US20240009279A1

US20240009279A1 - Treatment of patients with severe von willebrand disease undergoing elective surgery by administration of recombinant vwf

Info

Publication number: US20240009279A1
Application number: US18/316,988
Authority: US
Inventors: Miranda Chapman; Bruce Ewenstein; Bettina Ploder
Original assignee: Takeda Pharmaceutical Co Ltd
Current assignee: Takeda Pharmaceutical Co Ltd
Priority date: 2017-07-07
Filing date: 2023-05-12
Publication date: 2024-01-11
Also published as: RU2020105681A; JP2023085513A; AU2018298232A1; KR20200037251A; CA3069256A1; RU2766118C2; US20220168397A1; EP3648787A1; WO2019010496A1; CN111565741A; CO2020001314A2; JP2020526527A; US20190091298A1; BR112020000322A2; RU2020105681A3; JP7326246B2

Abstract

The present invention relates to method for pretreating a subject with severe von Willebrand disease prior to a surgical procedure comprising administering to the subject a dose ranging from about 20 IU/kg to about 60 IU/kg rVWF between about 12 hours and about 24 hours prior to the surgical procedure, and wherein Factor VIII is not administered with the rVWF prior to the surgical procedure.

Description

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No. 17/545,870, filed Dec. 8, 2021, which is a continuation of U.S. application Ser. No. 16/030,653, filed on Jul. 9, 2018, which claims priority to U.S. Provisional Patent Application No. 62/546,999, filed on Aug. 17, 2017, and U.S. Provisional Patent Application No. 62/530,024, filed on Jul. 7, 2017, which are hereby incorporated by reference in their entirety.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM, LISTING APPENDIX SUBMITTED ON A COMPACT DISK

The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML file, created on May 10, 2023, is named 008073-5186-U502 Sequence Listing.xml and is 24,576 bytes in size.

BACKGROUND OF THE INVENTION

Coagulation diseases, such as von Willebrand Disease (VWD) generally result from a deficiency in the coagulation cascade. von Willebrand Disease (VWD) refers to the group of diseases caused by a deficiency of von Willebrand factor. Von Willebrand factor helps blood platelets clump together and stick to the blood vessel wall, which is necessary for normal blood clotting.
von Willebrand disease (VWD) is the most common inherited bleeding disorder, with an estimated prevalence rate of 1% (Veyradier A, et al., Medicine (Baltimore). 2016, 95(11):e3038). However, excluding milder forms of the disease, only about 1/10,000 patients actually require treatment. Current treatment for these coagulopathies includes a replacement therapy using pharmaceutical preparations comprising the normal coagulation factor.
VWF is a glycoprotein circulating in plasma as a series of multimers ranging in size from about 500 to 20,000 kD. The full length of cDNA of VWF has been cloned; the propolypeptide corresponds to amino acid residues 23 to 764 of the full length prepro-VWF (Eikenboom et al (1995) Haemophilia 1, 77 90). Multimeric forms of VWF are composed of 250 kD polypeptide subunits linked together by disulfide bonds. VWF mediates the initial platelet adhesion to the sub-endothelium of the damaged vessel wall, with the larger multimers exhibiting enhanced hemostatic activity. Multimerized VWF binds to the platelet surface glycoprotein Gp1bα, through an interaction in the Al domain of VWF, facilitating platelet adhesion. Other sites on VWF mediate binding to the blood vessel wall. Thus, VWF forms a bridge between the platelet and the vessel wall that is essential to platelet adhesion and primary hemostasis under conditions of high shear stress. Normally, endothelial cells secrete large polymeric forms of VWF and those forms of VWF that have a lower molecular weight arise from proteolytic cleavage. The multimers of exceptionally large molecular masses are stored in the Weibel-Pallade bodies of the endothelial cells and liberated upon stimulation by agonists such as thrombin and histamine.
For patients with VWD, it is recommended that they be treated with von Willebrand factor (VWF) replacement given the need for prolonged hemostasis, particularly in major surgery (Mannucci P M and Franchini M., Haemophilia, 2017, 23(2):182-187; 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). Plasma-derived VWF therapies contain factor VIII (FVIII) and have the potential for FVIII accumulation with repeated dosing. VONVENDI® (von Willebrand factor [recombinant], Shire, Westlake Village, CA) is the first and only recombinant VWF (rVWF) concentrate (Turecek P L, et al. Hamostaseologie. 2009; 29(suppl 1):532-38; Mannucci P M, et al. Blood, 2013; 122(5):648-657; Gill J C, et al. Blood, 2015; 126(17):2038-2046).

