Treatment of gastrointestinal bleeding in patients with severe von Willebrand disease by administration of recombinant VWF

Abstract

To provide a novel method for treating gastrointestinal bleeding in a subject with severe von Willebrand Disease (VWD).SOLUTION: The present invention relates to a method for treating gastrointestinal bleeding in a subject with severe von Willebrand Disease (VWD), comprising administering to the subject at least one dose of recombinant von Willebrand Factor (rVWF) ranging from about 40 IU / kg to about 100 IU / kg, wherein the first dose further comprises recombinant Factor VIII (rFVIII).SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 530,027, filed July 7, 2017, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Coagulation disorders, such as von Willebrand's disease (VWD), generally result from defects in the coagulation cascade. Von Willebrand's disease (VWD) refers to a group of disorders caused by a deficiency in von Willebrand's factor, which helps platelets clump together and stick to the walls of blood vessels, which is necessary for normal blood clotting.

[0003] Von Willebrand's disease (VWD) is the most common bleeding disorder with an estimated prevalence of 1% (Veyradier A, et al., Medicine (Baltimore). 2016, 95(11):e3038). However, except for milder forms of the disease, only about 1 in 10,000 patients actually require treatment. Current treatments for these clotting disorders include replacement therapy using pharmaceutical preparations containing normal clotting factors.

[0004] VWF is a glycoprotein that circulates in plasma as a series of multimers ranging in size from approximately 500 to 20,000 kD. The full length of the VWF cDNA has been cloned, with the propolypeptide corresponding to amino acid residues 23-764 of the full length preproVWF (Eikenboom et al (1995) Haemophilia 1,77 90). The multimeric form of VWF is composed of 250 kD polypeptide subunits linked together by disulfide bonds. VWF mediates initial platelet adhesion to the subendothelium of the injured vessel wall, and larger multimers exhibit enhanced hemostatic activity. Multimerized VWF binds to the platelet surface glycoprotein Gp1bα through interactions in the A1 domain of VWF, promoting platelet adhesion. Other sites on VWF mediate binding to the vessel wall. Thus, VWF forms a bridge between platelets and the vessel wall that is essential for platelet adhesion and primary hemostasis under conditions of high shear stress. Normally, endothelial cells secrete larger polymeric forms of VWF, and lower molecular weight forms of VWF result from proteolytic cleavage. The extraordinarily large molecular weight multimers are stored in the Weibel-Palade bodies of endothelial cells and are released upon stimulation by agonists such as thrombin and histamine.

[0005] For patients with VWD, treatment with von Willebrand factor (VWF) replacement is recommended, especially considering the need for long-term hemostasis during major surgery (Mannucci PM and Franchini M., Haemophilia, 2017, 23(2):182-187 (Non-Patent Document 3); National Institutes of Health. National Heart, Lung, and Blood Institute. The Diagnosis, Evaluation, and Management of von Willebrand Disease NIH Publication No. 08-5832; December, 2007 (Non-Patent Document 4)). Plasma-derived VWF therapeutics contain factor VIII (FVIII), and repeated dosing can lead to accumulation of FVIII. VONVENDI® (von Willebrand Factor [recombinant], Shire, Westlake Village, CA) is the first and only recombinant VWF (rVWF) concentrate (Turecek PL, et al. Hamostaseologie. 2009; 29(suppl 1):S32-38 (Non-Patent Document 5); Mannucci PM, et al. Blood, 2013; 122(5):648-657 (Non-Patent Document 6); Gill JC, et al. Blood, 2015; 126(17):2038-2046 (Non-Patent Document 7)).

[0006] Gastrointestinal (GI) bleeding events occur in up to 20% of patients with von Willebrand disease (VWD) and are observed in association with angiodysplastic lesions in 2%-4% of patients with VWD. GI bleeding is closely associated with higher molecular weight of von Willebrand factor (VWF) and the absence of ultra-large multimers (ULM), which are most frequently seen in patients with VWD types 2A and 3. Resolution of GI bleeding usually requires higher doses and longer durations of therapy with plasma-derived VWF replacement concentrates compared to bleeding at other sites, and even then treatment may not be successful. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Veyradier A,et al.,Medicine(Baltimore).2016,95(11):e3038 [Non-Patent Document 2] Eikenboom et al (1995) Haemophilia 1,77 90 [Non-Patent Document 3] Mannucci PM and Franchini M.,Haemophilia,2017,23(2):182-187 [Non-Patent Document 4] National Institutes of Health.National Heart,Lung,and Blood Institute.The Diagnosis,Evaluation,and Management of von Willebrand Disease NIH Publication No.08-5832;December,2007 [Non-Patent Document 5] Turecek PL,et al.Hamostaseologie.2009;29(suppl 1):S32-38 [Non-Patent Document 6] Mannucci PM,et al.Blood,2013;122(5):648-657 [Non-Patent Document 7] Gill JC,et al.Blood,2015;126(17):2038-2046 Summary of the Invention

[0008] The present invention provides a method for treating gastrointestinal bleeding in a patient with severe von Willebrand disease (VWD), comprising administering to the subject at least one dose of recombinant von Willebrand factor (rVWF) in the range of about 40 IU / kg to about 100 IU / kg, the first dose further comprising recombinant factor VIII (rFVIII).

[0009] In some embodiments, the rFVIII is administered at a dose of about 20 IU / kg to about 50 IU / kg.

[0010] In some embodiments, the method includes administering to the subject a second dose of recombinant von Willebrand factor (rVWF) in the range of about 40 IU / kg to about 100 IU / kg, wherein the second dose does not include recombinant factor VIII (rFVIII).

[0011] In some embodiments, the ratio of rVWF to FVIII is about 1.5:0.8. In some embodiments, the ratio of rVWF to FVIII is about 1.3:1. In some embodiments, the ratio of rVWF to FVIII is about 1.1:0.8. In some embodiments, the ratio of rVWF to FVIII is about 1.5:1. In some embodiments, the ratio of rVWF to FVIII is about 1.1:1.2.

[0012] In some embodiments, rVWF is administered every 8 to 12 hours.

[0013] In some embodiments, 40-60 IU / kg of rVWF is administered and the gastrointestinal bleeding is minor or moderate gastrointestinal bleeding.

[0014] In some embodiments, 40-80 IU / kg of rVWF is administered and the gastrointestinal bleeding is heavy or severe gastrointestinal bleeding.

[0015] In some embodiments, the rVWF is administered every 8-12 hours for about 3 to about 7 days.

[0016] In some embodiments, 40-60 IU / kg of the rVWF is administered every 8-12 hours for about 3 to about 7 days, and the gastrointestinal bleeding is minor or moderate gastrointestinal bleeding.

[0017] In some embodiments, 40-80 IU / kg of the rVWF is administered every 8-12 hours for about 3 to about 7 days, and the gastrointestinal bleeding is heavy or severe gastrointestinal bleeding.

[0018] In some embodiments, the subject has type 3 VWD. In some embodiments, the subject has severe type 1 VWD. In some embodiments, the subject has severe type 2 VWD.

[0019] In some embodiments, the subject has been treated for at least one bleeding event within the past 12 months. In some embodiments, the subject has been treated for more than one bleeding event within the past 12 months.

[0020] [The present invention 1001] 1. A method for treating gastrointestinal bleeding in a subject suffering from severe von Willebrand disease (VWD), comprising administering to the subject at least one dose of recombinant von Willebrand factor (rVWF) in the range of 40 IU / kg to about 100 IU / kg, wherein a first dose further comprises recombinant factor VIII (rFVIII). [The present invention 1002] The method of treatment of the present invention 1001, wherein said rFVIII is administered at a dose of about 20 IU / kg to about 50 IU / kg. [The present invention 1003] The method of treatment of the present invention 1001, wherein the method further comprises administering to the subject a second dose of recombinant von Willebrand factor (rVWF) in the range of about 40 IU / kg to about 100 IU / kg, wherein the second dose does not include recombinant factor VIII (rFVIII). [The present invention 1004] The method of treatment of the present invention 1001, wherein the ratio of rVWF to FVIII is about 1.5:0.8. [The present invention 1005] The method of treatment of the present invention 1001, wherein the ratio of rVWF to FVIII is about 1.3:1. [The present invention 1006] The method of treatment of the present invention 1001, wherein the ratio of rVWF to FVIII is about 1.1:0.8. [The present invention 1007] 1001. The method of treatment of the present invention, wherein the ratio of rVWF to FVIII is about 1.5:1. [The present invention 1008] The method of treatment of the present invention 1001, wherein the ratio of rVWF to FVIII is about 1.1:1.2. [The present invention 1009] 1001. The method of treatment of the present invention, wherein said rVWF is administered every 8 to 12 hours. [The present invention 1010] The method for the treatment according to any one of claims 1001 to 1009, wherein 40 to 60 IU / kg of the rVWF is administered, and the gastrointestinal bleeding is slight or moderate gastrointestinal bleeding. [The present invention 1011] The method for the treatment of any of claims 1001 to 1009, wherein the rVWF is 40 to 80 IU / kg of rVWF, and the gastrointestinal bleeding is massive or severe gastrointestinal bleeding. [The present invention 1012] The method of any one of claims 1001 to 1009, wherein the rVWF is administered every 8 to 12 hours for about 3 to about 7 days. [The present invention 1013] The method for treatment according to any one of claims 1001 to 1009, wherein 40 to 60 IU / kg of the rVWF is administered every 8 to 12 hours for about 3 to about 7 days, and the gastrointestinal bleeding is minor or moderate gastrointestinal bleeding. [The present invention 1014] The method for treatment according to any one of claims 1001 to 1009, wherein 40 to 80 IU / kg of the rVWF is administered every 8 to 12 hours for about 3 to about 7 days, and the gastrointestinal bleeding is massive or severe gastrointestinal bleeding. [The present invention 1015] The method for treatment according to any one of claims 1001 to 1014, wherein the subject has type 3 VWD. [The present invention 1016] The method of any one of claims 1001 to 1014, wherein the subject has severe type 1 VWD. [The present invention 1017] The method of any one of claims 1001 to 1014, wherein the subject has severe type 2 VWD. [The present invention 1018] Any of the methods of claims 1001 to 1017, wherein the subject has been treated for at least one bleeding event within the past 12 months. [The present invention 1019] Any of the methods of claims 1001 to 1017, wherein the subject has been treated for more than one bleeding event within the past 12 months. Other objects, advantages and embodiments of the present invention will become apparent from the following detailed description. [Brief description of the drawings]

[0021] [Figure 1] Hemostatic efficiency of rVWF for the treatment of GI bleeding. BE is bleeding event, GI is gastrointestinal. Four patients had a total of six GI bleeding episodes. Minor and moderate BE were rated "good" if 1-2 more injections than predicted were required to control the bleeding episode and no additional VWF-containing product was required. Major BE was rated "good" if less than 1.5-fold more injections than predicted were required to control the bleeding episode and no additional VWF-containing product was required. Minor, moderate, and major BE were rated "excellent" if the number of injections given was equal to or less than the predicted number needed to treat the BE and no additional VWF-containing product was required. [Figure 2-1] The VWF nucleic acid and amino acid sequences are shown. [Figure 2-2] 1 shows the VWF nucleic acid and amino acid sequences. [Figure 2-3] The VWF nucleic acid and amino acid sequences are shown. [Figure 2-4] 1 shows the VWF nucleic acid and amino acid sequences. [Figure 2-5]1 shows the VWF nucleic acid and amino acid sequences. [Figure 2-6] 1 shows the VWF nucleic acid and amino acid sequences. [Figure 2-7] 1 shows the VWF nucleic acid and amino acid sequences. [Figure 2-8] 1 shows the VWF nucleic acid and amino acid sequences. [Figure 2-9] 1 shows the VWF nucleic acid and amino acid sequences. [Figure 2-10] 1 shows the VWF nucleic acid and amino acid sequences. [Figure 2-11] 1 shows the VWF nucleic acid and amino acid sequences. [Figure 2-12] 1 shows the VWF nucleic acid and amino acid sequences. [Figure 2-13] 1 shows the VWF nucleic acid and amino acid sequences. [Figure 2-14] 1 shows the VWF nucleic acid and amino acid sequences. [Figure 2-15] 1 shows the VWF nucleic acid and amino acid sequences. [Figure 2-16] 1 shows the VWF nucleic acid and amino acid sequences. [Figure 2-17] 1 shows the VWF nucleic acid and amino acid sequences. [Figure 2-18] 1 shows the VWF nucleic acid and amino acid sequences. [Figure 2-19] 1 shows the VWF nucleic acid and amino acid sequences. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Detailed Description of the Invention Introduction The present invention provides a method for treating gastrointestinal bleeding in a patient suffering from severe von Willebrand disease (VWD), comprising administering to the subject at least one dose of recombinant von Willebrand factor (rVWF) in the range of 40 IU / kg to about 100 IU / kg, wherein a first dose further comprises recombinant factor VIII (rFVIII).

[0023] The disclosure of PCT Application Publication No. WO2012 / 171031 is hereby incorporated by reference in its entirety for all purposes.

[0024] definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context dictates otherwise. Thus, for example, a reference to "an antibody" includes a plurality of such antibodies, a reference to "a host cell" includes a reference to one or more host cells and equivalents thereof known to those of skill in the art, and so forth. It is further noted that the claims may be drafted to exclude any element. Thus, this description is intended to serve as a guidepost for use of exclusive terminology such as "solely," "only," or "negative" limitations with regard to the recitation of claim elements.

[0025] Before the present invention is further described, it is to be understood that the invention is not limited to particular embodiments described, since such embodiments may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0026] As used herein, "rVWF" refers to recombinant VWF.

[0027] As used herein, "rFVIII" refers to recombinant FVIII.

[0028] The term "recombinant," when used with reference to, for example, a cell, or a nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein, or the alteration of a naturally occurring nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes that are not found in the native (non-recombinant) form of the cell, or expresses naturally occurring genes that are aberrantly expressed, under-expressed, or not expressed at all.

[0029] As used herein, "recombinant VWF" includes VWF obtained by recombinant DNA technology. In certain embodiments, the VWF protein of the present invention may include constructs, such as those prepared as in WO1986 / 06096 published October 23, 1986, and U.S. Patent Application Serial No. 07 / 559,509, filed July 23, 1990 in the name of Ginsburg et al., which are incorporated herein by reference for methods of producing recombinant VWF. The VWF in the present invention may include all possible forms, including monomeric and multimeric forms. It is also understood that the present invention encompasses different forms of VWF that are used in combination. For example, the VWF of the present invention may include different multimers, different derivatives, and both biologically active and non-biologically active derivatives.

[0030] In the context of the present invention, recombinant VWF includes any member of the VWF family, such as from a mammal, e.g., primate, human, monkey, rabbit, pig, rodent, mouse, rat, hamster, gerbil, dog, cat, etc., and biologically active derivatives thereof. Active mutant and variant VWF proteins are also included, as are functional fragments and fusion proteins of VWF proteins. Furthermore, the VWF of the present invention may further comprise a label that facilitates purification, detection, or both. The VWF described herein may further be modified with a therapeutic moiety or a moiety suitable for imaging in vitro or in vivo.

[0031] As used herein, "plasma-derived VWF (pdVWF)" includes all forms of the protein, including mature VWF obtained from a mammal, that has the property of in vivo stabilization, e.g., binding, of at least one FVIII molecule.

[0032] "Very polymeric VWF" or "high molecular weight VWF" refers to VWF that contains at least 10 subunits, or 12, 14, or 16 subunits, up to about 20, 22, 24, 26 subunits, or more. The term "subunit" refers to a monomer of VWF. As known in the art, it is usually a dimer of VWF that polymerizes to form higher multimers (see Turecek et al., Semin. Thromb. Hemost. 2010, 36(5):510-521, which is hereby incorporated by reference in its entirety for all purposes, particularly for all teachings related to multimer analysis of VWF).

[0033] As used herein, "factor VIII" or "FVIII" refers to any form of factor VIII molecule having typical characteristics of blood coagulation factor VIII, including all modified forms of factor VIII, whether endogenous to the patient, derived from plasma, or produced by the use of recombinant DNA techniques. Factor VIII (FVIII) exists naturally and in therapeutic preparations as a heterogeneous distribution of polypeptides resulting from a single gene product (e.g., Andersson et al., Proc. Natl. Acad. Sci. USA, 83:2979-2983 (1986)). Commercially available examples of therapeutic preparations containing factor VIII include those sold under the trade names HEMOFIL M, ADVATE, and RECOMBINATE (available from Baxter Healthcare Corporation, Deerfield, Ill., USA).

[0034] As used herein, "plasma FVIII activity" and "in vivo FVIII activity" are used interchangeably. In vivo FVIII activity, measured using standard assays, may be endogenous FVIII activity, the activity of therapeutically administered FVIII (recombinant or plasma-derived), or both endogenous and administered FVIII activity. Similarly, "plasma FVIII" refers to endogenous FVIII or administered recombinant or plasma-derived FVIII.

[0035] As used herein, "von Willebrand's disease" refers to a group of diseases caused by a deficiency of von Willebrand's factor. Von Willebrand's factor helps platelets clump together and stick to the walls of blood vessels, which is necessary for normal blood clotting. As further detailed herein, there are several types of von Willebrand's disease, including types 1, 2A, 2B, 2M, and 3.

[0036] The terms "isolated," "purified," or "biologically pure" refer to a material that is substantially or essentially free from components that normally accompany it when found in the native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide electrophoresis or high performance liquid chromatography. VWF is the predominant species present in the preparation and is substantially purified. The term "purified" in some embodiments indicates that the nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. In other embodiments, this means that the nucleic acid or protein is at least 50% pure, more preferably at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more pure. "Purify" or "purification" in other embodiments means that at least one contaminant is removed from the composition being purified. In this sense, purification does not necessarily require that the purified compound is homogeneous, e.g., 100% pure.

[0037] As used herein, "administration" (and all grammatical equivalents) includes intravenous, intramuscular, subcutaneous, oral, as a suppository, topical contact, intraperitoneal, intralesional, or intranasal administration, or implantation of a sustained release device, such as a mini-osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal routes (e.g., buccal, intranasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarticular, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like.

[0038] The term "therapeutically effective amount or dose" or "therapeutically sufficient amount or dose" or "effective or sufficient amount or dose" refers to a dose that produces the therapeutic effect for which it is administered. For example, a therapeutically effective amount of a drug useful for treating hemophilia may be an amount that can prevent or alleviate one or more symptoms associated with hemophilia. The exact dose will depend on the therapeutic purpose and will be ascertained by one of skill in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0039] As used herein, the terms "patient" and "subject" are used interchangeably and refer to a mammal, preferably a human, having or susceptible to a disease.

[0040] As used herein, the term "about" refers to an approximate range of ±10% of a specified value. For example, the term "about 20%" encompasses a range of 18-22%.

[0041] As used herein, the term "half-life" refers to the period of time it takes for the amount of a substance being degraded (or clearance from a sample or patient) to decrease by half.

[0042] I. Recombinant von Willebrand factor (rVWF) The present invention utilizes compositions comprising von Willebrand factor (rVWF) for pre-treatment of subjects with severe VWD undergoing surgery, including but not limited to major, minor, or oral surgery.

[0043] In certain embodiments, the VWF proteins of the present invention may include constructs, such as those prepared as in WO1986 / 06096, published October 23, 1986, and U.S. Patent Application Serial No. 07 / 559,509, filed July 23, 1990 in the name of Ginsburg et al., which are incorporated herein by reference for methods of producing recombinant VWF. VWF useful in the present invention includes all possible forms, including monomeric and multimeric forms. One particularly useful form of VWF is a homomultimer of at least two VWFs. The VWF protein may be a biologically active derivative, or, when used only as a stabilizer for FVIII, the VWF may be in a form that is not biologically active. It is also understood that the present invention encompasses different forms of VWF used in combination. For example, the compositions useful in the present invention may include different multimers, different derivatives, and both biologically active and non-biologically active derivatives.

[0044] In primary hemostasis, VWF acts as a bridge between platelets and certain components of the extracellular matrix, such as collagen. The biological activity of VWF in this process can be measured by various in vitro assays (Turecek et al., Semin. Thromb. Hemost. 28:149-160, 2002). The ristocetin cofactor assay is based on the aggregation of fresh or formalin-fixed platelets induced by the antibiotic ristocetin in the presence of VWF.

[0045] The degree of platelet aggregation depends on the VWF concentration and can be measured turbidimetrically, for example, by using an aggregometer (Weiss et al., J. Clin. Invest. 52:2708-2716, 1973; Macfarlane et al., Thromb. Diath. Haemorrh. 34:306-308, 1975). The second method is a collagen binding assay based on ELISA technology (Brown et Bosak, Thromb. Res. 43:303-311, 1986; Favaloro, Thromb. Haemost. 83:127-135, 2000). Microtiter plates are coated with type I or type III collagen. VWF is then bound to the collagen surface and subsequently detected using an enzyme-labeled polyclonal antibody. The last step is the substrate reaction, which can be monitored photometrically using an ELISA reader. As provided herein, the specific ristocetin cofactor activity of the VWF of the present invention (VWF:RCo) is generally described in mU / μg of VWF measured using an in vitro assay.

[0046] An advantage of the rVWF compositions of the invention compared to pdVWF is that rVWF exhibits a higher specific activity than pdVWF, in some embodiments, the rVWF of the invention has a specific activity of at least about 20, 22.5, 25, 27.5, 30, 32.5, 35, 37.5, 40, 42.5, 45, 47.5, 50, 52.5, 55, 57.5, 60, 62.5, 65, 67.5, 70, 72.5, 75, 77.5, 80, 82.5, 85, 87.5, 90, 92.5, 95, 97.5, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150 mU / μg or more.

[0047] The rVWF of the present invention is highly multimeric, comprising about 10 to about 40 subunits. In further embodiments, the multimeric rVWF produced using the methods of the present invention comprises about 10 to 30, 12 to 28, 14 to 26, 16 to 24, 18 to 22, 20 to 21 subunits. In further embodiments, the rVWF exists in multimers of various sizes, ranging from dimers to multimers of more than 40 subunits (>10 million daltons). The largest multimers provide multiple binding sites that can interact with both platelet receptors and subendothelial matrix sites of injury, and are the most hemostatically active form of VWF. Upon application of ADAMTS13, the ultra-large rVWF multimers are cleaved over time, but during production (typically by expression in cell culture), the rVWF composition of the present invention is generally not exposed to ADAMTS13 and retains its highly multimeric structure.

[0048] In one embodiment, the rVWF composition used in the methods described herein has a distribution of rVWF oligomers characterized in that 95% of the oligomers have between 6 subunits and 20 subunits. In another embodiment, the rVWF composition has a distribution of rVWF oligomers characterized in that 95% of the oligomers have a range of subunits selected from variants 458-641 found in Table 2 of International Publication No. WO 2012 / 171031, which is incorporated by reference in its entirety for all purposes.

[0049] In one embodiment, the rVWF composition can be characterized according to the percentage (%) of rVWF molecules present in a particular higher order rVWF multimer or larger multimer. For example, in one embodiment, at least 20% of the rVWF molecules in the rVWF composition used in the methods described herein are present in an oligomeric complex of at least 10 subunits. In another embodiment, at least 20% of the rVWF molecules in the rVWF composition used in the methods described herein are present in an oligomeric complex of at least 12 subunits. In yet another embodiment, the rVWF composition used in the methods provided herein has a minimum percentage (e.g., has at least X%) of rVWF molecules present in a particular higher order rVWF multimer or larger multimer (e.g., multimer of at least Y subunits) according to any one of variants 134-457 found in Tables 3-5, which are incorporated herein by reference in their entirety for all purposes.

[0050] In accordance with the above, the rVWF composition (with or without FVIII) administered to a subject generally comprises a significant percentage (%) of high molecular weight (HMW) rVWF multimers. In further embodiments, the HMW rVWF multimer composition comprises at least 10%-80% rVWF decamers or higher multimers. In further embodiments, the composition comprises about 10-95%, 20-90%, 30-85%, 40-80%, 50-75%, 60-70% decamers or higher multimers. In further embodiments, the HMW rVWF multimer composition comprises at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% decamers or higher multimers.

[0051] The evaluation of the number and proportion of rVWF multimers can be performed using methods known in the art, including but not limited to methods using electrophoresis and size exclusion chromatography to separate VWF multimers by size, as discussed, for example, by Cumming et al. (J Clin Pathol. 1993 May; 46(5): 470-473, which is incorporated herein by reference in its entirety for all purposes, particularly for all teachings related to the evaluation of VWF multimers). Such teachings may further include immunoblotting techniques (such as Western blots) that detect chemiluminescence after immunoblotting the gel with a radiolabeled antibody against VWF (see, for example, Wen et al., (1993), J. Clin. Lab. Anal., 7: 317-323, which is incorporated herein by reference in its entirety for all purposes, particularly for all teachings related to the evaluation of VWF multimers). Additional assays for VWF include the VWF:antigen (VWF:Ag), VWF:ristocetin cofactor (VWF:RCof), and VWF:collagen binding activity assays (VWF:CBA), which are often used to diagnose and classify von Willebrand disease (see, e.g., Favaloro et al., Pathology, 1997, 29(4):341-456, which is incorporated herein by reference in its entirety for all purposes, and in particular for all teachings relating to assays for VWF).

[0052] In further embodiments, the higher order rVWF multimers of the invention are stable for about 1 to about 90 hours after administration. In still further embodiments, the higher order rVWF multimers are stable for about 5 to 80, 10 to 70, 15 to 60, 20 to 50, 25 to 40, 30 to 35 hours after administration. In still further embodiments, the higher order rVWF multimers are stable for at least 3, 6, 12, 18, 24, 36, 48, 72 hours after administration. In certain embodiments, the stability of the rVWF multimers is assessed in vitro.

[0053] In one embodiment, the higher order rVWF multimers used in the compositions and methods provided herein have a half-life of at least 12 hours from administration. In another embodiment, the higher order rVWF multimers have a half-life of at least 24 hours from administration. In yet another embodiment, the higher order rVWF multimers have a half-life selected from variants 642-1045 found in Table 6 of International Publication No. WO2012 / 171031, which is incorporated by reference in its entirety for all purposes.

[0054] In certain aspects, the rVWF (recombinant or plasma-derived) used according to the present invention is not modified by any conjugation, post-translational or covalent modification. In certain embodiments, the rVWF of the present invention is not modified by water-soluble polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, polysialic acid, hydroxyethyl starch, polycarbohydrate moieties, etc.

[0055] In other aspects, the rVWF (recombinant or plasma-derived) used according to the present invention is modified through conjugation, post-translational modification, or covalent modification, including modification of N- or C-terminal residues and selected side chains, e.g., at free sulfhydryl groups, primary amines, and hydroxyl groups. In one embodiment, a water-soluble polymer is linked (directly or via a linker) to the protein through a lysine group or other primary amine. In one embodiment, the rVWF protein of the present invention may be modified by conjugation of a water-soluble polymer, including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, polysialic acid, hydroxyethyl starch, polycarbohydrate moieties, and the like.

[0056] Water-soluble polymers that can be used to modify rVWF and / or FVIII include linear or branched structures. The conjugated polymers may be directly attached to the coagulation proteins of the present invention or alternatively may be attached through a linking moiety. Non-limiting examples of protein conjugation with water-soluble polymers can be found in U.S. Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, and 4,179,337, as well as Abuchowski and Davis "Enzymes as Drugs," Holcenberg and Roberts, Eds., pp. 367-383, John Wiley and Sons, New York (1981), and Hermanson G., Bioconjugate Techniques 2nd Ed., Academic Press, Inc. 2008.

[0057] Protein conjugation may be performed by several techniques well known in the art, see for example Hermanson G., Bioconjugate Techniques 2nd Ed., Academic Press, Inc. 2008. Examples include linkages through peptide bonds between a carboxyl group on either the coagulation protein or the water-soluble polymer moiety and an amine group on the other, or through ester linkages between a carboxyl group on one side and a hydroxyl group on the other. Another linkage by which the coagulation proteins of the invention may be conjugated to water-soluble polymeric compounds is via a Schiff base between a free amino group on the polymer moiety that reacts with an aldehyde group formed at the non-reducing end of the polymer by periodate oxidation (Jennings and Lugowski, J. Immunol. 1981; 127: 1011-8; Femandes and Gregonradis, Biochim Biophys Acta. 1997; 1341; 26-34). The resulting Schiff base is reacted with NaCNBH 3 The amines can be stabilized by specific reduction with NH after prior oxidation to form secondary amines.4 The generation of a terminal free amino group on the polymer by reductive amination with Cl. Bifunctional reagents can be used to link two amino groups or two hydroxyl groups. For example, a polymer containing an amino group can be coupled to the amino group of a coagulation protein using a reagent such as BS3 (Bis(sulfosuccinimidyl)suberate / Pierce, Rockford, Ill.). In addition, heterobifunctional crosslinking reagents such as Sulfo-EMCS (N-ε-maleimidocaproyloxy)sulfosuccinimide ester / Pierce) can be used to link, for example, amine and thiol groups. In other embodiments, aldehyde reactive groups, such as PEG alkoxide and diethyl acetal of bromoacetaldehyde, PEG and DMSO and acetic anhydride, and PEG chloride and phenoxide of 4-hydroxybenzaldehyde, succinimidyl active ester, activated PEG dithiocarbonate, 2,4,5-trichlorophenyl chloroformate, and P-nitrophenyl chloroformate activated PEG, can be used for conjugation of coagulation proteins.

[0058] In some embodiments, the rVWF used in the methods of the invention is matured in vitro with furin. In a further embodiment, the furin is recombinant furin.

[0059] In a further aspect, the rVWF used in the methods of the invention is produced by expression in mammalian cell culture using methods known in the art. In a particular embodiment, the mammalian culture comprises CHO cells. In an exemplary embodiment, the rVWF of the invention comprises rVWF protein isolated from a CHO cell expression system. In a further embodiment, the propeptide removal is mediated in vitro through exposure of pro-VWF to furin, and in yet a further embodiment, the furin used for propeptide removal is recombinant furin. In yet a further embodiment, there is no fully glycosylated / ABO blood group glycan.

