, 98, 100-110, 118-121, 123-124, 131, 134, 138-142, and 144-145. TFPI can contain 5 or more, 8 or more, 10 or more, 12 or more, or 15 or more of these residues, including fragments of TFPI containing 5 or more, 8 or more, 10 or more, 12 or more, or 15 or more of these residues.

[0109] The positions of specific amino acid residues in TFPI are numbered according to SEQ ID NO:2 (human TFPIα K1K2K3). However, the present invention is not limited to SEQ ID NO:2. Corresponding residues from other TFPI homologs, isoforms, variants, or fragments can be identified according to sequence alignment or structural alignment known in the art. For example, alignment can be performed manually or using well-known sequence alignment programs such as ClustalW2 or "BLAST2Sequences" with default parameters. For example, Arg107 in SEQ ID NO:2 corresponds to Arg104 in mouse TFPI K1K2 (SEQ ID NO:4).

[0110] As used herein, a "TFPI antagonist antibody" (also interchangeably referred to as a "TFPI antibody" or an "anti-TFPI antibody") refers to an antibody capable of binding to TFPI and inhibiting the biological activity of TFPI and / or downstream pathways mediated by TFPI signaling. TFPI antagonist antibodies encompass antibodies that block, antagonize, inhibit or reduce (including significantly do so) the biological activity of TFPI, including downstream pathways mediated by TFPI signaling, such as ligand binding and / or eliciting a cellular response to TFPI. For purposes of the present invention, it will be clearly understood that the term "TFPI antagonist antibody" encompasses all previously identified terms, titles, and functional states and characteristics, whereby TFPI itself, the biological activity of TFPI (including but not limited to its ability to mediate any aspect of blood clotting), or the consequences of the biological activity, are substantially abolished, reduced, or neutralized to a meaningful degree. In some embodiments, the TFPI antagonist antibody binds to TFPI and prevents TFPI from binding to and / or inhibiting the tissue factor (TF) / factor VIIa complex. In other embodiments, the TFPI antibody binds to TFPI and prevents TFPI from binding to and / or inhibiting factor Xa. Examples of TFPI antagonist antibodies are provided herein.

[0111] An "effective amount" of a drug, formulation, compound, or pharmaceutical composition is an amount sufficient to produce a beneficial or desired result, including a clinical result such as alleviation or mitigation of a targeted pathological condition. An effective amount may be administered in one or more administrations. For purposes of the present invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to treat, ameliorate, or reduce the intensity of a targeted pathological condition. As understood in a clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if a desired result can or is achieved in combination with one or more other agents.

[0112] A "therapeutically effective amount" refers to an amount effective at dosages and for periods of time necessary to achieve a desired therapeutic result, which in the context of anti-TFPI antibodies includes the treatment or prophylactic prevention of a targeted pathological condition, e.g., bleeding disorders such as hemophilia A, B, or C. It should be noted that dosage values ​​may vary with the severity of the condition to be alleviated. It should be further understood that for any particular subject, a specific dosing regimen should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the composition, and that the dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions. Similarly, a therapeutically effective amount of an antibody or antibody portion may vary depending on factors such as the individual's condition, age, sex, and weight, the ability of the antibody or antibody portion to elicit a desired response in the individual, and the desired route of administration of the antibody formulation. A therapeutically effective amount is also an amount in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects.

[0113] Therapeutically effective amounts of anti-TFPI antibodies are described, for example, in PCT / IB2019 / 058597, filed October 9, 2019 (claiming priority to US62 / 802,401 and US62 / 744,481), which is incorporated herein in its entirety. In some embodiments, the therapeutically effective amount comprises an initial dose of about 300 mg, followed by subsequent weekly (i.e., once per week) doses of about 150 mg. In some embodiments, the therapeutically effective amount comprises an initial dose of about 150 mg, followed by subsequent weekly (i.e., once per week) doses of about 150 mg. In some embodiments, the therapeutically effective amount comprises an initial dose of about 300 mg, followed by subsequent weekly (i.e., once per week) doses of about 300 mg. In some embodiments, the therapeutically effective amount comprises an initial dose of about 450 mg, followed by subsequent weekly (i.e., once per week) doses of about 450 mg.

[0114] As used herein, the term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or slow down (alleviate) the targeted pathological condition (e.g., any condition that would benefit from treatment with an antibody). This includes chronic and acute disorders or diseases, including pathological conditions that predispose the mammal to the disorder in question. Those in need of treatment include those already with the condition, those susceptible to the condition, or those in need of prevention of the condition. As used herein, "treatment" is an approach to obtain a beneficial or desired clinical result, including, but not limited to, one or more of the following: reduction in severity, alleviation of one or more symptoms associated with a pathological condition.

[0115] As used herein, the term "subject" or "individual" for purposes of treatment includes any subject, preferably a subject in need of treatment of a target pathological condition (e.g., hemophilia). For purposes of prophylaxis, the subject is any subject, preferably a subject at risk of or predisposed to developing a target pathological condition. The term "subject" is intended to include organisms, e.g., prokaryotes and eukaryotes. Examples of subjects include mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In certain aspects of the invention, the subject is a human.

[0116] As used herein, the term "polynucleotide" or "nucleic acid", used interchangeably herein, refers to a polymeric form of nucleotides, either ribonucleotides or deoxynucleotides, or modified forms of either type of nucleotide, and can be in single-stranded and double-stranded form. A "polynucleotide" or "nucleic acid" sequence includes its complement unless otherwise specified. As used herein, the term "isolated polynucleotide" or "isolated nucleic acid" refers to a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, and because of its origin or source of derivation, an isolated polynucleotide has one to three of the following: (1) the "isolated polynucleotide" is not associated with all or a portion of a polynucleotide found in nature, (2) is operably linked to a polynucleotide that is not linked in nature, or (3) does not occur in nature as part of a larger sequence.

[0117] Reference herein to a value or parameter with "about" includes (and describes) aspects directed to the value or parameter itself. For example, a statement referring to "about X" includes the statement of "X." Numeric ranges include the numerical values ​​that define the range. Generally speaking, the term "about" refers to any value of the variable that is within the stated value of the variable and within experimental error of the stated value (e.g., within a 95% confidence interval of the mean) or within 10% of the stated value, whichever is greater.

[0118] When aspects or embodiments of the invention are described in terms of a Markush or other alternative group, the invention encompasses not only the entire group recited as a whole, but also each member of the group individually, and all possible subgroups of the main group, including the main group absent one or more group elements. The invention also envisions the explicit exclusion of any one or more of the group elements in the claimed invention.

[0119] When introducing elements of the invention or preferred embodiments thereof, the articles "a," "an," "the," and "said" are intended to mean that there are one or more elements. The terms "comprising," "comprise," "comprises," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements. Whenever an embodiment is described herein with the word "comprising," it is understood that analogous embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0120] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, shall control. Unless otherwise required by context, singular terms shall include plurals and plural terms shall include the singular.

[0121] Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. The materials, methods, and examples are illustrative only and are not intended to be limiting.

[0122] Anti-TFPI antibody preparations The present application is based on the surprising and unexpected observation that a formulation comprising a histidine buffer and having a pH of 5.8 slows degradation, reduces aggregate formation and improves stability of anti-TFPI antibodies compared to other IgG1 antibodies, such as anti-VEGF antibodies.

[0123] Thus, in one aspect, provided herein is a formulation comprising about 15 mg / mL to about 250 mg / mL of a tissue factor pathway inhibitor (TFPI) antibody, a buffer, a polyol, a surfactant, a chelating agent, the formulation having a pH of about 5.0 to about 6.0. In some embodiments, the formulation comprises about 15 mg / mL to about 250 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), a buffer, a polyol, a surfactant, a chelating agent, the formulation having a pH of about 5.0 to about 6.0, the epitope comprising residues Ile105, Arg107, and Leu131 based on the numbering of SEQ ID NO:2. The formulations described herein preferably have an extended shelf life of at least about 1 month, about 4 months, about 6 months, about 12 months, about 24 months, about 36 months, about 48 months, or about 60 months (e.g., at about -70°C, about -20°C, about 5°C, about 25°C, or about 40°C).

[0124] In some embodiments, the formulation comprises at least one TFPI antagonist antibody (i.e., an anti-TFPI antibody). Exemplary TFPI antagonist antibodies include, but are not limited to, those described in WO2017 / 029583, WO2010 / 017196, WO2011 / 109452, WO2014 / 144577, WO2010 / 072687, WO2012 / 001087, WO2014 / 140240, and WO2015 / 007880, each of which is incorporated herein by reference in its entirety.

[0125] In some embodiments, the TFPI antagonist antibody is selected from the group consisting of TFPI 106 (also known as PF-06741086), TFPI-23, TFPI-107, concizumab (also known as mAb-2021, hz4F36), 2A8 and 2A8-200 (see, e.g., US20170073428).

[0126] In some embodiments, there may be two or more antibodies. There may be at least one, at least two, at least three, at least four, at least five, or more different antibodies. Generally, the two or more different antibodies have complementary activities that do not adversely affect each other. One or more antibodies may also be used in combination with other agents that help to enhance and / or complement the effectiveness of the antibody.

[0127] In some embodiments, the antibody specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), the epitope including residues Ile105, Arg107, and Leu131 based on the numbering of SEQ ID NO:2. In some embodiments, the anti-TFPI antibody does not bind to Kunitz domain 1 (K1) of TFPI. In some embodiments, the epitope further includes residues Cys106, Gly108, Cys130, Leu131, and Gly132 based on the numbering of SEQ ID NO:2. In some embodiments, the epitope further includes residues Asp102, Arg112, Tyr127, Gly129, Met134, and Glu138 based on the numbering of SEQ ID NO:2. In some embodiments, the epitope does not include E100, E101, P103, Y109, T111, Y113, F114, N116, Q118, Q121, C122, E123, R124, F125, K126, and L140 based on the numbering of SEQ ID NO: 2. In some embodiments, the epitope does not include D31, D32, P34, C35, K36, E100, E101, P103, Y109, K126, and G128 based on the numbering of SEQ ID NO: 2.

[0128] In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising: (a) VH complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 13; (b) a VH complementarity determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 14; and (c) VH complementarity determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO:15.

[0129] In some embodiments, the antibody comprises a VH comprising an amino acid sequence at least 90%, at least 95%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 18, and 20. In some embodiments, the antibody comprises a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 18, and 20. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 20.