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods of pre-treatment for a patient with severe von Willebrand disease prior to surgery by administering 20-60 IU/kg recombinant von Willebrand Factor (rVWF) to the patient between 12 hours and 24 hours, e.g., 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 12 hours and 24 hours, 14 hours and 24 hours, 16 and 24 hours, 18 hours and 24 hours, or 20 hours and 24 hours prior to the surgical procedure, and not administering Factor VIII (FVIII) with the rVWF prior to the surgical procedure. In some embodiments, the method of pre-treating further comprises administering to the subject 5-90 IU/kg rVWF 1 hour prior to surgery. In some embodiments, the subject is administered 70-200 IU rVWF after the surgery, either with or without the pre-treatment described above. In some cases, the surgical procedure is selected from a group consisting of major surgery, minor surgery, and oral surgery.
In some embodiments, the subject is administered 35-60 IU/kg rVWF between 12 hours and 24 hours prior to a major surgical procedure. In other embodiments, the subject is administered 15-90 IU/kg rVWF 1 hour prior to major surgical procedure. In another embodiment, the subject is administered 150-220 IU/kg rVWF after a major surgical procedure. In some instances, the subject undergoing a major surgical procedure is administered a total dosage of 220-320 IU/kg. In some instances, when the surgical procedure is a major surgical procedure and the pre-treatment comprises administering at least two approximately equal doses of rVWF prior to the surgical procedure. By “approximately equal” as used herein refers to doses that have concentrations within 1-15%, 2-14%, 3-13%, 4-12%, 5-11%, 6-10%, 7-9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% of each other.
In some embodiments, the subject is administered 50-60 IU/kg rVWF between 12 hours and 24 hours prior a minor surgical procedure. In other embodiments, the subject is administered 5-50 IU/kg rVWF 1 hour prior to minor surgery. In another embodiment, the subject is administered 70-150 IU/kg rVWF after a minor surgical procedure. In some instances, the subject undergoing a minor surgical procedure is administered a total dosage of 100-220 IU/kg. In some instances, when the surgical procedure is a minor surgical procedure, the pre-treatment comprises administering at least two doses of rVWF prior to the surgical procedure, wherein the first dose is larger than the second dose.
In some embodiments, the subject is administered 20-40 IU/kg rVWF between 12 hours and 24 hours prior to an oral surgical procedure. In other embodiments, the subject is administered 20-50 IU/kg rVWF 1 hour prior to the oral surgical procedure. In another embodiment, the subject is administered 10-50 IU/kg rVWF during the oral surgical procedure. In another embodiment, the subject is administered 70-150 IU/kg rVWF after an oral surgical procedure. In some instances, the subject undergoing an oral surgical procedure is administered a total dosage of 70-190 IU/kg. In some instances, when the surgical procedure is an oral surgical procedure and the pre-treatment comprises administering at least two approximately equal doses of rVWF prior to the surgical procedure.
Other objects, advantages and embodiments of the invention will be apparent from the detailed description following.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows overall hemostatic efficiency (primary endpoint) in the study patients.

FIG. 2 shows hemostatic efficiency (secondary endpoint) in the study patients.

FIG. 3 shows baseline demographics and clinical characteristics.

FIG. 4 shows PK parameters for VWF:RCo (n=11).

FIG. 5A and FIG. 5B shows mean VWF:RCo and Endogenous FVIII:C Levels in Response to rVWF 50±5 IU rVWF:RCo/kg in all Patients with VWD With PK data analyzed (n=11) (FIG. 5A), and the subset of patients with type 3 VWD (n=5) (FIG. 5B).

FIG. 6A-1 -FIG. 6C-7 show VWF nucleic acid and amino acid sequences.