[0060] In yet further embodiments, the rVWF used in the methods and compositions of the present invention is expressed in a suitable eukaryotic host system. Examples of eukaryotic cells include, but are not limited to, mammalian cells such as CHO, COS, HEK293, BHK, SK-Hep, and HepG2, insect cells such as SF9 cells, SF21 cells, S2 cells, and High Five cells, and yeast cells such as Saccharomyces or Schizosaccharomyces cells. In one embodiment, the VWF can be expressed in yeast cells, insect cells, avian cells, mammalian cells, and the like, such as in a human cell line, a hamster cell line, or a mouse cell line. In one particular embodiment, the cell line is a CHO, BHK, or HEK cell line. Typically, mammalian cells, such as CHO cells from a continuous cell line, can be used to express the VWF of the present invention.

[0061] In certain embodiments, the nucleic acid sequence comprising the sequence encoding VWF can be a vector. The vector can be delivered by virus or can be a plasmid. The nucleic acid sequence encoding protein can be a specific gene or a biologically functional part thereof. In one embodiment, the protein is at least the biologically active part of VWF. A wide variety of vectors can be used for the expression of VWF, and these can be selected from eukaryotic expression vectors. Examples of vectors for eukaryotic expression include: (i) for expression in yeast, vectors such as pAO, pPIC, pYES, pMET, which use promoters such as AOX1, GAP, GAL1, AUG1, etc.; (ii) for expression in insect cells, vectors such as pMT, pAc5, pIB, pMIB, pBAC, etc., which use promoters such as PH, p10, MT, Ac5, OpIE2, gp64, polh, etc.; and (iii) for expression in mammalian cells, vectors such as pSVL, pCMV, pRc / RSV, pcDNA3, pBPV, etc., which use promoters such as CMV, SV40, EF-1, UbC, RSV, ADV, BPV, and β-actin, and vectors derived from viral systems such as vaccinia virus, adeno-associated virus, herpes virus, retrovirus, etc.

[0062] In some embodiments of the invention, the nucleic acid sequence further comprises other sequences suitable for controlled expression of proteins, such as promoter sequences, enhancers, TATA boxes, transcription initiation sites, polylinkers, restriction sites, polyA sequences, protein processing sequences, selection markers, and the like, which are commonly known to those skilled in the art.

[0063] In certain embodiments, the cell culture methods of the present invention may include the use of microcarriers. In some embodiments, the cell culture of the embodiments may be carried out in large bioreactors under conditions suitable for high cell density and large specific cell surface area to achieve protein expression. One means to provide such growth conditions is the use of microcarriers for cell culture in stirred tank bioreactors. The concept of cell growth on microcarriers was first described by van Wezel (van Wezel, AL, Nature 216:64-5 (1967)) and allows cells to attach to the surface of small solid particles suspended in the growth medium. These methods provide a high surface area to volume ratio, which results in efficient nutrient utilization. Furthermore, for expression of secreted proteins in eukaryotic cell lines, the increased surface area to volume ratio allows higher secretion levels, which results in higher protein yields in the culture supernatant. Finally, these methods facilitate the scale-up of eukaryotic expression cultures.

[0064] The cells expressing VWF may be bound to spherical or porous microcarriers during the growth of the cell culture. The microcarriers may be selected from the group of microcarriers based on dextran, collagen, plastic, gelatin, and cellulose, as well as others described in Butler (1988. In: Spier & Griffiths, Animal Cell Biotechnology 3:283-303). It is also possible to grow the cells to biomass on spherical microcarriers and subculture the cells when they reach the final fermenter biomass and before the production of the protein expressed on the porous microcarriers (or vice versa). Suitable spherical microcarriers may include smooth microcarriers, such as Cytodex™ 1, Cytodex™ 2, and Cytode™ 3 (GE Healthcare), and macroporous microcarriers, such as Cytopore™ 1, Cytopore™ 2, Cytoline™ 1, and Cytoline™ 2 (GE Healthcare).

[0065] In certain embodiments, rVWF is expressed in cells cultured in a cell culture medium that produces high molecular weight rVWF. The terms "cell culture solution", "cell culture medium(s)", and "cell culture supernatant" refer to aspects of the cell culture process that are generally known in the art. In the context of the present invention, the cell culture solution may include cell culture medium and cell culture supernatant. The cell culture medium is added exogenously to the cell culture solution, optionally together with supplements, to provide nutrients and other components for the cultivation of cells expressing VWF. The cell culture supernatant refers to the cell culture solution that contains nutrients and other components from the cell culture medium as well as products released, metabolized, and / or secreted from the cells during the cultivation. In further embodiments, the medium may be animal protein-free and chemically defined. Methods for preparing animal protein-free, chemically defined media are known in the art, e.g., in US 2008 / 0009040 and US 2007 / 0212770, both of which are incorporated herein for all purposes, particularly for all teachings related to cell culture media. "Protein-free" and related terms refer to proteins that are foreign to the cells in culture or that originate from a source other than the cells in culture, which naturally shed proteins during growth. In another embodiment, the media is polypeptide-free. In another embodiment, the media is serum-free. In another embodiment, the media is animal protein-free. In another embodiment, the media is animal component-free. In another embodiment, the media contains proteins, e.g., animal proteins from serum, such as fetal bovine serum. In another embodiment, the media is exogenously supplemented with recombinant proteins. In another embodiment, the proteins are derived from animals that are certified pathogen-free. The term "chemically defined" as used herein is intended to mean that the media is free of any supplements that are not defined, e.g., animal components, organ, gland, plant, or yeast extracts. Thus, each component of a chemically defined medium is precisely defined. In a preferred embodiment, the medium is animal component free and protein free.

[0066] In a further embodiment, after purification from mammalian cell culture, rFVIII is reconstituted before administration. In yet a further embodiment, rVWF is treated with furin before or after reconstitution. In a further embodiment, furin is recombinant furin. In yet a further embodiment, the rVWF of the present invention is not exposed to ADAMTS13, so that super-large (i.e., containing 10 or more subunits) are present in the rVWF composition of the present invention.

[0067] In certain aspects, the rVWF used in the methods of the invention is contained in a formulation containing a buffer, a sugar and / or sugar alcohol (including, but not limited to, trehalose and mannitol), a stabilizer (such as glycine), and a surfactant (such as polysorbate 80). In a further embodiment, for formulations containing rFVIII, the formulation may further comprise sodium, histidine, calcium, and glutathione.

[0068] In one embodiment, the formulation containing rVWF is lyophilized prior to administration. Lyophilization is performed using techniques common in the art and should be optimized for the composition being developed [Tang et al., Pharm Res. 21:191-200. (2004), and Chang et al., Pharm Res. 13:243-9 (1996)].

[0069] The method of preparing the pharmaceutical formulation may include one or more of the following steps: adding a stabilizing agent as described herein to the mixture before lyophilization; adding at least one agent selected from a bulking agent, an osmolality adjusting agent, and a surfactant, each of which is described herein, to the mixture before lyophilization. The lyophilized formulation, in one embodiment, is composed of at least one or more of a buffer, a bulking agent, and a stabilizing agent. In this embodiment, the use of a surfactant is evaluated and selected when aggregation during the lyophilization step or during reconstitution is an issue. An appropriate buffer is included to maintain the formulation in a stable pH range during lyophilization.

[0070] Standard reconstitution practice for lyophilized material is to add back a volume of purified water or sterile water for injection (WFI) (typically equal to the volume removed during lyophilization), although dilute solutions of antibiotics may be used in the manufacture of pharmaceutical products for parenteral administration [Chen, Drug Development and Industrial Pharmacy, 18:1311-1354 (1992)]. Thus, a method for preparing a reconstituted recombinant VWF composition is provided, comprising the step of adding a diluent to the lyophilized recombinant VWF composition of the invention.

[0071] The lyophilized material may be reconstituted as an aqueous solution with a variety of aqueous carriers, such as sterile water for injection, water containing a preservative for multiple dose use, or water containing an appropriate amount of surfactant (e.g., aqueous suspensions containing the active compound mixed with excipients suitable for the manufacture of suspensions). In various embodiments, such excipients are suspending agents, such as, but not limited to, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia, and dispersing or wetting agents are naturally occurring phosphatides, such as, but not limited to, lecithin, or condensation products of alkylene oxides with fatty acids, such as, but not limited to, polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, such as, but not limited to, heptadecaethylene-oxycetanol, or condensation products of ethylene oxide with fatty acids and partial esters derived from hexitols, such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with fatty acids and partial esters derived from hexitol anhydrides, such as, but not limited to, polyethylene sorbitan monooleate. In various embodiments, the aqueous suspension also contains one or more preservatives, such as, but not limited to, ethyl, or n-propyl, p-hydroxybenzoate.

[0072] In certain embodiments, the compositions of the invention are liquid formulations for administration with the use of a syringe or other reservoir. In further embodiments, these liquid formulations are made from lyophilized materials as described herein that are reconstituted as an aqueous solution.

[0073] In a further embodiment, the composition of the present invention further comprises one or more pharma- ceutically acceptable carriers. The term "pharmacologically" or "pharmacologically" acceptable refers to molecular entities and compositions that are stable, inhibit proteolysis, such as aggregation and cleavage products, as described below, and do not produce allergic or other adverse reactions when administered using routes known in the art. "Pharmaceutically acceptable carriers" include any and all clinically useful solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., including the agents disclosed above.

[0074] II. Production of recombinant VWF The free mature recombinant von Willebrand factor (rVWF) of the present invention can be produced by recombinant. Those skilled in the art will recognize useful methods for expressing recombinant proteins in host cells. In some examples, the method includes expressing a nucleic acid sequence encoding rVWF in a host cell, such as a CHO cell, and culturing the resulting host cell under certain conditions to produce rVWF, preproVWF, proVWF, etc.

[0075] In certain embodiments, the nucleic acid sequence comprising the sequence encoding VWF can be an expression vector. The vector can be delivered by a virus or can be a plasmid. The nucleic acid sequence encoding a protein can be a specific gene or a biologically functional part thereof. In one embodiment, the protein is at least the biologically active part of VWF. The nucleic acid sequence can further comprise other sequences suitable for regulated expression of the protein, such as promoter sequences, enhancers, TATA boxes, transcription initiation sites, polylinkers, restriction sites, polyA sequences, protein processing sequences, selection markers, and the like, which are generally known to those skilled in the art.

[0076] A wide variety of vectors can be used for the expression of VWF, and these can be selected from eukaryotic expression vectors. Examples of vectors for eukaryotic expression include: (i) for expression in yeast, vectors such as pAO, pPIC, pYES, pMET, using promoters such as AOX1, GAP, GAL1, AUG1, etc.; (ii) for expression in insect cells, vectors such as pMT, pAc5, pIB, pMIB, pBAC, etc., using promoters such as PH, p10, MT, Ac5, OpIE2, gp64, polh, etc.; and (iii) for expression in mammalian cells, vectors such as pSVL, pCMV, pRc / RSV, pcDNA3, pBPV, etc., using promoters such as CMV, SV40, EF-1, UbC, RSV, ADV, BPV, and β-actin, and vectors derived from viral systems such as vaccinia virus, adeno-associated virus, herpes virus, retrovirus, etc.

[0077] In a further aspect, the rVWF used in the method of the present invention is produced by expression in a mammalian cell culture using methods known in the art. In a particular embodiment, the mammalian culture comprises CHO cells. In a further embodiment, the rVWF is co-expressed with recombinant factor VIII (rFVIII) in the same culture. In such an embodiment, the rVWF and rFVIII are purified together (co-purified) or separately using methods known in the art. In another embodiment, the rVWF is expressed in a culture that does not contain rFVIII.

[0078] In some embodiments, rVWF is expressed and isolated from a suitable eukaryotic host system. Examples of eukaryotic cells include, but are not limited to, mammalian cells such as CHO, COS, HEK293, BHK, SK-Hep, and HepG2, insect cells such as SF9 cells, SF21 cells, S2 cells, and High Five cells, and yeast cells such as Saccharomyces or Schizosaccharomyces cells. In one embodiment, VWF can be expressed in yeast cells, insect cells, avian cells, mammalian cells, and the like, such as in a human cell line, a hamster cell line, or a mouse cell line. In one particular embodiment, the cell line is a CHO, BHK, or HEK cell line. Typically, mammalian cells, such as CHO cells from a continuous cell line, can be used to express the VWF of the present invention. In one particular case, the VWF protein is expressed and isolated from a CHO cell expression system.

[0079] VWF can be produced in a cell culture system or according to any art-recognized cell culture method. In some embodiments, cell culture can be performed in large bioreactors under conditions suitable for high cell density and large specific cell surface area to achieve protein expression. One means to provide such growth conditions is the use of microcarriers for cell culture in stirred tank bioreactors. The concept of cell growth on microcarriers was first described by van Wezel (van Wezel, AL, Nature, 1967, 216:64-5) and allows cells to attach to the surface of small solid particles suspended in the growth medium. These methods provide a high surface area to volume ratio, which results in efficient nutrient utilization. Furthermore, for expression of secreted proteins in eukaryotic cell lines, the increased surface area to volume ratio allows higher secretion levels, which results in higher protein yields in the culture supernatant. Finally, these methods facilitate the scale-up of eukaryotic expression cultures.

[0080] Cells expressing VWF can be bound to spherical or porous microcarriers during cell culture growth. Microcarriers include microcarriers based on dextran, collagen, plastic, gelatin, and cellulose, as well as Butler (1988. In: Spier & Griffiths, Animal Cell Biotechnology 3:283-303). It is also possible to grow cells to biomass on spherical microcarriers and subculture the cells when they reach final fermenter biomass and before the production of expressed protein on porous microcarriers (or vice versa). Suitable spherical microcarriers can include smooth microcarriers, such as Cytodex™ 1, Cytodex™ 2, and Cytodex™ 3 (GE Healthcare), and macroporous microcarriers, such as Cytopore™ 1, Cytopore™ 2, Cytoline™ 1, and Cytoline™ 2 (GE Healthcare).

[0081] In further embodiments, the VWF propeptide is cleaved from non-mature VWF in vitro through exposure of pro-VWF to furin. In some embodiments, the furin used for propeptide cleavage is recombinant furin.

[0082] In certain embodiments, rVWF is expressed in cells cultured in a cell culture medium that produces high molecular weight rVWF. The terms "cell culture solution", "cell culture medium(s)", and "cell culture supernatant" refer to aspects of the cell culture process that are generally known in the art. In the context of the present invention, the cell culture solution may include cell culture medium and cell culture supernatant. The cell culture medium is added exogenously to the cell culture solution, optionally together with supplements, to provide nutrients and other components for the cultivation of cells expressing VWF. The cell culture supernatant refers to the cell culture solution that contains nutrients and other components from the cell culture medium as well as products released, metabolized, and / or secreted from the cells during the cultivation. In further embodiments, the medium may be animal protein-free and chemically defined. Methods for preparing animal protein-free, chemically defined media are known in the art, e.g., in US 2006 / 0094104, US 2007 / 0212770, and US 2008 / 0009040, both of which are incorporated herein for all purposes, particularly for all teachings related to cell culture media. "Protein-free" and related terms refer to proteins that are foreign to the cells in culture or that originate from a source other than the cells in culture, which naturally shed proteins during growth. In another embodiment, the medium is polypeptide-free. In another embodiment, the medium is serum-free. In another embodiment, the medium is animal protein-free. In another embodiment, the medium is animal component-free. In another embodiment, the medium contains proteins, e.g., animal proteins from serum, such as fetal bovine serum. In another embodiment, the medium is exogenously supplemented with recombinant proteins. In another embodiment, the proteins are derived from certified pathogen-free animals. The term "chemically defined" as used herein means that the medium does not contain any undefined supplements, such as, for example, animal components, organs, glands, plants, or yeast extracts. Thus, each component of a chemically defined medium is precisely defined. In a preferred embodiment, the medium is animal component-free and protein-free.

[0083] In certain embodiments, the culture of cells expressing VWF may be maintained for at least about 7 days, or at least about 14 days, 21 days, 28 days, or at least about 5 weeks, 6 weeks, 7 weeks, or at least about 2 months, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 months, or more. The cell density at which the cell culture is maintained for the production of recombinant VWF protein will depend on the culture conditions and medium used for protein expression. One of skill in the art will be able to easily determine the optimal cell density for a cell culture producing VWF. In one embodiment, the culture is maintained at a cell density of about 0.5×10 6 ~4×10 7 In another embodiment, the cell density is maintained at about 1.0×10 6 ~Approx. 1.0×10 7 In another embodiment, the cell density is maintained at about 1.0×10 6 ~Approx. 4.0×10 6 In another embodiment, the cell density is maintained at about 1.0×10 6 ~Approx. 4.0×10 6 In yet another embodiment, the cell density is maintained at about 2.0×10 cells / ml for an extended period of time. 6 ~Approx. 4.0×10 6 , or approximately 1.0 × 10 6 ~Approx. 2.5×10 6 , or about 1.5 × 10 6 ~Approx. 3.5×10 6 or any other similar range of concentrations for extended periods of time. After a suitable period in cell culture, the rVWF can be isolated from the expression system using methods known in the art.

[0084] In a particular embodiment, the cell density of the continuous cell culture for producing rVWF is 2.5×10 6 In another specific embodiment, the cell density is maintained at a concentration not exceeding 2.0×10 cells / ml for an extended period of time. 6 cells / ml, not exceeding 1.5 x 10 6 Not to exceed 1.0 x 10 cells / ml6 Not to exceed 0.5 x 10 cells / ml 6 In one embodiment, the cell density is maintained at a concentration of no more than 1.5×10 cells / ml or less. 6 cells / ml~2.5×10 6 Maintained in cells / ml.

[0085] In one embodiment of the above cell culture, the cell culture solution comprises a media supplement comprising copper, as described, for example, in U.S. Patent No. 8,852,888 and U.S. Patent No. 9,409,971, which are incorporated by reference in their entirety for all purposes, particularly for all teachings relating to cell culture methods and compositions for the production of recombinant VWF.

[0086] The polynucleotide and amino acid sequences of prepro-VWF are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively, and are available under GenBank accession numbers NM_000552 (Homo sapiens von Willebrand factor (VWF) mRNA) and NP_000543, respectively. The amino acid sequence corresponding to the mature VWF protein is shown in SEQ ID NO:3 (corresponding to amino acid sequence 764-2813 of the full-length prepro-VWF amino acid sequence). In some embodiments, the VWF exhibits at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity to the sequence of SEQ ID NO:3. In some embodiments, the rVWF of the invention exhibits at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity to SEQ ID NO:3. See, for example, U.S. Patent No. 8,597,910, U.S. Patent Publication No. 2016 / 0129090, and FIG.

[0087] A useful form of rVWF has at least the property of in vivo stabilization, e.g., binding, of at least one factor VIII (FVIII) molecule, and optionally has a pharmacologically acceptable glycosylation pattern. Particular examples include VWF that does not contain the A2 domain and is therefore resistant to proteolysis (Lankhof et al., Thromb. Haemost. 77:1008-1013, 1997) and the VWF fragment from Val449 to Asn730, which contains the glycoprotein lb binding domain and binding sites for collagen and heparin (Pietu et al., Biochem. Biophys. Res. Commun. 164:1339-1347, 1989). The determination of the ability of VWF to stabilize at least one FVIII molecule is, in one embodiment, performed in a VWF-deficient mammal according to methods known in the art.

[0088] The rVWF of the present invention can be produced by any method known in the art. One particular example is disclosed in International Publication No. WO 86 / 06096, published October 23, 1986, and US Patent Application No. 07 / 559,509, filed July 23, 1990, which are incorporated herein by reference for methods of producing recombinant VWF. Thus, methods are known in the art for (i) producing recombinant DNA by genetic engineering, e.g., via reverse transcription of RNA and / or amplification of DNA, (ii) introducing recombinant DNA into prokaryotic or eukaryotic cells by transfection, e.g., via electroporation or microinjection, (iii) culturing transformed cells, e.g., in a continuous or batch mode, (iv) expressing VWF, e.g., constitutively or upon induction, and (v) isolating VWF, e.g., from the culture medium or by harvesting transformed cells, and subsequently (vi) obtaining the produced rVWF, e.g., via anion exchange or affinity chromatography. In one embodiment, recombinant VWF is produced in transformed host cells using recombinant DNA techniques well known in the art.For example, the sequence coding for the polypeptide can be excised from DNA using suitable restriction enzymes.Alternatively, in another embodiment, DNA molecule is synthesized using chemical synthesis techniques such as phosphoramidate method.In yet another embodiment, a combination of these techniques is used.

[0089] The present invention also provides a vector encoding the polypeptide of the present invention in a suitable host. The vector comprises a polynucleotide encoding the polypeptide operably linked to a suitable expression control sequence. Methods for effecting this operably linkage, either before or after the polynucleotide is inserted into the vector, are well known. Expression control sequences include promoters, activators, enhancers, operators, ribosome binding sites, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved in the control of transcription or translation. The resulting vector with the polynucleotide therein is used to transform a suitable host. This transformation may be carried out using methods well known in the art.

[0090] Any of a large number of available and well-known host cells may be used in the practice of the present invention. The selection of a particular host is determined by several art-recognized factors, including, for example, compatibility with the selected expression vector, toxicity of the peptide encoded by the DNA molecule, transformation rate, ease of peptide recovery, expression characteristics, biosafety, and cost. These factors must be balanced with the understanding that not all host cells are equally effective in expressing a particular DNA sequence. Within these general guidelines, useful microbial host cells include, but are not limited to, bacteria, yeast and other fungi, insects, plants, mammalian (including human) cultured cells, or other hosts known in the art.

[0091] The transformed host cells are cultured under conventional fermentation conditions to express the desired compound. Such fermentation conditions are well known in the art. Finally, the polypeptide is purified from the medium or the host cells themselves by methods well known in the art.

[0092] Depending on the host cell utilized to express the compounds of the present invention, carbohydrate (oligosaccharide) groups are optionally attached to sites known to be glycosylation sites in proteins. Generally, O-linked oligosaccharides are attached to serine (Ser) or threonine (Thr) residues while N-linked oligosaccharides are attached to asparagine (Asn) residues when part of the sequence Asn-X-Ser / Thr, where X can be any amino acid except proline. X is preferably one of the 19 naturally occurring amino acids, not counting proline. The structures of the N-linked and O-linked oligosaccharides and sugar residues found in each type are different. One type of sugar commonly found in both N-linked and O-linked oligosaccharides is N-acetylacetylneuraminic acid (referred to as sialic acid). Sialic acid is usually the terminal residue of both N-linked and O-linked oligosaccharides and, due to its negative charge, in one embodiment confers acidity to the glycosylated compound. Such a site(s) may be incorporated into the linker of the compounds of the invention and are preferably glycosylated by the cell during recombinant production of the polypeptide compound (e.g., in mammalian cells such as CHO, BHK, COS, etc.) In other embodiments, such sites are glycosylated by synthetic or semi-synthetic procedures known in the art.

[0093] In some embodiments, sialylation (also referred to as sialylation) may be performed on a column as part of the purification procedures described herein (including anion exchange, cation exchange, size exclusion, and / or immunoaffinity methods). In some embodiments, sialylation increases the stability of rVWF compared to non-sialylated rVWF. In some embodiments, sialylation increases the stability of rVWF in the blood circulation (e.g., after administration to a subject) compared to non-sialylated rVWF. In some embodiments, the increased stability of salivated rVWF results in a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more increase compared to non-sialylated rVWF. In some embodiments, sialylation increases the half-life of rVWF compared to non-sialylated rVWF. In some embodiments, sialylation increases the half-life of rVWF in the blood circulation (e.g., after administration to a subject) compared to a non-sialylated rVWF. In some embodiments, the increased half-life of sialylated rVWF results in an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to a non-sialylated rVWF. In some embodiments, the increased half-life of sialylated rVWF results in an rVWF that is stable in the blood circulation for 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 24 hours or more (e.g., after administration to a subject) compared to a non-sialylated rVWF. In some embodiments, sialylation increases the number of 2,3 sialylations and / or 2,6 sialylations. In some embodiments, sialylation is increased by adding a 2,3 sialyltransferase and / or a 2,6 sialyltransferase and CMP-NANA (cytidine-5'-monophospho-N-acetylneuraminic acid, sodium salt) as an additional buffering step. In some embodiments, sialylation is increased by adding a 2,3 sialyltransferase and CMP-NANA (cytidine-5'-monophospho-N-acetylneuraminic acid, sodium salt) as an additional buffering step.In some embodiments, 2,3 sialylation is increased by adding a 2,3 sialyltransferase and CMP-NANA (cytidine-5'-monophospho-N-acetylneuraminic acid, sodium salt) as an additional buffering step.

[0094] In some embodiments, 2,6 sialylation is increased by adding 2,6 sialyltransferase and CMP-NANA (cytidine-5'-monophospho-N-acetylneuraminic acid, sodium salt) as an additional buffering step. In some embodiments, 2,3 sialylation and / or 2,6 sialylation is increased by adding 2,3 sialyltransferase and / or 2,6 sialyltransferase and CMP-NANA (cytidine-5'-monophospho-N-acetylneuraminic acid, sodium salt) as an additional buffering step. In some embodiments, CMP-NANA is chemically or enzymatically modified to move the modified sialic acid to a potential-free position. In some embodiments, sialylation is carried out by loading rVWF onto the resin, washing with one or more buffers described herein to deplete unwanted impurities, applying one or more buffers containing sialyltransferase and CMP-NANA at concentrations that allow additional sialylation, and washing with one or more buffers to deplete excess sialylation reagent, and eluting the enhanced rVWF (e.g., rVWF with increased sialylation) with one or more buffers. In some embodiments, the sialylation process is carried out as part of a cation exchange, anion exchange, size exclusion, or immunoaffinity purification method as described herein.

[0095] Alternatively, the compounds are made by synthetic methods, for example using solid phase synthesis techniques. Suitable techniques are well known in the art and include those described in Merrifield (1973), Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.), Merrifield (1963), J. Am. Chem. Soc. 85: 2149, Davis et al. (1985), Biochem. Intl. 10: 394-414, Stewart and Young (1969), Solid Phase Peptide Synthesis, U.S. Patent No. 3,941,763, Finn et al. (1976), The Proteins (3rd ed.) 2: 105-253, and Erickson et al. (1976), The Proteins (3rd ed.) 2: 257-527'. Solid phase synthesis is the preferred technique for making individual peptides because it is a cost-effective method for making small peptides.

[0096] Fragments, variants, and analogs of VWF can be produced according to methods well known in the art. Polypeptide fragments can be prepared using, but not limited to, enzymatic cleavage (e.g., trypsin, chymotrypsin) and recombinant means to generate polypeptide fragments having specific amino acid sequences. Polypeptide fragments can be generated that contain regions of the protein that have a specific activity, such as the multimerization domain or any other identifiable VWF domain known in the art.

[0097] Methods for making polypeptide analogs are also well known. Amino acid sequence analogs of polypeptides can be substitution, insertion, addition, or deletion analogs. Deletion analogs, including fragments of polypeptides, lack one or more residues of the native protein that are not essential for function or immunogenic activity. Insertion analogs involve, for example, the addition of an amino acid(s) at a non-terminal point in the polypeptide. This analog may include, for example, but is not limited to, the insertion of an immunoreactive epitope or simply a single residue. Addition analogs, including fragments of polypeptides, include the addition of one or more amino acids at either or both ends of the protein, including, for example, fusion proteins. Combinations of the above analogs are also contemplated.

[0098] Substitution analogs are typically designed to replace one wild-type amino acid with another amino acid at one or more sites in a protein, and to modify one or more properties of the polypeptide without completely losing other functions or properties. In one embodiment, the substitution is a conservative substitution. A "conservative amino acid substitution" is a substitution of an amino acid sequence with an amino acid having a side chain or similar chemical properties. Similar amino acids for making conservative substitutions include those with acidic side chains (glutamic acid, aspartic acid), basic side chains (arginine, lysine, histidine), polar amine side chains (glutamine, asparagine), hydrophobic aliphatic side chains (leucine, isoleucine, valine, alanine, glycine), aromatic side chains (phenylalanine, tryptophan, tyrosine), small side chains (glycine, alanine, serine, threonine, methionine), or aliphatic hydroxyl side chains (serine, threonine).

[0099] In one embodiment, analogs are substantially homologous or substantially identical to the recombinant VWF from which they are derived. Analogs include those that retain at least some of the biological activity of the wild-type polypeptide, such as blood clotting activity.

[0100] Contemplated polypeptide variants include, but are not limited to, polypeptides that have been chemically modified by techniques such as ubiquitination, glycosylation, including polysialylation (or polysialylation), conjugation to therapeutic or diagnostic agents, labeling, covalent polymeric linkages such as pegylation (derivatization with polyethylene glycol), introduction of non-hydrolyzable bonds, and chemically synthesized insertion or substitution of amino acids that do not normally occur in human proteins, such as ornithine. The variants retain the same or essentially the same binding properties as the unmodified molecules of the invention. Such chemical modifications may include direct or indirect (e.g., via a linker) attachment of an agent to the VWF polypeptide. In the case of indirect attachment, it is contemplated that the linker may be hydrolyzable or non-hydrolyzable.

[0101] The preparation of a PEGylated polypeptide analog, in one aspect, involves (a) reacting a polypeptide with polyethylene glycol (such as a reactive ester or aldehyde derivative of PEG) under conditions that result in the conjugated construct polypeptide becoming conjugated to one or more PEG groups, and (b) obtaining a reaction product(s). In general, optimal reaction conditions for the acylation reaction are determined based on known parameters and the desired outcome. For example, the higher the PEG:protein ratio, the higher the percentage of poly-PEGylated product. In some embodiments, the conjugated construct has a single PEG moiety at the N-terminus. Polyethylene glycol (PEG) may be conjugated to blood clotting factors to, for example, provide a longer half-life in vivo. The PEG group may be of any convenient molecular weight, linear or branched. The average molecular weight of the PEG ranges from about 2 kilodaltons ("kD") to about 100 kDa, about 5 kDa to about 50 kDa, or about 5 kDa to about 10 kDa. In certain embodiments, the PEG group is attached to the blood clotting factor via acylation or reductive alkylation to a reactive group on the blood clotting factor (e.g., an aldehyde, amino, or ester group) through a natural or engineered reactive group on the PEG moiety (e.g., an aldehyde, amino, thiol, or ester group), or by any other technique known in the art.