[0130] In some embodiments, the antibody comprises a light chain variable region (VL) comprising: (a) a VL complementarity determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO:8, (b) a VL complementarity determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO:9, and (c) a VL complementarity determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO:10.

[0131] In some embodiments, the antibody comprises a VL comprising an amino acid sequence at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 11. In some embodiments, the antibody comprises a VL comprising the amino acid sequence of SEQ ID NO:11.

[0132] In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 21. In some embodiments, the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 12.

[0133] In some embodiments, the antibody comprises: (i) a heavy chain variable region (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15; and (ii) a light chain variable region (VL) comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10.

[0134] In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 18, and a VL comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19, and a light chain comprising the amino acid sequence of SEQ ID NO:12.

[0135] Exemplary antibodies of the invention were deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, USA on July 22, 2015. Plasmid vector mAb-TFPI-106VH, having ATCC accession number PTA-122329, contains a DNA insert encoding the heavy chain variable region of antibody TFPI-106, and plasmid vector mAb-TFPI-106VL, having ATCC accession number PTA-122328, contains a DNA insert encoding the light chain variable region of antibody TFPI-106.

[0136] In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 16, and a VL comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a light chain comprising the amino acid sequence of SEQ ID NO: 12.

[0137] In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 20, and a VL comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 21, and a light chain comprising the amino acid sequence of SEQ ID NO: 12.

[0138] In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 23 and a light chain comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ ID NO: 24.

[0139] In some embodiments, the antibody has a serum half-life of at least 25 hours, at least 29 hours, at least 30 hours, at least 35 hours, at least 40 hours, at least 50 hours, at least 55 hours, at least 60 hours, at least 65 hours, at least 70 hours, at least 75 hours, at least 80 hours, at least 85 hours, at least 90 hours, at least 95 hours, at least 100 hours, at least 105 hours, at least 110 hours, at least 115 hours, at least 120 hours, or at least 125 hours. In some embodiments, the antibody has a serum half-life of at least 25 hours, at least 29 hours, or at least 30 hours. In some embodiments, the antibody has a serum half-life of at least 29 hours. In some embodiments, the antibody has a serum half-life of at least 30 hours. In some embodiments, the antibody has a serum half-life of at least 115 hours, at least 120 hours, or at least 125 hours.

[0140] In some embodiments, the antibody is at about 5×10 -7 M to approx. 5 x 10 -11 Binding affinity of M (K D In some embodiments, the antibody has a -8 M to approx. 1×10 -10 K for M (0.1 to 10 nm) D In some embodiments, the antibody has a K of 1 nM or less, 500 pM or less, 250 pM or less, 200 pM or less, 100 pM or less, 50 pM or less, 20 pM or less, or 10 pM or less. D In some embodiments, the antibodies have a K in the low pM range (i.e., 100 pM or less). D In some aspects, K Dis measured by surface plasmon resonance. In some aspects, surface plasmon resonance can be measured using Biacore. In some aspects, SPR can be measured using Biacore with captured antibody and human TFPI in solution phase.

[0141] In some aspects, the subcutaneous bioavailability of the antibody may be at least 10% compared to the intravenous bioavailability. In some aspects, the subcutaneous bioavailability of the antibody may be at least 15% compared to the intravenous bioavailability. In some aspects, the subcutaneous bioavailability of the antibody may be at least 20% compared to the intravenous bioavailability. In some aspects, the subcutaneous bioavailability of the antibody may be at least 25% compared to the intravenous bioavailability. In some aspects, the subcutaneous bioavailability of the antibody may be at least 27% compared to the intravenous bioavailability. In some aspects, the subcutaneous bioavailability of the antibody may be at least 30% compared to the intravenous bioavailability. In some aspects, the subcutaneous bioavailability of the antibody may be at least 35% compared to the intravenous bioavailability. In some aspects, the subcutaneous bioavailability of the antibody may be at least 40% compared to the intravenous bioavailability. In some aspects, the subcutaneous bioavailability of the antibody may be at least 50% compared to the intravenous bioavailability. In some aspects, the subcutaneous bioavailability of the antibody may be at least 60% compared to the intravenous bioavailability. In some aspects, the subcutaneous bioavailability of the antibody may be at least 70% compared to the intravenous bioavailability. In some aspects, the subcutaneous bioavailability of the antibody may be at least 80% compared to the intravenous bioavailability. In some aspects, the subcutaneous bioavailability of the antibody may be at least 90% compared to the intravenous bioavailability. In some aspects, the subcutaneous bioavailability of the antibody may be at least 99% compared to the intravenous bioavailability.

[0142] The antibody may be present in the formulation at a concentration ranging from about 0.1 mg / mL to about 250 mg / mL, from about 15 mg / mL to 250 mg / mL, from about 20 mg / mL to about 175 mg / mL, or from about 25 mg / mL to about 160 mg / mL. For example, in some embodiments, the concentration of the antibody is about 0.5 mg / mL, about 1 mg / mL, about 2 mg / mL, about 2.5 mg / mL, about 3 mg / mL, about 3.5 mg / mL, about 4 mg / mL, about 4.5 mg / mL, about 5 mg / mL, about 5.5 mg / mL, about 6 mg / mL, about 6.5 mg / mL, about 7 mg / mL, about 7.5 mg / mL, about 8 mg / mL, about 8.5 mg / mL, about 9 mg / mL, about 9.5 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, about 20 mg / mL, about 21 mg / mL, about 22 mg / mL, about 23 mg / mL, about 24 mg / mL, about 25 mg / mL, about 26 mg / mL, about 27 mg / mL, about 28 mg / mL, about 29 mg / mL, about 30 mg / mL, about 31 mg / mL, about 32 mg / mL, about 33 mg / mL, about 34 mg / mL, about 35 mg / mL, about 36 mg / mL, about 37 mg / mL, about 38 mg / mL, about 39 mg / mL, about 40 mg / mL, about 40 mg / mL, about 41 mg / mL, about 42 mg mg / mL, approximately 14 mg / mL, approximately 15 mg / mL, approximately 16 mg / mL, approximately 17 mg / mL, approximately 18 mg / mL, approximately 19 mg / mL, approximately 20 mg / mL, approximately 21 mg / mL, approximately 22 mg / mL, approximately 23 mg / mL, approximately 24 mg / mL, approximately 2 5mg / mL, approximately 26mg / mL, approximately 27mg / mL, approximately 28mg / mL, approximately 29mg / mL, approximately 30mg / mL, approximately 31mg / mL, approximately 32mg / mL, approximately 33mg / mL, approximately 34mg / mL, approximately 35mg / mL, approximately 36mg / mL, approximately 3 7mg / mL, approximately 38mg / mL, approximately 39mg / mL, approximately 40mg / mL, approximately 41mg / mL, approximately 42mg / mL, approximately 43mg / mL, approximately 44mg / mL, approximately 45mg / mL, approximately 46mg / mL, approximately 47mg / mL, approximately 48mg / mL, approximately 49mg / mL, approximately 50mg / mL, approximately 51mg / mL, approximately 52mg / mL, approximately 53mg / mL, approximately 54mg / mL, approximately 55mg / mL, approximately 56mg / mL, approximately 57mg / mL, approximately 58mg / mL, approximately 59mg / mL, approximately 60mg / mL, approximately 70mg / mL, approximately 80mg / mL, approximately 90mg / mL, approximately 100mg / mL, approximately 101mg / mL, approximately 102mg / mL, approximately 102.5mg / mL, approximately 103mg / mL, approximately 103.5mg / mL, approximately 104mg / mL, approximately 104.5m g / mL, approximately 105 mg / mL, approximately 105.5 mg / mL, approximately 106 mg / mL, approximately 106.5 mg / mL, approximately 107 mg / mL, approximately 107.5 mg / mL, approximately 108 mg / mL, approximately 108.5 mg / mL, approximately 109 mg / mL, approximately 109.5mg / mL, approx. 110mg / mL, approx. 111mg / mL, approx. 112mg / mL, approx. 113mg / mL, approx. 114mg / mL, approx. 115mg / mL, approx. 116mg / mL , about 117 mg / mL, about 118 mg / mL, about 119 mg / mL, about 120 mg / mL, about 121 mg / mL, about 122 mg / mL, about 123 mg / mL, about 124 mg / mL, approximately 125mg / mL, approximately 126mg / mL, approximately 127mg / mL, approximately 128mg / mL, approximately 129mg / mL, approximately 130mg / mL, approximately 131mg / mL, approximately 1 32mg / mL, approx. 133mg / mL, approx. 134mg / mL, approx. 135mg / mL, approx. 136mg / mL, approx. 137mg / mL, approx. 138mg / mL, approx. 139mg / mL , about 140 mg / mL, about 141 mg / mL, about 142 mg / mL, about 143 mg / mL, about 144 mg / mL, about 145 mg / mL, about 146 mg / mL, about 147 mg / mL, about 148 mg / mL, about 149 mg / mL, about 150 mg / mL, about 151 mg / mL, about 152 mg / mL, about 153 mg / mL, about 154 mg / mL, about 155 mg / mL, about 156 mg / mL, about 157 mg / mL, about 158 ​​mg / mL, about 159 mg / mL, about 160 mg / mL, about 170 mg / mL, about 180 mg / mL, about 190 mg / mL, about 200 mg / mL, about 210 mg / mL, about 220 mg / mL, about 230 mg / mL, about 240 mg / mL, or about 250 mg / mL. In some embodiments, the concentration of the antibody in the formulation is about 100 mg / mL, about 115 mg / mL, about 150 mg / mL, or about 158 ​​mg / mL. In some embodiments, the concentration of the antibody in the formulation is 150 mg / mL. In some embodiments, the concentration of the antibody in the formulation is 155 mg / mL. In some embodiments, the concentration of the antibody in the formulation is 158 mg / mL.

[0143] According to the invention, the buffer (e.g., histidine or succinate buffer) provides the formulation with a near-physiological pH to reduce pain or the risk of anaphylactic-like side effects upon injection, and also provides enhanced antibody stability and resistance to aggregation, oxidation and fragmentation.

[0144] The buffer may be, for example, but is not limited to, acetate, succinate (e.g., disodium succinate hexahydrate), gluconate, citrate, histidine, acetic acid, phosphate, phosphoric acid, ascorbate, tartaric acid, maleic acid, glycine, lactate, lactic acid, ascorbic acid, imidazole, bicarbonate and carbonic acid, succinic acid, sodium benzoate, benzoic acid, gluconate, edetate, acetate, malate, imidazole, tris, phosphate, and mixtures thereof. In some embodiments, the buffer is histidine or succinate. In some embodiments, the succinate buffer comprises disodium succinate hexahydrate (basic form) and / or succinic acid or mixtures thereof. Preferably, the buffer is histidine, and the histidine buffer comprises L-histidine, L-histidine monohydrochloride (also called L-histidine monohydrochloride monohydrate and / or L-histidine hydrochloride monohydrate), or a mixture thereof.