DETAILED DESCRIPTION OF THE INVENTION

Introduction

The present invention provides methods for pretreating a patient with severe von Willebrand disease prior to surgery by administering recombinant von Willebrand Factor (rVWF) to the patient 45-60 IG/kg rVWF without administering Factor VIII with the rVWF prior to the surgical procedure. In some cases, the surgical procedure is selected from a group consisting of major surgery, minor surgery, and oral surgery.
The disclosure of PCT Application Publication No. WO2012/171031 is herein incorporated by reference in its entirety for all purposes.

Definitions

As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an antibody” includes a plurality of such antibodies and reference to “a host cell” includes reference to one or more host cells and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.
Before the invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such 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.
The term “pre-propeptide VWF,” “prepro-VWF” or “pro-VWF” refers to a non-mature VWF polypeptide comprising a signal peptide of about 22 amino acid residues, a VWF propeptide of about 741 amino acid residues, and a mature VWF subunit of about 2050 amino acid residues. Pro-VWF subunits can dimerize through disulfide bonds near their carboxyl termini in the endoplasmic reticulum to form tail-to tail dimers which are then transported to the Golgi. In the Golgi, additional head-to-head disulfide bonds are formed near the amino-termini of the subunits, thereby forming multimers. Proteolytic cleavage of the VWF propeptide occurs via the processing protease furin, thus producing a mature VWF/VWF-PP complex. When “r” is included prior to the VWF designation, this refers to the recombinant version. In some embodiments, the methods described herein apply to recombinant VWF (rVWF).
The term “VWF complex” or “mat-VWF/VWF-PP complex” refers to a non-covalently linked heterodimeric structure comprising a mature VWF subunit and VWF propeptide. The VWF complex can be generated as a product of furin cleavage between the propeptide portion and mature VWF portion of the pre-propeptide VWF. When “r” is included prior to the VWF designation, this refers to the recombinant version. In some embodiments, the methods described herein apply to recombinant VWF (rVWF). As used herein. “rVWF” refers to recombinant VWF.
As used herein, “rFVIII” refers to recombinant FVIII.
The term “recombinant” when used with reference, e.g., to a cell, or 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 native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
As used herein, “recombinant VWF” includes VWF obtained via recombinant DNA technology. In certain embodiments, VWF proteins of the invention can comprise a construct, for example, prepared as in WO 1986/06096 published on Oct. 23, 1986 and U.S. patent application Ser. No. 07/559,509, filed on Jul. 23, 1990, in the name of Ginsburg et al., which is incorporated herein by reference with respect to the methods of producing recombinant VWF. The VWF in the present invention can include all potential forms, including the monomeric and multimeric forms. It should also be understood that the present invention encompasses different forms of VWF to be used in combination. For example, the VWF of the present invention may include different multimers, different derivatives and both biologically active derivatives and derivatives not biologically active.
In the context of the present invention, the recombinant VWF embraces any member of the VWF family from, for example, a mammal such as a primate, human, monkey, rabbit, pig, rodent, mouse, rat, hamster, gerbil, canine, feline, and biologically active derivatives thereof. Mutant and variant VWF proteins having activity are also embraced, as are functional fragments and fusion proteins of the VWF proteins. Furthermore, the VWF of the invention may further comprise tags that facilitate purification, detection, or both. The VWF described herein may further be modified with a therapeutic moiety or a moiety suitable imaging in vitro or in vivo.