[0102] Methods for preparing polysialylated polypeptides are described in U.S. Patent Publication No. 20060160948, Fernandes et Gregoriadis; Biochim. Biophys. Acta 1341:26-34, 1997, and Saenko et al., Haemophilia 12:42-51, 2006. Briefly, 0.1 M NaIO 4 A solution of colominic acid (CA) containing is stirred in the dark at room temperature to oxidize CA. The activated CA solution is dialyzed in the dark, for example against 0.05M sodium phosphate buffer at pH 7.2, and this solution is added to the rVWF solution and incubated in the dark at room temperature for 18 hours with gentle shaking. The free reagent is optionally isolated from the rVWF-polysialic acid conjugate, for example by ultrafiltration / diafiltration. The conjugation of rVWF with polysialic acid is achieved using glutaraldehyde as a cross-linking reagent (Migneault et al., Biotechniques 37:790-796, 2004).

[0103] In another embodiment, it is also contemplated that preproVWF and proVWF polypeptides provide therapeutic utility in the formulations of the invention. For example, U.S. Patent No. 7,005,502 describes pharmaceutical preparations containing substantial amounts of proVWF that induce thrombin generation in vitro. In addition to biologically active fragments, variants, or other analogs of recombinant naturally occurring mature VWF, the invention contemplates the use of recombinant biologically active fragments, variants, or analogs of preproVWF (as set forth in SEQ ID NO:2) or proVWF polypeptide (amino acid residues 23-764 of SEQ ID NO:2) in the formulations described herein.

[0104] Polynucleotides encoding fragments, variants, and analogs can be easily generated by those skilled in the art to encode biologically active fragments, variants, or analogs of naturally occurring molecules that have the same or similar biological activity as the naturally occurring molecules. In various embodiments, these polynucleotides are prepared using PCR techniques, digestion / ligation of DNA coding molecules, and the like. Thus, those skilled in the art can use any method known in the art, including but not limited to site-directed mutagenesis, to generate single base changes in DNA strands, resulting in modified codons and missense mutations. As used herein, the expression "moderately stringent hybridization conditions" refers to, for example, hybridization in 50% formamide at 42°C, and washing in 0.1x SSC, 0.1% SDS at 60°C. Those skilled in the art will understand that these conditions will vary based on the length and GC nucleotide base content of the sequences to be hybridized. Standard formulas in the art are appropriate for determining the exact hybridization conditions. See Sambrook et al., 9.47-9.51 in Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989).

[0105] A. VWF multimers The evaluation of the number and percentage (%) of rVWF multimers can be performed using methods known in the art, including but not limited to methods using electrophoresis and size exclusion chromatography to separate VWF multimers by size, as discussed, for example, by Cumming et al. (J Clin Pathol., 1993 May;46(5):470-473, which is incorporated herein by reference in its entirety for all purposes, particularly for all teachings related to the evaluation of VWF multimers). Such teachings may further include immunoblotting techniques (such as Western blots) that detect chemiluminescence after immunoblotting the gel with a radiolabeled antibody against VWF (see, for example, Wen et al., J.Clin.Lab.Anal., 1993,7:317-323, which is incorporated herein by reference in its entirety for all purposes, particularly for all teachings related to the evaluation of VWF multimers). Further assays for VWF include VWF:antigen (VWF:Ag), VWF:ristocetin cofactor (VWF:RCof), and VWF:collagen binding activity assays (VWF:CBA), which are often used to diagnose and classify von Willebrand's disease (see, e.g., Favaloro et al., Pathology, 1997, 29(4):341-456; Sadler, JE, Annu Rev Biochem, 1998, 67:395-424; and Turecek et al., Semin Thromb Hemost, 2010, 36:510-521, which are incorporated herein by reference in their entirety for all purposes, and in particular for all teachings relating to assays for VWF). In some embodiments, the rVWF obtained using the present method comprises any multimer pattern present in the loading sample of rVWF. In some embodiments, the rVWF obtained using this method comprises the physiologically occurring multimer pattern and the extra-large VWF multimer pattern.

[0106] b. VWF assay In primary hemostasis, VWF acts as a bridge between platelets and certain components of the extracellular matrix, such as collagen. The biological activity of VWF in this process can be measured by various in vitro assays (Turecek et al., Semin Thromb Hemost, 2010, 36:510-521).

[0107] VWF:Ristocetin cofactor (VWF:RCof) assay is based on the aggregation of fresh or formalin-fixed platelets induced by antibiotic ristocetin in the presence of VWF. The degree of platelet aggregation depends on the VWF concentration and can be measured by turbidimetry, for example, by using an aggregometer (Weiss et al., J.Clin.Invest., 1973,52:2708-2716; Macfarlane et al., Thromb.Diath.Haemorrh., 1975,34:306-308). As provided herein, the specific ristocetin cofactor activity of VWF of the present invention (VWF:RCo) is generally described in mU / μg of VWF measured using an in vitro assay.

[0108] In some embodiments, the rVWF purified according to the methods of the invention has a specific activity of at least about 20, 22.5, 25, 27.5, 30, 32.5, 35, 37.5, 40, 42.5, 45, 47.5, 50, 52.5, 55, 57.5, 60, 62.5, 65, 67.5, 70, 72.5, 75, 77.5, 80, 82.5, 85, 87.5, 90, 92.5, 95, 97.5, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150 mU / μg or more. In some embodiments, the rVWF used in the methods described herein has a specific activity of between 20 mU / μg and 150 mU / μg. In some embodiments, the rVWF has a specific activity of 30 mU / μg to 120 mU / μg. In some embodiments, the rVWF has a specific activity of 40 mU / μg to 90 mU / μg. In some embodiments, the specific activity of the rVWF is selected from variants 1 to 133 found in Table 3 below.

[0109] Table 3. Exemplary embodiments of the specific activity of rVWF found in the compositions and used in the methods provided herein. TIFF0007641327000001.tif177148Var.=Variant

[0110] The rVWF of the present invention is highly multimeric, comprising about 10 to about 40 subunits. In further embodiments, the multimeric rVWF produced using the methods of the present invention comprises about 10 to 30, 12 to 28, 14 to 26, 16 to 24, 18 to 22, 20 to 21 subunits. In some embodiments, the rVWF exists in multimers of various sizes, ranging from dimers to multimers of more than 40 subunits (>10 million daltons). The largest multimers provide multiple binding sites that can interact with both platelet receptors and subendothelial matrix sites of injury and are the most hemostatically active form of VWF. In some embodiments, the rVWF of the present invention comprises ultra-large multimers (ULM). In general, high and very large multimers are believed to be the most effective for hemostasis (see, e.g., Turecek, P., Hamostaseologie, (Vol. 37): Supplement 1, pages S15-S25 (2017)). In some embodiments, the rVWF is between 500 kDa and 20,000 kDa. In some embodiments, any desired multimer pattern can be obtained using the described methods. In some embodiments, when using anion exchange and / or cation exchange methods, the pH, conductivity, and / or counterion concentration of the buffer or gradient buffer during one or more wash step(s) can be manipulated to obtain the desired multimer pattern. In some embodiments, size exclusion chromatography methods can then be used to obtain the desired multimer pattern using collection criteria. In some embodiments, the described multimer pattern includes very large multimers. In some embodiments, the ultra-large multimers are at least 10,000 kDa, at least 11,000 kDa, at least 12,000 kDa, at least 13,000 kDa, at least 14,000 kDa, at least 15,000 kDa, at least 16,000 kDa, at least 17,000 kDa, at least 18,000 kDa, at least 19,000 kDa, at least 20,000 kDa. In some embodiments, the ultra-large multimers are between about 10,000 kDa and 20,000 kDa.In some embodiments, the ultra-large multimers are between about 11,000 kDa and 20,000 kDa. In some embodiments, the ultra-large multimers are between about 12,000 kDa and 20,000 kDa. In some embodiments, the ultra-large multimers are between about 13,000 kDa and 20,000 kDa. In some embodiments, the ultra-large multimers are between about 14,000 kDa and 20,000 kDa. In some embodiments, the ultra-large multimers are between about 15,000 kDa and 20,000 kDa. In some embodiments, the ultra-large multimers are between about 16,000 kDa and 20,000 kDa. In some embodiments, the ultra-large multimers are between about 17,000 kDa and 20,000 kDa. In some embodiments, the ultra-large multimers are between about 18,000 kDa and 20,000 kDa. In some embodiments, the ultra-large multimers are between about 19,000 kDa and 20,000 kDa. In some embodiments, the rVWF obtained using the method comprises any multimer pattern present in a loading sample of rVWF. In some embodiments, the rVWF obtained using the method comprises a physiologically occurring multimer pattern and an ultra-large VWF multimer pattern.

[0111] In some embodiments, an rVWF composition prepared by the purification methods described herein has a distribution of rVWF oligomers characterized in that 95% of the oligomers have between 6 subunits and 20 subunits. In some embodiments, an rVWF composition has a distribution of rVWF oligomers characterized in that 95% of the oligomers have a range of subunits selected from variants 458-641 found in Table 4.

[0112] Table 4. Exemplary embodiments of the distribution of rVWF oligomers found in the compositions and used in the methods provided herein. TIFF0007641327000002.tif163150TIFF0007641327000003.tif70150Var.=Variant

[0113] In some embodiments, the rVWF composition prepared by the methods provided herein can be characterized according to the percentage of rVWF molecules present in a particular higher order rVWF multimer or larger multimer. For example, in one embodiment, at least 20% of the rVWF molecules in the rVWF composition used in the methods described herein are present in an oligomeric complex of at least 10 subunits. In another embodiment, at least 20% of the rVWF molecules in the rVWF composition used in the methods described herein are present in an oligomeric complex of at least 12 subunits. In yet other embodiments, the rVWF composition used in the methods provided herein has a minimum percentage (e.g., at least X%) of rVWF molecules present in a particular higher order rVWF multimer or larger multimer (e.g., multimer of at least Y subunits) according to any one of variants 134-457 found in Tables 5-7.

[0114] Table 5. Exemplary embodiments of the percentage of rVWF molecules present in specific higher order rVWF multimers or larger multimers found in the compositions provided herein and used in the methods. TIFF0007641327000004.tif101132Var.=Var

[0115] Table 6. Exemplary embodiments of the percentage of rVWF molecules present in specific higher order rVWF multimers or larger multimers found in the compositions provided herein and used in the methods. TIFF0007641327000005.tif101132Var.=Var

[0116] Table 7. Exemplary embodiments of the percentage of rVWF molecules present in specific higher order rVWF multimers or larger multimers found in the compositions provided herein and used in the methods. TIFF0007641327000006.tif99132Var.=Variant

[0117] In accordance with the above, the rVWF comprises a significant percentage (%) of high molecular weight (HMW) rVWF multimers. In further embodiments, the HMW rVWF multimer composition comprises at least 10%-80% rVWF decamers or higher multimers. In further embodiments, the composition comprises about 10-95%, 20-90%, 30-85%, 40-80%, 50-75%, 60-70% decamers or higher multimers. In further embodiments, the HMW rVWF multimer composition comprises at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% decamers or higher multimers.

[0118] The evaluation of the number and percentage (%) of rVWF multimers can be performed using methods known in the art, including but not limited to methods using electrophoresis and size exclusion chromatography to separate rVWF multimers by size, as discussed, for example, by Cumming et al. (J Clin Pathol. 1993 May; 46(5): 470-473, which is incorporated herein by reference in its entirety for all purposes, particularly for all teachings related to the evaluation of rVWF multimers). Such teachings may further include immunoblotting techniques (such as Western blots) that detect chemiluminescence after immunoblotting the gel with a radiolabeled antibody against VWF (see, for example, Wen et al., (1993), J. Clin. Lab. Anal., 7: 317-323, which is incorporated herein by reference in its entirety for all purposes, particularly for all teachings related to the evaluation of rVWF multimers). Additional assays for VWF include the VWF:antigen (VWF:Ag), VWF:ristocetin cofactor (VWF:RCof), and VWF:collagen binding activity assays (VWF:CBA), which are often used to diagnose and classify von Willebrand's disease (see, e.g., Favaloro et al., Pathology, 1997, 29(4):341-456, which is incorporated herein by reference in its entirety for all purposes, and in particular for all teachings relating to assays for VWF).

[0119] In some embodiments, the ratio of rFVIII procoagulant activity (IU rFVIII:C) to rVWF ristocetin cofactor activity (IU rVWF:RCo) for rVWF prepared according to the method of the invention is 3:1 to 1:5. In further embodiments, the ratio is 2:1 to 1:4. In yet further embodiments, the ratio is 5:2 to 1:4. In further embodiments, the ratio is 3:2 to 1:3. In yet further embodiments, the ratio is about 1:1, 1:2, 1:3, 1:4, 1:5, 2:1, 2:3, 2:4, 2:5, 3:1, 3:2, 3:4, or 3:5. In further embodiments, the ratio is 1:1 to 1:2. In still further embodiments, the ratio is 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, or 2:1. In certain embodiments, the ratio of rFVIII procoagulant activity (IU rFVIII:C) to rVWF ristocetin cofactor activity (IU rVWF:RCo) in the compositions useful for the methods described herein is selected from variants 1988-2140 found in Table 8.

[0120] Table 8. Exemplary embodiments of the ratio of rFVIII procoagulant activity (IU rFVIII:C) to rVWF ristocetin cofactor activity (IU rVWF:RCo) in the compositions and used in the methods provided herein. TIFF0007641327000007.tif118153TIFF0007641327000008.tif196153Var.=Variant

[0121] In further embodiments, the higher order rVWF multimers of the invention are stable for about 1 to about 90 hours after administration. In still further embodiments, the higher order rVWF multimers are stable for about 5 to 80, 10 to 70, 15 to 60, 20 to 50, 25 to 40, 30 to 35 hours after administration. In still further embodiments, the higher order rVWF multimers are stable for at least 3, 6, 12, 18, 24, 36, 48, 72 hours after administration. In certain embodiments, the stability of the rVWF multimers is assessed in vitro.

[0122] In one embodiment, the higher order rVWF multimers used in the compositions and methods provided herein have a half-life of at least 12 hours after administration. In another embodiment, the higher order rVWF multimers have a half-life of at least 24 hours after administration. In yet another embodiment, the higher order rVWF multimers have a half-life selected from variants 642-1045 found in Table 9.

[0123] Table 9. Exemplary embodiments of the half-lives of higher order rVWF multimers found in compositions prepared by the methods provided herein. TIFF0007641327000009.tif162139TIFF0007641327000010.tif231139TIFF0007641327000011.tif109139Var.=Variant

[0124] In some embodiments, the pro-VWF and / or purified rVWF purified according to the invention is not modified by any conjugation, post-translational or covalent modification. In certain embodiments, the pro-VWF and / or purified rVWF of the invention is not modified with water-soluble polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, polysialic acid, hydroxyethyl starch, polycarbohydrate moieties, etc.

[0125] In some embodiments, the pro-VWF and / or purified rVWF purified according to the invention are modified through conjugation, post-translational modification, or covalent modification, including modification of N- or C-terminal residues and selected side chains, e.g., at free sulfhydryl groups, primary amines, and hydroxyl groups. In one embodiment, the water-soluble polymer is linked (directly or via a linker) to the protein by a lysine group or other primary amine. In some embodiments, the pro-VWF and / or purified rVWF of the invention may be modified by conjugation of a water-soluble polymer, including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, polysialic acid, hydroxyethyl starch, polycarbohydrate moieties, and the like.

[0126] Water-soluble polymers that may be used to modify pro-VWF and / or purified rVWF include linear and branched structures. The conjugated polymer may be directly attached to the coagulation protein of the invention or alternatively may be attached through a linking moiety. Non-limiting examples of protein conjugation with water-soluble polymers can be found in U.S. Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, and 4,179,337, as well as Abuchowski and Davis "Enzymes as Drugs," Holcenberg and Roberts, Eds., pp. 367-383, John Wiley and Sons, New York (1981), and Hermanson G., Bioconjugate Techniques 2nd Ed., Academic Press, Inc. 2008.

[0127] Protein conjugation may be performed by several techniques well known in the art, see for example Hermanson G., Bioconjugate Techniques 2nd Ed., Academic Press, Inc. 2008. Examples include linkages through peptide bonds between a carboxyl group on either the coagulation protein or the water-soluble polymer moiety and an amine group on the other, or through ester linkages between a carboxyl group on one and a hydroxyl group on the other. Another linkage by which the coagulation proteins of the invention can be conjugated to water-soluble polymeric compounds is via a Schiff base between a free amino group on the polymer moiety that reacts with an aldehyde group formed at the non-reducing end of the polymer by periodate oxidation (Jennings and Lugowski, J. Immunol. 1981; 127: 1011-8; Femandes and Gregonradis, Biochim Biophys Acta. 1997; 1341; 26-34). The resulting Schiff base is reacted with NaCNBH 3 The amines can be stabilized by specific reduction with NH after prior oxidation to form secondary amines. 4The generation of a terminal free amino group on the polymer by reductive amination with Cl. Bifunctional reagents can be used to link two amino groups or two hydroxyl groups. For example, a polymer containing an amino group can be coupled to the amino group of a coagulation protein using a reagent such as BS3 (Bis(sulfosuccinimidyl)suberate / Pierce, Rockford, Ill.). In addition, heterobifunctional crosslinking reagents such as Sulfo-EMCS (N-ε-maleimidocaproyloxy)sulfosuccinimide ester / Pierce) can be used to link, for example, amine and thiol groups. In other embodiments, aldehyde reactive groups, such as PEG alkoxide and diethyl acetal of bromoacetaldehyde, PEG and DMSO and acetic anhydride, and PEG chloride and phenoxide of 4-hydroxybenzaldehyde, succinimidyl active ester, activated PEG dithiocarbonate, 2,4,5-trichlorophenyl chloroformate, and P-nitrophenyl chloroformate activated PEG, can be used for conjugation of coagulation proteins.

[0128] Another method for measuring the biological activity of VWF is the collagen binding assay based on ELISA technology (Brown and Bosak, Thromb. Res., 1986, 43:303-311; Favaloro, Thromb. Haemost., 2000, 83 127-135). Microtiter plates are coated with type I or III collagen. VWF is then bound to the collagen surface and subsequently detected using an enzyme-labeled polyclonal antibody. The final step is the substrate reaction, which can be monitored photometrically using an ELISA reader.

[0129] Von Willebrand factor (VWF:Ag) immunoassays are immunoassays that measure the concentration of VWF protein in plasma. They give no indication of VWF function. Several methods exist for measuring VWF:Ag, including both enzyme-linked immunosorbent assays (ELISAs) or automated latex immunoassays (LIAs). Many laboratories now use fully automated latex immunoassays. In the past, laboratories used a variety of techniques, including the Laurell electroimmunoassay "Laurell Rockets," but these are rarely used in most laboratories today.

[0130] III. Kit As an additional aspect, the present invention includes a kit comprising one or more lyophilized compositions packaged in a manner that facilitates their use for administration to a subject. In one embodiment, such a kit includes a pharmaceutical formulation (e.g., a composition comprising a therapeutic protein or peptide) as described herein packaged in a container, such as a sealed bottle or vessel, with a label attached to the container or included in the package that describes the use of the compound or composition in practicing the method. In one embodiment, the pharmaceutical formulation is packaged in a container such that the amount of head space of the container (e.g., the amount of air between the liquid formulation and the top of the container) is very low. Preferably, the amount of head space is negligible (e.g., nonexistent). In one embodiment, the kit includes a first container that includes a therapeutic protein or peptide composition and a second container that has a physiologically acceptable reconstitution solution for the composition. In one aspect, the pharmaceutical formulation is packaged in a unit dosage form. The kit may further include a device suitable for administration of the pharmaceutical formulation according to a particular route of administration. Preferably, the kit includes a label that describes the use of the pharmaceutical formulation.

[0131] IV. rVWF for methods of treating GI bleeding in patients with severe VWD One advantage of administering rVWF to patients with severe VWD for preparation for surgery is that the higher specific activity of rVWF compared to pdVWF allows flexibility in the amount of rVWF administered and the number of times it is re-administered to the subject. As will be appreciated, and as discussed in more detail herein, the co-administered FVIII may be recombinant or plasma-derived.

[0132] Single or multiple administrations of rVWF are administered with the dosage level and pattern selected by the treating physician. For disease prevention or treatment, the appropriate dosage will depend on the type of disease being treated (e.g., von Willebrand's disease), the severity and course of the disease, whether the drug is given for prophylactic or therapeutic purposes, previous therapy, the patient's medical history and response to the drugs, and the discretion of the attending physician.

[0133] In some embodiments, rVWF is administered to a subject prior to surgery in the range of 20-60 IU / kg, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 20-60, 35-70, 20-40, 35-60, 45-60, 45-55, 45-50, 50-60, 55-60, or 50-55 IU / kg. In some embodiments, rVWF is administered 12 to 24 hours, e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, 12 to 24 hours, 14 to 24 hours, 16 to 24 hours, 18 to 24 hours, or 20 to 24 hours, prior to surgery. In some embodiments, factor VIII (FVIII) is not administered with rVWF prior to surgery.

[0134] In some embodiments, rVWF is administered to the subject one hour prior to surgery in the range of 5-90 IU / kg, e.g., 5-90, 5-50, 10-90, 15-90, 20-90, 30-90, 40-90, 50-90, 60-90, 70-90, 80-90, 5-80, 10-70, 20-60, 30-50, 35-60, 5-50, 5-40, 5-30.5-20, 10-90, 10-50, or 20-40 IU / kg. In other embodiments, rVWF is administered after surgery at a dose of 70-200 IU / kg, e.g., 70-200, 80-200, 90-200, 100-200, 110-200, 120-200, 130-200, 130-200, 140-200, 150-200, 160-200, 170-200, 180-200, 190-200, 70-170, 80-180, 60-160, 50-150, 40-140, 30, 130, 20-120, 10-110, 70-100, or 70-90 IU / kg. In some cases, the surgical procedure is selected from the group consisting of major surgery, minor surgery, or oral surgery.

[0135] In some embodiments, a subject is administered 35-60 IU / kg of rVWF 12-24 hours prior to major surgery. In other embodiments, a subject is administered 15-90 IU / kg of rVWF 1 hour prior to major surgery. In another embodiment, a subject is administered 150-220 IU / kg of rVWF after major surgery. In some examples, a subject undergoing major surgery is administered a total dose of 220-320 IU / kg.

[0136] In some embodiments, a subject is administered 50-60 IU / kg of rVWF 12-24 hours prior to minor surgery. In other embodiments, a subject is administered 5-50 IU / kg of rVWF 1 hour prior to minor surgery. In another embodiment, a subject is administered 70-150 IU / kg of rVWF after minor surgery. In some examples, a subject undergoing minor surgery is administered a total dose of 100-220 IU / kg.

[0137] In some embodiments, a subject is administered 20-40 IU / kg of rVWF 12-24 hours prior to oral surgery. In other embodiments, a subject is administered 20-50 IU / kg of rVWF 1 hour prior to oral surgery. In another embodiment, a subject is administered 10-50 IU / kg of rVWF during oral surgery. In another embodiment, a subject is administered 20-50 IU / kg of rVWF after oral surgery. In some examples, a total dose of 70-190 IU / kg is administered to a subject undergoing oral surgery.

[0138] The composition of rVWF can be included in a pharmaceutical formulation as described herein. Such formulations can be administered orally, topically, transdermally, parenterally, by inhalation spray, vaginally, rectally, or by intracranial injection. The term parenteral as used herein includes subcutaneous injection, intravenous, intramuscular, intracisternal injection, or infusion techniques. Administration by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonary injection, and / or surgical implantation at a specific site is also contemplated. In general, the composition is essentially free of pyrogens and other impurities that may be harmful to the recipient.

[0139] In one embodiment, the formulation of the present invention is administered initially by bolus, followed by continuous infusion to maintain therapeutic circulating levels of the drug product. In another example, the compound of the present invention is administered as a one-off dose. Those skilled in the art will be able to easily optimize effective dosages and administration regimens as determined by good medical practice and individual patient conditions. The route of administration may be, but is not limited to, intravenous, intraperitoneal, subcutaneous, or intramuscular. The frequency of administration will depend on the pharmacokinetic parameters of the drug and the route of administration. The optimal pharmaceutical formulation will be determined by those skilled in the art depending on the route of administration and the desired dosage. See, for example, Remington's Pharmaceutical Sciences, 18th Ed., 1990, Mack Publishing Co., Easton, Pa. 18042 pages 1435-1712. This disclosure is incorporated herein by reference in its entirety for all purposes, particularly for all teachings regarding formulations, routes of administration, and dosages for pharmaceutical products. Such formulations affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the administered drug. Depending on the route of administration, the appropriate dose is calculated according to body weight, body surface area, or organ size. Appropriate dosages can be ascertained by using established assays for determining blood concentration dosages in combination with appropriate dose-response data. The final dosage regimen is determined by the attending physician, taking into account various factors that modify the action of the drug, such as the specific activity of the drug, the severity of the injury and the patient's response, the patient's age, condition, weight, sex, and dietary habits, the severity of any infection, the time of administration, and other clinical factors. By way of example, a typical dose of the recombinant VWF of the present invention is about 50 IU / kg, which is equivalent to 500 μg / kg. As studies are conducted, more information will become available regarding appropriate dosage levels and duration of treatment for various diseases and conditions.

[0140] The practice of the present invention may employ conventional techniques and procedures within the skill of the art in organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry, and immunology, unless otherwise indicated. Such conventional techniques include polymer array synthesis, hybridization, ligation, and detection of hybridization using labels. Specific illustrations of suitable techniques can be obtained by reference to the following examples of this specification. However, it goes without saying that other equivalent conventional procedures can also be used. Such conventional techniques and descriptions can be found in standard laboratory manuals, such as Genome Analysis: A Laboratory Manual Series (Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A Laboratory Manual, PCR Primer: A Laboratory Manual, and Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press), Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, Highly stabilized York, Gait, "Oligonucleotide Synthesis: A Practical Approach" 1984, IRL Press, London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry 3rd Ed., W.H. Freeman Pub., Highly stabilized York, NY, and Berg et al. (2002) Biochemistry, 5th Ed., W.H. Freeman Pub., Highly stabilized York, NY, all of which are incorporated herein by reference in their entirety for all purposes.

[0141] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context dictates otherwise. Thus, for example, "a polymerase" refers to an agent or mixture of such agents, reference to "the method" includes references to equivalent steps and methods known to those skilled in the art, and so forth.

[0142] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context dictates otherwise. Thus, for example, "a polymerase" refers to an agent or mixture of such agents, reference to "the method" includes references to equivalent steps and methods known to those skilled in the art, and so forth.

[0143] Unless otherwise specified, 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. All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing the devices, compositions, formulations, and methods described in these publications and that can be used in connection with the inventions described herein.

[0144] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, to one-tenth of the unit of the lower limit, as well as any other stated or intervening value in that stated range, is encompassed within the invention, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, which are also encompassed within the invention, subject to any specific excluded limits in the stated range. Where a stated range includes one or both of its upper and lower limits, ranges excluding either or both of those included upper and lower limits are also encompassed within the invention.

[0145] In the above description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of ordinary skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the present invention.

[0146] Although the present invention will be described primarily with reference to specific embodiments, it is anticipated that other embodiments will be apparent to those skilled in the art upon reading this disclosure, and such embodiments are intended to be encompassed by the methods of the present invention.

[0147] a. Lyophilized VWF preparation The present invention also provides a formulation of rVWF for use in the therapeutic methods provided herein. In some embodiments, the rVWF composition is used for the manufacture of a pharmaceutical composition. In some embodiments, the rVWF can be formulated into a lyophilized formulation.

[0148] In some embodiments, formulations containing the VWF polypeptides of the present invention are lyophilized after purification and before administration to a subject. Lyophilization is performed using techniques common in the art and should be optimized for the composition being developed (Tang et al., Pharm Res. 21:191-200, (2004), and Chang et al., Pharm Res. 13:243-9 (1996)).

[0149] The lyophilization cycle, in one embodiment, consists of three steps: freezing, primary drying, and secondary drying (AP Mackenzie, Phil Trans R Soc London, Ser B, Biol 278:167 (1977)). In the freezing step, the solution is cooled to initiate the formation of ice. This step also involves the crystallization of the bulking agent. The ice is sublimated in the primary drying step, which is carried out by using a vacuum and introducing heat to reduce the chamber pressure below the vapor pressure of the ice to promote sublimation. Finally, the absorbed or bound water is removed in the secondary drying step under reduced chamber pressure and high shelf temperature. This process produces a material known as a lyophilized cake. The cake can then be reconstituted with either sterile water or a diluent suitable for injection.

[0150] The lyophilization cycle not only determines the final physical state of the excipients, but also influences other parameters such as reconstitution time, appearance, stability, and final water content. The composition structure in the frozen state undergoes several transitions (e.g., glass transition, wetting, and crystallization) that occur at specific temperatures, and this structure can be used to understand and optimize the lyophilization process. The glass transition temperature (Tg and / or Tg') can provide information about the physical state of the solute and can be determined by differential scanning calorimetry (DSC). Tg and Tg' are important parameters that need to be taken into account when designing the lyophilization cycle. For example, Tg' is important for primary drying. Furthermore, in the dry state, the glass transition temperature provides information about the storage temperature of the final product.

[0151] b. Pharmaceutical preparations and excipients in general Excipients are additives that confer or enhance the stability and delivery of drug products (e.g., proteins). Regardless of the reason for their inclusion, excipients are essential components of the formulation and therefore need to be safe and well tolerated by patients. For protein drugs, the choice of excipients is particularly important as it can affect both the efficacy and immunogenicity of the drug. Thus, protein formulations need to be developed with the appropriate selection of excipients that provide suitable stability, safety, and marketability.

[0152] In one embodiment, the lyophilized formulation is composed of at least one or more of buffers, bulking agents, and stabilizers. In this embodiment, the use of surfactants is evaluated and selected when aggregation during the lyophilization step or during reconstitution is an issue. Appropriate buffers are included to maintain the formulation in a stable pH range during lyophilization. A comparison of the excipient compounds contemplated for liquid and lyophilized protein formulations is provided in Table 10.