[0145] The concentration of the buffer can range from about 0.1 millimolar (mM) to about 100 mM. Preferably, the concentration of the buffer is from about 0.5 mM to about 50 mM, more preferably from about 1 mM to about 30 mM, and even more preferably from about 1 mM to about 25 mM. Preferably, the concentration of the buffer is about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, or about 100 mM. In some embodiments, the buffer is histidine at a concentration of about 20 mM. In some embodiments, the buffer is succinate at a concentration of about 20 mM.

[0146] The concentration of the buffer can also range from about 0.01 mg / mL to about 30 mg / mL, from about 0.1 mg / mL to about 5 mg / mL, or from about 0.5 mg / mL to about 4 mg / mL. For example, the concentration of the buffer can be about 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, about 0.04 mg / mL, about 0.05 mg / mL, about 0.06 mg / mL, about 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, about 0.10 mg / mL, 0.11 mg / mL, 0.12 mg / mL, 0.13 mg / mL, about 0.14 mg / mL, about 0.15 mg / mL, about 0.16 mg / mL, about 0.17 mg / mL, 0.18 mg / mL, 0.19 mg / mL, Approx. 0.20mg / mL, approx. 0.25mg / mL, approx. 0.3mg / mL, approx. 0.4mg / mL, approx. 0.5mg / mL, approx. 0.6mg / mL, approx. 0.7mg / mL, approx. 0.8mg / mL, approx. 0.9mg / mL, approx. 1.0mg / mL, approx. 2.0mg / mL, approximately 3.0mg / mL, approximately 4.0mg / mL, approximately 5.0mg / mL, approximately 6.0mg / mL, approximately 7.0mg / mL, approximately 8.0mg / mL, approximately 9.0mg / mL, approximately 10.0mg / mL, approximately 11.0mg / mL, approximately 12 The concentration of the histidine buffer is about 0.5-5.0 mg / mL. In some embodiments, the formulation comprises about 0.5-5.0 mg / mL of histidine buffer. In some embodiments, the buffer is a histidine buffer comprising about 0.1-1.5 mg / mL of L-histidine and about 1-3 mg / mL of L-histidine monohydrochloride. In some embodiments, the buffer is a histidine buffer containing 1.12 mg / mL L-histidine and 2.67 mg / mL L-histidine monohydrochloride. In some embodiments, the buffer is a succinate buffer containing about 0.5-5.0 mg / mL disodium succinate hexahydrate and about 0.1-1.0 mg / mL succinic acid.In some embodiments, the buffer is a succinate buffer comprising about 4.08 mg / mL disodium succinate hexahydrate and about 0.58 mg / mL succinic acid. In some embodiments, the buffer is a succinate buffer comprising about 2.362 mg / mL succinic acid.

[0147] In some embodiments, the polyol may have a molecular weight of, for example, but is not limited to, less than about 600 kD (e.g., in the range of about 120 to about 400 kD) and contain multiple hydroxyl groups including sugars (e.g., reducing and non-reducing sugars or mixtures thereof, sugars or carbohydrates), sugar alcohols, sugar acids, or salts or mixtures thereof. Examples of non-reducing sugars include, but are not limited to, sucrose, trehalose, and mixtures thereof. In some embodiments, the polyol is mannitol, trehalose, sorbitol, erythritol, isomalt, lactitol, maltitol, xylitol, glycerol, lactitol, propylene glycol, polyethylene glycol, inositol, or mixtures thereof. In other embodiments, the polyol may be, for example, but is not limited to, a monosaccharide, disaccharide, or polysaccharide, or a mixture of any of the foregoing. The sugar or carbohydrate can be, for example, but is not limited to, fructose, glucose, mannose, sucrose, sorbose, xylose, lactose, maltose, sucrose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch, water soluble glucan, or mixtures thereof.

[0148] In some embodiments, the polyol is selected from the group consisting of mannitol, trehalose, sorbitol, erythritol, isomalt, lactitol, maltitol, xylitol, glycerol, lactitol, propylene glycol, polyethylene glycol, inositol, fructose, glucose, mannose, sucrose, sorbose, xylose, lactose, maltose, sucrose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch, water-soluble glucan, or mixtures thereof. In some embodiments, the polyol is sucrose or trehalose. In some embodiments, the polyol is sucrose.

[0149] The concentration of the polyol in the formulation ranges from about 1 mg / mL to about 300 mg / mL, from about 1 mg / mL to about 200 mg / mL, or from about 1 mg / mL to about 120 mg / mL. Preferably, the concentration of the polyol in the formulation is from about 50 mg / mL to about 120 mg / mL, from about 60 mg / mL to about 110 mg / mL, or from about 80 mg / mL to about 90 mg / mL. For example, the concentration of the polyol in the formulation may be about 0.5 mg / mL, about 1 mg / mL, about 2 mg / mL, about 2.5 mg / mL, about 3 mg / mL, about 3.5 mg / mL, about 4 mg / mL, about 4.5 mg / mL, about 5 mg / mL, about 5.5 mg / mL, about 6 mg / mL, about 6.5 mg / mL, about 7 mg / mL, about 7.5 mg / mL, about 8 mg / mL, about 8.5 mg / mL, about 9 mg / mL, about 9.5 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, Approximately 13mg / mL, approximately 14mg / mL, approximately 15mg / mL, approximately 16mg / mL, approximately 17mg / mL, approximately 18mg / mL, approximately 19mg / mL, approximately 20mg / mL, approximately 21mg / mL, approximately 22mg / mL, approximately 23mg / mL, approximately 24m g / mL, approximately 25 mg / mL, approximately 26 mg / mL, approximately 27 mg / mL, approximately 28 mg / mL, approximately 29 mg / mL, approximately 30 mg / mL, approximately 31 mg / mL, approximately 32 mg / mL, approximately 33 mg / mL, approximately 34 mg / mL, approximately 35 mg / mL, Approximately 36mg / mL, approximately 37mg / mL, approximately 38mg / mL, approximately 39mg / mL, approximately 40mg / mL, approximately 41mg / mL, approximately 42mg / mL, approximately 43mg / mL, approximately 44mg / mL, approximately 45mg / mL, approximately 46mg / mL, approximately 47m g / mL, approximately 48 mg / mL, approximately 49 mg / mL, approximately 50 mg / mL, approximately 51 mg / mL, approximately 52 mg / mL, approximately 53 mg / mL, approximately 54 mg / mL, approximately 55 mg / mL, approximately 56 mg / mL, approximately 57 mg / mL, approximately 58 mg / mL, Approximately 59mg / mL, approximately 60mg / mL, approximately 65mg / mL, approximately 70mg / mL, approximately 75mg / mL, approximately 80mg / mL, approximately 81mg / mL, approximately 82mg / mL, approximately 83mg / mL, approximately 84mg / mL, approximately 85mg / mL, approximately 86m g / mL, approximately 87 mg / mL, approximately 88 mg / mL, approximately 89 mg / mL, approximately 90 mg / mL, approximately 91 mg / mL, approximately 92 mg / mL, approximately 93 mg / mL, approximately 94 mg / mL, approximately 95 mg / mL, approximately 96 mg / mL, approximately 97 mg / mL,Approximately 98 mg / mL, approximately 99 mg / mL, approximately 100 mg / mL, approximately 101 mg / mL, approximately 102 mg / mL, approximately 103 mg / mL, approximately 104 mg / mL, approximately 105 mg / mL, approximately 106 mg / mL, approximately 107 mg / mL, approximately 108 mg / mL, approximately 109 mg / mL, approximately 110 mg / mL, approximately 111 m g / mL, approximately 112 mg / mL, approximately 113 mg / mL, approximately 114 mg / mL, approximately 115 mg / mL, approximately 116 mg / mL, approximately 117 mg / mL, approximately 118 mg / mL, approximately 119 mg / mL, approximately 120 mg / mL, approximately 121 mg / mL, approximately 122 mg / mL, approximately 123 mg / mL, approximately 124 mg / mL , about 125 mg / mL, about 126 mg / mL, about 127 mg / mL, about 128 mg / mL, about 129 mg / mL, about 130 mg / mL, about 131 mg / mL, about 132 mg / mL, about 133 mg / mL, about 134 mg / mL, about 135 mg / mL, about 136 mg / mL, about 137 mg / mL, about 138 mg / mL, about 139 mg / mL, about 140 mg / mL, about 141 mg / mL, about 142 mg / mL, about 143 mg / mL, about 144 mg / mL, about 145 mg / mL, about 146 mg / mL, about 147 mg / mL, about 148 mg / mL, about 149 mg / mL, or about 150 mg / mL.

[0150] In some embodiments, the polyol is sucrose at a concentration of about 1 mg / mL to about 300 mg / mL, about 1 mg / mL to about 200 mg / mL, or about 1 mg / mL to about 120 mg / mL. Preferably, the concentration of sucrose in the formulation is about 50 mg / mL to about 120 mg / mL, about 60 mg / mL to about 110 mg / mL, or about 80 mg / mL to about 90 mg / mL. In some embodiments, the concentration of sucrose in the formulation is about 85 mg / mL. In some embodiments, the concentration of sucrose in the formulation is about 84 mg / mL. In some embodiments, the concentration of sucrose in the formulation is about 90 mg / mL. In some embodiments, the polyol is trehalose at a concentration of about 84 mg / mL or about 85 mg / mL or about 90 mg / mL.

[0151] The surfactant used in the present invention can change the surface tension of the liquid antibody formulation. In certain embodiments, the surfactant reduces the surface tension of the liquid antibody formulation. In yet other embodiments, the surfactant can contribute to improving the stability of any antibody in the formulation. The surfactant can also reduce the aggregation of the formulated antibody (e.g., during transportation and storage) and / or minimize the formation of particulates in the formulation and / or reduce adsorption (e.g., adsorption to the container). For example, the surfactant can also improve the stability of the antibody during and after freeze / thaw cycles. Surfactants include, for example, polysorbates, poloxamers, tritons, sodium dodecyl sulfate, sodium lauryl sulfate, sodium octyl glucoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, lauroamidopropyl-betaine, cocamidopropyl-betaine ... The surfactant may be, but is not limited to, amidopropyl-betaine, myristamidopropyl-betaine, palmidopropyl-betaine, isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmidopropyl-dimethylamine, isostearamidopropyl-dimethylamine, sodium methyl cocoyl-taurate, disodium methyl oleyl-taurate, dihydroxypropyl PEG5 linoleic ammonium chloride, polyethylene glycol, polypropylene glycol, and mixtures thereof. The surfactant may be, but is not limited to, polysorbate 20, polysorbate 21, polysorbate 40, polysorbate 60, polysorbate 61, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, PEG3350, and mixtures thereof.