As used herein, “plasma-derived VWF (pdVWF)” includes all forms of the protein found in blood including the mature VWF obtained from a mammal having the property of in vivo-stabilizing, e.g. binding, of at least one FVIII molecule.
The term “highly multimeric VWF” or “high molecular weight VWF” refers to VWF comprising at least 10 subunits, or 12, 14, or 16 subunits, to about 20, 22, 24 or 26 subunits or more. The term “subunit” refers to a monomer of VWF. As is known in the art, it is generally dimers of VWF that polymerize to form the larger order 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 and in particular for all teachings regarding multimer analysis of VWF).
As used herein, the term “factor VIII” or “FVIII” refers to any form of factor VIII molecule with the typical characteristics of blood coagulation factor VIII, whether endogenous to a patient, derived from blood plasma, or produced through the use of recombinant DNA techniques, and including all modified forms of factor VIII. Factor VIII (FVIII) exists naturally and in therapeutic preparations as a heterogeneous distribution of polypeptides arising from a single gene product (see, 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 of HEMOFIL M, ADVATE, and RECOMBINATE (available from Baxter Healthcare Corporation, Deerfield, Ill., U.S.A.).
As used herein, “plasma FVIII activity” and “in vivo FVIII activity” are used interchangeably. The in vivo FVIII activity measured using standard assays may be endogenous FVIII activity, the activity of a 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.
As used herein “von Willebrand Disease” refers to the group of diseases caused by a deficiency of von Willebrand factor. Von Willebrand factor helps blood platelets clump together and stick to the blood vessel wall, which is necessary for normal blood clotting. As described in further detail herein, there are several types of Von Willebrand disease including type 1, 2A, 2B, 2M and 3.
The terms “isolated,” “purified,” or “biologically pure” refer to material that is substantially or essentially free from components that normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. VWF is the predominant species present in a preparation is substantially purified. The term “purified” in some embodiments denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. In other embodiments, it 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 removing at least one contaminant from the composition to be purified. In this sense, purification does not require that the purified compound be homogenous, e.g., 100% pure.
As used herein, “administering” (and all grammatical equivalents) includes intravenous administration, intramuscular administration, subcutaneous administration, oral administration, administration as a suppository, topical contact, intraperitoneal, intralesional, or intranasal administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject. Administration is by any route including parenteral, and transmucosal (e.g., oral, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc.
The terms “therapeutically effective amount or dose” or “therapeutically sufficient amount or dose” or “effective or sufficient amount or dose” refer to a dose that produces therapeutic effects for which it is administered. For example, a therapeutically effective amount of a drug useful for treating hemophilia can be the amount that is capable of preventing or relieving one or more symptoms associated with hemophilia. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., 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).
As used herein, the terms “patient” and “subject” are used interchangeably and refer to a mammal (preferably human) that has a disease or has the potential of contracting a disease.
As used herein, the term “about” denotes an approximate range of plus or minus 10% from a specified value. For instance, the language “about 20%” encompasses a range of 18-22%.
As used herein, the term “half-life” refers to the period of time it takes for the amount of a substance undergoing decay (or clearance from a sample or from a patient) to decrease by half.