[0153] Table 1. Excipient compounds for lyophilized protein formulations TIFF0007641327000012.tif164128

[0154] A major challenge in developing formulations for proteins is to stabilize the product against the stresses of manufacturing, transportation, and storage. The role of formulation excipients is to provide stabilization against these stresses. Excipients are also used to reduce the viscosity of high concentration protein formulations, thus allowing their transportation and improving patient convenience. In general, excipients can be classified based on the mechanism by which they stabilize proteins against various chemical and physical stresses. Some excipients are used to mitigate the effects of specific stresses or to modulate the special susceptibility of certain proteins. Other excipients have a more general effect on the physical and covalent stability of proteins. The excipients described herein are organized either by their chemical type or by their functional role in the formulation. A brief description of the stabilization measures is provided upon discussion of each excipient type.

[0155] Given the teachings and guidance provided herein, one of skill in the art will understand the amounts or ranges of excipients that can be included in any particular formulation to obtain a biopharmaceutical formulation of the invention that promotes retention of biopharmaceutical (e.g., protein) stability. For example, the amounts and types of salts that can be included in a biopharmaceutical formulation of the invention are selected based on the desired osmolality (e.g., isotonic, hypotonic, or hypertonic) of the final solution, as well as the amounts and osmolality of other components to be included in the formulation.

[0156] As an example, about 5% sorbitol can be included to obtain isotonicity, while about 9% sucrose excipient is required to obtain isotonicity. The selection of the amount and concentration range of one or more excipients that can be included in the biopharmaceutical formulation of the present invention is exemplified above with reference to salts, polyols, and sugars. However, those skilled in the art will understand that the considerations described herein and further exemplified with reference to specific excipients are equally applicable to all types and combinations of excipients, including, for example, salts, amino acids, other isotonicity agents, surfactants, stabilizers, bulking agents, cryoprotectants, lyoprotectants, antioxidants, metal ions, chelating agents, and / or preservatives.

[0157] Additionally, when a particular excipient is reported as a molar concentration, one of skill in the art will recognize that the equivalent percent (%) w / v of the solution is also contemplated (e.g., (grams of substance in solution sample / mL of solution) x 100%).

[0158] Of course, those skilled in the art will understand that the concentrations of excipients described herein share interdependencies in a particular formulation. For example, the concentration of bulking agent may be reduced when, for example, protein concentration is high, or when, for example, stabilizer concentration is high. In addition, those skilled in the art will recognize that the concentration of stabilizer may be adjusted accordingly (e.g., a "tonicity-increasing" amount of stabilizer may be used) to maintain the isotonicity of a particular formulation without bulking agent. Common excipients are known in the art and can be found in Powell et al., Compendium of Excipients fir Parenteral Formulations (1998), PDA J.Pharm.Sci.Technology, 52:238-311.

[0159] c. Pharmaceutical buffers and buffering agents The stability of pharmacologically active protein formulations is usually observed to be maximum in a narrow pH range. This pH range of optimal stability needs to be identified early during preformulation testing. Several approaches, such as accelerated stability testing and calorimetric screening testing, are useful in this endeavor (Remmele RL Jr., et al., Biochemistry, 38(16):5241-7(1999)). Once the formulation is finalized, the protein needs to be manufactured and maintained throughout its shelf life. Thus, buffers are almost always used to control the pH of the formulation.

[0160] The buffering capacity of a buffer species is greatest at a pH equal to its pKa and decreases as the pH increases or decreases away from this value. Ninety percent of the buffering capacity is within one pH unit of its pKa. Buffering capacity also increases proportionally with increasing concentration of the buffering agent.

[0161] Several factors must be considered when selecting a buffer. First, the buffer species and its concentration must be defined based on its pKa and the desired formulation pH. It is equally important to ensure that the buffer is compatible with the protein and other formulation excipients and does not catalyze any degradation reactions. A third important aspect to consider is the stinging and irritation that the buffer may induce upon administration. For example, citrate is known to cause stinging upon injection (Laursen T, et al., Basic Clin Pharmacol Toxicol., 98(2):218-21(2006)). The potential for stinging and irritation is greater for drugs administered via subcutaneous (SC) or intramuscular (IM) routes, where the drug solution remains at the site for a relatively longer time than when administered via IV routes, where the formulation is rapidly diluted in the blood upon administration. For formulations administered via direct IV infusion, the total amount of buffer (and any other formulation components) must be monitored. Particular caution should be exercised with potassium ions administered in the form of potassium phosphate buffers, which can cause cardiovascular effects in patients (Hollander-Rodriguez JC, et al., Am. Fam. Physician., 73(2):283-90 (2006)).

[0162] Buffers for lyophilized formulations require additional consideration. Some buffers, such as sodium phosphate, can change pH by crystallizing from the protein amorphous phase during freezing. Other common buffers, such as acetate and imidazole, can change the pH of the formulation during lyophilization or after reconstitution by subliming or evaporating during the lyophilization process.

[0163] The buffer system present in the composition is selected to be physiologically compatible and to maintain the desired pH of the pharmaceutical formulation. In one embodiment, the pH of the solution is between pH 2.0 and pH 12.0. For example, the pH of the solution may be 2.0, 2.3, 2.5, 2.7, 3.0, 3.3, 3.5, 3.7, 4.0, 4.3, 4.5, 4.7, 5.0, 5.3, 5.5, 5.7, 6.0, 6.3, 6.5, 6.7, 7.0, 7.3, 7.5, 7.7, 8.0, 8.3, 8.5, 8.7, 9.0, 9.3, 9.5, 9.7, 10.0, 10.3, 10.5, 10.7, 11.0, 11.3, 11.5, 11.7, or 12.0.

[0164] The pH buffering compound may be present in any amount suitable for maintaining the pH of the formulation at a predetermined level. In one embodiment, the pH buffering concentration is 0.1 mM to 500 mM (1 M). For example, it is contemplated that the pH buffering agent is at least 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 500 mM.

[0165] Exemplary pH buffering agents used to buffer the formulations provided herein include, but are not limited to, organic acids, glycine, histidine, glutamate, succinate, phosphate, acetate, citrate, Tris, HEPES, and amino acids or mixtures of amino acids, such as, but not limited to, histidine and glycine. In one embodiment of the present invention, the buffering agent is citrate.

[0166] d. Pharmaceutical Stabilizers and Bulking Agents In one embodiment of the pharmaceutical preparation, a stabilizer (or a combination of stabilizers) is added to prevent or reduce storage-induced aggregation and chemical degradation.If the solution is hazy or turbid upon reconstitution, this means that protein has precipitated or at least aggregated.The term "stabilizer" refers to an excipient that can prevent aggregation or physical degradation, including chemical degradation (e.g., autolysis, deamidation, oxidation, etc.) in aqueous state. Contemplated stabilizers include, but are not limited to, sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, glucosamine, fructose, mannitol, sorbitol, glycine, arginine HCL, poly-hydroxy compounds, e.g., polysaccharides such as dextran, starch, hydroxyethyl starch, cyclodextrin, N-methylpyrrolidone, cellulose, and hyaluronic acid, sodium chloride (Carpenter et al., Develop. Biol. Standard 74:225, (1991)). In the present formulations, the stabilizer is incorporated at a concentration of about 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 700, 900, or 1000 mM. In one embodiment of the present invention, mannitol and trehalose are used as stabilizers.

[0167] If desired, the formulation also includes an appropriate amount of bulking agent and osmolality adjusting agent. Bulking agents include, but are not limited to, mannitol, glycine, sucrose, polymers such as dextran, polyvinylpyrrolidone, carboxymethylcellulose, lactose, sorbitol, trehalose, or xylitol. In one embodiment, the bulking agent is mannitol. Bulking agents are incorporated at a concentration of about 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 700, 900, or 1000 mM.

[0168] e. Pharmaceutical surfactants Proteins have a strong tendency to interact with surfaces, which makes them susceptible to absorption and denaturation at air-liquid, virus-liquid, and liquid-liquid (silicone oil) interfaces. This degradation pathway has been observed to be inversely dependent on protein concentration, resulting in either the formation of soluble and insoluble protein aggregates or loss of protein from solution due to absorption to surfaces. In addition to container surface absorption, surface-induced degradation is exacerbated by physical agitation, such as that experienced during product distribution and handling.

[0169] Surfactants are commonly used in protein formulations to prevent surface-induced degradation. Surfactants are amphiphilic molecules that have the ability to outcompete proteins for interfacial positions. The hydrophobic portion of the surfactant molecule occupies the interfacial positions (e.g., air / liquid), while the hydrophilic portion of the molecule remains oriented toward the bulk solvent. At sufficient concentrations (typically around the detergent's critical micelle concentration), the surface layer of the surfactant molecule acts to prevent protein molecules from absorbing at the interface, thereby minimizing surface-induced degradation. Surfactants contemplated herein include, but are not limited to, fatty acid esters of sorbitan polyethoxylates, such as polysorbate 20 and polysorbate 80. The two differ only in the length of the aliphatic chain that confers hydrophobic properties to the molecule (C-12 and C-18, respectively). Thus, polysorbate 80 has a higher surface activity and a lower critical micelle concentration than polysorbate 20.

[0170] Surfactants can also affect the thermodynamic conformational stability of proteins. Here again, the effect of a given surfactant excipient will be protein specific. For example, polysorbates have been shown to decrease the stability of some proteins and increase the stability of others. Detergent destabilization of proteins can be theoretically explained in terms of the hydrophobic tails of the detergent molecules that may be involved in specific binding with partially or fully unfolded protein states. These types of interactions can result in a shift in the conformational equilibrium toward more unfolded protein states (e.g., complementing the binding polysorbates to increase the exposure of hydrophobic portions of the protein molecule). Alternatively, if the native state of a protein exhibits some hydrophobic surface, binding of the surfactant to the native state may stabilize that conformation.

[0171] Another aspect of polysorbates is that they are inherently susceptible to oxidative degradation. Often, as raw materials, they contain sufficient amounts of peroxides to cause oxidation of protein residue side chains, especially methionine. The possibility of oxidative damage due to the addition of stabilizers emphasizes the point that the minimum effective concentration of excipients should be used in the formulation. For surfactants, the effective concentration for a given protein will depend on the mechanism of stabilization.

[0172] Surfactants are also added in appropriate amounts to prevent surface-related aggregation phenomena during freezing and drying (Chang, B, J. Pharm. Sci. 85:1325, (1996)). Thus, exemplary surfactants include, but are not limited to, anionic, cationic, nonionic, zwitterionic, and amphoteric surfactants, including those derived from naturally occurring amino acids. Anionic surfactants include, but are not limited to, sodium lauryl sulfate, sodium dioctyl sulfosuccinate, and sodium dioctyl sulfonate, chenodeoxycholic acid, N-lauroyl sarcosine sodium salt, lithium dodecyl sulfate, 1-octanesulfonic acid sodium salt, sodium cholate hydrate, sodium deoxycholate, and sodium glycodeoxycholic acid salt. Cationic surfactants include, but are not limited to, benzalkonium chloride or benzethonium chloride, cetylpyridinium chloride monohydrate, and hexadecyltrimethylammonium bromide. Zwitterionic surfactants include, but are not limited to, CHAPS, CHAPSO, SB3-10, and SB3-12. Nonionic surfactants include, but are not limited to, digitonin, Triton X-100, Triton X-114, TWEEN-20, and TWEEN-80. Surfactants also include, but are not limited to, lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 40, 50, and 60, glycerol monostearate, polysorbate 40, 60, 65, and 80, soy lecithin, and other phospholipids such as dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylglycerol (DMPG), dimyristoylphosphatidylcholine (DMPC), and (dioleylphosphatidylglycerol)DOPG; sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. Thus, compositions comprising these surfactants, either individually or as a mixture in different ratios, are further provided. In one embodiment of the present invention, the surfactant is TWEEN-80. In the present formulation, the surfactant is incorporated at a concentration of about 0.01 to about 0.5 g / L.In the provided formulations, the surfactant concentration is 0.005, 0.01, 0.02, 0.03, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 g / L.

[0173] f. Pharmaceutical salts Salts are often added to increase the ionic strength of the formulation, which can be important for protein solubility, physical stability, and isotonicity. Salts can affect the physical stability of proteins in a variety of ways. Ions can stabilize the native state of proteins by binding to charged residues on the surface of the protein. Alternatively, salts can stabilize the denatured state by binding to peptide groups along the protein backbone (-CONH-). Salts can also stabilize the native conformation of proteins by blocking repulsive electrostatic interactions between residues within the protein molecule. Salts in protein formulations can also block attractive electrostatic interactions between protein molecules that can lead to protein aggregation and insolubility. In the provided formulations, the salt concentration is between 0.1, 1, 10, 20, 30, 40, 50, 80, 100, 120, 150, 200, 300, and 500 mM.

[0174] g. Other common excipient ingredients: Pharmaceutical amino acids Amino acids find versatile use in protein formulations as buffers, bulking agents, stabilizers, and antioxidants. Thus, in one embodiment, histidine and glutamic acid are used to buffer protein formulations in the pH ranges of 5.5-6.5 and 4.0-5.5, respectively. The imidazole group of histidine has a pKa of 6.0, and the carboxyl group of the glutamic acid side chain has a pKa of 4.3, making these amino acids suitable for buffering in the corresponding pH ranges. In such cases, glutamic acid is particularly useful. Histidine is commonly found in commercial protein formulations, and this amino acid is an alternative to citrate, a buffering agent known to cause stinging upon injection. Interestingly, histidine has also been reported to have a stabilizing effect on aggregation when used in liquid form at high concentrations, both in liquid and lyophilized form (Chen B, et al., Pharm Res., 20(12):1952-60(2003)). Histidine has also been observed by others to reduce the viscosity of formulations with high protein concentrations. However, in the same study, the authors observed increased aggregation and discoloration in histidine-containing formulations during freeze-thaw studies of antibodies in stainless steel containers. Another caveat of histidine is that it undergoes photo-oxidation in the presence of metal ions (Tomita M, et al., Biochemistry, 8(12):5149-60(1969)). The use of methionine as an antioxidant in formulations appears promising and has been observed to be effective against some oxidative stresses (Lam XM, et al., J Pharm ScL, 86(11):1250-5(1997)).

[0175] In various aspects, formulations are provided that include one or more of the amino acids glycine, proline, serine, arginine, and alanine, which have been shown to stabilize proteins by a mechanism of selective exclusion. Glycine is also a commonly used bulking agent in lyophilized formulations. Arginine has been shown to be effective in inhibiting aggregation and is used in both liquid and lyophilized formulations. In the formulations provided, the amino acid concentration is between 0.1, 1, 10, 20, 30, 40, 50, 80, 100, 120, 150, 200, 300, and 500 mM. In one embodiment of the present invention, the amino acid is glycine.

[0176] h. Other common excipient ingredients: Pharmaceutical antioxidants Oxidation of protein residues occurs from several different sources. In addition to adding specific antioxidants, prevention of oxidative protein damage involves careful control of a number of factors, such as atmospheric oxygen, temperature, light exposure, and chemical contamination, throughout the manufacturing process and storage of the product. Thus, the present invention contemplates the use of pharmaceutical antioxidants, including but not limited to reducing agents, oxygen / free radical scavengers, or chelating agents. The antioxidants in therapeutic protein formulations, in one embodiment, are water-soluble and remain active throughout the product shelf life. Reducing agents and oxygen / free radical scavengers act by scavenging reactive oxygen species in solution. Chelating agents, such as EDTA, are effective by binding trace metal contaminants that promote free radical formation. For example, EDTA has been utilized in liquid formulations of acidic fibroblast growth factor to inhibit metal ion-catalyzed oxidation of cysteine ​​residues.

[0177] In addition to the effectiveness of various excipients in preventing protein oxidation, there is concern that antioxidants themselves may induce other covalent or physical changes to proteins. For example, reducing agents can cause the disruption of intramolecular disulfide bonds that can lead to disulfide shuffling. In the presence of transition metal ions, ascorbic acid and EDTA have been shown to promote methionine oxidation in some proteins and peptides (Akers MJ, and Defelippis MR. Peptides and Proteins as Parenteral Solutions. In: Pharmaceutical Formulation Development of Peptides and Proteins. Sven Frokjaer, Lars Hovgaard, editors. Pharmaceutical Science. Taylor and Francis, UK (1999)); Fransson JR, / . Pharm. Sci. 86(9): 4046-1050 (1997); Yin J, et al., Pharm Res., 21(12): 2377-83 (2004)). Sodium thiosulfate has been reported to reduce the level of light- and temperature-induced methionine oxidation in rhuMab HER2, although the formation of thiosulfate-protein adducts was also reported in this study (Lam XM, Yang JY, et al., J Pharm Sci. 86(11):1250-5(1997)). Select the appropriate antioxidant according to the specific stress and sensitivity of the protein. Antioxidants contemplated in certain embodiments include, but are not limited to, reducing agents and oxygen / free radical scavengers, EDTA, and sodium thiosulfate.

[0178] i. Other common excipient ingredients: Pharmaceutical metal ions Generally, transition metal ions are undesirable in protein formulations because they can catalyze physical and chemical decomposition in proteins. However, certain metal ions are included in formulations where they are cofactors for proteins, and in suspension formulations of proteins where they form coordination complexes (e.g., zinc suspensions of insulin). Recently, the use of magnesium ions (10-120 mM) has been proposed to inhibit the isomerization of aspartic acid to isoaspartic acid (International Publication No. WO2004039337).

[0179] Two examples where metal ions confer stability or increased activity in proteins are human deoxyribonuclease (rhDNase, Pulmozyme®) and Factor VIII. In the case of rhDNase, Ca +2 ions (up to 100 mM) increased the enzyme's stability through specific binding sites (Chen B, et al., / Pharm Sci.,88(4):477-82(1999)). Indeed, removing calcium ions from solution using EGTA led to increased deamidation and aggregation. However, this effect was not observed in the presence of Ca +2 ions, and not the other divalent cation, Mg +2 , Mn +2 , and Zn +2 It was observed that Ca destabilized rhDNase. A similar effect was observed with factor VIII. +2 and Sr +2 ions stabilized the protein, but Mg +2 , Mn +2 , and Zn +2 , Cu +2 and Fe +2 Others, such as Fatouros, A., et al., Int. J. Pharm., 155, 121-131 (1997), destabilized the enzyme. In a separate study with factor VIII, Al +3A significant increase in the rate of aggregation was observed in the presence of ions (Derrick TS, et al., / .Pharm.Sci.,93(10):2549-57(2004)). The authors suggest that other excipients, such as buffer salts, often +3 The authors note that the drug is contaminated with ions, highlighting the need to use appropriate amounts of excipients in the formulated product.

[0180] j. Other common excipient ingredients: Pharmaceutical preservatives Preservatives are necessary when developing multi-use parenteral formulations that involve more than one withdrawal from the same container. Their main function is to inhibit microbial growth and ensure product sterility over the shelf life or use period of the drug product. Commonly used preservatives include, but are not limited to, benzyl alcohol, phenol, and m-cresol. Although preservatives have been used for many years, developing protein formulations containing preservatives can be difficult. Preservatives almost always have a destabilizing effect on proteins (aggregation), which is the main factor limiting their use in multi-dose protein formulations (Roy S, et al., J Pharm ScL, 94(2):382-96(2005)).

[0181] To date, most protein drugs have been formulated for single use only. However, when multi-dose formulations are possible, they have the added advantage of allowing for patient convenience and increased marketability. A good example is that of human growth hormone (hGH), where the development of a preserved formulation has led to the commercialization of a more convenient multi-use injection pen format. At least four such pen devices containing preserved formulations of hGH are currently on the market. Norditropin® (liquid, Novo Nordisk), Nutropin AQ® (liquid, Genentech), and Genotropin (lyophilized-dual chamber cartridge, Pharmacia & Upjohn) contain phenol, while Somatrope® (Eli Lilly) is formulated with m-cresol.

[0182] Several aspects must be considered during formulation development of a preserved dosage form. The effective preservative concentration in the drug product must be optimized. This requires testing a given preservative in the dosage form over a range of concentrations that impart antimicrobial efficacy without compromising protein stability. For example, three preservatives were screened in the development of a liquid formulation for interleukin-1 receptor (type I) using differential scanning calorimetry (DSC). The preservatives were ranked based on their impact on stability at concentrations commonly used in commercial products (Remmele RL Jr., et al., Pharm Res., 15(2):200-8 (1998)).

[0183] Developing liquid formulations containing preservatives is more challenging than lyophilized formulations. Lyophilized products can be lyophilized without preservatives and reconstituted with a preservative containing diluent at the time of use. This reduces the time the preservative is in contact with the protein and greatly minimizes the associated stability risks. In liquid formulations, the effectiveness and stability of the preservative must be maintained throughout the entire product shelf life (approximately 18-24 months). An important point to note is that the effectiveness of the preservative must be demonstrated in the final formulation containing the active drug and all excipient components.

[0184] Some preservatives may cause injection site reactions, which is another factor that needs to be considered when selecting a preservative. In a clinical trial focused on evaluating preservatives and buffers in Norditropin, lower pain perception was observed in formulations containing phenol and benzyl alcohol compared to formulations containing m-cresol (Kappelgaard AM, Horm Res. 62 Suppl 3: 98-103 (2004)). Interestingly, among the commonly used preservatives, benzyl alcohol has anesthetic properties (Minogue SC, and Sun DA., AnesthAnalg., 100 (3): 683-6 (2005)). In various embodiments, the use of a preservative provides benefits that outweigh any side effects.

[0185] K. Methods for preparing pharmaceutical formulations The present invention further contemplates a method for preparing a pharmaceutical formulation.

[0186] The method further includes one or more of the following steps: adding a stabilizing agent as described herein to the mixture prior to lyophilization; adding at least one agent selected from a bulking agent, an osmotic pressure adjusting agent, and a surfactant, each as described herein, to the mixture prior to lyophilization.

[0187] Standard reconstitution practice for lyophilized material is to add back a large amount of purified water or sterile water for injection (WFI) (typically equal to the volume removed during lyophilization), although dilute solutions of antibiotics may be used in the manufacture of pharmaceutical products for parenteral administration (Chen, Drug Development and Industrial Pharmacy, 18:1311-1354 (1992)). Thus, a method for preparing a reconstituted rVWF composition is provided, comprising adding a diluent to the lyophilized rVWF composition of the invention.

[0188] The lyophilized material may be reconstituted as an aqueous solution with a variety of aqueous carriers, for example, sterile water for injection, water containing a preservative for multiple dose use, or water containing an appropriate amount of surfactant (e.g., aqueous suspensions containing the active compound mixed with excipients suitable for the manufacture of aqueous suspensions). In various embodiments, such excipients are suspending agents, such as, but not limited to, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia, and dispersing or wetting agents are naturally occurring phosphatides, such as, but not limited to, lecithin, or condensation products of alkylene oxides with fatty acids, such as, but not limited to, polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, such as, but not limited to, heptadecaethyl-enoxycetanol, or condensation products of ethylene oxide with fatty acids and partial esters derived from hexitols, such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with fatty acids and partial esters derived from hexitol anhydrides, such as, but not limited to, polyethylene sorbitan monooleate. In various embodiments, the aqueous suspension also contains one or more preservatives, such as, but not limited to, ethyl, or n-propyl, p-hydroxybenzoate.

[0189] l. Exemplary rVWF Formulations for Administration In some embodiments, the method provides an enhanced formulation that allows for a final product with high potency (high rVWF concentration and enhanced long-term stability) to reduce the amount of treatment (100 IU / ml to 10000 IU / ml). In some embodiments, the rVWF concentration in the formulation for administration is about 100 IU / ml to 10000 IU / ml. In some embodiments, the rVWF concentration in the formulation for administration is about 500 IU / ml to 10000 IU / ml. In some embodiments, the rVWF concentration in the formulation for administration is about 1000 IU / ml to 10000 IU / ml. In some embodiments, the rVWF concentration in the formulation for administration is about 2000 IU / ml to 10000 IU / ml. In some embodiments, the rVWF concentration in the formulation for administration is about 3000 IU / ml to 10000 IU / ml. In some embodiments, the rVWF concentration in the formulation for administration is about 4000 IU / ml to 10000 IU / ml. In some embodiments, the rVWF concentration in the formulation for administration is about 5000 IU / ml to 10000 IU / ml. In some embodiments, the rVWF concentration in the formulation for administration is about 6000 IU / ml to 10000 IU / ml. In some embodiments, the rVWF concentration in the formulation for administration is about 7000 IU / ml to 10000 IU / ml. In some embodiments, the rVWF concentration in the formulation for administration is about 8000 IU / ml to 10000 IU / ml. In some embodiments, the rVWF concentration in the formulation for administration is about 9000 IU / ml to 10000 IU / ml.

[0190] In some embodiments, the formulation for administration includes one or more zwitterionic compounds, including, for example, amino acids such as histidine, glycine, and arginine. In some embodiments, the formulation for administration includes a component with amphiphilic properties, having at least one hydrophobic group and one hydrophilic group, including, for example, polysorbate 80, octylpyranoside, dipeptides, and / or amphipathic peptides. In some embodiments, the formulation for administration includes a non-reducing sugar or sugar alcohol or disaccharide, including, for example, sorbitol, mannitol, sucrose, or trehalose. In some embodiments, the formulation for administration includes a non-toxic water-soluble salt, including, for example, sodium chloride, that provides a physiological osmolality. In some embodiments, the formulation for administration has a pH in the range of 6.0 to 8.0. In some embodiments, the formulation for administration has a pH of about 6.0, about 6.5, about 7, about 7.5, or about 8.0. In some embodiments, the formulation for administration includes, for example, Ca 2+ , Mg 2+ , Zn 2+ , Mn 2+ and / or combinations thereof. In some embodiments, the formulation for administration has a physiological osmolality at the time of administration, has a pH of about 7.0, and comprises about 1 mM to about 50 mM glycine, about 1 mM to about 50 mM histidine, about 0 to about 300 mM sodium chloride (e.g., less than 300 mM sodium), about 0.01% to about 0.05% polysorbate 20 (or polysorbate 80), and about 0.5% to about 20% (w / w) sucrose.

[0191] In some embodiments, the formulation for administration can be lyophilized. In some embodiments, the formulation for administration is stable and can be stored in liquid form at about 2° C. to about 8° C. and about 18° C. to about 25° C. In some embodiments, the formulation for administration is stable and can be stored in liquid form at about 2° C. to about 8° C. In some embodiments, the formulation for administration is stable and can be stored in liquid form at about 18° C. to about 25° C.

[0192] V. Administration of rVWF for the Treatment of GI Bleeding in Patients with Severe VWD One advantage of administering rVWF to patients with severe VWD to treat GI bleeding episodes is that the higher specific activity of rVWF compared to pdVWF allows flexibility in the amount of rVWF administered and the number of times it is re-administered to a subject. As will be appreciated, and as discussed in more detail herein, co-administered FVIII may be recombinant or plasma-derived.

[0193] For administration of the composition to humans or test animals, in one embodiment, the composition comprises one or more pharma- ceutically acceptable carriers. The term "pharmacologically" or "pharmacologically" acceptable refers to molecular entities and compositions that are stable, inhibit proteolytic degradation, such as aggregation and cleavage products, as described below, and do not produce allergic or other adverse reactions when administered using routes well known in the art. "Pharmaceutically acceptable carriers" include any and all clinically useful solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., including the agents disclosed above.

[0194] Pharmaceutical preparations can be administered orally, topically, transdermally, parenterally, by inhalation spray, vaginally, rectally, or by intracranial injection. The term parenteral as used herein includes subcutaneous injection, intravenous, intramuscular, intracisternal injection, or infusion techniques. Administration by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, retrobulbar, and / or intrapulmonary injection at a specific site is also contemplated. Generally, compositions are essentially free of pyrogens and other impurities that may be harmful to the recipient.

[0195] Single or multiple administrations of rVWF are administered with the dosage level and pattern selected by the treating physician. For disease prevention or treatment, the appropriate dosage will depend on the type of disease being treated (e.g., von Willebrand's disease), the severity and course of the disease, whether the drug is given for prophylactic or therapeutic purposes, previous therapy, the patient's medical history and response to the drugs, and the discretion of the attending physician.

[0196] In some embodiments, rVWF is administered to the subject in the range of 40-100 IU / kg, e.g., 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 40-100, 40-80, 50-80, 60-80, 70-80, 40-50, 40-60, 40-70, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 IU / kg. In some embodiments, rVWF is administered at least once during a GI bleeding episode. In other embodiments, rVWF is administered two or more times, e.g., two, three, four, five, or more times, during a GI bleeding episode. In some examples, the subject is administered one or more infusions of rVWF. Each injection may include rVWF in the range of about 40-80 IU / kg, e.g., 40, 45, 50, 55, 60, 65, 70, 75, 80, 40-80, 50-80, 60-80, 70-80, 40-50, 40-60, 40-70, 40-50, 50-60, or 60-70 IU / kg. In some embodiments, the injection amounts may be substantially equal. For example, the first injection and the second injection may be substantially equal in amount. In some embodiments, the total dose of rVWF administered to a subject per bleeding episode is about 40-150 IU / kg, e.g., 40-150, 40-125, 40-100, 40-90, 40-75, 50-150, 50-100, 75-150, or 100-150 IU / kg.

[0197] In some embodiments, minor and moderate bleeding events required only 1-2 more infusions than predicted to control the bleeding episode and no additional VWF-containing product was required. In some embodiments, major bleeding events required less than 1.5 times more infusions than predicted to control the bleeding episode and no additional VWF-containing product was required. In some embodiments, minor, moderate, and major bleeding events had an actual number of infusions that was equal to or less than the predicted number needed to treat the bleeding event and no additional VWF-containing product was required.

[0198] In some embodiments, rVWF is administered at least once a day, at least twice a day, every 8-12 hours, etc. In some examples, rVWF is administered for a total of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, etc. In some embodiments, rVWF is administered every 8 hours, 9 hours, 10 hours, 11 hours, or every 12 hours. In some embodiments, rVWF is administered every 8-12 hours for about 3 days to about 7 days.

[0199] In some embodiments, recombinant factor VIII (rFVIII) is also administered to subjects with severe VWD to treat GI bleeding symptoms. In some cases, the administered treatment includes rVWF and rFVIII. In other cases, the administered treatment does not include rFVIII. In some embodiments, rFVIII is administered to the subject at a dose in the range of about 10-70 IU / kg, e.g., 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-30, 30-40, 40-50, 50-60, or 60-70 IU / kg. In some examples, rFVIII is administered at the initial (first) dose or initial (first) infusion. In some cases, rFVIII is not administered as part of the second dose or second infusion. In some embodiments, a subject with VWD experiencing a GI bleeding episode is administered a single infusion of rVWF and rFVIII, hi some embodiments, a second administration of rVWF is not administered together with FVIII.