[0152] The concentration of the surfactant is generally from about 0.01 mg / mL to about 10 mg / mL, from about 0.01 mg / mL to about 5.0 mg / mL, from about 0.01 mg / mL to about 2.0 mg / mL, from about 0.01 mg / mL to about 1.5 mg / mL, from about 0.01 mg / mL to about 1.0 mg / mL, from about 0.01 mg / mL to about 0.5 mg / mL, from about 0.01 mg / mL to about 0.4 ... 0.01 mg / mL to about 0.3 mg / mL, about 0.01 mg / mL to about 0.2 mg / mL, about 0.01 mg / mL to about 0.15 mg / mL, about 0.01 mg / mL to about 0.1 mg / mL, about 0.01 mg / mL to about 0.05 mg / mL, about 0.1 mg / mL to about 1 mg / mL, about 0.1 mg / mL to about 0.5 mg / mL, or about 0.1 mg / mL to about 0.3 mg / mL. More preferably, the concentration of the surfactant is about 0.05 mg / mL, about 0.06 mg / mL, about 0.07 mg / mL, about 0.08 mg / mL, about 0.09 mg / mL, about 0.1 mg / mL, about 0.15 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, or about 1 mg / mL.

[0153] In some embodiments, the polysorbate is at a concentration ranging from about 0.1 mg / mL to about 0.3 mg / mL, e.g., 0.2 mg / mL polysorbate 80. Chelators used in the present invention reduce the formation of reduced oxygen species, reduce the formation of acidic species (e.g., deamidation), reduce antibody aggregation, and / or reduce antibody fragmentation, and / or reduce antibody oxidation in the formulations of the present invention. For example, the chelator can be a multidentate ligand that forms at least one bond (e.g., covalent, ionic, or other) to a metal ion and acts as a stabilizer, forming complexes with species that might otherwise promote instability.

[0154] In some embodiments, the chelating agent may be selected from the group consisting of aminopolycarboxylic acids, hydroxyaminocarboxylic acids, N-substituted glycines, 2-(2-amino-2-oxoctyl)aminoethanesulfonic acid (BES), deferoxamine (DEF), citric acid, niacinamide, and deoxycholate, and mixtures thereof. In some embodiments, the chelating agent may be selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid 5 (DTPA), nitrilotriacetic acid (NTA), N-2-acetamido-2-iminodiacetic acid (ADA), bis(aminoethyl)glycol ether, N,N,N',N'-tetraacetic acid (EGTA), trans-diaminocyclohexanetetraacetic acid (DCTA), glutamic acid, and aspartic acid, N-hydroxyethyliminodiacetic acid (HIMDA), N,N-bis-hydroxyethylglycine (bicine), and N-(trishydroxymethylmethyl)10glycine (trishydroxymethylmethyl). sine), glycylglycine, sodium deoxycholate, ethylenediamine, propylenediamine, diethylenetriamine, triethylenetetraamine (triene), disodium edetate dihydrate (or disodium EDTA dihydrate or disodium EDTA salt), calcium EDTA, oxalic acid, malic acid, citric acid, citric acid monohydrate, and trisodium citrate dihydrate, 8-hydroxyquinolate, amino acids, histidine, cysteine, methionine, peptides, polypeptides, and proteins, and mixtures thereof. In some embodiments, the chelating agent is selected from the group consisting of salts of EDTA, including, but not limited to, dipotassium edetate, disodium edetate, calcium disodium edetate, sodium edetate, trisodium edetate, and potassium edetate, or mixtures thereof; a suitable salt of deferoxamine (DEF) is deferoxamine mesylate (DFM). The chelating agents used in the present invention, where possible, may exist as the free acid or free base form or salt form of the compound, and as the anhydrous, solvated or hydrated form of the compound or its corresponding salt.

[0155] In some embodiments, the chelating agent is EDTA. In some embodiments, the chelating agent is a salt of EDTA. In some embodiments, the chelating agent is edetate disodium dihydrate.

[0156] The concentration of the chelating agent generally ranges from about 0.01 mg / mL to about 50 mg / mL, from about 0.1 mg / mL to about 10.0 mg / mL, from about 5 mg / mL to about 15.0 mg / mL, from about 0.01 mg / mL to about 1.0 mg / mL, from about 0.02 mg / mL to about 0.5 mg / mL, or from about 0.025 mg / mL to about 0.075 mg / mL. More preferably, the concentration of the chelating agent is generally from about 0.01 mM to about 2.0 mM, from about 0.01 mM to about 1.5 mM, from about 0.01 mM to about 0.5 mM, from about 0.01 mM to about 0.4 mM, from about 0.01 mM to about 0.3 mM, from about 0.01 mM to about 0.2 mM, from about 0.01 mM to about 0.15 mM, from about 0.01 mM to about 0.1 mM, from about 0.01 mM to about 0.2 mM, from about 0.01 mM to about 0.15 mM, from about 0.01 mM to about 0.2 ... about 0.09 mM, about 0.01 mM to about 0.08 mM, about 0.01 mM to about 0.07 mM, about 0.01 mM to about 0.06 mM, about 0.01 mM to about 0.05 mM, about 0.01 mM to about 0.04 mM, about 0.01 mM to about 0.03 mM, about 0.01 mM to about 0.02 mM, about 0.02 to about 0.05 mM to about 0.01 mM. Preferably, the concentration of the chelating agent can be about 0.01 mg / mL, about 0.02 mg / mL, about 0.025 mg / mL, about 0.03 mg / mL, about 0.04 mg / mL, about 0.05 mg / mL, about 0.06 mg / mL, about 0.07 mg / mL, about 0.075 mg / mL, about 0.08 mg / mL, about 0.09 mg / mL, about 0.10 mg / mL, or about 0.20 mg / mL. More preferably, the concentration of the chelating agent is about 0.025 mg / mL, about 0.03 mg / mL, about 0.035 mg / mL, about 0.04 mg / mL, about 0.045 mg / mL, about 0.05 mg / mL, about 0.055 mg / mL, about 0.06 mg / mL, about 0.065 mg / mL, about 0.07 mg / mL, or about 0.075 mg / mL. Most preferably, the concentration of the chelating agent is about 0.05 mg / mL.

[0157] In some embodiments, the chelating agent is edetate disodium dihydrate at a concentration of about 0.05 mg / mL. In some embodiments, the chelating agent is ethylenediaminetetraacetic acid (EDTA) at a concentration of about 0.05 mg / mL.

[0158] In some embodiments, the pH may range from about pH 5.0 to about 6.6, preferably between about pH 5.0 and 6.5 or about 5.0 and 6.0, and most preferably between pH 5.2 and 5.8. In some embodiments, the pH of the formulations of the present invention may be within a range selected from between about any one of pH 5.2, 5.3, 5.4, 5.5, or 5.6 and about any one of pH 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, or 5.7. In some embodiments, the pH may be selected from any of the following pH values: about pH 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5. In some embodiments, the pH is pH 5.8+ / -0.5, and in some embodiments, the pH is pH 5.8+ / -0.3.

[0159] In some embodiments, the formulation may include a preservative. Preferably, the preservative is selected from phenol, m-cresol, benzyl alcohol, benzalkonium chloride, benzalthonium chloride, phenoxyethanol, and methylparaben.

[0160] The concentration of the preservative generally ranges from about 0.001 mg / mL to about 50 mg / mL, from about 0.005 mg / mL to about 15.0 mg / mL, from about 0.008 mg / mL to about 12.0 mg / mL, or from about 0.01 mg / mL to about 10.0 mg / mL. Preferably, the concentration of the preservative may be about 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL about 1.0 mg / mL, 2.0 mg / mL, 3.0 mg / mL, about 4.0 mg / mL, about 5.0 mg / mL, about 6.0 mg / mL, about 7.0 mg / mL, 8.0 mg / mL, 9.0 mg / mL about 9.1 mg / mL, about 9.2 mg / mL, 9.3 mg / mL, 9.4 mg / mL, 9.5 mg / mL, 9.6 mg / mL, 9.7 mg / mL, 9.8 mg / mL, 9.9 mg / mL, 10.0 mg / mL. Most preferably, the concentration of the preservative is about 0.1 mg / mL or 9.0 mg / mL.

[0161] In some embodiments, the formulation is preservative-free. In some embodiments, the antibody may be selected from the group consisting of monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, Fc, ScFv, etc.), chimeric antibodies, bispecific antibodies, heteroconjugate antibodies, single chain (ScFv), variants thereof, fusion proteins comprising antibody portions (e.g., domain antibodies), humanized antibodies, human antibodies, and other modified configurations of immunoglobulin molecules comprising an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. The antibody may be of murine, rat, human, or any other origin (including chimeric or humanized antibodies). In some embodiments, the antibody is human. Preferably, the antibody is isolated, and more preferably, it is substantially pure. If the antibody is an antibody fragment, it preferably retains the functional characteristics of the original antibody, i.e., ligand binding and / or antagonist or agonist activity.

[0162] In some embodiments, the antibody heavy chain constant region can be from any type of constant region, such as IgG, IgM, IgD, IgA, and IgE; and any isotype, such as IgG1, IgG2, IgG3, IgG4, etc. Preferably, the antibody is an IgG1 antibody.

[0163] According to further aspects of the present invention, there is provided a formulation comprising or consisting of: about 15 mg / mL to about 250 mg / mL of tissue factor pathway inhibitor (TFPI) antibody; about 1 mM to about 100 mM buffer; about 1 mg / mL to about 300 mg / mL of polyol; about 0.01 mg / mL to about 10 mg / mL of surfactant; about 0.01 mg / mL to about 50.0 mg / mL of chelating agent; wherein the formulation has a pH of about 5.0 to about 6.0. In some embodiments, the antibody comprises (i) a heavy chain variable region (VH) comprising: (a) a VH complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 13; (b) a VH complementarity determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 14; and (c) a VH complementarity determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 15; and (ii) a light chain variable region (VL) comprising: (a) a VL complementarity determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 8; (b) a VL complementarity determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 9; and (c) a VL complementarity determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-TFPI antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 18, and a VL region comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-TFPI antibody is TFPI-106 (also known as PF-06741086 or marstacimab). In some embodiments, the buffer is a histidine buffer, the polyol is sucrose or trehalose, the surfactant is a polysorbate (e.g., polysorbate 80), and / or the chelating agent is EDTA or disodium edetate dihydrate. In some embodiments, the buffer is a histidine buffer, the polyol is sucrose, the surfactant is polysorbate 80, and the chelating agent is disodium edetate dihydrate.