I. Recombinant Von Willebrand Factor (rVWF)

The present invention utilizes compositions comprising von Willebrand Factor (rVWF) for pretreatment of subject with severe VWD who are undergoing a surgical procedure, such as, but not limited to, major surgery, minor surgery, or oral surgery.
In certain embodiments, VWF proteins of the invention may comprise a construct, for example, prepared as in WO 1986/06096 published on Oct. 23, 1986 and U.S. patent application Ser. No. 07/559,509, filed on Jul. 23, 1990, in the name of Ginsburg et al., which is incorporated herein by reference with respect to the methods of producing recombinant VWF. The VWF useful for the present invention includes all potential forms, including the monomeric and multimeric forms. One particularly useful form of VWF are homo-multimers of at least two VWFs. The VWF proteins may be either a biologically active derivative, or when to be used solely as a stabilizer for FVIII the VWF may be of a form not biologically active. It should also be understood that the present invention encompasses different forms of VWF to be used in combination. For example, a composition useful for the present invention may include different multimers, different derivatives and both biologically active derivatives and derivatives not biologically active.
In primary hemostasis VWF serves as a bridge between platelets and specific components of the extracellular matrix, such as collagen. The biological activity of VWF in this process can be measured by different in vitro assays (Turecek et al., Semin. Thromb. Hemost. 28: 149-160, 2002). The ristocetin cofactor assay is based on the agglutination of fresh or formalin-fixed platelets induced by the antibiotic ristocetin in the presence of VWF.
The degree of platelet agglutination depends on the VWF concentration and can be measured by the turbidimetric method, e.g. by use of 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 the collagen binding assay, which is based on ELISA technology (Brown et Bosak, Thromb. Res. 43: 303-311, 1986; Favaloro, Thromb. Haemost. 83: 127-135, 2000). A microtiter plate is coated with type I or III collagen. Then the VWF is bound to the collagen surface and subsequently detected with an enzyme-labeled polyclonal antibody. The last step is the substrate reaction, which can be photometrically monitored with an ELISA reader. As provided herein, the specific Ristocetin Cofactor activity of the VWF (VWF:RCo) of the present invention is generally described in terms of mU/μg of VWF, as measured using in vitro assays.
An advantage of the rVWF compositions of the present invention over 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, 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 or more mU/μg.
The rVWF of the present invention is highly multimeric comprising about 10 to about 40 subunits. In further embodiments, the multimeric rVWF produced using methods of the present invention comprise about 10-30, 12-28, 14-26, 16-24, 18-22, 20-21 subunits. In further embodiments, the rVWF is present in multimers varying in size from dimers to multimers of over 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. Application of ADAMTS13 will cleave the ultra-large rVWF multimers over time, but during production (generally through expression in cell culture), rVWF compositions of the present invention are generally not exposed to ADAMTS13 and retain their highly multimeric structure.
In one embodiment, a 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 other embodiments, the a rVWF composition has a distribution of rVWF oligomers characterized in that 95% of the oligomers have a range of subunits selected from variations 458 to 641 found in Table 2 of WO 2012/171031, which is herein incorporated by reference in its entirety for all purposes.
In one embodiment, a rVWF composition can be characterized according to the percentage of rVWF molecules that are present in a particular higher order rVWF multimer or larger multimer. For example, in one embodiment, at least 20% of rVWF molecules in a 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 rVWF molecules in a rVWF composition used in the methods described herein are present in an oligomeric complex of at least 12 subunits. In yet other embodiments, a rVWF composition used in the methods provided herein has a minimal percentage (e.g., has at least X %) of rVWF molecules present in a particular higher-order rVWF multimer or larger multimer (e.g., a multimer of at least Y subunits) according to any one of variations 134 to 457 found in Table 3 to Table 5, which is herein incorporated by reference in its entirety for all purposes.
In accordance with the above, the rVWF composition administered to the subject (with or without FVIII) 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 order multimers. In further embodiments, the composition comprises about 10-95%, 20-90%, 30-85%, 40-80%, 50-75%, 60-70% decamers or higher order 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 order multimers.
Assessment of the number and percentage of rVWF multimers can be conducted using methods known in the art, including without limitation methods using electrophoresis and size exclusion chromatography methods to separate VWF multimers by size, for example as discussed by Cumming et al, (J Clin Pathol. 1993 May; 46(5): 470-473, which is hereby incorporated by reference in its entirety for all purposes and in particular for all teachings related to assessment of VWF multimers). Such techniques may further include immunoblotting techniques (such as Western Blot), in which the gel is immunoblotted with a radiolabeled antibody against VWF followed by chemiluminescent detection (see for example Wen et al., (1993), J. Clin. Lab. Anal., 7: 317-323, which is hereby incorporated by reference in its entirety for all purposes and in particular for all teachings related to assessment of VWF multimers). Further assays for VWF include VWF:Antigen (VWF:Ag), VWF:Ristocetin Cofactor (VWF:RCof), and VWF:Collagen Binding Activity assay (VWF:CBA), which are often used for diagnosis and classification of Von Willebrand Disease. (see for example Favaloro et al., Pathology, 1997, 29(4): 341-456, which is hereby incorporated by reference in its entirety for all purposes and in particular for all teachings related to assays for VWF).