[0200] In some embodiments of the method, when rVWF and FVIII are administered together, the ratio of rVWF to FVIII is about 1.5:0.8. In some embodiments of the method, when rVWF and FVIII are administered together, the ratio of rVWF to FVIII is about 1.3:1. In some embodiments of the method, when rVWF and FVIII are administered together, the ratio of rVWF to FVIII is about 1.1:0.8. In some embodiments of the method, when rVWF and FVIII are administered together, the ratio of rVWF to FVIII is about 1.5:1. In some embodiments of the method, when rVWF and FVIII are administered together, the ratio of rVWF to FVIII is about 1.1:1.2.

[0201] In some embodiments, when the gastrointestinal bleeding is light or moderate gastrointestinal bleeding, 40-60 IU / kg of rVWF is administered. In some embodiments, when the gastrointestinal bleeding is light or moderate gastrointestinal bleeding, about 40 IU / kg of rVWF is administered. In some embodiments, when the gastrointestinal bleeding is light or moderate gastrointestinal bleeding, about 45 IU / kg of rVWF is administered. In some embodiments, when the gastrointestinal bleeding is light or moderate gastrointestinal bleeding, about 50 IU / kg of rVWF is administered. In some embodiments, when the gastrointestinal bleeding is light or moderate gastrointestinal bleeding, about 55 IU / kg of rVWF is administered. In some embodiments, when the gastrointestinal bleeding is light or moderate gastrointestinal bleeding, about 60 IU / kg of rVWF is administered. In some embodiments, when the gastrointestinal bleeding is light or moderate, 40-60 IU / kg rVWF is administered every 8-12 hours for about 3 to about 7 days. In some embodiments, when the gastrointestinal bleeding is light or moderate, about 40 IU / kg rVWF is administered every 8-12 hours for about 3 to about 7 days. In some embodiments, when the gastrointestinal bleeding is light or moderate, about 45 IU / kg rVWF is administered every 8-12 hours for about 3 to about 7 days. In some embodiments, when the gastrointestinal bleeding is light or moderate, about 50 IU / kg rVWF is administered every 8-12 hours for about 3 to about 7 days. In some embodiments, when the GI bleeding is light or moderate, about 55 IU / kg rVWF is administered every 8-12 hours for about 3 to about 7 days. In some embodiments, when the GI bleeding is light or moderate, about 60 IU / kg rVWF is administered every 8-12 hours for about 3 to about 7 days. In some embodiments, when the GI bleeding is light or moderate, about 40-60 IU / kg rVWF is administered every 8 hours for about 3 to about 7 days.In some embodiments, when the GI bleeding is light or moderate GI bleeding, 40-60 IU / kg rVWF is administered every 9 hours for about 3 to about 7 days. In some embodiments, when the GI bleeding is light or moderate GI bleeding, 40-60 IU / kg rVWF is administered every 10 hours for about 3 to about 7 days. In some embodiments, when the GI bleeding is light or moderate GI bleeding, 40-60 IU / kg rVWF is administered every 11 hours for about 3 to about 7 days. In some embodiments, when the GI bleeding is light or moderate GI bleeding, 40-60 IU / kg rVWF is administered every 12 hours for about 3 to about 7 days. In some embodiments, when the GI bleeding is light or moderate GI bleeding, 40-60 IU / kg rVWF is administered every 8-12 hours for about 3 days. In some embodiments, when the GI bleeding is light or moderate GI bleeding, 40-60 IU / kg rVWF is administered every 8-12 hours for about 4 days. In some embodiments, when the GI bleeding is light or moderate GI bleeding, 40-60 IU / kg rVWF is administered every 8-12 hours for about 5 days. In some embodiments, when the GI bleeding is light or moderate GI bleeding, 40-60 IU / kg rVWF is administered every 8-12 hours for about 6 days. In some embodiments, when the GI bleeding is light or moderate GI bleeding, 40-60 IU / kg rVWF is administered every 8-12 hours for about 7 days. In some embodiments, when the GI bleeding is light or moderate GI bleeding, about 40, about 45, about 50, about 55, or about 60 IU / kg rVWF is administered every 8 hours, about 9 hours, about 10 hours, about 11 hours, or about 12 hours for about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days.

[0202] In some embodiments, about 40 IU / kg rVWF of the rVWF and the gastrointestinal bleeding is heavy or severe gastrointestinal bleeding. In some embodiments, about 45 IU / kg rVWF of the rVWF and the gastrointestinal bleeding is heavy or severe gastrointestinal bleeding. In some embodiments, about 50 IU / kg rVWF of the rVWF and the gastrointestinal bleeding is heavy or severe gastrointestinal bleeding. In some embodiments, about 55 IU / kg rVWF of the rVWF and the gastrointestinal bleeding is heavy or severe gastrointestinal bleeding. In some embodiments, about 60 IU / kg rVWF of the rVWF and the gastrointestinal bleeding is heavy or severe gastrointestinal bleeding. In some embodiments, about 65 IU / kg rVWF of the rVWF and the gastrointestinal bleeding is heavy or severe gastrointestinal bleeding. In some embodiments, about 70 IU / kg rVWF of the rVWF and when the gastrointestinal bleeding is heavy or severe gastrointestinal bleeding. In some embodiments, about 75 IU / kg rVWF of the rVWF and when the gastrointestinal bleeding is heavy or severe gastrointestinal bleeding. In some embodiments, about 80 IU / kg rVWF of the rVWF and when the gastrointestinal bleeding is heavy or severe gastrointestinal bleeding. In some embodiments, when the gastrointestinal bleeding is heavy or severe gastrointestinal bleeding, 40-80 IU / kg rVWF of the rVWF is administered every 8-12 hours for about 3 to about 7 days. In some embodiments, when the gastrointestinal bleeding is heavy or severe gastrointestinal bleeding, about 40 IU / kg rVWF of the rVWF is administered every 8-12 hours for about 3 to about 7 days. In some embodiments, when the gastrointestinal bleeding is heavy or severe, about 45 IU / kg of the rVWF is administered every 8-12 hours for about 3 to about 7 days. In some embodiments, when the gastrointestinal bleeding is heavy or severe, about 50 IU / kg of the rVWF is administered every 8-12 hours for about 3 to about 7 days. In some embodiments, when the gastrointestinal bleeding is heavy or severe, about 55 IU / kg of the rVWF is administered every 8-12 hours for about 3 to about 7 days.In some embodiments, when the gastrointestinal bleeding is heavy or severe gastrointestinal bleeding, about 60 IU / kg of the rVWF is administered every 8-12 hours for about 3 to about 7 days. In some embodiments, when the gastrointestinal bleeding is heavy or severe gastrointestinal bleeding, about 65 IU / kg of the rVWF is administered every 8-12 hours for about 3 to about 7 days. In some embodiments, when the gastrointestinal bleeding is heavy or severe gastrointestinal bleeding, about 70 IU / kg of the rVWF is administered every 8-12 hours for about 3 to about 7 days. In some embodiments, when the gastrointestinal bleeding is heavy or severe gastrointestinal bleeding, about 75 IU / kg of the rVWF is administered every 8-12 hours for about 3 to about 7 days. In some embodiments, when the gastrointestinal bleeding is heavy or severe, about 80 IU / kg of the rVWF is administered every 8-12 hours for about 3 to about 7 days. In some embodiments, when the gastrointestinal bleeding is heavy or severe, 40-80 IU / kg of the rVWF is administered every 8 hours for about 3 to about 7 days. In some embodiments, when the gastrointestinal bleeding is heavy or severe, 40-80 IU / kg of the rVWF is administered every 9 hours for about 3 to about 7 days. In some embodiments, when the gastrointestinal bleeding is heavy or severe, 40-80 IU / kg of the rVWF is administered every 10 hours for about 3 to about 7 days. In some embodiments, when the gastrointestinal bleeding is heavy or severe, 40-80 IU / kg of the rVWF is administered every 11 hours for about 3 to about 7 days. In some embodiments, when the gastrointestinal bleeding is heavy or severe, 40-80 IU / kg of the rVWF is administered every 12 hours for about 3 to about 7 days. In some embodiments, when the gastrointestinal bleeding is heavy or severe, 40-80 IU / kg of the rVWF is administered every 8 to 12 hours for about 3 days. In some embodiments, when the gastrointestinal bleeding is heavy or severe, 40-80 IU / kg of the rVWF is administered every 8 to 12 hours for about 4 days.In some embodiments, when the GI bleeding is heavy or severe GI bleeding, 40-80 IU / kg rVWF of the rVWF is administered every 8-12 hours for about 5 days. In some embodiments, when the GI bleeding is heavy or severe GI bleeding, 40-80 IU / kg rVWF of the rVWF is administered every 8-12 hours for about 6 days. In some embodiments, when the GI bleeding is heavy or severe GI bleeding, 40-80 IU / kg rVWF of the rVWF is administered every 8-12 hours for about 7 days. In some embodiments, when the gastrointestinal bleeding is heavy or severe gastrointestinal bleeding, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, or about 80 IU / kg of rVWF is administered every about 8 hours, about 9 hours, about 10 hours, about 11 hours, or about 12 hours for about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days.

[0203] Generally, type 1 VWD is represented by VWF:RCo less than 30 IU / dL, VWF:Ag less than 30 IU / dL, low or normal FVIII, and a VWF:RCo / VWF:Ag ratio greater than 0.5-0.7 IU / dL. Type 2A VWD is represented by VWF:RCo less than 30 IU / dL, VWF:Ag less than 30-200 IU / dL, low or normal FVIII, and a VWF:RCo / VWF:Ag ratio less than 0.5-0.7 IU / dL. Type 2B VWD is represented by VWF:RCo less than 30-200 IU / dL, VWF:Ag less than 30 IU / dL, low or normal FVIII, and a VWF:RCo / VWF:Ag ratio usually less than 0.5-0.7 IU / dL. Type 2M VWD is represented by VWF:RCo less than 30 IU / dL, VWF:Ag less than 30-200 IU / dL, low or normal FVIII, and a VWF:RCo / VWF:Ag ratio less than 0.5-0.7 IU / dL. Type 2N VWD is represented by VWF:RCo between 30-2000 IU / dL, VWF:Ag between 30-200 IU / dL, very low FVIII, and a VWF:RCo / VWF:Ag ratio greater than 0.5-0.7 IU / dL. Type 3 VWD is represented by VWF:RCo less than 3 IU / dL, VWF:Ag less than 3 IU / dL, extremely low (less than 10 IU / dL) FVIII, and no VWF:RCo / VWF:Ag ratio is applicable. Normal is represented by a VWF:RCo of 50-200 IU / dL, a VWF:Ag of 50-200 IU / dL, normal FVIII, and a VWF:RCo / VWF:Ag ratio of greater than 0.5-0.7 IU / dL. In some embodiments, the subject has type 3 VWD. In some embodiments, the subject has severe type 1 VWD. In some embodiments, the subject has severe type 2 VWD.

[0204] In some embodiments, the subject has been treated for at least one bleeding event within the past 12 months. In some embodiments, the subject has been treated for more than one bleeding event within the past 12 months.

[0205] In general, minor bleeding is defined as any acute or subacute clinically overt bleeding that does not meet the criteria for major bleeding and that has led to hospitalization for bleeding, physician-initiated medical or surgical treatment for bleeding, or a change in thrombotic therapy (including study drug) (Aristotle clinical definition); any other bleeding (excluding major and ICH) (RE-LY clinical definition); any overt bleeding that does not meet the criteria for major bleeding but requires medical intervention, extraordinary physician contact (visit or phone call), temporary interruption of study drug (i.e., dosing delay), pain, or impairment of daily activities (Rocket-AF clinical definition); or any bleeding that is more severe than 25 cm 2Clinically significant bleeding (Petro clinical definition), defined as skin hematoma >5 min, involuntary nosebleed >5 min duration, gross hematuria, involuntary rectal bleeding, gingival bleeding >5 min, any bleeding leading to hospitalization, any bleeding leading to transfusion of <2U, or any other bleeding deemed significant by the investigator; and / or minor bleeding events (Aristotle-J clinical definition), defined as acute or subacute, clinically overt, and not massive, and leading to hospitalization for bleeding, investigator-initiated medical or surgical treatment for bleeding, or change in thrombotic therapy, CRNM (clinically significant non-major bleeding), and acute clinical trial overt events that do not meet the criteria for either major or CRNM bleeding. See, e.g., Wells G, Coyle D, Cameron C, et al. Safety, Effectiveness, and Cost-Effectiveness of New Oral Anticoagulants Compared with Warfarin in Preventing Stroke and Other Cardiovascular Events in Patients with Atrial Fibrillation [Internet]. Ottawa (ON): Canadian Agency for Drugs and Technologies in Health; 2012 Apr 9.3, CLINICAL REVIEW. Available at the website www.ncbi.nlm.nih.gov / books / NBK169813 / .Minor bleeding may include events defined as not meeting the criteria for major or clinically significant bleeding; minor bleeding from a wound (injection site, nosebleed, or wound hematoma not requiring surgical decompression); overt bleeding that does not meet the criteria for major bleeding and is accompanied by one or more of the following: nosebleed lasting more than 5 minutes or requiring intervention, ecchymosis or hematoma >5 cm in greatest dimension, hematuria not associated with urinary catheter-related trauma, GI bleeding not associated with NG tube intubation or placement, wound hematoma or complication, subconjunctival hemorrhage requiring cessation of medication; GI or urinary minor bleeding and hematoma at the injection site; and / or overt bleeding that does not meet the criteria for major bleeding. See, e.g., Sobieraj DM, Coleman CI, Tongbram V, et al. Venous Thromboembolism Prophylaxis in Orthopedic Surgery [Internet]. Rockville (MD): Agency for Healthcare Research and Quality (US); 2012 Mar. (Comparative Effectiveness Reviews, No. 49.) Appendix F, Additional Evidence Tables. Available at the website www.ncbi.nlm.nih.gov / books / NBK92309 / .

[0206] In general, major hemorrhage is characterized by the International Society on Thrombosis and Haemostasis (ISTH) criteria and includes any life-threatening and / or fatal hemorrhage; symptomatic hemorrhage into a critical area or organ, and major hemorrhage divided into intracranial (intracranial, subdural) and extracranial (GI, non-GI) hemorrhage (RE-LY clinical definition); symptomatic hemorrhage into a critical anatomical site (Rocket-AF clinical definition); life-threatening retroperitoneal, intracranial, intraocular, or intrathecal hemorrhage; or hemorrhage requiring surgery (Artistotle-J clinical definition). Major bleeding events may include a decrease in hemoglobin of at least 20 g / L or transfusion of more than 2 units of whole blood (packed blood cells as mentioned in the definition of life-threatening bleeding; RE-LY definition of life-threatening bleeding: one or more of the following criteria: (1) fatal symptomatic intracranial hemorrhage; (2) a decrease in hemoglobin level of at least 5.0 g / L; (3) transfusion of at least 4 U of whole blood or packed blood cells; (4) associated with hypotension requiring the use of intravenous inotropes; or (5) requiring surgical intervention); a decrease in hemoglobin of more than 2 g / dL or transfusion of more than 2 units of whole blood / red blood cells (ISTH or Rocket-AF clinical definition); and / or bleeding requiring surgery or transfusion of 2 U or more or associated with symptoms of a decrease in hemoglobin of 2.0 g / L or more. See, e.g., Wells G, Coyle D, Cameron C, et al. Safety, Effectiveness, and Cost-Effectiveness of New Oral Anticoagulants Compared with Warfarin in Preventing Stroke and Other Cardiovascular Events in Patients with Atrial Fibrillation [Internet]. Ottawa (ON): Canadian Agency for Drugs and Technologies in Health; 2012 Apr 9.3, CLINICAL REVIEW. Available at the website www.ncbi.nlm.nih.gov / books / NBK169813 / .Major bleeding may include: clinically evident bleeding with a decrease in Hb of >20 g / L; clinically evident leading to transfusion of >2U of packed red blood cells or whole blood; fatal retroperitoneal, intracranial, intraocular, or intrathecal bleeding; bleeding requiring cessation of treatment or leading to reoperation; fatal retroperitoneal, intracranial, or intrathecal bleeding; bleeding involving any critical organ; bleeding leading to reoperation; overt bleeding with a Bleeding Index of ≥2; major bleeding from a wound (wound hematoma requiring surgical decompression) or major bleeding not related to the wound (gastrointestinal or intracerebral bleeding); clinically evident bleeding associated with either a decrease in Hb of >2 g / dL or the need for transfusion of >2U of RBCs; intracranial or retroperitoneal (resulting in permanent cessation of anticoagulants). See, e.g., Sobieraj DM, Coleman CI, Tongbram V, et al. Venous Thromboembolism Prophylaxis in Orthopedic Surgery [Internet]. Rockville (MD): Agency for Healthcare Research and Quality (US); 2012 Mar. (Comparative Effectiveness Reviews, No. 49.) Appendix F, Additional Evidence Tables. Available at the website www.ncbi.nlm.nih.gov / books / NBK92309 / .

[0207] The composition of rVWF can be included in a pharmaceutical formulation as described herein. Such formulations can be administered orally, topically, transdermally, parenterally, by inhalation spray, vaginally, rectally, or by intracranial injection. The term parenteral as used herein includes subcutaneous injection, intravenous, intramuscular, intracisternal injection, or infusion techniques. Administration by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonary injection, and / or surgical implantation at a specific site is also contemplated. In general, the composition is essentially free of pyrogens and other impurities that may be harmful to the recipient.

[0208] In one embodiment, the formulation of the present invention is administered initially by bolus, followed by continuous infusion to maintain therapeutic circulating levels of the drug product. In another example, the compound of the present invention is administered as a one-off dose. Those skilled in the art will be able to easily optimize effective dosages and administration regimens as determined by good medical practice and individual patient conditions. The route of administration may be, but is not limited to, intravenous, intraperitoneal, subcutaneous, or intramuscular. The frequency of administration will depend on the pharmacokinetic parameters of the drug and the route of administration. The optimal pharmaceutical formulation will be determined by those skilled in the art depending on the route of administration and the desired dosage. See, for example, Remington's Pharmaceutical Sciences, 18th Ed., 1990, Mack Publishing Co., Easton, Pa. 18042 pages 1435-1712. This disclosure is incorporated herein by reference in its entirety for all purposes, particularly for all teachings regarding formulations, routes of administration, and dosages for pharmaceutical products. Such formulations affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the administered drug. Depending on the route of administration, the appropriate dose is calculated according to body weight, body surface area, or organ size. Appropriate dosages may be ascertained by using established assays for determining blood concentration dosages in combination with appropriate dose-response data. The final dosage regimen is determined by the attending physician, taking into account various factors that modify the action of the drug, such as the specific activity of the drug, the severity of the injury and the patient's response, the patient's age, condition, weight, sex, and dietary habits, the severity of any infection, the time of administration, and other clinical factors. By way of example, a typical dose of the recombinant VWF of the present invention is about 50 IU / kg, which is equivalent to 500 μg / kg. As studies are conducted, more information will become available regarding appropriate dosage levels and duration of treatment for various diseases and conditions.

[0209] The practice of the present invention may employ conventional techniques and procedures within the skill of the art in organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry, and immunology, unless otherwise indicated. Such conventional techniques include polymer array synthesis, hybridization, ligation, and detection of hybridization using labels. Specific illustrations of suitable techniques can be obtained by reference to the following examples of this specification. However, it goes without saying that other equivalent conventional procedures can also be used. Such conventional techniques and descriptions can be found in standard laboratory manuals, such as Genome Analysis: A Laboratory Manual Series (Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A Laboratory Manual, PCR Primer: A Laboratory Manual, and Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press), Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, Highly stabilized York, Gait, "Oligonucleotide Synthesis: A Practical Approach" 1984, IRL Press, London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry 3rd Ed., W.H. Freeman Pub., Highly stabilized York, NY, and Berg et al. (2002) Biochemistry, 5th Ed., W.H. Freeman Pub., Highly stabilized York, NY, all of which are incorporated herein by reference in their entirety for all purposes.

[0210] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context dictates otherwise. Thus, for example, "a polymerase" refers to an agent or mixture of such agents, reference to "the method" includes references to equivalent steps and methods known to those skilled in the art, and so forth.

[0211] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context dictates otherwise. Thus, for example, "a polymerase" refers to an agent or mixture of such agents, reference to "the method" includes references to equivalent steps and methods known to those skilled in the art, and so forth.

[0212] Unless otherwise specified, 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. All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing the devices, compositions, formulations, and methods described in these publications and that can be used in connection with the inventions described herein.

[0213] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, to the tenth of the unit of the lower limit, as well as any other stated or intervening value in that stated range, is encompassed within the invention, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, which are also encompassed within the invention, subject to any specific excluded limits in the stated range. Where the stated range includes one or both of those upper and lower limits, ranges excluding either or both of those included upper and lower limits are also encompassed within the invention.

[0214] In the above description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of ordinary skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the present invention.

[0215] Although the present invention will be described primarily with reference to specific embodiments, it is anticipated that other embodiments will be apparent to those skilled in the art upon reading this disclosure, and such embodiments are intended to be encompassed by the methods of the present invention. EXAMPLES

[0216] Example 1: Treatment of GI bleeding episodes with recombinant von Willebrand factor in patients with severe von Willebrand disease: Subanalysis from a central Phase 3 on-demand study Introduction Gastrointestinal (GI) bleeding events occur in up to 20% of patients with von Willebrand disease (VWD) and are observed in association with angiodysplastic lesions in 2% to 4% of patients with VWD (1-3). GI bleeding is closely associated with the higher molecular weight of von Willebrand factor (VWF) and the absence of ultra-large multimers (ULM), which are most frequently seen in patients with type 2A and type 3 VWD (4). Resolution of GI bleeding usually requires higher doses and longer durations of therapy with plasma-derived VWF replacement concentrates compared to bleeding at other sites, and even then treatment may be unsuccessful (5). VONVENDI (von Willebrand factor [recombinant], Baxalta US Inc., Westlake Village, CA) is a recombinant VWF (rVWF) concentrate in which the most hemostatically effective VWF multimer, ULM, is preserved because it is not exposed to ADAMTS13 during production (6).

[0217] the purpose A central phase 3 clinical trial of rVWF evaluated its efficacy and safety with and without recombinant factor VIII (rFVIII) (ADVATE [Antihemophilic Factor (Recombinant)], Baxalta US Inc., Westlake Village, CA) for the treatment of bleeding in patients with severe VWD.(7) This subanalysis was performed using data from patients who experienced a GI bleeding event while participating in the central clinical trial.

[0218] method A phase 3 prospective randomized clinical trial (NCT01410227) to evaluate patient demographics, GI bleeding characteristics, hemostatic efficacy, timing of treatment and bleeding resolution, and dosing of rVWF ± rFVIII. The study population included men and women aged 18-65 years with type 3 or severe type 1 or 2 VWD and treated for 1 or more bleeding events within 12 months prior to enrollment. On-demand treatment of bleeding: Light / moderate bleeding: 40-60 IU / kg rVWF, heavy / severe bleeding: up to 80 IU / kg rVWF every 8-12 hours for 3-7 days.

[0219] An initial dose of rVWF was co-administered with rFVIII at a ratio of rVWF:rFVIII of 1.3:1±0.2, after which rVWF was administered alone, provided that hemostatic FVIII:C levels were achieved.

[0220] Hemostatic effectiveness was assessed on a 4-point scale (none=4, moderate=3, good=2, excellent=1).

[0221] Adverse events were monitored throughout the study.

[0222] result A total of 192 bleeding events were treated with rVWF during the study and evaluated for hemostatic efficacy. Hemostatic efficacy was rated in each case as either excellent (96.9%) or good (3.1%). Four patients with type 3 VWD, with a median age of 32.5 years, had a total of six GI bleeding events (Table 1).

[0223] Table 1. GI Bleeding Patient Demographic Subgroups TIFF0007641327000013.tif31143GI = gastrointestinal, VWD = von Willebrand disease.

[0224] Table 2 Bleeding characteristics and efficacy in GI bleeding subgroups TIFF0007641327000014.tif47150GI=Gastrointestinal tract. * Number of days from bleeding onset to first injection. †Time from first infusion of rVWF to resolution of bleeding episodes.

[0225] Table 3. Use of rVWF and rFVIII in GI bleeding subgroups TIFF0007641327000015.tif57158GI = gastrointestinal, rFVIII = recombinant factor VIII, rVWF = recombinant von Willebrand factor. * Time from end of rVWF infusion 1 to start of rVWF infusion 2.

[0226] Table 4. Adverse events in GI bleeding subgroups TIFF0007641327000016.tif36128GI=Gastrointestinal tract. * Tachycardia, dysgeusia, and paresthesias at the injection site.

[0227] Of the six GI bleeds, two each were reported as minor, moderate, and major / severe (Table 2). Sixty-seven percent of GI bleeds (4 / 6) required only one infusion of rVWF to successfully treat the bleed, and 33% of GI bleeds (2 / 6) required two infusions to achieve hemostasis. The median time to resolution known for four of the six bleeds was 8.3 hours (range, 1.8-18.6 hours). One hundred percent of GI bleeds treated with rVWF had excellent (83% [5 / 6]) or good (17% [1 / 6]; Figure 1) hemostatic efficacy.

[0228] There were a total of 28 adverse events in the four patients who had GI bleeding (Table 4). Three potentially related nonserious adverse events occurred in one patient (tachycardia, dysgeusia, and paresthesia at the infusion site). Serious adverse events included GI bleeding and constipation in one patient each, none of which were considered related to study drug.

[0229] This GI bleed was considered a serious adverse event because it arose from two chronic ulcers with recent evidence of bleeding and, as per the study protocol, was considered by the investigator to have occurred in a healthy individual under the same circumstances. The use of rVWF and rFVIII is shown in Table 3. The median dose per infusion was 58.7 IU / kg (range 53.5-60.5 IU / kg) for rVWF and 33.3 IU / kg (range 19.3-49.4 IU / kg) for rFVIII. The median total dose per bleed was 60.0 IU / kg (range 53.6-121.0 IU / kg) for rVWF and 33.3 IU / kg (range 19.3-49.4 IU / kg) for rFVIII.

[0230] conclusion In a subanalysis of a pivotal phase 3 trial, rVWF was safe and effective for on-demand treatment of GI bleeding in patients with severe VWD.

[0231] Of the six GI bleeds (two minor, two moderate, and two major / severe), hemostatic efficacy was rated excellent in five (83%) and good in one (17%). A single infusion of rVWF successfully treated four (67%) GI bleeds (one minor, two moderate, and one major / severe). Time to resolution of GI bleed was available for four patients and ranged from 1.8 to 18.6 hours (median 8.3 hours).

[0232] These findings from a small cohort of patients justify further evaluation of the role of rVWF in the treatment of GI bleeding and angiodysplasia in a larger population of patients with VWD.

[0233] Due to the emerging association between vascular dysplasia and the absence of high molecular weight VWF multimers, rVWF, due to its high ULM content, may be of particular benefit in this patient population ( 8 , 9 ).

[0234] References TIFF0007641327000017.tif41130

[0235] Example 2: Pharmacokinetics, safety, and efficacy of recombinant von Willebrand factor (rVWF) in the treatment of bleeding overview: The objectives of this Phase 3 study are to evaluate the pharmacokinetics of rVWF:rFVIII and rVWF, as well as the safety and efficacy of rVWF:rFVIII and rVWF in treating bleeding events in subjects with severe hereditary von Willebrand disease (VWD). TIFF0007641327000018.tif179147TIFF0007641327000019.tif176147

[0236] Key Evaluation Criteria Primary Criteria: Primary Outcome #1 Percentage of participants successfully treated for treated bleeding episodes [Timeframe: rVWF: 12 months from first infusion of rFVIII or rVWF]

[0237] Treatment success was defined as the degree of control of bleeding episodes (BE) using a mean efficacy assessment score of less than 2.5 for BE in participants treated with the study drug (recombinant von Willebrand factor [rVWF] with or without recombinant factor VIII [rFVIII]) during the study period. Assessment scores used were: excellent = 1 - actual infusions less than or equal to the predicted number of infusions needed to treat BE, no additional VWF needed (total BE); good = 2 - more than 1-2 infusions (low / moderate BE) or less than 1.5 infusions (high-volume BE) than predicted needed to control BE, no additional VWF needed (total BE); moderate = 3 - more than 3 infusions (low / moderate BE) or more than 1.5 infusions (high-volume BE) than predicted needed to control BE, no additional VWF needed (total BE); none = 4 - severe uncontrolled bleeding or no change in the intensity of bleeding, additional VWF needed. Participants in the full analysis set for whom the primary efficacy assessment was available (prospective - excluding gastrointestinal bleeding) were included.

[0238] Secondary Criteria Secondary Criteria:Secondary Item #1 Percentage of treated bleeding episodes with excellent or good efficacy rating [Timeframe: rVWF: 12 months from first infusion of rFVIII or rVWF]

[0239] Efficacy assessments of "excellent" or "good" for control of bleeding episodes (BE) with the study drug (recombinant von Willebrand factor [rVWF] with or without recombinant factor VIII [rFVIII]) were defined as follows: excellent - actual infusions were less than or equal to the predicted number of infusions needed to treat BE, and no additional von Willebrand factor (VWF) was needed (total BE); good - more than 1-2 infusions (low / moderate BE) or less than 1.5 infusions (high BE) more than predicted needed to control BE, and no additional VWF was needed (total BE). The dataset included predicted BE from prospective studies treated with the study drug, for which efficacy assessments were available from participants in the full analysis set.

[0240] Secondary Criteria:Secondary Item #2 Percentage of treated bleeding episodes with excellent or good efficacy, excluding gastrointestinal bleeding [Time frame: rVWF: 12 months from first infusion of rFVIII or rVWF]

[0241] An efficacy assessment of "excellent" or "good" for control of bleeding episodes (BE) with the study drug (recombinant von Willebrand factor [rVWF] with or without recombinant factor VIII [rFVIII]) was defined as follows: excellent - actual infusions were less than or equal to the predicted number of infusions needed to treat BE, and no additional von Willebrand factor (VWF) was needed (total BE); good - more than 1-2 infusions (minor / moderate BE) or less than 1.5 infusions (major BE) more than predicted needed to control BE, and no additional VWF was needed (total BE). The dataset included BE, excluding prospectively predicted gastrointestinal (GI) bleeding, treated with the study drug, for which efficacy assessments were available from participants in the full analysis set.