[0164] In some embodiments, the formulations of anti-TFPI antibody drug substances disclosed herein may be stored in sterile ethylene vinyl acetate (EVA) bags with ethylene vinyl acetate monolithic (EVAM) product contact surfaces. In some embodiments, the formulations of anti-TFPI antibody drug substances disclosed herein may be stored in sterile high density polyethylene bottles (HDPE). In some embodiments, the formulations of anti-TFPI antibody drug substances disclosed herein may be stored in stainless steel containers. In some embodiments, the formulations of anti-TFPI antibody pharmaceuticals disclosed herein may be stored in sterile vials or pre-filled syringes (PFS). In some embodiments, the formulations of anti-TFPI antibody pharmaceuticals disclosed herein may be stored in pre-filled pens (PFP).

[0165] According to further aspects of the invention, there are provided formulations comprising or consisting of: about 100 mg / mL, about 110 mg / mL, about 115 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, about 158 ​​mg / mL, about 160 mg / mL, or about 200 mg / mL of a tissue factor pathway inhibitor antibody (e.g., a human anti-TFPI antibody); about 1 mM to about 100 mM buffer; about 1 mg / mL to about 300 mg / mL of a polyol; about 0.01 mg / mL to about 10 mg / mL of a surfactant; about 0.01 mg / mL to about 1.0 mg / mL of a chelating agent; wherein the formulation has a pH of about 5.0 to about 6.0. In some embodiments, the antibody comprises (i) a heavy chain variable region (VH) comprising: (a) a VH complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 13; (b) a VH complementarity determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 14; and (c) a VH complementarity determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 15; and (ii) a light chain variable region (VL) comprising: (a) a VL complementarity determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 8; (b) a VL complementarity determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 9; and (c) a VL complementarity determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-TFPI antibody is TFPI-106 (also known as PF-06741086 or marstacimab).

[0166] According to further aspects of the invention, there is provided a formulation comprising or consisting of: about 50 mg / mL to about 160 mg / mL of a tissue factor pathway inhibitor antibody (e.g., a human anti-TFPI antibody); about 20 mM buffer; about 1 mg / mL to about 120 mg / mL of a polyol; about 0.1 mg / mL to about 0.3 mg / mL of a surfactant; about 0.025 mg / mL to about 0.075 mg / mL of a chelating agent; wherein the formulation has a pH of about 5.0 to about 6.0. In some embodiments, the antibody comprises (i) a heavy chain variable region (VH) comprising: (a) a VH complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 13; (b) a VH complementarity determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 14; and (c) a VH complementarity determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 15; and (ii) a light chain variable region (VL) comprising: (a) a VL complementarity determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 8; (b) a VL complementarity determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 9; and (c) a VL complementarity determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-TFPI antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 18, and a VL region comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-TFPI antibody is TFPI-106 (also known as PF-06741086 or marstacimab). In some embodiments, the buffer is a histidine buffer, the polyol is sucrose, the chelating agent is edetate disodium dihydrate, and / or the surfactant is polysorbate 80. In some embodiments, the concentration of the antibody is 100 mg / mL, 115 mg / mL, 150 mg / mL, or 158 mg / mL.

[0167] In some embodiments, an aqueous formulation is provided comprising or consisting of: about 150 mg / mL of a tissue factor pathway inhibitor antibody (e.g., a human anti-TFPI antibody); about 20 mM histidine buffer; about 85 mg / mL of sucrose; about 0.2 mg / mL of polysorbate 80; about 0.05 mg / mL of edetate disodium dihydrate; wherein the formulation has a pH of about 5.8. In some embodiments, the antibody comprises (i) a heavy chain variable region (VH) comprising: (a) a VH complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 13; (b) a VH complementarity determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 14; and (c) a VH complementarity determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 15; and (ii) a light chain variable region (VL) comprising: (a) a VL complementarity determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 8; (b) a VL complementarity determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 9; and (c) a VL complementarity determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-TFPI antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 18, and a VL region comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-TFPI antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19, and a light chain comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-TFPI antibody is TFPI-106 (also known as PF-06741086 or marstacimab).

[0168] In some embodiments, the formulations described herein have a shelf life of at least about 1 month, about 3 months, about 6 months, about 12 months, about 18 months, about 24 months, about 30 months, about 36 months, about 42 months, about 48 months, or about 60 months or more (e.g., at -20°C, 5°C, 25°C, or 40°C). For example, in some embodiments, the formulations of the present invention have a shelf life of at least about 1 month, about 3 months, about 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, or more (e.g., at -20°C, 5°C, 25°C, or 40°C). In some embodiments, the formulations described herein have a shelf life of at least about 24 months at 5±3° C.

[0169] In some embodiments, the formulations described herein have less than about 7% HMMS (e.g., as measured by size exclusion HPLC) at up to 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months at 40° C. / 75% RH. In some embodiments, the formulations described herein have less than about 3% HMMS (e.g., as measured by size exclusion HPLC) at up to 1 month, 2 months, or 3 months at 40° C. In some embodiments, the formulations described herein have less than about 2% HMMS (e.g., as measured by size exclusion HPLC) at up to 1 month at 40° C.

[0170] In some embodiments, the formulations described herein have less than about 2% HMMS (e.g., as measured by size exclusion HPLC) at up to 24 months at 5±3° C. In some embodiments, the formulations described herein have less than about 1% HMMS (e.g., as measured by size exclusion HPLC) at up to 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 12 months, 18 months, or 24 months at 5±3° C. In some embodiments, the formulations described herein have less than about 2% fragments (e.g., as measured by reduced capillary gel electrophoresis) at up to 24 months at 5±3° C.

[0171] Unless otherwise stated, concentrations listed herein are at ambient conditions, i.e., 25° C. and atmospheric pressure. Methods for using anti-TFPI antibody preparations The formulations described herein are useful in a variety of applications, including, but not limited to, therapeutic treatment methods comprising administering a formulation of the invention to a subject in need thereof.

[0172] Exemplary therapeutic uses of the formulations of the present invention include shortening bleeding time in a subject in need thereof, treating or preventing blood clotting disorders or blood disorders (e.g., hemophilia A, hemophilia B, hemophilia C, von Willebrand's disease (vWD), factor VII deficiency, factor VIII, factor IX, or factor XI deficiency), treating or preventing thrombocytopenia, and treating or preventing platelet disorders (disorders of platelet function or number). The formulations described herein may also be used to treat uncontrollable bleeding (e.g., uncontrollable bleeding in indications such as trauma and hemorrhagic stroke). The formulations described herein may also be used for prophylactic treatments (e.g., to treat or prevent bleeding prior to surgery).

[0173] In particular, the formulations described herein can be used to treat coagulation defects or deficiencies or coagulation disorders. For example, the formulations described herein can be used to reduce or inhibit the interaction of TFPI with FXa, or to reduce TFPI-dependent inhibition of TF / FVIIa / FXa activity.

[0174] Thus, in some embodiments, the subject suffers from or is susceptible to a blood clotting disorder or blood disorder, such as: In some embodiments, the subject suffers from or is susceptible to hemophilia A, B, or C. In some embodiments, the subject suffers from or is susceptible to hemophilia A or B. In some embodiments, the subject suffers from or is susceptible to hemophilia A and has neutralizing antibodies (i.e., inhibitors) to clotting factor VIII. In some embodiments, the subject suffers from or is susceptible to hemophilia B and has neutralizing antibodies (i.e., inhibitors) to clotting factor IX. In some embodiments, the subject suffers from or is susceptible to hemophilia C and has neutralizing antibodies (i.e., inhibitors) to clotting factor XI. In some embodiments, the subject suffers from or is susceptible to von Willebrand's disease (vWD). In some embodiments, the subject suffers from or is susceptible to a platelet disorder. In some embodiments, the subject has or is susceptible to a factor VII deficiency. In some embodiments, the subject has or is susceptible to a factor XI deficiency.

[0175] The formulations comprising the TFPI antagonist antibody or antigen-binding portion described herein can be used in combination with coagulants.The present invention provides for separate, simultaneous, or sequential administration of coagulants and the formulations of the present invention.In some embodiments, the formulations described herein further comprise coagulants.Examples of coagulants include, but are not limited to, factor VIIa, factor VIII, factor IX, tranexamic acid, and bypass agents (e.g., anti-inhibitor coagulant complex or FEIBA).

[0176] In some aspects, there is provided a use of a formulation of the invention for the manufacture of a medicament for reducing bleeding time. In some embodiments, the formulation of the present invention can be directly administered into the bloodstream, muscle, tissue, fat, or viscera of a subject. Means suitable for parenteral administration include intravenous, intraocular, intravitreal, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, intraosseous, intradermal, and subcutaneous. Apparatus suitable for parenteral administration include needle (including microneedle, microprojection, dissolvable needle, and other micropore forming technology) injectors, needle-free injectors, and injection technology. In some embodiments, the formulation of the present invention is administered intravenously or subcutaneously to a subject. In some embodiments, the formulation of the present invention is administered subcutaneously to a subject. In some embodiments, the formulation of the present invention is administered intravenously to a subject.

[0177] In some embodiments, the administration pattern of the formulation of the present invention comprises administration of a dosage of the formulation once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, once every fifteen weeks, once every twenty weeks, once every twenty-five weeks, or once every twenty-six weeks. In some embodiments, the formulations described herein are administered once a month, once every two months, once every three months, once every four months, once every five months, or once every six months. In some embodiments, the formulations described herein are administered daily or weekly (i.e., once a week). The progress of this therapy is easily monitored by conventional techniques and assays. The administration schedule can vary over time.

[0178] In some embodiments, the formulations described herein are administered at an initial antibody dose of about 300 mg, followed by subsequent weekly (i.e., once per week) doses of about 150 mg. In some embodiments, the formulations described herein are administered weekly (i.e., once per week) at an antibody dose of about 150 mg. In some embodiments, the formulations described herein are administered weekly (i.e., once per week) at an antibody dose of about 300 mg. In some embodiments, the formulations described herein are administered weekly (i.e., once per week) at an antibody dose of about 450 mg.

[0179] For purposes of the present invention, the appropriate dosage of the drug will depend on the antibody used, the type and severity of the disease being treated, whether the drug is administered for prophylactic or therapeutic purposes, previous treatments, the patient's medical history and response to the drug, and the discretion of the attending physician. Typically, the clinician will administer the drug until a dosage is reached that achieves the desired result. Dosages can be determined empirically.

[0180] The following examples are offered for illustrative purposes only and are not intended to limit the scope of the invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

[0181] Working Example EXAMPLES

[0182] Stability test of anti-TFPI antibody This example demonstrates the stability of formulations containing anti-TFPI antibodies in high density polyethylene (HDPE) bottles, vials, and pre-filled syringes.

[0183] Anti-TFPI antibody at 100 mg / mL, 115 mg / mL, and 150 mg / mL was prepared in 20 mM histidine, pH 5.8, 85 mg / mL sucrose, 0.05 mg / mL edetate disodium dihydrate, 0.2 mg / mL polysorbate 80. In this example, the anti-TFPI antibody used has a heavy chain variable region having the amino acid sequence of SEQ ID NO:18, and a light chain variable region having the amino acid sequence of SEQ ID NO:11.

[0184] Anti-TFPI antibody at 115 mg / mL was packaged in HDPE bottles and stored at -20±5°C and 5±3°C for at least 3 months (ie, 12 weeks). Anti-TFPI antibodies at 100 mg / mL and 150 mg / mL were filled into type I glass vials, sealed with coated stoppers, capped with aluminum seals, and stored in an inverted orientation at 5±3°C, 25°C / 60% relative humidity (RH), and 40°C / 75% relative humidity (RH) for at least 3 months (i.e., 12 weeks). Additionally, anti-TFPI antibodies at 150 mg / mL were filled into 2.25 mL and 1 mL type I glass prefilled syringes (PFS), stoppered with coated plunger stoppers, and stored in a horizontal orientation at 5±3°C, 25°C / 60% RH, and 40°C / 75% RH for at least 3 months (i.e., 12 weeks).

[0185] Samples were then analyzed for quality attributes routinely used to monitor protein degradation. For example, analysis consisted of size-exclusion high-performance liquid chromatography (SE-HPLC) to assess high molecular weight species (HMMS), reducing and non-reducing capillary gel electrophoresis (CGE) to assess purity and fragmentation, and image capillary isoelectric focusing (iCE) to assess charge heterogeneity, including acidic, basic, and major species. Analysis also included evaluation of the relative potency of anti-TFPI antibodies using an inhibition assay.

[0186] Specific analytical procedures, including compendial and non-compendial methods, were used to evaluate the properties, purity, and relative potency of the anti-TFPI antibodies. A description of the analytical procedures is provided below. The results are summarized in Tables 1-8.

[0187] Appearance (Transparency and Color): The drug substance is evaluated for transparency and color according to current European Pharmacopoeia procedures, Ph.Eur.2.2.1 and Ph.Eur.2.2.2, respectively.

[0188] pH: Current USP Procedure <791> Analyze the pH of the drug substance according to: Protein Concentration by UV: Using a spectrophotometer, determine the concentration of test samples using absorbance at 280 nm. Use the specific absorption coefficient or absorptivity (a280) of PF-06741086 (shown below) to calculate concentration. a280 = 1.45 mL mg -1 cm -1 Imaged capillary isoelectric focusing (iCE): To assess charge heterogeneity, imaged capillary isoelectric focusing (iCE) is used. iCE separates protein species based on charge differences in a pH gradient generated by ampholytes under the influence of an electric field and is used to monitor the charge heterogeneity of the products. Protein charge species are focused in a capillary under a DC voltage and detected at 280 nm by imaging the entire capillary.

[0189] Size Exclusion HPLC (SE-HPLC): The SE-HPLC method is used to determine the purity of the product. The test sample is diluted and injected into a size exclusion column. The content of high molecular weight species (HMMS) and monomer is reported as a percentage of the total area of ​​all protein-related peaks.

[0190] Reduced Capillary Gel Electrophoresis (CGE): This method is used to determine reduced protein purity. Samples are denatured with SDS and heated in the presence of a reducing agent. Proteins are reduced to heavy and light chains, which are electrophoretically separated in a capillary containing a sieving medium and detected using UV spectroscopy. Separation allows quantification of the separated heavy and light chains and size-matched impurities. Purity is reported as a percentage of the total heavy and light chains.

[0191] Non-reducing capillary gel electrophoresis (CGE): This method is used to determine the purity of intact proteins. Samples are denatured with SDS and heated in the presence of an alkylating agent. Proteins are electrophoretically separated in a capillary containing a sieving medium and detected using UV spectroscopy. Separation allows quantification of separated intact IgG and size-matched impurities. Purity is reported as a percentage of IgG.

[0192] Lysyl-endoproteinase (Lys-C) mapping: This method is used to determine the rate of oxidation in anti-TFPI antibodies localized to Fc peptides containing methionine residues. Lysyl-endoproteinase (Lys-C) is used to digest samples of anti-TFPI antibodies into peptide fragments. The resulting peptide fragments are separated by reversed-phase HPLC. Separation of the Fc-peptides and oxidized Fc-peptides allows the rate of oxidation of anti-TFPI antibodies to be measured and monitored.

[0193] Biological activity by inhibition assay: The inhibition assay method is used to evaluate the in vitro biological activity of PF-06741086. The inhibition assay shows that PF-06741086 can inhibit the formation of the TFPI:FXa complex, resulting in an increase in free human coagulation factor Xa (FXa), thus making free FXa available for coagulation activity.

[0194] TFPI is added to the microtiter plate. Dilutions of PF-06741086 reference material and test samples are added to the microtiter plate to allow the antibody to bind to its target. FXa is then added to the microtiter plate and allowed to bind to available TFPI that is not bound by PF-06741086, forming a TFPI:FXa complex. Free FXa is detected using the chromogenic substrate Spectrozyme FXa. After addition of the substrate, the colorimetric reaction is measured spectrophotometrically.

[0195] Methods of data analysis include nonlinear curve fitting and evaluation of parallelism between standard and sample curves. Sample potency is determined from the shift in IC50 between parallel curves. The specific activity of the sample, expressed in units per PF-06741086, is calculated by multiplying the relative potency value by the number of units assigned to 1 milligram (mg) of reference material.

[0196] [Table 1]

[0197] [Table 2]

[0198] [Table 3]

[0199] [Table 4]

[0200] [Table 5]

[0201] [Table 6]

[0202] [Table 7]

[0203] [Table 8]

[0204] Data from this study show that high concentrations (100 mg / mL-150 mg / mL) of anti-TFPI antibody are stable in a formulation containing 20 mM histidine, 85 mg / mL sucrose, 0.05 mg / mL edetate disodium dihydrate, and 0.2 mg / mL polysorbate 80, pH 5.8. Results also show that different container / closure systems (HDPE bottles, vials, PFS) do not affect the stability of anti-TFPI antibody.

[0205] The study evaluated intended storage conditions of 5 ± 3 °C, accelerated storage conditions of 25 °C / 60% RH, and heat stress storage conditions of 40 °C / 75% RH. At long-term storage conditions of 5 ± 3 °C, anti-TFPI antibodies were shown to be stable for up to 24 months, with no significant differences observed in product quality attributes.

[0206] A small increase in %HMMS is observed after 3 months at 25°C / 60% RH and after 1 month at 40°C / 75% RH. After 3 months at 40°C / 75% RH, degradation was observed, but the magnitude is less than that seen with other IgG1 antibodies, such as anti-VEGF antibodies. Table 9 compares the %HMMS at 40°C / 75% RH data for anti-TFPI and anti-VEGF antibodies. A qualified product-specific SE-HPLC method was used to quantify the %HMMS for each antibody.

[0207] [Table 9]

[0208] Despite degradation, anti-TFPI antibodies maintain activity in a formulation of 20 mM histidine, 85 mg / mL sucrose, 0.05 mg / mL edetate disodium dihydrate, 0.2 mg / mL polysorbate 80, pH 5.8, for up to 24 months at 25°C / 60% RH and for 12 months at 40°C / 75% RH. EXAMPLES

[0209] Formulation robustness stability testing of anti-TFPI antibodies This example illustrates the stability of anti-TFPI antibody at 150 mg / mL in a target formulation (control formulation) and anti-TFPI antibody formulated with high or low excipient levels and pH. The formulations evaluated are shown in Table 10. The concentration of each component was confirmed by analytical testing. In this example, the anti-TFPI antibody (TFPI-106) used has a heavy chain variable region having the amino acid sequence of SEQ ID NO: 18 and a light chain variable region having the amino acid sequence of SEQ ID NO: 11.

[0210] [Table 10]

[0211] The formulations were stored for 6 months at the intended storage conditions of 5 ± 3 °C, accelerated storage conditions of 25 °C / 60% RH, and heat stress storage conditions of 40 °C / 75% RH. Samples were then analyzed for quality attributes routinely used to monitor protein degradation using methods described herein. For example, analysis consisted of size-exclusion high performance liquid chromatography (SE-HPLC) to assess high molecular weight species (HMMS), reducing and non-reducing capillary gel electrophoresis (CGE) to assess purity and fragmentation, and image capillary isoelectric focusing (iCE) to assess charge heterogeneity, such as acidic, basic, and major species. Analysis also included evaluation of the relative potency of anti-TFPI antibodies using inhibition assays. Specific analytical procedures, including compendial and non-commendatory methods, were used to assess the characteristics, purity, and relative potency of anti-TFPI antibodies. The results are summarized in Tables 11-19.

[0212] [Table 11]

[0213] [Table 12]

[0214] [Table 13]

[0215] [Table 14]

[0216] [Table 15]

[0217] [Table 16]

[0218] [Table 17]

[0219] [Table 18]

[0220] [Table 19]

[0221] The data presented in Tables 11-19 and described below demonstrate that anti-TFPI antibody formulations containing either high or low excipients or pH levels are stable after 6 months under both real-time stability conditions.

[0222] exterior After 6 months of storage at 2-8°C and 25°C (Table 18), no significant changes in color, clarity, and visible particles were observed for most formulations (A-E). After 6 months of storage at 2-8°C and 25°C, a significant increase in visible particulates was observed for formulation F without PS80.