[0242] Secondary Criteria:Secondary Item #3 Number of infusions of rVWF:rFVIII and / or rVWF per bleeding episode [Time frame: 12 months from first infusion of rVWF:rFVIII or rVWF]

[0243] The actual number of infusions of recombinant von Willebrand factor:recombinant factor VIII (rVWF:rFVIII) and / or rVWF required to treat bleeding episodes (BE). BE was to be treated first with an infusion of rVWF:rFVIII, followed by rVWF with or without rFVIII based on FVIII levels, if available. If FVIII levels were not available, individual participant PK data were used to determine rFVIII dose. The dataset included prospectively predicted BE treated with the investigational drug for which efficacy assessments were available from participants in the full analysis set.

[0244] Secondary Criteria:Secondary Item #4 Number of units of rVWF:rFVIII and / or rVWF per bleeding episode [Time frame: 12 months from first infusion of rVWF:rFVIII or rVWF]

[0245] Units are provided as the actual dose [IU / kg] of recombinant von Willebrand factor:recombinant factor VIII (rVWF:rFVIII) and / or rVWF required to treat bleeding episodes (BE). BE was to be treated initially with an infusion of rVWF:rFVIII, followed by rVWF with or without rFVIII based on FVIII levels, if available. When FVIII levels were not available, individual participant PK data were used to determine rFVIII dose. The dataset included prospectively predicted BE treated with investigational drug with known lot number, for which efficacy assessments from participants in the full analysis set were available.

[0246] Secondary Criteria:Secondary Item #5 Percentage of participants who develop inhibitory antibodies to FVIII [Time frame: rVWF: 12 months from first infusion of rFVIII or rVWF]

[0247] The development of neutralizing antibodies (inhibitors) to factor VIII (FVIII) was assessed by the Nijmegen modification of the Bethesda assay. A positive FVIII inhibitor test was defined as 0.4 or more Bethesda units / mL (BU / mL) by the Nijmegen modification of the Bethesda assay, confirmed by a second test on an independent sample obtained 2-4 weeks after the first test. Category titles include the number of participants [N] who provided data for that category.

[0248] Secondary Criteria:Secondary Item #6 Percentage of participants who develop inhibitory antibodies to VWF [Time frame: from signing of informed consent to 12 months after first infusion of rVWF:rFVIII or rVWF]

[0249] Neutralizing antibodies (inhibitors) to von Willebrand factor ristocetin cofactor (VWF:RCo), VWF collagen binding (VWF:CB), and VWF factor VIII binding (VWF:FVIIIB) activities were measured using the Nijmegen modification of the Bethesda assay. One Bethesda unit (BU) is therefore defined as the amount of inhibitor that reduced the measured activity in the assay to 50% of the activity of the negative control sample. The assay was validated using human plasma samples from two type 3 VWD patients with low (1-2 BU / mL) and high (approximately 10 BU / mL) titers of inhibitors, and plasma samples from non-human primates immunized with human rVWF (>100 BU / mL). Category titles include the number [N] of participants who provided data for that category.

[0250] Secondary Criteria:Secondary Item #7 Percentage of participants who develop binding antibodies to VWF [Time frame: from signing of informed consent to 12 months after first infusion of rVWF:rFVIII or rVWF]

[0251] The presence of total binding anti-VWF antibodies was determined by enzyme-linked immunosorbent assay (ELISA) using polyclonal anti-human immunoglobulin (Ig) antibodies (IgG, IgM, and IgA). Category titles include the number of participants [N] who provided data for that category.

[0252] Secondary Criteria:Secondary Item #8 Percentage of participants who develop binding antibodies to CHO [Time frame: from signing of informed consent to 12 months after first infusion of rVWF:rFVIII or rVWF]

[0253] The presence of total anti-CHO antibodies was determined by measuring total immunoglobulin (Ig) antibodies (IgG, IgA, IgM) against Chinese hamster ovary (CHO) proteins using an enzyme-linked immunosorbent assay (ELISA). Category titles include the number of participants [N] who provided data for that category.

[0254] Secondary Criteria:Secondary Item #9 Percentage of participants who develop binding antibodies to rFurin [Time frame: from signing of informed consent to 12 months after first infusion of rVWF:rFVIII or rVWF]

[0255] The presence of total anti-rFurin binding antibodies was determined by measuring total immunoglobulin (Ig) antibodies (IgG, IgA, IgM) against the rFurin protein using an enzyme-linked immunosorbent assay (ELISA). Category titles include the number of participants [N] who provided data for that category.

[0256] Secondary Criteria: Secondary Item #10 Percentage of participants developing binding antibodies to mouse immunoglobulin [Time frame: from signing of informed consent to 12 months after first infusion of rVWF:rFVIII or rVWF]

[0257] The presence of total anti-mouse (IgG) binding antibodies was determined using an enzyme-linked immunosorbent assay (ELISA). Category titles include the number of participants [N] who provided data for that category.

[0258] Secondary Criteria:Secondary Item #11 Percentage (%) of participants who experienced a thrombotic event [Time frame: from signing of informed consent to 12 months after first infusion of rVWF:rFVIII or rVWF]

[0259] Secondary Criteria: Secondary Item #12 Number of study drug-related adverse events, including clinically significant changes in laboratory parameters and vital signs [Time frame: rVWF: 12 months from first infusion of rFVIII or rVWF]

[0260] Adverse events (AEs) related to the study drug (recombinant von Willebrand factor [rVWF] with or without recombinant factor VIII [rFVIII]) should be described. Include only laboratory parameters (hematology and clinical chemistry) and vital signs (physical examination, ECG) with clinically significant findings recorded as AEs. Categories are presented as severity-organ system class-preferred term severity: serious adverse events (SAEs), non-serious adverse events (nsAEs). Organ System Classes: Cardiac Disorders (CARD); General Disorders and Administration Site Conditions (GEN); Investigations (INV); Nervous System Disorders (NERV); Skin and Subcutaneous Tissue Disorders (SKN); Vascular Disorders (VAS). Category titles include the number [N] of AEs in that category.

[0261] Secondary Criteria:Secondary Item #13 Number of participants with study drug-related adverse events, including clinically significant changes in laboratory parameters and vital signs [Timeframe: rVWF: 12 months from first infusion of rFVIII or rVWF]

[0262] Describe the number of participants with adverse events (AEs) related to the study drug (recombinant von Willebrand factor [rVWF] with or without recombinant factor VIII [rFVIII]). Include only laboratory parameters (hematology and clinical chemistry) and vital signs (physical examination, ECG) with clinically significant findings recorded as AEs. Categories presented as severity-organ system class-preferred term severity: serious adverse events (SAEs), non-serious adverse events (nsAEs). Organ System Classes: Cardiac Disorders (CARD); General Disorders and Administration Site Conditions (GEN); Laboratory Investigations (INV); Nervous System Disorders (NERV); Skin and Subcutaneous Tissue Disorders (SKN); Vascular Disorders (VAS).

[0263] Secondary Outcome Scale:Secondary Outcome #14 Number of study drug-related infusion-related adverse events, including clinically significant changes in laboratory parameters and vital signs [Time frame: rVWF: 12 months from first infusion of rFVIII or rVWF]

[0264] Adverse events (AEs) related to the study drug (recombinant von Willebrand factor [rVWF] with or without recombinant factor VIII [rFVIII]) are described. Include only laboratory parameters (hematology and clinical chemistry) and vital signs (physical examination, ECG) with clinically significant findings recorded as AEs. Categories are presented as severity-organ system class-preferred term severity: serious adverse events (SAEs), non-serious adverse events (nsAEs). Organ System Classes: Cardiac Disorders (CARD); General Disorders and Administration Site Conditions (GEN); Laboratory Investigations (INV); Nervous System Disorders (NERV); Skin and Subcutaneous Tissue Disorders (SKN); Vascular Disorders (VAS).

[0265] Secondary Criteria:Secondary Item #15 PK50-VWF: Area under the plasma concentration / time curve from time 0 to infinity (AUC0-∞ / dose) of RCo [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0266] Area under the plasma concentration curve (AUC) from time 0 to infinity for von Willebrand factor ristocetin cofactor (VWF:RCo) following infusion of 50 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) (ratio 1.3:1 ± 0.2) [rVWF:rFVIII] administered with 38.5 IU / kg recombinant factor VIII (rFVIII), or 50 IU / kg VWF:RCo rVWF [rVWF] administered with saline (placebo) for subjects in the PK50 treatment groups (treatment groups 1 and 2). Category titles include the number of participants [N] who provided data for that category.

[0267] Secondary Criteria:Secondary Item #16 PK50-VWF: Area under the plasma concentration / time curve from time 0 to 96 hours (AUC0-96h / dose) of RCo [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes, and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28±3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0268] Area under the plasma concentration curve (AUC) of von Willebrand factor ristocetin cofactor (VWF:RCo) from time 0 to 96 hours following infusion of 50 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) (ratio 1.3:1 ± 0.2) [rVWF:rFVIII] administered with 38.5 IU / kg recombinant factor VIII (rFVIII) or 50 IU / kg VWF:RCo rVWF [rVWF] administered with saline (placebo) for participants in the PK50 treatment groups (treatment groups 1 and 2). Category titles include the number of participants [N] who provided data for that category.

[0269] Secondary Criteria: Secondary Item #17 PK50-VWF: Mean residence time of RCo [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours post-infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0270] Mean residence time (MRT) of von Willebrand factor ristocetin cofactor (VWF:RCo) following infusion of 50 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) administered with 38.5 IU / kg recombinant factor VIII (rFVIII) (ratio 1.3:1 ± 0.2) [rVWF:rFVIII] or 50 IU / kg VWF:RCo rVWF [rVWF] administered with saline (placebo) for participants in the PK50 treatment groups (Arms 1 and 2). Category titles include the number of participants [N] who provided data for that category.

[0271] Secondary Criteria:Secondary Item #18 PK50-VWF: Clearance of RCo [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours post-infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0272] Clearance (CL) of von Willebrand factor ristocetin cofactor (VWF:RCo) following infusion of 50 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) (ratio 1.3:1 ± 0.2) [rVWF:rFVIII] administered with 38.5 IU / kg recombinant factor VIII (rFVIII) or 50 IU / kg VWF:RCo rVWF [rVWF] administered with saline (placebo) for participants in the PK50 treatment groups (Arms 1 and 2). Category titles include the number of participants [N] who provided data for that category.

[0273] Secondary Criteria:Secondary Item #19 PK50-VWF: Stepwise recovery of RCo [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours post-infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0274] Stepwise recovery (IR) at maximum plasma concentration of von Willebrand factor ristocetin cofactor (VWF:RCo) following infusion of 50 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo) administered with 38.5 IU / kg recombinant factor VIII (rFVIII) (ratio 1.3:1 ± 0.2) [rVWF:rFVIII] or 50 IU / kg VWF:RCo rVWF [rVWF] administered with saline (placebo) for participants in the PK50 treatment groups (treatment groups 1 and 2). Category titles include the number of participants [N] who provided data for that category.

[0275] Secondary Criteria:Secondary Item #20 Elimination half-life of PK50-VWF:Co [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours post-infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0276] Elimination phase half-life (T1 / 2) of von Willebrand factor ristocetin cofactor (VWF:RCo) following infusion of 50 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) (ratio 1.3:1 ± 0.2) [rVWF:rFVIII] administered with 38.5 IU / kg recombinant FVIII (rFVIII) or 50 IU / kg VWF:RCo rVWF [rVWF] administered with saline (placebo) for participants in the PK50 treatment groups (Arms 1 and 2). Category titles include the number of participants [N] who provided data for that category.

[0277] Secondary Criteria:Secondary Item #21 PK50-VWF: Volume of distribution of RCo at steady state [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0278] Volume of distribution (Vss) at steady state of von Willebrand factor ristocetin cofactor (VWF:RCo) following infusion of 50 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) (ratio 1.3:1 ± 0.2) [rVWF:rFVIII] administered with 38.5 IU / kg recombinant factor VIII (rFVIII) or 50 IU / kg VWF:RCo rVWF [rVWF] administered with saline (placebo) for participants in the PK50 treatment groups (Arms 1 and 2). Category titles include the number of participants [N] who provided data for that category.

[0279] Secondary Criteria: Secondary Item #22 PK50-VWF: Area under the plasma concentration / time curve from time 0 to infinity (AUC0-∞ / dose) of Ag [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0280] Area under the plasma concentration curve (AUC) of von Willebrand factor antigen (VWF:Ag) from time 0 to infinity following infusion of 50 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) (ratio 1.3:1 ± 0.2) [rVWF:rFVIII] administered with 38.5 IU / kg recombinant factor VIII (rFVIII), or 50 IU / kg VWF:RCo rVWF [rVWF] administered with saline (placebo) for participants in the PK50 treatment groups (treatment groups 1 and 2). Category titles include the number of participants [N] who provided data for that category.

[0281] Secondary Criteria: Secondary Item #23 PK50-VWF: Area under the plasma concentration / time curve from time 0 to 96 hours of Ag (AUC0-96h / dose) [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes, and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28±3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0282] Area under the plasma concentration curve (AUC) of von Willebrand factor antigen (VWF:Ag) from time 0 to 96 hours following infusion of 50 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) (ratio 1.3:1 ± 0.2) [rVWF:rFVIII] administered with 38.5 IU / kg recombinant factor VIII (rFVIII), or 50 IU / kg VWF:RCo rVWF [rVWF] administered with saline (placebo) for participants in the PK50 treatment groups (treatment groups 1 and 2). Category titles include the number of participants [N] who provided data for that category.

[0283] Secondary Criteria:Secondary Item #24 PK50-VWF:Ag mean residence time [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours post-infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0284] Mean residence time (MRT) of von Willebrand factor antigen (VWF:Ag) following infusion of 50 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) (ratio 1.3:1 ± 0.2) [rVWF:rFVIII] administered with 38.5 IU / kg recombinant factor VIII (rFVIII) or 50 IU / kg VWF:RCo rVWF [rVWF] administered with saline (placebo) for participants in the PK50 treatment groups (Arms 1 and 2). Category titles include the number of participants [N] who provided data for that category.

[0285] Secondary Criteria:Secondary Item #25 PK50-VWF:Ag clearance [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours post-infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0286] Von Willebrand factor antigen (VWF:Ag) clearance (CL) following infusion of 50 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) (ratio 1.3:1 ± 0.2) [rVWF:rFVIII] administered together with 38.5 IU / kg recombinant factor VIII (rFVIII) or 50 IU / kg VWF:RCo rVWF [rVWF] administered together with saline (placebo) for participants in the PK50 treatment groups (treatment groups 1 and 2). Category titles include the number of participants [N] who provided data for that category.

[0287] Secondary Criteria:Secondary Item #26 PK50-VWF:Ag stepwise recovery [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours post-infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0288] Stepwise recovery (IR) at maximum plasma concentration of von Willebrand factor antigen (VWF:Ag) following infusion of 50 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) (ratio 1.3:1 ± 0.2) [rVWF:rFVIII] administered with 38.5 IU / kg recombinant factor VIII (rFVIII) or 50 IU / kg VWF:RCo rVWF [rVWF] administered with saline (placebo) for participants in the PK50 treatment groups (treatment groups 1 and 2). Category titles include the number of participants [N] who provided data for that category.

[0289] Secondary Criteria:Secondary Item #27 PK50-VWF:Ag elimination half-life [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours post-infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0290] Elimination phase half-life (T1 / 2) of von Willebrand factor antigen (VWF:Ag) following infusion of 50 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) (ratio 1.3:1 ± 0.2) [rVWF:rFVIII] administered with 38.5 IU / kg recombinant FVIII (rFVIII) or 50 IU / kg VWF:RCo rVWF [rVWF] administered with saline (placebo) for participants in the PK50 treatment groups (Arms 1 and 2). Category titles include the number of participants [N] who provided data for that category.

[0291] Secondary Criteria:Secondary Item #28 PK50-VWF: Ag steady-state volume of distribution [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours post-infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0292] Steady-state volume of distribution (Vss) of von Willebrand factor antigen (VWF:Ag) following infusion of 50 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) (ratio 1.3:1 ± 0.2) [rVWF:rFVIII] administered with 38.5 IU / kg recombinant factor VIII (rFVIII), or 50 IU / kg VWF:RCo rVWF [rVWF] administered with saline (placebo) for participants in the PK50 treatment groups (treatment groups 1 and 2). Category titles include the number of participants [N] who provided data for that category.

[0293] Secondary Criteria:Secondary Item #29 PK50-VWF: Area under the plasma concentration / time curve from time 0 to infinity (AUC0-∞ / dose) of CB [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0294] Area under the plasma concentration curve (AUC) from time 0 to infinity for von Willebrand factor collagen binding (VWF:CB) following infusion of 50 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) (ratio 1.3:1 ± 0.2) [rVWF:rFVIII] administered with 38.5 IU / kg recombinant factor VIII (rFVIII) or 50 IU / kg VWF:RCo rVWF [rVWF] administered with saline (placebo) for participants in the PK50 treatment groups (treatment groups 1 and 2). Category titles include the number of participants [N] who provided data for that category.

[0295] Secondary Criteria:Secondary Item #30 PK50-VWF: Area under the plasma concentration / time curve from time 0 to 96 hours (AUC0-96h / dose) of CB [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28±3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0296] Area under the plasma concentration curve (AUC) of von Willebrand factor collagen binding (VWF:CB) from time 0 to 96 hours following infusion of 50 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) (ratio 1.3:1 ± 0.2) [rVWF:rFVIII] administered with 38.5 IU / kg recombinant factor VIII (rFVIII), or 50 IU / kg VWF:RCo rVWF [rVWF] administered with saline (placebo) for participants in the PK50 treatment groups (treatment groups 1 and 2). Category titles include the number of participants [N] who provided data for that category.

[0297] Secondary Criteria:Secondary Item #31 PK50-VWF: Mean residence time of CB [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0298] Von Willebrand factor collagen binding (VWF:CB) mean residence time (MRT) following infusion of 50 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) (ratio 1.3:1 ± 0.2) [rVWF:rFVIII] administered with 38.5 IU / kg recombinant factor VIII (rFVIII) or 50 IU / kg VWF:RCo rVWF [rVWF] administered with saline (placebo) for participants in the PK50 treatment groups (Arms 1 and 2). Category titles include the number of participants [N] who provided data for that category.

[0299] Secondary Criteria:Secondary Item #32 PK50-VWF: Clearance of CB [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours post-infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0300] Clearance (CL) of von Willebrand factor collagen-bound (VWF:CB) following infusion of 50 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) (ratio 1.3:1 ± 0.2) [rVWF:rFVIII] administered together with 38.5 IU / kg recombinant factor VIII (rFVIII) or 50 IU / kg VWF:RCo rVWF [rVWF] administered together with saline (placebo) for participants in the PK50 treatment groups (treatment groups 1 and 2). Category titles include the number of participants [N] who provided data for that category.

[0301] Secondary Criteria:Secondary Item #33 PK50-VWF: Stepwise recovery of CB [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours post-infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0302] Stepwise recovery (IR) at maximum plasma concentration of von Willebrand factor collagen-bound (VWF:CB) following infusion of 50 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) (ratio 1.3:1 ± 0.2) [rVWF:rFVIII] administered together with 38.5 IU / kg recombinant factor VIII (rFVIII) or 50 IU / kg VWF:RCo rVWF [rVWF] administered together with saline (placebo) for participants in the PK50 treatment groups (treatment groups 1 and 2). Category titles include the number of participants [N] who provided data for that category.

[0303] Secondary Criteria:Secondary Item #34 PK50-VWF: Elimination half-life of CB [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours post-infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0304] Elimination phase half-life (T1 / 2) of von Willebrand factor collagen bound (VWF:CB) following infusion of 50 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) (ratio 1.3:1 ± 0.2) [rVWF:rFVIII] administered with 38.5 IU / kg recombinant FVIII (rFVIII) or 50 IU / kg VWF:RCo rVWF [rVWF] administered with saline (placebo) for participants in the PK50 treatment groups (Arms 1 and 2). Category titles include the number of participants [N] who provided data for that category.

[0305] Secondary Criteria:Secondary Item #35 PK50-VWF: Volume of distribution at steady state of CB [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0306] Steady-state volume of distribution (Vss) of von Willebrand factor collagen-bound (VWF:CB) following infusion of 50 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) (ratio 1.3:1 ± 0.2) [rVWF:rFVIII] administered with 38.5 IU / kg recombinant factor VIII (rFVIII) or 50 IU / kg VWF:RCo rVWF [rVWF] administered with saline (placebo) for participants in the PK50 treatment groups (Arms 1 and 2). Category titles include the number of participants [N] who provided data for that category.

[0307] Secondary Criteria:Secondary Item #36 PK50-Area under the plasma concentration / time curve from time 0 to infinity (AUC0-∞ / dose) of FVIII:C [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes, and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0308] Area under the plasma concentration curve (AUC) from time 0 to infinity for factor VIII activity (FVIII:C) following infusion of 50 IU / kg recombinant von Willebrand factor:von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) (ratio 1.3:1 ± 0.2) [rVWF:rFVIII] administered with 38.5 IU / kg recombinant factor VIII (rFVIII), or 50 IU / kg VWF:RCo rVWF [rVWF] administered with saline (placebo) for participants in the PK50 treatment groups (treatment groups 1 and 2). Category titles include the number of participants [N] who provided data for that category.

[0309] Secondary Criteria:Secondary Item #37 PK50-FVIII:C area under the plasma concentration / time curve from time 0 to 96 hours (AUC0-96h / dose) [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes, and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28±3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0310] Area under the plasma concentration curve (AUC) of factor VIII activity (FVIII:C) from time 0 to 96 hours following infusion of 50 IU / kg recombinant von Willebrand factor:von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) (ratio 1.3:1 ± 0.2) [rVWF:rFVIII] administered together with 38.5 IU / kg recombinant factor VIII (rFVIII) or 50 IU / kg VWF:RCo rVWF [rVWF] administered together with saline (placebo) for participants in the PK50 treatment groups (treatment groups 1 and 2). Category titles include the number of participants [N] who provided data for that category.

[0311] Secondary Criteria:Secondary Item #38 Mean residence time of PK50-FVIII:C [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0312] Mean residence time (MRT) of factor VIII activity (FVIII:C) following infusion of 50 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) (ratio 1.3:1 ± 0.2) [rVWF:rFVIII] administered together with 38.5 IU / kg recombinant factor VIII (rFVIII) for participants in the PK50 treatment groups (treatment groups 1 and 2).

[0313] Secondary Criteria:Secondary Item #39 PK50-FVIII:C Clearance [Time Frame: Pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0314] Clearance (CL) of factor VIII activity (FVIII:C) following infusion of 50 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) (ratio 1.3:1 ± 0.2) [rVWF:rFVIII] administered together with 38.5 IU / kg recombinant factor VIII (rFVIII) for participants in the PK50 treatment groups (treatment groups 1 and 2).

[0315] Secondary Criteria:Secondary Item #40 Stepwise recovery of PK50-FVIII:C [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours post-infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0316] Stepwise recovery (IR) at maximum plasma concentration of factor VIII activity (FVIII:C) following infusion of 50 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) (ratio 1.3:1 ± 0.2) [rVWF:rFVIII] administered together with 38.5 IU / kg recombinant factor VIII (rFVIII) for participants in the PK50 treatment groups (treatment groups 1 and 2).

[0317] Secondary Criteria:Secondary Item #41 Elimination half-life of PK50-FVIII:C [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours post-infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0318] Elimination phase half-life (T1 / 2) of factor VIII activity (FVIII:C) following infusion of 50 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) (ratio 1.3:1 ± 0.2) [rVWF:rFVIII] administered together with 38.5 IU / kg recombinant FVIII (rFVIII) for participants in the PK50 treatment groups (treatment groups 1 and 2).

[0319] Secondary Criteria:Secondary Item #42 Volume of distribution at steady state for PK50-FVIII:C [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0320] Steady-state volume of distribution (Vss) of factor VIII activity (FVIII:C) following infusion of 50 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) (ratio 1.3:1 ± 0.2) [rVWF:rFVIII] administered together with 38.5 IU / kg recombinant factor VIII (rFVIII) for participants in the PK50 treatment groups (treatment groups 1 and 2).

[0321] Secondary Criteria:Secondary Item #43 PK80-VWF: Area under the plasma concentration / time curve from time 0 to infinity (AUC0-∞ / dose) of RCo [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0322] Area under the plasma concentration curve (AUC) of von Willebrand factor ristocetin cofactor (VWF:RCo) from time 0 to infinity following infusion of 80 IU / kg recombinant von Willebrand factor:von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) [rVWF] for participants in the PK80 arm (participants from Arm 3 with PK80 data only). PK assessments performed at the first infusion of 80 IU / kg rVWF [PK1] and the second infusion of 80 IU / kg rVWF after the participant had been treated on demand for bleeding symptoms for at least 6 months after their first infusion of study drug [PK2]. Category titles include the number of participants who provided data for that category [N].

[0323] Secondary Criteria:Secondary Item #44 PK80-VWF: Area under the plasma concentration / time curve from time 0 to 96 hours (AUC0-96h / dose) of RCo [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes, and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28±3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0324] Area under the plasma concentration curve (AUC) of von Willebrand factor ristocetin cofactor (VWF:RCo) from time 0 to 96 hours following infusion of 80 IU / kg recombinant von Willebrand factor:von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) [rVWF] for participants in the PK80 arm (participants from Arm 3 with PK80 data only). PK assessments performed at the first infusion of 80 IU / kg rVWF [PK1] and the second infusion of 80 IU / kg rVWF after the participant had been treated on demand for bleeding symptoms for at least 6 months after their first infusion of study drug [PK2]. Category titles include the number of participants who provided data for that category [N].

[0325] Secondary Criteria:Secondary Item #45 PK80-VWF: Mean residence time of RCo [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours post-infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0326] Mean residence time (MRT) of von Willebrand factor ristocetin cofactor (VWF:RCo) following infusion of 80 IU / kg recombinant von Willebrand factor:von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) [rVWF] for participants in the PK80 arm (participants from Arm 3 with PK80 data only). PK assessments performed at the first infusion of 80 IU / kg rVWF [PK1] and the second infusion of 80 IU / kg rVWF after the participant had been treated on demand for bleeding symptoms for at least 6 months after their first infusion of study drug [PK2]. Category titles include the number of participants who provided data for that category [N].

[0327] Secondary Criteria:Secondary Item #46 PK80-VWF: Clearance of RCo [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours post-infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0328] Clearance (CL) of von Willebrand factor ristocetin cofactor (VWF:RCo) following infusion of 80 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) [rVWF] for participants in the PK80 arm (participants from Arm 3 with PK80 data only). PK assessments performed at the first infusion of 80 IU / kg rVWF [PK1] and the second infusion of 80 IU / kg rVWF after participants had been treated on demand for bleeding symptoms for at least 6 months after their first infusion of study drug [PK2]. Category titles include the number of participants who provided data for that category [N].

[0329] Secondary Criteria:Secondary Item #47 PK80-VWF: Stepwise recovery of RCo [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours post-infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0330] Stepwise recovery (IR) area under the plasma concentration curve (AUC) from time 0 to infinity at maximum plasma concentration of von Willebrand factor ristocetin cofactor (VWF:RCo) following an infusion of 80 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) [rVWF] for participants in the PK80 arm (participants from arm 3 with PK80 data only). PK assessments performed at the first infusion of 80 IU / kg rVWF [PK1] and the second infusion of 80 IU / kg rVWF after the participant had been treated on demand for bleeding symptoms for at least 6 months after their first infusion of study drug [PK2]. Category titles include the number of participants who provided data for that category [N].

[0331] Secondary Criteria:Secondary Item #48 Elimination half-life of PK80-VWF:Co [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0332] Elimination phase half-life (T1 / 2) of von Willebrand factor ristocetin cofactor (VWF:RCo) following infusion of 80 IU / kg recombinant von Willebrand factor:von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) [rVWF] for participants in the PK80 arm (participants from Arm 3 with PK80 data only). PK assessments performed at the first infusion of 80 IU / kg rVWF [PK1] and the second infusion of 80 IU / kg rVWF after the participant had been treated on demand for bleeding symptoms for at least 6 months after their first infusion of study drug [PK2]. Category titles include the number of participants who provided data for that category [N].

[0333] Secondary Criteria:Secondary Item #49 PK80-VWF: Volume of distribution of RCo at steady state [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0334] Volume of distribution at steady state (Vss) of von Willebrand factor ristocetin cofactor (VWF:RCo) following an infusion of 80 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) [rVWF] for participants in the PK80 arm (participants from Arm 3 with PK80 data only). PK assessments performed at the first infusion of 80 IU / kg rWVF [PK1] and the second infusion of 80 IU / kg rVWF after the participant had been treated on demand for bleeding symptoms for at least 6 months after the first infusion of their study drug. PK assessments performed at the first infusion of 80 IU / kg rWVF [PK1] and the second infusion of 80 IU / kg rVWF after the participant had been treated on demand for bleeding symptoms for at least 6 months after the first infusion of their study drug [PK2]. Category titles include the number of participants [N] who provided data for that category.

[0335] Secondary Criteria: Secondary Item #50 PK80-VWF: Area under the plasma concentration / time curve from time 0 to infinity (AUC0-∞ / dose) of Ag [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0336] Area under the plasma concentration curve (AUC) of von Willebrand factor antigen (VWF:Ag) from time 0 to infinity following infusion of 80 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) [rVWF] for participants in the PK80 arm (participants from Arm 3 with PK80 data only). PK assessments performed at the first infusion of 80 IU / kg rVWF [PK1] and the second infusion of 80 IU / kg rVWF after the participant had been treated on demand for bleeding symptoms for at least 6 months after their first infusion of study drug [PK2]. Category titles include the number of participants who provided data for that category [N].

[0337] Secondary Criteria: Secondary Item #51 PK80-VWF:Area under the plasma concentration / time curve from time 0 to 96 hours (AUC0-96h / dose) of Ag [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28±3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0338] Area under the plasma concentration curve (AUC) of von Willebrand factor antigen (VWF:Ag) from time 0 to 96 hours following infusion of 80 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) [rVWF] for participants in the PK80 arm (participants from Arm 3 with PK80 data only). PK assessments performed at the first infusion of 80 IU / kg rVWF [PK1] and the second infusion of 80 IU / kg rVWF after participants had been treated on demand for bleeding symptoms for at least 6 months after their first infusion of study drug [PK2]. Category titles include the number of participants who provided data for that category [N].

[0339] Secondary Criteria: Secondary Item #52 Mean residence time of PK80-VWF:Ag [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0340] Mean residence time (MRT) of von Willebrand factor antigen (VWF:Ag) following infusion of 80 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) [rVWF] for participants in the PK80 treatment arm (participants from Arm 3 with PK80 data only). PK assessments performed at the first infusion of 80 IU / kg rVWF [PK1] and the second infusion of 80 IU / kg rVWF after participants had been treated on demand for bleeding symptoms for at least 6 months after their first infusion of study drug [PK2]. Category titles include the number of participants who provided data for that category [N].

[0341] Secondary Criteria:Secondary Item #53 PK80-VWF:Ag clearance [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours post-infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0342] Clearance (CL) of von Willebrand factor antigen (VWF:Ag) following infusion of 80 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) [rVWF] for participants in the PK80 arm (participants from Arm 3 with PK80 data only). PK assessments performed at the first infusion of 80 IU / kg rVWF [PK1] and the second infusion of 80 IU / kg rVWF after participants had been treated on demand for bleeding symptoms for at least 6 months after their first infusion of study drug [PK2]. Category titles include the number of participants who provided data for that category [N].

[0343] Secondary Criteria:Secondary Item #54 Stepwise recovery of PK80-VWF:Ag [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours post-infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0344] Stepwise recovery (IR) area under the plasma concentration curve (AUC) from time 0 to infinity at maximum plasma concentration of von Willebrand factor antigen (VWF:Ag) following infusion of 80 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) [rVWF] for participants in the PK80 arm (participants from arm 3 with PK80 data only). PK assessments performed at the first infusion of 80 IU / kg rVWF [PK1] and the second infusion of 80 IU / kg rVWF after the participant had been treated on demand for bleeding symptoms for at least 6 months after their first infusion of study drug [PK2]. Category titles include the number of participants who provided data for that category [N].

[0345] Secondary Criteria:Secondary Item #55 Elimination half-life of PK80-VWF:Ag [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours post-infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0346] Elimination phase half-life (T1 / 2) of von Willebrand factor antigen (VWF:Ag) following infusion of 80 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) [rVWF] for participants in the PK80 treatment arm (participants from Arm 3 with PK80 data only). PK assessments performed at the first infusion of 80 IU / kg rVWF [PK1] and the second infusion of 80 IU / kg rVWF after participants had been treated on demand for bleeding symptoms for at least 6 months after their first infusion of study drug [PK2]. Category titles include the number of participants who provided data for that category [N].

[0347] Secondary Criteria: Secondary Item #56 PK80-VWF:Ag distribution volume at steady state [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0348] Steady-state volume of distribution (Vss) of von Willebrand factor antigen (VWF:Ag) following infusion of 80 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) [rVWF] for participants in the PK80 arm (participants from Arm 3 with PK80 data only). PK assessments performed at the first infusion of 80 IU / kg rVWF [PK1] and the second infusion of 80 IU / kg rVWF after participants had been treated on demand for bleeding symptoms for at least 6 months after their first infusion of study drug [PK2]. Category titles include the number of participants who provided data for that category [N].

[0349] Secondary Criteria:Secondary Item #57 PK80-VWF: Area under the plasma concentration / time curve from time 0 to infinity (AUC0-∞ / dose) of CB [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0350] Area under the plasma concentration curve (AUC) of von Willebrand factor collagen binding (VWF:CB) from time 0 to infinity following infusion of 80 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) [rVWF] for participants in the PK80 arm (participants from Arm 3 with PK80 data only). PK assessments performed at the first infusion of 80 IU / kg rVWF [PK1] and the second infusion of 80 IU / kg rVWF after the participant had been treated on demand for bleeding symptoms for at least 6 months after their first infusion of study drug [PK2]. Category titles include the number of participants who provided data for that category [N].

[0351] Secondary Criteria:Secondary Item #58 PK80-VWF: Area under the plasma concentration / time curve from time 0 to 96 hours (AUC0-96h / dose) of CB [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28±3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0352] Area under the plasma concentration curve (AUC) of von Willebrand factor collagen binding (VWF:CB) from time 0 to 96 hours following infusion of 80 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) [rVWF] for participants in the PK80 arm (participants from Arm 3 with PK80 data only). PK assessments performed at the first infusion of 80 IU / kg rVWF [PK1] and the second infusion of 80 IU / kg rVWF after participants had been treated on demand for bleeding symptoms for at least 6 months after their first infusion of study drug [PK2]. Category titles include the number of participants who provided data for that category [N].

[0353] Secondary Criteria: Secondary Item #59 PK80-VWF: Mean residence time of CB [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0354] Mean residence time (MRT) of von Willebrand factor collagen binding (VWF:CB) following infusion of 80 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) [rVWF] for participants in the PK80 treatment arm (participants from Arm 3 with PK80 data only). PK assessments performed at the first infusion of 80 IU / kg rWVF [PK1] and the second infusion of 80 IU / kg rVWF after participants had been treated on demand for bleeding symptoms for at least 6 months after their first infusion of study drug [PK2]. Category titles include the number of participants who provided data for that category [N].

[0355] Secondary Criteria: Secondary Item #60 PK80-VWF:Clearance of CB [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours post-infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0356] Clearance (CL) of von Willebrand factor collagen-bound (VWF:CB) following infusion of 80 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) [rVWF] for participants in the PK80 arm (participants from Arm 3 with PK80 data only). PK assessments performed at the first infusion of 80 IU / kg rWVF [PK1] and the second infusion of 80 IU / kg rVWF after participants had been treated on demand for bleeding symptoms for at least 6 months after their first infusion of study drug [PK2]. Category titles include the number of participants who provided data for that category [N].

[0357] Secondary Criteria:Secondary Item #61 PK80-VWF: Stepwise recovery of CB [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours post-infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0358] Stepwise recovery (IR) area under the plasma concentration curve (AUC) from time 0 to infinity at maximum plasma concentration of von Willebrand factor collagen bound (VWF:CB) following infusion of 80 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) [rVWF] for participants in the PK80 arm (participants from arm 3 with PK80 data only). PK assessments performed at the first infusion of 80 IU / kg rVWF [PK1] and the second infusion of 80 IU / kg rVWF after the participant had been treated on demand for bleeding symptoms for at least 6 months after their first infusion of study drug [PK2]. Category titles include the number of participants who provided data for that category [N].

[0359] Secondary Criteria: Secondary Item #62 Elimination half-life of PK80-VWF:CB [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours post-infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0360] Elimination phase half-life (T1 / 2) of von Willebrand factor collagen bound (VWF:CB) following infusion of 80 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) [rVWF] for participants in the PK80 treatment arm (participants from Arm 3 with PK80 data only). PK assessments performed at the first infusion of 80 IU / kg rVWF [PK1] and the second infusion of 80 IU / kg rVWF after participants had been treated on demand for bleeding symptoms for at least 6 months after their first infusion of study drug [PK2]. Category titles include the number of participants who provided data for that category [N].

[0361] Secondary Criteria: Secondary Item #63 PK80-VWF: Volume of distribution at steady state of CB [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0362] Steady-state volume of distribution (Vss) of von Willebrand factor collagen bound (VWF:CB) following infusion of 80 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) [rVWF] for participants in the PK80 treatment arm (participants from Arm 3 with PK80 data only). PK assessments performed at the first infusion of 80 IU / kg rVWF [PK1] and the second infusion of 80 IU / kg rVWF after participants had been treated on demand for bleeding symptoms for at least 6 months after their first infusion of study drug [PK2]. Category titles include the number of participants who provided data for that category [N].

[0363] Secondary Criteria: Secondary Item #64 Area under the plasma concentration / time curve from time 0 to infinity (AUC0-∞ / dose) of PK80-FVIII:C [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes, and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0364] Area under the plasma concentration curve (AUC) of factor VIII activity (FVIII:C) from time 0 to infinity following infusion of 80 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) [rVWF] for participants in the PK80 arm (participants from Arm 3 with PK80 data only). PK assessments performed at the first infusion of 80 IU / kg rVWF [PK1] and the second infusion of 80 IU / kg rVWF after the participant had been treated on demand for bleeding symptoms for at least 6 months after their first infusion of study drug [PK2]. Category titles include the number of participants who provided data for that category [N].

[0365] Secondary Criteria:Secondary Item #65 Area under the plasma concentration / time curve from time 0 to 96 hours (AUC0-96h / dose) of PK80-FVIII:C [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes, and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28±3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0366] Area under the plasma concentration curve (AUC) of factor VIII activity (FVIII:C) from time 0 to 96 hours following infusion of 80 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) [rVWF] for participants in the PK80 arm (participants from Arm 3 with PK80 data only). PK assessments performed at the first infusion of 80 IU / kg rVWF [PK1] and the second infusion of 80 IU / kg rVWF after the participant had been treated on demand for bleeding symptoms for at least 6 months after their first infusion of study drug [PK2]. Category titles include the number of participants who provided data for that category [N].

[0367] Secondary Criteria: Secondary Item #66 PK80 - Within-participant PK ratios of VWF:RCo, VWF:Ag, and VWF:CB at baseline and 6 months [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0368] Area under the plasma concentration curve (AUC) from time 0 to infinity per dose (AUC0-∞ / dose) for von Willebrand factor ristocetin cofactor (VWF:RCo), von Willebrand factor antigen (VWF:Ag), and von Willebrand factor collagen binding (VWF:CB). Each parameter was compared between two PK assessments after infusion of 80 IU / kg recombinant von Willebrand factor: von Willebrand factor ristocetin cofactor (VWF:RCo rVWF) [rVWF] for participants in the PK80 treatment group (participants from treatment group 3 with PK80 data only). The PK assessment was performed at the first infusion of 80 IU / kg rWF [PK1], and the second infusion of 80 IU / kg rVWF [PK2] after the participant had been treated on demand for bleeding symptoms for at least 6 months after their first infusion of study drug. Data for this endpoint, i.e., PK1 and PK2, were available for 13 participants.

[0369] Eligibility Criteria 18-65 years old (adults, seniors), all genders.

[0370] Inclusion Criteria: Participants must have been diagnosed with the following: Type 1 (von Willebrand factor: ristocetin cofactor activity (VWF:RCo) less than 20 IU / dL), or Type 2A (VWF:RCo <20 IU / dL), Type 2B (diagnosed by phenotype), Type 2N (Factor VIII activity (FVIII:C) <10% and previously confirmed genetics), Type 2M, or Type 3 (von Willebrand factor antigen (VWF:Ag) ≤ 3 IU / dL), or - Severe von Willebrand disease (VWD) with a history of requiring replacement therapy with von Willebrand factor concentrates to control bleeding

[0371] Participants enrolled in treatment for bleeding episodes were required to have a documented history of at least one bleeding episode requiring VWF clotting factor replacement therapy in the 12 months prior to enrollment.

[0372] Participants were required to have a Karnofsky score of 60% or higher.

[0373] Participants must be at least 18 years of age and no older than 65 years of age at the time of enrollment.

[0374] If female of childbearing potential, participants must present a negative pregnancy test.

[0375] Participants agree to use acceptable contraception for the duration of the study.

[0376] Participants are willing and able to comply with the requirements of the clinical trial protocol.

[0377] Exclusion criteria: Participants with a diagnosis of pseudo-VWD or another inherited or acquired coagulation disorder other than VWD (e.g., qualitative and numerical platelet disorders or elevated PT / international normalized ratio [INR] >1.4).

[0378] Participants with a confirmed history of VWF:RCo half-life <6 hours.

[0379] Participants with a history or presence of VWF inhibitors at screening.

[0380] Participants with a history or presence of a factor VIII (FVIII) inhibitor with a titer of ≥0.4 BU (by Nijmegen assay) or ≥0.6 BU (by Bethesda assay).

[0381] Participants with known hypersensitivity to any of the components of the study drug, such as mouse or hamster proteins.

[0382] Participants with a history of immune disorders except seasonal allergic rhinitis / conjunctivitis, mild asthma, food allergies, or animal allergies.

[0383] Participants with a history of thromboembolic events.

[0384] Absolute CD4 count 200 / mm 3 Participants who were HIV positive were less than .

[0385] Participants were diagnosed with cardiovascular disease (New York Heart Association [NYHA] class 1 to 4).

[0386] Participants with acute illness at screening (e.g., influenza, influenza-like syndrome, allergic rhinitis / conjunctivitis, non-seasonal asthma).

[0387] Participants had a diagnosis of significant liver disease as evidenced by any of the following: serum alanine aminotransferase (ALT) five times the upper limit of normal, hypoalbuminemia, portal hypertension (e.g., presence of splenomegaly unexplained otherwise, previous history of esophageal varices).

[0388] Participants with diagnosed kidney disease with creatinine levels of 2 mg / dL or higher.

[0389] Participants who are judged by the investigator to have clinically significant comorbidities (e.g., uncontrolled hypertension) that may pose additional risk to the participant.

[0390] Participants who had received treatment with immunomodulatory medications, excluding topical treatments (e.g., ointments, nasal sprays), within 30 days prior to enrollment.

[0391] Participants who were pregnant or breastfeeding at the time of enrollment.

[0392] Participants who have participated in another clinical trial involving an IP or investigational device within 30 days prior to enrollment, or who plan to participate in another clinical trial involving an investigational drug or investigational device during the course of this study.

[0393] Participants with a history of drug or alcohol abuse within 2 years prior to enrollment.

[0394] Participants with progressive, life-threatening disease and / or life expectancy less than 3 months.

[0395] Participants identified by the investigator as unable or unwilling to cooperate with study procedures.

[0396] Participants with mental conditions and / or uncooperative behavior that would prevent them from understanding the nature, scope, and possible outcomes of the study.

[0397] Participants who are incarcerated or forcibly detained pursuant to regulations and / or legal orders.

[0398] Pre-allocation details Forty-nine participants provided informed consent and were screened for the study, of which 37 received the study drug. Reasons for discontinuation included six screening failures, three participants withdrawing consent, one physician decision, and one participant receiving high-dose rFVIII for oral treatment, which was closed in the treatment arm for which this participant was eligible.

[0399] report group TIFF0007641327000020.tif214151TIFF0007641327000021.tif170151

[0400] Participant Flow: General Testing TIFF0007641327000022.tif77151

[0401] Baseline characteristics Baseline consisted of all participants in the study [N=37], totalling four treatment arms described in participant flow (arm 1: PK50 + treatment [N=8], arm 2: PK50 only [N=8], arm 3: PK80 + treatment [N=15], arm 4: treatment only [N=6]).

[0402] result: Evaluation criteria Primary Criteria: Primary Outcome #1 1. Primary outcome: Percentage of participants successfully treated for treated bleeding episodes [Time frame: rVWF: 12 months from first infusion of rFVIII or rVWF] TIFF0007641327000023.tif113148

[0403] report group TIFF0007641327000024.tif44148

[0404] Measurements TIFF0007641327000025.tif41148

[0405] Statistical analysis of the proportion of participants successfully treated for treated bleeding episodes1 TIFF0007641327000026.tif189148

[0406] Secondary Criteria:Secondary Item #1 2. Secondary: Percentage of treated bleeding episodes with excellent or good efficacy rating [Time frame: rVWF: 12 months from first infusion of rFVIII or rVWF]

[0407] Table 10: Secondary endpoint #1 TIFF0007641327000027.tif109148

[0408] (Table 11) Report group TIFF0007641327000028.tif44148

[0409] (Table 12) Measurement values TIFF0007641327000029.tif48148 No statistical analysis was provided regarding the proportion of treated bleeding episodes with an efficacy rating of "excellent" or "good."

[0410] Secondary Criteria:Secondary Item #2 3. Secondary: Percentage of treated bleeding episodes with excellent or good efficacy scores, excluding gastrointestinal bleeding [Time frame: rVWF: 12 months from first infusion of rFVIII or rVWF]

[0411] Table 13: Secondary endpoint #2 TIFF0007641327000030.tif124148

[0412] (Table 14) Report group TIFF0007641327000031.tif44148

[0413] (Table 15) Measurement values TIFF0007641327000032.tif55153 No statistical analysis was provided on the proportion of treated bleeding episodes with an efficacy rating of "excellent" or "good," excluding gastrointestinal bleeding.

[0414] Secondary Criteria:Secondary Item #3 4. Secondary outcome: Number of infusions of rVWF:rFVIII and / or rVWF per bleeding episode [Time frame: 12 months from first infusion of rVWF:rFVIII or rVWF]

[0415] Table 16: Secondary endpoint #3 TIFF0007641327000033.tif109148

[0416] (Table 17) Report group TIFF0007641327000034.tif44148

[0417] (Table 18) Measurement values TIFF0007641327000035.tif48151 rVWF per bleeding episode: No statistical analysis of the number of infusions of rFVIII and / or rVWF was provided.

[0418] Secondary Criteria:Secondary Item #4 5. Secondary: Number of units of rVWF:rFVIII and / or rVWF per bleeding episode [Time frame: 12 months from first infusion of rVWF:rFVIII or rVWF]

[0419] Table 19: Secondary endpoint #4 TIFF0007641327000036.tif109148

[0420] (Table 20) Report group TIFF0007641327000037.tif44148

[0421] (Table 21) Measurement values TIFF0007641327000038.tif48153 rVWF per bleeding episode: No statistical analysis of units of rFVIII and / or rVWF was provided.

[0422] Secondary Criteria:Secondary Item #5 6. Secondary endpoint: Percentage of participants who developed inhibitory antibodies to FVIII [Time frame: rVWF: 12 months from first infusion of rFVIII or rVWF]

[0423] Table 22 Secondary endpoint #5 TIFF0007641327000039.tif88148

[0424] (Table 23) Report group TIFF0007641327000040.tif37148

[0425] (Table 24) Measurement values TIFF0007641327000041.tif59148No statistical analysis was provided regarding the percentage of participants developing inhibitory antibodies to FVIII.

[0426] Secondary Criteria:Secondary Item #6 7. Secondary outcome: Percentage of participants who develop inhibitory antibodies to VWF [Time frame: from signing of informed consent to 12 months after first infusion of rVWF:rFVIII or rVWF]

[0427] Table 25: Secondary endpoint #6 TIFF0007641327000042.tif124148

[0428] (Table 26) Report group TIFF0007641327000043.tif37151

[0429] (Table 27) Measurement values TIFF0007641327000044.tif59144No statistical analysis was provided regarding the proportion of participants developing inhibitory antibodies to VWF.

[0430] Secondary Criteria:Secondary Item #7 8. Secondary outcome: Percentage of participants who develop binding antibodies to VWF [Time frame: from signing of informed consent to 12 months after first infusion of rVWF:rFVIII or rVWF]

[0431] (Table 28) Secondary endpoint #7 TIFF0007641327000045.tif73150

[0432] (Table 29) Report group TIFF0007641327000046.tif44150

[0433] (Table 30) Measurement values TIFF0007641327000047.tif59144No statistical analysis was provided regarding the percentage of participants developing binding antibodies to VWF.

[0434] Secondary Criteria:Secondary Item #8 9. Secondary outcome: Percentage of participants who develop binding antibodies to CHO [Time frame: from signing of informed consent to 12 months after first infusion of rVWF:rFVIII or rVWF]

[0435] (Table 30) Secondary endpoint #9 TIFF0007641327000048.tif80150

[0436] (Table 31) Report group TIFF0007641327000049.tif37141

[0437] (Table 32) Measurement values TIFF0007641327000050.tif59132No statistical analysis was provided regarding the percentage of participants developing binding antibodies to CHO.

[0438] Secondary Criteria:Secondary Item #9 10. Secondary outcome: Percentage of participants who develop binding antibodies to rFurin [Time frame: from signing of informed consent to 12 months after first infusion of rVWF:rFVIII or rVWF]

[0439] (Table 33) Secondary endpoint #9 TIFF0007641327000051.tif80164

[0440] (Table 34) Report group TIFF0007641327000052.tif37164

[0441] (Table 35) Measurement values TIFF0007641327000053.tif59146r No statistical analysis was provided regarding the percentage of participants developing binding antibodies to furin.

[0442] Secondary Criteria: Secondary Item #10 11. Secondary outcome: Percentage of participants who develop binding antibodies to mouse immunoglobulins [Time frame: from signing of informed consent to 12 months after first infusion of rVWF:rFVIII or rVWF]

[0443] (Table 36) Secondary endpoint #10 TIFF0007641327000054.tif66162

[0444] (Table 37) Report group TIFF0007641327000055.tif37162

[0445] (Table 38) Measurement values TIFF0007641327000056.tif59162 No statistical analysis was provided regarding the percentage of participants developing binding antibodies to mouse immunoglobulin.

[0446] Secondary Criteria:Secondary Item #11 12. Secondary outcomes: Percentage (%) of participants who experienced a thrombotic event [Time frame: from signing of informed consent to 12 months after first infusion of rVWF:rFVIII or rVWF]

[0447] (Table 39) Secondary endpoint #11 TIFF0007641327000057.tif51162

[0448] (Table 40) Report group TIFF0007641327000058.tif37162

[0449] (Table 41) Measurement values TIFF0007641327000059.tif33162No statistical analysis was provided regarding the percentage of participants who experienced thrombotic events.

[0450] Secondary Criteria: Secondary Item #12 13. Secondary outcomes: Number of drug-related adverse events, including clinically significant changes in laboratory parameters and vital signs [Time frame: rVWF: 12 months from first infusion of rFVIII or rVWF]

[0451] (Table 42) Secondary endpoint #12 TIFF0007641327000060.tif117162

[0452] (Table 43) Report group TIFF0007641327000061.tif37162

[0453] (Table 44) Measurement values TIFF0007641327000062.tif117164 No statistical analysis was provided regarding the number of drug-related adverse events, including clinically significant changes in laboratory parameters and vital signs.

[0454] Secondary Criteria:Secondary Item #13 14. Secondary outcomes: Number of participants with treatment-related adverse events, including clinically significant changes in laboratory parameters and vital signs [Time frame: rVWF: 12 months from first infusion of rFVIII or rVWF]

[0455] (Table 45) Secondary endpoint #13 TIFF0007641327000063.tif102167

[0456] (Table 46) Report group TIFF0007641327000064.tif37154

[0457] (Table 47) Measurement values TIFF0007641327000065.tif103158 No statistical analysis was provided regarding the number of participants with drug-related adverse events, including clinically significant changes in laboratory parameters and vital signs.

[0458] Secondary Outcome Scale:Secondary Outcome #14 15. Secondary outcomes: Number of study drug-related infusion-related adverse events, including clinically significant changes in laboratory parameters and vital signs [Time frame: rVWF: 12 months from first infusion of rFVIII or rVWF]

[0459] (Table 48) Secondary endpoint #14 TIFF0007641327000066.tif109148

[0460] (Table 49) Report group TIFF0007641327000067.tif37162

[0461] (Table 50) Measurement values TIFF0007641327000068.tif110159 No statistical analysis was provided regarding the number of infusion-related adverse events, including clinically significant changes in laboratory parameters and vital signs.

[0462] Secondary Criteria:Secondary Item #15 16. Secondary Item: PK50-VWF: Area under the plasma concentration / time curve from time 0 to infinity (AUC0-∞ / dose) of RCo [Time Frame: Pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0463] (Table 51) Secondary endpoint #15 TIFF0007641327000069.tif132162

[0464] (Table 52) Report group TIFF0007641327000070.tif73148

[0465] (Table 53) Measurement values TIFF0007641327000071.tif81158PK50-VWF: No statistical analysis was provided for the area under the plasma concentration / time curve from time 0 to infinity (AUC0-∞ / dose) of RCo.

[0466] Secondary Criteria:Secondary Item #16 17. Secondary Item: PK50-VWF: Area under the plasma concentration / time curve from time 0 to 96 hours (AUC0-96h / dose) of RCo [Time Frame: Pre-infusion, followed by PK assessments at 15, 30, and 60 minutes, and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28±3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0467] Table 54 Secondary Endpoint #16 TIFF0007641327000072.tif139162

[0468] (Table 55) Report group TIFF0007641327000073.tif73148

[0469] (Table 56) Measurement values TIFF0007641327000074.tif81158PK50-VWF: No statistical analysis was provided for the area under the plasma concentration / time curve from time 0 to 96 hours (AUC0-96h / dose) of RCo.

[0470] Secondary Criteria: Secondary Item #17 18. Secondary: PK50-VWF: Mean residence time of RCo [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours post-infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0471] (Table 57) Secondary endpoint #17 TIFF0007641327000075.tif124162

[0472] (Table 58) Report group TIFF0007641327000076.tif73148

[0473] (Table 59) Measurement values TIFF0007641327000077.tif73137PK50-VWF:No statistical analysis of mean residence time of RCo was provided.

[0474] Secondary Criteria:Secondary Item #18 19. Secondary Item: PK50-VWF: Clearance of RCo [Timeframe: Pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment timeframe of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0475] (Table 60) Secondary endpoint #18 TIFF0007641327000078.tif124162

[0476] (Table 61) Report group TIFF0007641327000079.tif81148

[0477] (Table 62) Measurement values TIFF0007641327000080.tif73128PK50-VWF:No statistical analysis of RCo clearance was provided.

[0478] Secondary Criteria:Secondary Item #19 20. Secondary: PK50-VWF: Stepwise recovery of RCo [Timeframe: Pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours post-infusion. PK assessment timeframe of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0479] (Table 63) Secondary endpoint #19 TIFF0007641327000081.tif124162

[0480] (Table 64) Report group TIFF0007641327000082.tif81148

[0481] (Table 65) Measurement values TIFF0007641327000083.tif73156PK50-VWF:No statistical analysis of the stepwise recovery rate of RCo was provided.

[0482] Secondary Criteria:Secondary Item #20 21. Secondary: Elimination half-life of PK50-VWF:Co [Timeframe: Pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment timeframe of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0483] (Table 66) Secondary endpoint #20 TIFF0007641327000084.tif124162

[0484] (Table 67) Report group TIFF0007641327000085.tif81148

[0485] (Table 68) Measurement values TIFF0007641327000086.tif73143No statistical analysis of the elimination half-life of PK50-VWF:Co was provided.

[0486] Secondary Criteria:Secondary Item #21 22. Secondary: PK50-VWF: Volume of distribution of RCo at steady state. [Timeframe: Pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment timeframe of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0487] (Table 69) Secondary endpoint #21 TIFF0007641327000087.tif117161

[0488] (Table 70) Report group TIFF0007641327000088.tif81148

[0489] (Table 71) Measurement values TIFF0007641327000089.tif73157PK50-VWF:No statistical analysis of steady-state volume of distribution of RCo was provided.

[0490] Secondary Criteria: Secondary Item #22 23. Secondary Item: PK50-VWF:Area under the plasma concentration / time curve from time 0 to infinity (AUC0-∞ / dose) of Ag [Time frame: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0491] (Table 72) Secondary endpoint #23 TIFF0007641327000090.tif132162

[0492] (Table 73) Report group TIFF0007641327000091.tif81148

[0493] (Table 74) Measurement values TIFF0007641327000092.tif81159PK50-VWF:No statistical analysis was provided for the area under the plasma concentration / time curve from time 0 to infinity (AUC0-∞ / dose) of Ag.

[0494] Secondary Criteria: Secondary Item #23 24. Secondary Item: PK50-VWF:Ag Area Under the Plasma Concentration / Time Curve from Time 0 to 96 Hours (AUC0-96h / Dose) [Time Frame: Pre-infusion, followed by PK assessments at 15, 30, and 60 minutes, and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28±3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0495] (Table 75) Secondary endpoint #24 TIFF0007641327000093.tif132154

[0496] (Table 76) Report group TIFF0007641327000094.tif81148

[0497] (Table 77) Measurement values TIFF0007641327000095.tif81158 No statistical analysis was provided for the area under the plasma concentration / time curve from time 0 to 96 hours (AUC0-96h / dose) of PK50-VWF:Ag.

[0498] Secondary Criteria:Secondary Item #24 25. Secondary: PK50-Mean residence time of VWF:Ag [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0499] (Table 78) Secondary endpoint #24 TIFF0007641327000096.tif124162

[0500] (Table 79) Report group TIFF0007641327000097.tif81148

[0501] (Table 80) Measurement values TIFF0007641327000098.tif73128PK50-VWF:No statistical analysis of Ag mean residence time was provided.

[0502] Secondary Criteria:Secondary Item #25 26. Secondary Item: PK50-VWF:Ag Clearance [Time Frame: Pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0503] (Table 81) Secondary endpoint #25 TIFF0007641327000099.tif124162

[0504] (Table 82) Report group TIFF0007641327000100.tif81148

[0505] (Table 83) Measurement values TIFF0007641327000101.tif73131No statistical analysis of PK50-VWF:Ag clearance was provided.

[0506] Secondary Criteria:Secondary Item #26 27. Secondary: PK50-Stepwise recovery of VWF:Ag [Timeframe: Pre-infusion, followed by PK assessments at 15, 30, and 60 minutes, and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment timeframe of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0507] (Table 84) Secondary endpoint #26 TIFF0007641327000102.tif124162

[0508] (Table 85) Report group TIFF0007641327000103.tif81148

[0509] (Table 86) Measurement values TIFF0007641327000104.tif73156 No statistical analysis of stepwise recovery of PK50-VWF:Ag was provided.

[0510] Secondary Criteria:Secondary Item #27 28. Secondary: Elimination half-life of PK50-VWF:Ag [Timeframe: Pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment timeframe of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0511] (Table 87) Secondary endpoint #27 TIFF0007641327000105.tif124162

[0512] (Table 88) Report group TIFF0007641327000106.tif81148

[0513] (Table 89) Measurement values TIFF0007641327000107.tif73135No statistical analysis of PK50-VWF:Ag elimination half-life was provided.

[0514] Secondary Criteria:Secondary Item #28 29. Secondary: PK50-VWF:Ag volume of distribution at steady state [Time frame: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0515] (Table 90) Secondary endpoint #28 TIFF0007641327000108.tif124162

[0516] (Table 91) Report group TIFF0007641327000109.tif81148

[0517] (Table 92) Measurement values TIFF0007641327000110.tif73139Statistical analysis of steady-state volume of distribution of PK50-VWF:Ag is not provided.