[0223] After 6 months of storage at 40°C, significant changes in solution color were observed for all formulations (A–F). This result is expected when stored under accelerated stress conditions based on previous stability studies. These effects were most pronounced for formulations C and E, which contained higher excipient concentrations compared to the target formulation. No significant changes in solution clarity were observed. For formulations A–E, occasional visible particles were observed, which may be attributed to sample handling. After 6 months at 40°C, a significant increase in visible particles was observed in formulation F, which does not contain PS80, although the increase was less rapid than at 2–8°C and 25°C storage conditions. This finding highlights the importance of including PS80 in pharmaceutical formulations.

[0224] pH After storage at 2-8°C, 25°C, and 40°C for 6 months (Table 11), no significant change in pH was observed for all formulations (A-F).

[0225] Protein Concentration by UV After 6 months of storage at 2-8°C, 25°C, and 40°C (Table 12), protein concentrations remained within the specification of 150 mg / mL ± 15 mg / mL. A small increase in protein concentration was observed for most formulations and storage conditions, which may be due to assay variability.

[0226] Size Exclusion Chromatography (SE-HPLC) After storage at 2-8°C for 6 months (Table 13), no significant changes were observed in %HMMS and %monomer for all formulations (A-F). It was noted that peaks corresponding to low molecular weight species were not consistently observed across all formulations during storage at 2-8°C.

[0227] After 6 months of storage at 25°C, a slight increase of 1-2% in both %HMMS and %LMMS and a corresponding decrease in %monomer were observed for all formulations (A-F). All %HMMS results remained below the 5% acceptance criterion.

[0228] After 6 months of storage at 40°C, significant increases in %HMMS and %LMMS and corresponding decreases in %monomer were observed for all formulations (A-F). Formulations B (low excipients / high pH) and C (high excipients / low pH) showed the greatest decrease in %monomer, approximately 20%. These results are expected under accelerated stress conditions based on previous stability studies.

[0229] Non-reducing capillary gel electrophoresis (nrCGE) After storage at 2-8°C for 6 months (Table 14), no significant changes were observed in % IgG, % Fragments, and % Others for all formulations (A-F).

[0230] After 6 months of storage at 25°C, a slight increase of 2-3% in fragments and a corresponding decrease in %IgG were observed in all formulations (A-F). No other significant changes were observed.

[0231] After 6 months of storage at 40°C, a significant increase in fragments of >25% and a corresponding decrease in %IgG was observed in all formulations (A-F). The largest increase in %fragments of >30% was observed in formulations B (low excipients / high pH) and C (high excipients / low pH). This result is consistent with the SE-HPLC results. Small increases in %Other were observed in all formulations.

[0232] Reduced Capillary Gel Electrophoresis (rCGE) After storage at 2-8°C for 6 months (Table 15), no significant changes were observed in % heavy + light chain (HC+LC), % fragments, and % other for all formulations (A-F).

[0233] After 6 months of storage at 25°C, a slight increase in fragments of 1–2% and a corresponding decrease in %HC+LC was observed for all formulations (A–F). An additional slight increase of 1% was observed only for formulations B and E.

[0234] After 6 months of storage at 40°C, a significant increase in fragments of approximately 10% and a corresponding decrease in %HC+LC was observed in all formulations (A-F). The largest increase in %fragments was observed in formulation C (high excipients / low pH). Most formulations (A, C, D, and F) showed a small increase in %others of 2-3%. In contrast, a significant increase in %others of 7-8% was observed in formulations B and E.

[0235] Isoelectric focusing capillary electrophoresis (iCE) After 6 months of storage at 2–8°C (Table 16), a slight increase in % acidic and a corresponding decrease in % major species was observed for all formulations (A–F), while no significant change in % basic species was observed.

[0236] After 6 months of storage at 25°C, a significant increase of 10-20% acidic species and a corresponding decrease in % major species was observed in all formulations. A small increase of 2-3% in basic species was observed across most formulations. High pH formulations B and E showed the largest increase in % acidic species, approximately 18%. This finding may be explained by the pH dependence of asparagine deamidation, which has been described in detail elsewhere. 9 After 6 months of storage at 40°C, a significant increase of approximately 60% acidic species and a corresponding decrease in % major species was observed in all formulations. The largest increase in % acidic species was observed in formulation E, which can be explained by the pH dependence of asparagine deamidation. A net decrease in % basic species was observed in all formulations when comparing T=0 and T=6 month samples.

[0237] Methionine oxidation After 6 months of storage at 2-8°C (Table 17), no significant change in %MetOx was observed for all formulations (A-F). After 6 months of storage at 25°C, a slight increase in MetOx of 1-2% was observed for all formulations (A-F). After 6 months of storage at 40°C, a significant increase in MetOX of approximately 10% was observed for all formulations. The largest increase in %MetOX of approximately 20% was observed for formulation C (high excipients / low pH).

[0238] Inhibition bioassay After 6 months of storage at 2-8°C and 25°C (Table 19), no significant changes in relative potency were observed for all formulations. After 6 months of storage at 40°C, a trend toward decreased relative potency was observed for formulations A, C, D, and E, but all results remained within the acceptance criteria.

[0239] Thus, although degradation is observed, anti-TFPI antibodies maintain activity for 6 months under accelerated storage conditions of 25° C. / 60% RH and heat stress storage conditions of 40° C. / 75% RH in all formulations (Table 19).

[0240] Mixing test The agitation test was performed by filling 1 mL of formulations A-F into a long syringe with a 1 mL fixed needle. The syringe was then subjected to 48 hours of agitation stress. The agitation test was performed at ambient room temperature (approximately 22°C) with the syringe positioned horizontally and agitated using an orbital shaker set at 300 rpm. Time zero (T0) controls were not exposed to shaking stress.

[0241] After 48 h of stirring at 300 rpm, no significant changes were observed in all formulations (A-F) by SE-HPLC, nrCGE, rCGE, iCE, and methionine oxidation. A significant increase in visible particles was observed in formulation F, which does not contain PS80, confirming the importance of PS80 in DP formulations. No changes in appearance were observed in most formulations (A-E).