[0518] Secondary Criteria:Secondary Item #29 30. Secondary Item: PK50-VWF: Area under the plasma concentration / time curve from time 0 to infinity (AUC0-∞ / dose) of CB [Timeframe: Pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment timeframe of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0519] (Table 93) Secondary endpoint #30 TIFF0007641327000111.tif139148

[0520] (Table 94) Report group TIFF0007641327000112.tif73148

[0521] (Table 95) Measurement values TIFF0007641327000113.tif80151PK50-VWF: No statistical analysis was provided for the area under the plasma concentration / time curve from time 0 to infinity (AUC0-∞ / dose) of CB.

[0522] Secondary Criteria:Secondary Item #30 31. Secondary Item: PK50-VWF: Area under the plasma concentration / time curve from time 0 to 96 hours (AUC0-96h / dose) of CB [Timeframe: Pre-infusion, followed by PK assessments at 15, 30, and 60 minutes, and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment timeframe of 28±3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0523] (Table 96) Secondary endpoint #30 TIFF0007641327000114.tif132148

[0524] (Table 97) Report group TIFF0007641327000115.tif73148

[0525] (Table 98) Measurement values TIFF0007641327000116.tif80148PK50-VWF: No statistical analysis was provided for the area under the plasma concentration / time curve from time 0 to 96 hours (AUC0-96h / dose) of CB.

[0526] Secondary Criteria:Secondary Item #31 32. Secondary: PK50-VWF:CB mean residence time [Timeframe: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment timeframe of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0527] (Table 99) Secondary endpoint #31 TIFF0007641327000117.tif124148

[0528] (Table 100) Report group TIFF0007641327000118.tif73150

[0529] (Table 101) Measurement values TIFF0007641327000119.tif73128PK50-VWF:No statistical analysis of the mean residence time of CB was provided.

[0530] Secondary Criteria:Secondary Item #32 33. Secondary Item: PK50-VWF:Clearance of CB [Timeframe: Pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment timeframe of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0531] (Table 102) Secondary endpoint #32 TIFF0007641327000120.tif124148

[0532] (Table 103) Report group TIFF0007641327000121.tif73148

[0533] (Table 104) Measurement values TIFF0007641327000122.tif73128PK50-VWF:No statistical analysis of CB clearance was provided.

[0534] Secondary Criteria:Secondary Item #33 34. Secondary: PK50-VWF: Stepwise recovery of CB [Timeframe: Pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment timeframe of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0535] (Table 105) Secondary endpoint #33 TIFF0007641327000123.tif124150

[0536] (Table 106) Report group TIFF0007641327000124.tif73151

[0537] (Table 107) Measurement values TIFF0007641327000125.tif73128PK50-VWF:No statistical analysis of the stepwise recovery rate of CB was provided.

[0538] Secondary Criteria:Secondary Item #34 35. Secondary: PK50-VWF:CB elimination half-life [Timeframe: Pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment timeframe of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0539] (Table 108) Secondary endpoint #34 TIFF0007641327000126.tif124150

[0540] (Table 109) Report group TIFF0007641327000127.tif73150

[0541] (Table 110) Measurement values TIFF0007641327000128.tif73128PK50-VWF:No statistical analysis of the elimination half-life of CB was provided.

[0542] Secondary Criteria:Secondary Item #35 36. Secondary: PK50-VWF: Volume of distribution at steady state for CB [Timeframe: Pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment timeframe of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0543] (Table 111) Secondary endpoint #35 TIFF0007641327000129.tif124151

[0544] (Table 112) Report group TIFF0007641327000130.tif73151

[0545] (Table 113) Measurement values TIFF0007641327000131.tif73128PK50-VWF:No statistical analysis of the steady-state volume of distribution of CB was provided.

[0546] Secondary Criteria:Secondary Item #36 37. Secondary Item: PK50-Area under the plasma concentration / time curve from time 0 to infinity (AUC0~∞ / dose) of FVIII:C [Time frame: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0547] (Table 114) Secondary endpoint #36 TIFF0007641327000132.tif132151

[0548] (Table 115) Report group TIFF0007641327000133.tif73151

[0549] (Table 116) Measurement values TIFF0007641327000134.tif80156 No statistical analysis was provided for the area under the plasma concentration / time curve from time 0 to infinity (AUC0-∞ / dose) of PK50-FVIII:C.

[0550] Secondary Criteria:Secondary Item #37 38. Secondary Item: PK50-Area under the plasma concentration / time curve from time 0 to 96 hours (AUC0-96h / dose) of FVIII:C [Time frame: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes, and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0551] (Table 116) Secondary endpoint #37 TIFF0007641327000135.tif132151

[0552] (Table 117) Report group TIFF0007641327000136.tif73151

[0553] (Table 118) Measurement values TIFF0007641327000137.tif80153No statistical analysis was provided for the area under the plasma concentration / time curve from time 0 to 96 hours (AUC0-96h / dose) of PK50-FVIII:C.

[0554] Secondary Criteria:Secondary Item #38 39. Secondary Item: PK50-Mean residence time of FVIII:C [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0555] (Table 119) Secondary endpoint #38 TIFF0007641327000138.tif102150

[0556] (Table 120) Report group TIFF0007641327000139.tif73150

[0557] (Table 121) Measurement values TIFF0007641327000140.tif41128No statistical analysis of the mean residence time of PK50-FVIII:C was provided.

[0558] Secondary Criteria:Secondary Item #39 40. Secondary Item: PK50-Clearance of FVIII:C [Time Frame: Pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0559] (Table 122) Secondary endpoint #39 TIFF0007641327000141.tif102151

[0560] (Table 123) Report group TIFF0007641327000142.tif73151

[0561] (Table 124) Measurement values TIFF0007641327000143.tif41128No statistical analysis of PK50-FVIII:C clearance was provided.

[0562] Secondary Criteria:Secondary Item #40 41. Secondary: Phased recovery of PK50-FVIII:C [Timeframe: Pre-infusion, followed by PK assessments at 15, 30, and 60 minutes, and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment timeframe of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0563] (Table 125) Secondary endpoint #40 TIFF0007641327000144.tif102151

[0564] (Table 126) Report group TIFF0007641327000145.tif22128

[0565] (Table 127) Measurement values TIFF0007641327000146.tif41128No statistical analysis of stepwise recovery rates of PK50-FVIII:C was provided.

[0566] Secondary Criteria:Secondary Item #41 42. Secondary: PK50-FVIII:C elimination half-life [Timeframe: Pre-infusion, followed by PK assessments at 15, 30, and 60 minutes, and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment timeframe of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0567] (Table 128) Secondary endpoint #41 TIFF0007641327000147.tif102151

[0568] (Table 129) Report group TIFF0007641327000148.tif73150

[0569] (Table 130) Measurement values TIFF0007641327000149.tif41128No statistical analysis of the elimination half-life of PK50-FVIII:C was provided.

[0570] Secondary Criteria:Secondary Item #42 43. Secondary: PK50-FVIII:C volume of distribution at steady state [Time frame: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0571] (Table 131) Secondary endpoint #42 TIFF0007641327000150.tif102151

[0572] (Table 132) Report group TIFF0007641327000151.tif73151

[0573] (Table 133) Measurement values TIFF0007641327000152.tif41128No statistical analysis of steady-state volume of distribution of PK50-FVIII:C was provided.

[0574] Secondary Criteria:Secondary Item #43 44. Secondary: PK80-VWF: Area under the plasma concentration / time curve from time 0 to infinity (AUC0-∞ / dose) of RCo [Time frame: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0575] (Table 134) Secondary endpoint #43 TIFF0007641327000153.tif139150

[0576] (Table 135) Report group TIFF0007641327000154.tif44151

[0577] (Table 136) Measurement values TIFF0007641327000155.tif81151PK80-VWF: No statistical analysis was provided for the area under the plasma concentration / time curve from time 0 to infinity (AUC0-∞ / dose) of RCo.

[0578] Secondary Criteria:Secondary Item #44 45. Secondary: PK80-VWF: Area under the plasma concentration / time curve from time 0 to 96 hours (AUC0-96h / dose) of RCo [Time frame: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0579] (Table 137) Secondary endpoint #44 TIFF0007641327000156.tif139150

[0580] (Table 136) Report group TIFF0007641327000157.tif44150

[0581] (Table 137) Measurement values TIFF0007641327000158.tif81151PK80-VWF: No statistical analysis was provided for the area under the plasma concentration / time curve from time 0 to 96 hours (AUC0-96h / dose) of RCo.

[0582] Secondary Criteria:Secondary Item #45 46. ​​Secondary: Mean residence time of PK80-VWF:RCo [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours post-infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0583] (Table 138) Secondary endpoint #45 TIFF0007641327000159.tif124150

[0584] (Table 139) Report group TIFF0007641327000160.tif44151

[0585] (Table 140) Measurement values TIFF0007641327000161.tif73128PK80-VWF:No statistical analysis of mean residence time of RCo was provided.

[0586] Secondary Criteria:Secondary Item #46 47. Secondary: PK80-VWF: Clearance of RCo [Timeframe: Pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment timeframe of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0587] (Table 141) Secondary endpoint #47 TIFF0007641327000162.tif124151

[0588] (Table 142) Report group TIFF0007641327000163.tif44151

[0589] (Table 143) Measurement values TIFF0007641327000164.tif73128PK80-VWF:No statistical analysis of RCo clearance was provided.

[0590] Secondary Criteria:Secondary Item #47 48. Secondary: PK80-VWF: Stepwise recovery of RCo [Timeframe: Pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment timeframe of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0591] (Table 144) Secondary endpoint #47 TIFF0007641327000165.tif132151

[0592] (Table 145) Report group TIFF0007641327000166.tif44151

[0593] (Table 146) Measurement values TIFF0007641327000167.tif73128PK80-VWF:No statistical analysis of the stepwise recovery rate of RCo was provided.

[0594] Secondary Criteria:Secondary Item #48 49. Secondary: Elimination half-life of PK80-VWF:Co [Timeframe: Pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment timeframe of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0595] (Table 147) Secondary endpoint #48 TIFF0007641327000168.tif124151

[0596] (Table 148) Report group TIFF0007641327000169.tif44151

[0597] (Table 149) Measurement values TIFF0007641327000170.tif73128No statistical analysis of the elimination half-life of PK80-VWF:Co was provided.

[0598] Secondary Criteria:Secondary Item #49 50. Secondary: PK80-VWF: Volume of distribution of RCo at steady state. [Timeframe: Pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment timeframe of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0599] (Table 150) Secondary endpoint #50 TIFF0007641327000171.tif153151

[0600] (Table 151) Report group TIFF0007641327000172.tif44151

[0601] (Table 152) Measurement values TIFF0007641327000173.tif73128PK80-VWF:No statistical analysis of steady-state volume of distribution of RCo was provided.

[0602] Secondary Criteria: Secondary Item #50 51. Secondary: Area under the plasma concentration / time curve from time 0 to infinity (AUC0-∞ / dose) of PK80-VWF:Ag [Time frame: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0603] (Table 153) Secondary endpoint #50 TIFF0007641327000174.tif139151

[0604] (Table 154) Report group TIFF0007641327000175.tif44151

[0605] (Table 155) Measurement values TIFF0007641327000176.tif80151 PK80-VWF: No statistical analysis was provided for the area under the plasma concentration / time curve from time 0 to infinity (AUC0-∞ / dose) of Ag.

[0606] Secondary Criteria: Secondary Item #51 52. Secondary: Area under the plasma concentration / time curve from time 0 to 96 hours (AUC0-96h / dose) of PK80-VWF:Ag [Time frame: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes, and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0607] (Table 156) Secondary endpoint #51 TIFF0007641327000177.tif139151

[0608] (Table 157) Report group TIFF0007641327000178.tif44151

[0609] (Table 158) Measurement values TIFF0007641327000179.tif81151 No statistical analysis was provided for the area under the plasma concentration / time curve from time 0 to 96 hours (AUC0-96h / dose) of PK80-VWF:Ag.

[0610] Secondary Criteria: Secondary Item #52 53. Secondary: Mean residence time of PK80-VWF:Ag [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0611] (Table 159) Secondary endpoint #52 TIFF0007641327000180.tif124151

[0612] (Table 160) Report group TIFF0007641327000181.tif44151

[0613] (Table 161) Measurement values TIFF0007641327000182.tif73128PK80-VWF:No statistical analysis of Ag mean residence time was provided.

[0614] Secondary Criteria:Secondary Item #53 54. Secondary: Clearance of PK80-VWF:Ag [Timeframe: Pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment timeframe of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0615] (Table 162) Secondary endpoint #54 TIFF0007641327000183.tif124151

[0616] (Table 163) Report group TIFF0007641327000184.tif44151

[0617] (Table 164) Measurement values TIFF0007641327000185.tif73128No statistical analysis of PK80-VWF:Ag clearance was provided.

[0618] Secondary Criteria:Secondary Item #54 55. Secondary: Phased recovery of PK80-VWF:Ag [Timeframe: Pre-infusion, followed by PK assessments at 15, 30, and 60 minutes, and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment timeframe of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0619] (Table 165) Secondary endpoint #54 TIFF0007641327000186.tif132151

[0620] (Table 166) Report group TIFF0007641327000187.tif44151

[0621] (Table 167) Measurement values TIFF0007641327000188.tif73128PK80-VWF:No statistical analysis of Ag stepwise recovery was provided.

[0622] Secondary Criteria:Secondary Item #55 56. Secondary: Elimination half-life of PK80-VWF:Ag [Timeframe: Pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment timeframe of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0623] (Table 168) Secondary endpoint #56 TIFF0007641327000189.tif124151

[0624] (Table 169) Report group TIFF0007641327000190.tif44151

[0625] (Table 170) Measurement values TIFF0007641327000191.tif73128No statistical analysis of PK80-VWF:Ag elimination half-life was provided.

[0626] Secondary Criteria: Secondary Item #56 57. Secondary: Volume of distribution of PK80-VWF:Ag at steady state [Time frame: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0627] (Table 171) Secondary endpoint #57 TIFF0007641327000192.tif132151 (Table 172) Report group TIFF0007641327000193.tif44150

[0628] (Table 173) Measurement values TIFF0007641327000194.tif73128 No statistical analysis of steady-state volume of distribution of PK80-VWF:Ag was provided.

[0629] Secondary Criteria:Secondary Item #57 58. Secondary: PK80-VWF: Area under the plasma concentration / time curve from time 0 to infinity (AUC0-∞ / dose) of CB [Time frame: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0630] (Table 174) Secondary endpoint #57 TIFF0007641327000195.tif139151

[0631] (Table 175) Report group TIFF0007641327000196.tif44151

[0632] (Table 176) Measurement values TIFF0007641327000197.tif81153PK80-VWF: No statistical analysis was provided for the area under the plasma concentration / time curve from time 0 to infinity (AUC0-∞ / dose) of CB.

[0633] Secondary Criteria:Secondary Item #58 59. Secondary: PK80-VWF: Area under the plasma concentration / time curve from time 0 to 96 hours (AUC0-96h / dose) of CB [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes, and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0634] (Table 177) Secondary endpoint #58 TIFF0007641327000198.tif139151

[0635] (Table 178) Report group TIFF0007641327000199.tif44151

[0636] (Table 188) Measurement values TIFF0007641327000200.tif81151PK80-VWF: No statistical analysis was provided for the area under the plasma concentration / time curve from time 0 to 96 hours (AUC0-96h / dose) of CB.

[0637] Secondary Criteria: Secondary Item #59 60. Secondary: Mean residence time of PK80-VWF:CB [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0638] (Table 189) Secondary endpoint #59 TIFF0007641327000201.tif124151

[0639] (Table 190) Report group TIFF0007641327000202.tif44151

[0640] (Table 191) Measurement values TIFF0007641327000203.tif73128PK80-VWF:No statistical analysis of the mean residence time of CB was provided.

[0641] Secondary Criteria: Secondary Item #60 61. Secondary Item: PK80-VWF:Clearance of CB [Timeframe: Pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment timeframe of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0642] (Table 192) Secondary endpoint #60 TIFF0007641327000204.tif132151

[0643] (Table 193) Report group TIFF0007641327000205.tif44151

[0644] (Table 194) Measurement values TIFF0007641327000206.tif73128PK80-VWF:No statistical analysis of CB clearance was provided.

[0645] Secondary Criteria:Secondary Item #61 62. Secondary: Phased recovery of PK80-VWF:CB [Timeframe: Pre-infusion, followed by PK assessments at 15, 30, and 60 minutes, and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment timeframe of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0646] (Table 195) Secondary endpoint #61 TIFF0007641327000207.tif132151

[0647] (Table 196) Report group TIFF0007641327000208.tif44151

[0648] (Table 197) Measurement values TIFF0007641327000209.tif73128PK80-VWF:No statistical analysis of the stepwise recovery rate of CB was provided.

[0649] Secondary Criteria: Secondary Item #62 63. Secondary: Elimination half-life of PK80-VWF:CB [Timeframe: Pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment timeframe of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0650] (Table 198) Secondary endpoint #62 TIFF0007641327000210.tif124151

[0651] (Table 199) Report group TIFF0007641327000211.tif44150

[0652] (Table 200) Measurement values TIFF0007641327000212.tif73128PK80-VWF:No statistical analysis of the elimination half-life of CB was provided.

[0653] Secondary Criteria: Secondary Item #63 64. Secondary: PK80-VWF:CB steady state volume of distribution [Timeframe: Pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment timeframe of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0654] (Table 201) Secondary endpoint #63 TIFF0007641327000213.tif124151

[0655] (Table 202) Report group TIFF0007641327000214.tif44151

[0656] (Table 203) Measurement values TIFF0007641327000215.tif73128PK80-VWF:No statistical analysis of the steady-state volume of distribution of CB was provided.

[0657] Secondary Criteria: Secondary Item #64 65. Secondary: Area under the plasma concentration / time curve from time 0 to infinity (AUC0-∞ / dose) of PK80-FVIII:C [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0658] (Table 204) Secondary endpoint #65 TIFF0007641327000216.tif132151

[0659] (Table 205) Report group TIFF0007641327000217.tif44151

[0660] (Table 206) Measurement values TIFF0007641327000218.tif81151 No statistical analysis was provided for the area under the plasma concentration / time curve from time 0 to infinity (AUC0-∞ / dose) of PK80-FVIII:C.

[0661] Secondary Criteria:Secondary Item #65 66. Secondary: Area under the plasma concentration / time curve from time 0 to 96 hours (AUC0-96h / dose) of PK80-FVIII:C [Time frames: pre-infusion, followed by PK assessments at 15, 30, and 60 minutes, and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment time frame of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0662] (Table 207) Secondary endpoint #65 TIFF0007641327000219.tif132151

[0663] (Table 208) Report group TIFF0007641327000220.tif44151

[0664] (Table 209) Measurement values TIFF0007641327000221.tif81153 No statistical analysis was provided for the area under the plasma concentration / time curve from time 0 to 96 hours (AUC0-96h / dose) of PK80-FVIII:C.

[0665] Secondary Criteria: Secondary Item #66 67. Secondary Item: PK80 - Within-participant PK ratios of VWF:RCo, VWF:Ag, and VWF:CB at baseline and 6 months. [Timeframe: Pre-infusion, followed by PK assessments at 15, 30, and 60 minutes, and 3, 6, 12, 24, 30, 48, 72, and 96 hours after infusion. PK assessment timeframe of 28 ± 3 days after first study drug infusion, including PK assessments for both infusion and washout]

[0666] (Table 210) Secondary endpoint #66 TIFF0007641327000222.tif161151

[0667] (Table 211) Description of the population TIFF0007641327000223.tif34151

[0668] (Table 212) Report group TIFF0007641327000224.tif44151

[0669] (Table 213) Measurement values TIFF0007641327000225.tif95151PK80 - No statistical analysis was provided for within-participant PK ratios of VWF:RCo, VWF:Ag, and VWF:CB at baseline and 6 months.

[0670] The above examples are provided to give those of skill in the art a complete disclosure and description of how to make and use the embodiments of the compositions, systems, and methods of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the above-described modes for carrying out the invention that are obvious to those of skill in the art are intended to be within the scope of the following claims. All patents and publications mentioned in this specification are indicative of the level of skill in the art to which this invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference was individually incorporated by reference in its entirety.

[0671] All headings and section names are used for clarity and reference purposes only and should not be construed as limiting in any manner For example, those skilled in the art will understand the utility of combining various aspects from different headings and sections, as appropriate, in accordance with the spirit and scope of the invention described herein.

[0672] All references cited in this specification are hereby incorporated by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0673] As will be apparent to those skilled in the art, many modifications and variations of this application can be made without departing from its spirit and scope. The specific embodiments and examples described herein are offered by way of example only, and this application is limited only by the terms of the appended claims, along with the full scope of equivalents to which the scope of the patent application is entitled.

[0674] Sequence information SUBSTITUTE SEQUENCE LISTING <110> TAKEDA PHARMACEUTICAL COMPANY LIMITED <120> TREATMENT OF GASTROINTESTINAL BLEEDING IN PATIENTS WITH SEVERE VON WILLEBRAND DISEASE BY ADMINISTRATION OF RECOMMENDED VWF <150> US 62 / 530,027 <151> 2017-07-07 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 8833 <212> DNA <213> Artificial Sequence <220> <223> prepro-VWF <400> 1 agctcacagc tattgtggtg ggaaagggag ggtggttggt ggatgtcaca gcttgggctt 60 tatctccccc agcagtgggg actccacagc ccctgggcta cataacagca agacagtccg 120 gagctgtagc agacctgatt gagcctttgc agcagctgag agcatggcct agggtgggcg 180 gcaccattgt ccagcagctg agttcccag ggaccttgga gatagccgca gccctcattt 240 gcaggggaag atgattcctg ccagatttgc cggggtgctg cttgctctgg ccctcattttt 300 gccagggacc ctttgtgcag aaggaactcg cggcaggtca tccacggccc gatgcagcct 360 ttcggaagt gacttcgtca acacctttga tgggagcatg tacagctttg cgggatactg 420 cagttacctc ctggcagggg gctgccagaa acgctccttc tcgattattg gggacttcca 480 gaatggcaag agagtgagcc tctccgtgta tcttggggaa ttttttgaca tccatttgtt 540 tgtcaatggt accgtgacac agggggacca aagagtctcc atgccctatg cctccaaagg 600 gctgtatcta gaaactgagg ctgggtacta caagctgtcc ggtgaggcct atggctttgt 660 ggccaggatc gatggcagcg gcaactttca agtcctgctg tcagacagat acttcaacaa 720 gacctgcggg ctgtgtggca actttaacat ctttgctgaa gatgacttta tgacccaaga 780 agggaccttg acctcggacc cttatgactt tgccaactca tgggctctga gcagtggaga 840 acagtggtgt gaacgggcat ctcctcccag cagctcatgc aacatctcct ctggggaaat 900 gcagaagggc ctgtgggagc agtgccagct tctgaagagc acctcggtgt ttgcccgctg 960 ccaccctctg gtggaccccg agccttttgt ggccctgtgt gagaagactt tgtgtgagtg 1020 tgctgggggg ctggagtgcg cctgccctgc cctcctggag tacgcccgga cctgtgccca 1080 ggagggaatg gtgctgtacg gctggaccga ccacagcgcg tgcagcccag tgtgccctgc 1140 tggtatggag tataggcagt gtgtgtcccc ttgcgccagg acctgccaga gcctgcacat 1200 caatgaaatg tgtcaggagc gatgcgtgga tggctgcagc tgccctgagg gacagctcct 1260 ggatgaaggc ctctgcgtgg agagcaccga gtgtccctgc gtgcattccg gaaagcgcta 1320 ccctcccggc acctccctct ctcgagactg caacacctgc atttgccgaa acagccagtg 1380 gatctgcagc aatgaagaat gtccagggga gtgccttgtc acaggtcaat cacacttcaa 1440 gagctttgac aacagatact tcaccttcag tgggatctgc cagtacctgc tggcccggga 1500 ttgccaggac cactccttct ccattgtcat tgagactgtc cagtgtgctg atgaccgcga 1560 cgctgtgtgc acccgctccg tcaccgtccg gctgcctggc ctgcacaaca gccttgtgaa 1620 actgaagcat ggggcaggag ttgccatgga tggccaggac gtccagctcc ccctcctgaa 1680 aggtgacctc cgcatccagc atacagtgac ggcctccgtg cgcctcagct acggggagga 1740 cctgcagatg gactgggatg gccgcgggag gctgctggtg aagctgtccc ccgtctatgc 1800 cgggaagacc tgcggcctgt gtgggaatta caatggcaac cagggcgacg acttccttac 1860 cccctctggg ctggcggagc cccgggtgga ggacttcggg aacgcctgga agctgcacgg 1920 ggactgccag gacctgcaga agcagcacag cgatccctgc gccctcaacc cgcgcatgac 1980 caggttctcc gaggaggcgt gcgcggtcct gacgtccccc acattcgagg cctgccatcg 2040 tgccgtcagc ccgctgccct acctgcggaa ctgccgctac gacgtgtgct cctgctcgga 2100 cggccgcgag tgcctgtgcg gcgccctggc cagctatgcc gcggcctgcg cggggagagg 2160 cgtgcgcgtc gcgtggcgcg agccaggccg ctgtgagctg aactgcccga aaggccaggt 2220 gtacctgcag tgcgggaccc cctgcaacct gacctgccgc tctctctctt acccggatga 2280 ggaatgcaat gaggcctgcc tggagggctg cttctgcccc ccagggctct acatggatga 2340 gaggggggac tgcgtgccca aggcccagtg cccctgttac tatgacggtg agatcttcca 2400 gccagaagac atcttctcag accatcacac catgtgctac tgtgaggatg gcttcatgca 2460 ctgtaccatg agtggagtcc ccggaagctt gctgcctgac gctgtcctca gcagtcccct 2520 gtctcatcgc agcaaaagga gcctatcctg tcggcccccc atggtcaagc tggtgtgtcc 2580 cgctgacaac ctgcgggctg aagggctcga gtgtaccaaa acgtgccaga actatgacct 2640 ggagtgcatg agcatgggct gtgtctctgg ctgcctctgc cccccgggca tggtccggca 2700 tgagaacaga tgtgtggccc tggaaaggtg tccctgcttc catcagggca aggagtatgc 2760 ccctggagaa acagtgaaga ttggctgcaa cacttgtgtc tgtcgggacc ggaagtggaa 2820 ctgcacagac catgtgtgtg atgccacgtg ctccacgatc ggcatggccc actacctcac 2880 cttcgacggg ctcaaatacc tgttccccgg ggagtgccag tacgttctgg tgcaggatta 2940 ctgcggcagt aaccctggga cctttcggat cctagtgggg aataagggat gcagccaccc 3000 ctcagtgaaa tgcaagaaac gggtcaccat cctggtggag ggaggagaga ttgagctgtt 3060 tgacggggag gtgaatgtga agaggcccat gaaggatgag actcactttg aggtggtgga 3120 gtctggccgg tacatcattc tgctgctggg caaagccctc tccgtggtct ...

Claims

1. A pharmaceutical composition for treating minor, mild, or moderate gastrointestinal bleeding in a subject suffering from severe von Willebrand disease (VWD), comprising recombinant von Willebrand factor (rVWF), characterized in that the pharmaceutical composition is used to administer to the subject two doses of rVWF, each in the range of about 40 IU / kg to about 60 IU / kg, the two doses comprising a first dose and a second dose, the first dose further comprising recombinant factor VIII (rFVIII) in the range of about 20-50 IU / kg.

2. A medicament for treating minor, mild, or moderate gastrointestinal bleeding in a subject suffering from severe von Willebrand disease (VWD), comprising recombinant von Willebrand factor (rVWF), characterized in that the medicament is used to administer to the subject a dose of rVWF in the range of about 40 IU / kg to about 60 IU / kg, and a dose of recombinant factor VIII (rFVIII) in the range of about 20-50 IU / kg.

3. A pharmaceutical composition for treating minor, mild or moderate gastrointestinal bleeding in a subject suffering from severe von Willebrand disease (VWD), comprising recombinant von Willebrand factor (rVWF), characterized in that the pharmaceutical composition is used to administer to the subject at least one dose of rVWF and at least one dose of recombinant factor VIII (rFVIII), wherein the total dose of rVWF administered to the subject per bleeding episode is about 40-150 IU / kg, and the total dose of rFVIII administered to the subject per bleeding episode is less than 50 IU / kg.

4. A pharmaceutical for treating minor, mild, or moderate gastrointestinal bleeding in a subject suffering from severe von Willebrand disease (VWD), comprising recombinant von Willebrand factor (rVWF), characterized in that the pharmaceutical is used to administer to the subject two doses of rVWF, each ranging from about 40 IU / kg to about 100 IU / kg, and one dose of recombinant factor VIII (rFVIII), wherein the two doses comprise a first dose and a second dose, and the period between the first dose of rVWF and the second dose of rVWF is longer than 20 hours.

5. The method of claim 1 , wherein the second dose does not contain recombinant factor VIII (rFVIII).

6. The method of claim 1 or 3, wherein the rVWF is administered every 8 to 12 hours.

7. The pharmaceutical according to any one of claims 1 to 5, characterized in that the pharmaceutical is used so that 40 to 60 IU / kg of rVWF is administered per dose.

8. The method according to claim 3, wherein the rVWF is administered every 8 to 12 hours for about 3 to about 7 days.

9. The method according to claim 3, wherein the medicament is used such that 40 to 60 IU / kg of rVWF is administered every 8 to 12 hours for about 3 to about 7 days.

10. The method of any one of claims 1 to 9, wherein the subject has type 3 VWD.

11. The method of any one of claims 1 to 9, wherein the subject has severe type 1 VWD.

12. The method of any one of claims 1 to 9, wherein the subject has severe type 2 VWD.

13. The method of any one of claims 1 to 12, wherein the subject has been treated for at least one bleeding event within the past 12 months.

14. The method of any one of claims 1 to 12, wherein the subject has been treated for more than one bleeding event within the past 12 months.

15. The method of any one of claims 1 to 12, wherein the rVWF and the rFVIII are administered sequentially.

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