[0242] conclusion The objective of this study was to establish the design region and demonstrate that product quality is robust to variations in formulation composition and agitation. At the intended storage temperatures of 2–8°C, formulation variability had minimal impact on DP stability for most formulations (A–E) after 6 months of storage. A significant increase in visible and microscopic particles was observed in the formulation without PS80 (F), confirming the importance of PS80 in DP formulations. At 25°C and 40°C, changes in product quality were observed from 3 months at 25°C and 1 month at 40°C, which is expected under accelerated stress conditions based on previous stability studies. Results from storage at 40°C suggest that protein aggregation reaches a maximum in the corners of the design region (B and C) and that deamidation reaches a maximum at high pH (B and E). All formulations demonstrated robustness to agitation stress, except for formulation F, which does not contain PS80. These results indicate robustness of the DP to variations in excipient levels outside the range expected in the process. The formulation evaluated was comparable to the formulation containing the target excipient levels (control formulation A), thus demonstrating the robustness of the formulation. Without being limited thereto, the present invention includes the following aspects. [Aspect 1] A formulation comprising about 15 mg / mL to about 250 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), a buffer, a polyol, a surfactant, and a chelating agent, wherein the formulation has a pH of about 5.0 to about 6.0, and the epitope comprises residues Ile105, Arg107, and Leu131, based on the numbering of SEQ ID NO:2. [Aspect 2] The formulation of embodiment 1, wherein the buffer is a histidine or succinate buffer. [Aspect 3] The formulation of embodiment 1 or 2, wherein the concentration of the buffer is from about 0.1 mM to about 100 mM. [Aspect 4] The formulation according to any one of aspects 1 to 3, wherein the polyol is sucrose. [Aspect 5] The formulation of any one of aspects 1-4, wherein the concentration of polyol is from about 1 mg / mL to about 300 mg / mL. [Aspect 6] A formulation according to any one of aspects 1 to 5, wherein the surfactant is a polysorbate. [Aspect 7] The formulation of embodiment 6, wherein the polysorbate is polysorbate 80 (PS80). [Aspect 8] The formulation of any one of the preceding aspects, wherein the concentration of the surfactant is from about 0.01 mg / mL to about 10 mg / mL. [Aspect 9] The formulation according to any one of aspects 1 to 8, wherein the chelating agent is edetate disodium dihydrate or ethylenediaminetetraacetic acid (EDTA). [Aspect 10] The formulation of any one of aspects 1-9, wherein the concentration of the chelating agent is from about 0.01 mg / mL to about 1.0 mg / mL. [Aspect 11] The antibody, (i) a heavy chain variable region (VH) comprising: (a) a VH complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 13; (b) a VH complementarity determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 14; and (c) a VH complementarity determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 15; and (ii) a light chain variable region (VL) comprising: (a) a VL complementarity determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO:8; (b) a VL complementarity determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO:9; and (c) a VL complementarity determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO:10. 11. The formulation according to any one of aspects 1 to 10, comprising: [Aspect 12] The formulation of any one of aspects 1 to 11, wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:18, and a VL comprising the amino acid sequence of SEQ ID NO:11. [Aspect 13] The formulation of any one of embodiments 1-12, wherein the antibody comprises a VH sequence encoded by an insert present in a plasmid deposited under ATCC Accession No. PTA-122329, and a VL sequence encoded by an insert present in a plasmid deposited under ATCC Accession No. PTA-122328. [Aspect 14] The formulation according to any one of aspects 1 to 13, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:19 and a light chain comprising the amino acid sequence of SEQ ID NO:12. [Aspect 15] an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI) at about 15 mg / mL to about 250 mg / mL; about 1 mM to about 40 mM buffer; about 1 mg / mL to about 120 mg / mL of polyol; from about 0.01 mg / mL to about 1 mg / mL of a surfactant; About 0.01 mg / mL to about 1.0 mg / mL of a chelating agent A formulation comprising: Where: The formulation has a pH of about 5.0 to about 6.0; The epitope comprises residues Ile105, Arg107, and Leu131, based on the numbering of SEQ ID NO:2. [Aspect 16] an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI) at about 15 mg / mL to about 250 mg / mL; about 1 mM to about 40 mM buffer; about 1 mg / mL to about 120 mg / mL of polyol; from about 0.01 mg / mL to about 1 mg / mL of a surfactant; About 0.01 mg / mL to about 1.0 mg / mL of a chelating agent A formulation comprising: Where: The formulation has a pH of about 5.0 to about 6.0; The antibody comprises: (i) a heavy chain variable region (VH) comprising: (a) a VH complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 13; (b) a VH complementarity determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 14; and (c) a VH complementarity determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 15; and (ii) a light chain variable region (VL) comprising: (a) a VL complementarity determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 8; (b) a VL complementarity determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 9; and (c) a VL complementarity determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 10. [Aspect 17] 17. The formulation of any one of aspects 1-16, wherein the concentration of the antibody is about 100 mg / mL, about 115 mg / mL, about 150 mg / mL, or about 158 ​​mg / mL. [Aspect 18] 1. A pharmaceutical formulation comprising 150 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 20 mM histidine buffer, 85 mg / mL sucrose, 0.2 mg / mL polysorbate 80, 0.05 mg / mL edetate disodium dihydrate, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:18 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:11, and the formulation has a pH of 5.8. [Aspect 19] 1. A pharmaceutical formulation comprising 150 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 20 mM histidine buffer, 85 mg / mL sucrose, 0.2 mg / mL polysorbate 80, 0.05 mg / mL edetate disodium dihydrate, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:19 and a light chain comprising the amino acid sequence of SEQ ID NO:12, and the formulation has a pH of 5.8. [Aspect 20] 1. A pharmaceutical formulation comprising 150 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 20 mM histidine buffer, 85 mg / mL sucrose, 0.2 mg / mL polysorbate 80, 0.05 mg / mL edetate disodium dihydrate, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:17 and a light chain comprising the amino acid sequence of SEQ ID NO:12, and wherein the formulation has a pH of 5.8. [Aspect 21] 1. A pharmaceutical formulation comprising 150 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 20 mM histidine buffer, 85 mg / mL sucrose, 0.2 mg / mL polysorbate 80, 0.05 mg / mL edetate disodium dihydrate, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:21 and a light chain comprising the amino acid sequence of SEQ ID NO:12, and wherein the formulation has a pH of 5.8. [Aspect 22] 1. A pharmaceutical formulation comprising about 50 mg / mL to about 250 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 20 mM histidine buffer, 85 mg / mL sucrose, 0.2 mg / mL polysorbate 80, 0.05 mg / mL edetate disodium dihydrate, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:23 and a light chain comprising the amino acid sequence of SEQ ID NO:22, and the formulation has a pH of 5.8. [Aspect 23] 1. A pharmaceutical formulation comprising about 50 mg / mL to about 250 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 20 mM histidine buffer, 85 mg / mL sucrose, 0.2 mg / mL polysorbate 80, 0.05 mg / mL edetate disodium dihydrate, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:25 and a light chain comprising the amino acid sequence of SEQ ID NO:24, and the formulation has a pH of 5.8. [Aspect 24] 24. The formulation of any one of the preceding aspects, wherein the formulation has a shelf life at 5±3° C. of at least about 24 months. [Aspect 25] The formulation of any one of embodiments 1-23, wherein the formulation has less than about 7% HMMS (e.g., as measured by size exclusion HPLC) at 40° C. / 75% RH for up to 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months. [Aspect 26] The formulation of any one of aspects 1-23, wherein the formulation has less than about 3% HMMS (e.g., as measured by size exclusion HPLC) at 40° C. for up to 1 month, 2 months, or 3 months. [Aspect 27] The formulation of any one of aspects 1-23, wherein the formulation has less than about 2% HMMS (e.g., as measured by size exclusion HPLC) at 40° C. for up to 1 month. [Aspect 28] A method of reducing bleeding time, comprising the step of administering to a subject in need thereof a therapeutically effective amount of the formulation according to any one of aspects 1-27. [Aspect 29] A method for treating or preventing a disorder in blood coagulation or a bleeding disorder, comprising the step of administering to a subject in need thereof a therapeutically effective amount of a formulation according to any one of aspects 1-27. [Aspect 30] 1. A method for treating or preventing hemophilia A, B, or C comprising administering to a subject in need thereof A method comprising the step of administering a therapeutically effective amount of the formulation of any one of aspects 1-27. [Aspect 31] A method for treating or preventing von Willebrand's disease (vWD), comprising administering to a subject in need thereof a therapeutically effective amount of the formulation of any one of aspects 1-27. [Aspect 32] A method for reducing an activity of TFPI, comprising the step of administering to a subject in need thereof a therapeutically effective amount of the formulation according to any one of aspects 1-27. [Aspect 33] The method of any one of aspects 28-32, wherein the formulation is administered to the subject subcutaneously or intravenously. [Aspect 34] The method according to any one of aspects 28 to 33, wherein the subject is a human. [Aspect 35] 28. Use of a formulation according to any one of aspects 1 to 27 for the manufacture of a medicament for the treatment of a bleeding disorder in a subject. [Aspect 36] 30. Use of a formulation according to any one of aspects 1 to 27 for the manufacture of a medicament for the treatment of hemophilia A, B, or C in a subject.

[0243] [Table 20-1]

[0244] [Table 20-2]

[0245] [Table 20-3]

[0246] [Table 20-4]

[0247] [Table 20-5]

[0248] The various features and aspects of the invention mentioned in each section above apply mutatis mutandis to other sections as appropriate. Thus, features specified in one section may be combined with features specified in other sections as appropriate. All references cited herein, including patents, patent applications, papers, textbooks, and cited sequence accession numbers, and references cited therein, are incorporated herein by reference in their entirety. In the event that one or more of the incorporated documents and similar materials differ or conflict with this application in any respect, including but not limited to defined terms, term usage, techniques described, etc., this application shall control.

Claims

1. An antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI) at 50 mg / mL to 250 mg / mL. 10 mM to 40 mM histidine buffer; 64 mg / mL to 106 mg / mL sucrose; 0.1 mg / mL to 0.3 mg / mL polysorbate 80 (PS80); 0.038 mg / mL to 0.063 mg / mL of edetate disodium dihydrate or ethylenediaminetetraacetic acid (EDTA) A formulation comprising: Where: The formulation has a pH of 5.2 to 6.4; The antibody comprises (i) a heavy chain variable region (VH) comprising: (a) a VH complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 13; (b) a VH complementarity determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 14; and (c) a VH complementarity determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 15; and (ii) a light chain variable region (VL) comprising: (a) a VL complementarity determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO:8; (b) a VL complementarity determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO:9; and (c) a VL complementarity determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO:

10.

13. A formulation comprising:

2. 2. The formulation of claim 1, wherein the concentration of the antibody is 100 mg / mL, 115 mg / mL, 150 mg / mL, or 158 mg / mL.

3. The formulation of any one of claims 1 to 2, wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:18, and a VL comprising the amino acid sequence of SEQ ID NO:

11.

4. 4. The formulation of any one of claims 1-3, wherein the antibody comprises a VH sequence encoded by an insert present in a plasmid deposited under ATCC Accession No. PTA-122329 and a VL sequence encoded by an insert present in a plasmid deposited under ATCC Accession No. PTA-122328.

5. The formulation of any one of claims 1 to 4, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:19 and a light chain comprising the amino acid sequence of SEQ ID NO:

12.

6. A formulation comprising 150 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 20 mM histidine buffer, 85 mg / mL sucrose, 0.2 mg / mL polysorbate 80, and 0.05 mg / mL edetate disodium dihydrate, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:18 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:11, and the formulation has a pH of 5.

8.

7. A formulation comprising 150 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 20 mM histidine buffer, 85 mg / mL sucrose, 0.2 mg / mL polysorbate 80, and 0.05 mg / mL edetate disodium dihydrate, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:19 and a light chain comprising the amino acid sequence of SEQ ID NO:12, and the formulation has a pH of 5.

8.

8. A formulation comprising 150 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 20 mM histidine buffer, 85 mg / mL sucrose, 0.2 mg / mL polysorbate 80, and 0.05 mg / mL edetate disodium dihydrate, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 17 and a light chain comprising the amino acid sequence of SEQ ID NO: 12, and the formulation has a pH of 5.

8.

9. A formulation comprising 150 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 20 mM histidine buffer, 85 mg / mL sucrose, 0.2 mg / mL polysorbate 80, and 0.05 mg / mL edetate disodium dihydrate, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:21 and a light chain comprising the amino acid sequence of SEQ ID NO:12, and the formulation has a pH of 5.

8.

10. A formulation comprising 50 mg / mL to 250 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 20 mM histidine buffer, 85 mg / mL sucrose, 0.2 mg / mL polysorbate 80, and 0.05 mg / mL edetate disodium dihydrate, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:23 and a light chain comprising the amino acid sequence of SEQ ID NO:22, and the formulation has a pH of 5.

8.

11. A formulation comprising 50 mg / mL to 250 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 20 mM histidine buffer, 85 mg / mL sucrose, 0.2 mg / mL polysorbate 80, and 0.05 mg / mL edetate disodium dihydrate, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:25 and a light chain comprising the amino acid sequence of SEQ ID NO:24, and the formulation has a pH of 5.

8.

12. 12. The formulation of any one of claims 1 to 11, wherein the formulation has a shelf life of at least 24 months at 5±3°C.

13. 12. The formulation of any one of claims 1 to 11, wherein the formulation has less than 7% high molecular weight species (HMMS) as measured by size-exclusion high performance liquid chromatography (SE-HPLC) for up to 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months at 40°C / 75% relative humidity (RH).

14. 12. The formulation of any one of claims 1 to 11, wherein the formulation has less than 3% HMMS as measured by SE-HPLC at 40°C for up to 1 month, 2 months, or 3 months.

15. 12. The formulation of any one of claims 1 to 11, wherein the formulation has less than 2% HMMS as measured by SE-HPLC at 40°C for up to 1 month.

16. A formulation according to any one of claims 1 to 15 for reducing bleeding time in a subject.

17. The formulation according to any one of claims 1 to 15 for treating or preventing a disorder in blood clotting or a bleeding disorder in a subject.

18. The formulation of any one of claims 1 to 15 for treating or preventing hemophilia A, B or C in a subject.

19. The formulation of any one of claims 1 to 15 for treating or preventing von Willebrand's disease (vWD) in a subject.

20. The formulation of any one of claims 1 to 15 for reducing the activity of TFPI in a subject.

21. The formulation of any one of claims 16 to 20, which is administered subcutaneously or intravenously to a subject.

22. The formulation according to any one of claims 16 to 20, wherein the subject is a human.

23. Use of a formulation according to any one of claims 1 to 15 for the manufacture of a medicament for the treatment of a bleeding disorder in a subject.

24. 16. Use of a formulation according to any one of claims 1 to 15 for the manufacture of a medicament for the treatment of hemophilia A, B or C in a subject.

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