In further embodiments, higher order rVWF multimers of the invention are stable for about 1 to about 90 hours post-administration. In still further embodiments, the higher order rVWF multimers are stable for about 5-80, 10-70, 15-60, 20-50, 25-40, 30-35 hours post-administration. In yet further embodiments, the higher order rVWF multimers are stable for at least 3, 6, 12, 18, 24, 36, 48, 72 hours post-administration. In certain embodiments the stability of the rVWF multimers is assessed in vitro.
In one embodiment, higher order rVWF multimers used in the compositions and methods provided herein have a half-life of at least 12 hour post administration. In another embodiment, the higher order rVWF multimers have a half-life of at least 24 hour post administration. In yet other embodiments, the higher order rVWF multimers have a half-life selected from variations 642 to 1045 found in Table 6 of WO 2012/171031, which is herein incorporated by reference in its entirety for all purposes.
In specific aspects, the rVWF (recombinant or plasma derived) used in accordance with the present invention are not modified with any conjugation, post-translation or covalent modifications. In particular embodiments, the rVWF of the present invention is not modified with a water soluble polymer, including without limitation, a polyethylene glycol (PEG), a polypropylene glycol, a polyoxyalkylene, a polysialic acid, hydroxyl ethyl starch, a poly-carbohydrate moiety, and the like.
In other aspects, the rVWF (recombinant or plasma derived) used in accordance with the present invention is modified through conjugation, post-translation modification, or covalent modification, including modifications of the N- or C-terminal residues as well as modifications of selected side chains, for example, at free sulfhydryl-groups, primary amines, and hydroxyl-groups. In one embodiment, a water soluble polymer is linked to the protein (directly or via a linker) by a lysine group or other primary amine. In one embodiment, the rVWF proteins of the present invention may be modified by conjugation of a water soluble polymer, including without limitation, a polyethylene glycol (PEG), a polypropylene glycol, a polyoxyalkylene, a polysialic acid, hydroxyl ethyl starch, a poly-carbohydrate moiety, and the like.
Water soluble polymers that may be used to modify the rVWF and/or FVIII include linear and branched structures. The conjugated polymers may be attached directly to the coagulation proteins 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 in 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.
Protein conjugation may be performed by a number of well-known techniques in the art, for example, see Hermanson G., Bioconjugate Techniques 2nd Ed., Academic Press, Inc. 2008. Examples include linkage through the peptide bond between a carboxyl group on one of either the coagulation protein or water-soluble polymer moiety and an amine group of the other, or an ester linkage between a carboxyl group of one and a hydroxyl group of the other. Another linkage by which a coagulation protein of the invention could be conjugated to a water-soluble polymer compound is via a Schiff base, between a free amino group on the polymer moiety being reacted 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; Fernandes and Gregonradis, Biochim Biophys Acta. 1997; 1341; 26-34). The generated Schiff Base can be stabilized by specific reduction with NaCNBH₃to form a secondary amine. An alternative approach is the generation of terminal free amino groups on the polymer by reductive amination with NH₄Cl after prior oxidation. Bifunctional reagents can be used for linking two amino or two hydroxyl groups. For example a polymer containing an amino group can be coupled to an amino group of the coagulation protein with reagents like BS3 (Bis(sulfosuccinimidyl)suberate/Pierce, Rockford, Ill.). In addition heterobifunctional cross linking reagents like Sulfo-EMCS (N-.epsilon.-Maleimidocaproyloxy) sulfosuccinimide ester/Pierce) can be used for instance to link amine and thiol groups. In other embodiments, an aldehyde reactive group, such as PEG alkoxide plus diethyl acetal of bromoacetaldehyde; PEG plus DMSO and acetic anhydride, and PEG chloride plus the phenoxide of 4-hydroxybenzaldehyde, succinimidyl active esters, activated dithiocarbonate P E.G., 2,4,5-trichlorophenylcloroformate and P-nitrophenylcloroformate activated PE.G., may be used in the conjugation of a coagulation protein.
In some aspects, the rVWF used in methods of the present invention has been matured in vitro with furin. In further embodiments, the furin is recombinant furin.
In further aspects, the rVWF used in the methods of the present invention are produced by expression in a mammalian cell culture using methods known in the art. In particular embodiments, 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 the pro-VWF to furin—in a still further embodiment, the Furin used for propeptide removal is recombinant furin. In as yet further embodiment, fully glycosylated/ABO blood group glycans are absent.
In yet further embodiments, the rVWF used in methods and compositions of the present invention by expression in a suitable eukaryotic host system. Examples of eukaryotic cells include, without limitation, mammalian cells, such as CHO, COS, HEK 293, BHK, SK-Hep, and HepG2; insect cells, e.g., SF9 cells, SF21 cells, S2 cells, and High Five cells; and yeast cells, e.g., Saccharomyces or Schizosaccharomyces cells. In one embodiment, the VWF can be expressed in yeast cells, insect cells, avian cells, mammalian cells, and the like. For example, in a human cell line, a hamster cell line, or a murine cell line. In one particular embodiment, the cell line is a CHO, BHK, or HEK cell line. Typically, mammalian cells, e.g., CHO cell from a continuous cell line, can be used to express the VWF of the present invention.
In certain embodiments, the nucleic acid sequence comprising a sequence coding for VWF can be a vector. The vector can be delivered by a virus or can be a plasmid. The nucleic acid sequence coding for the protein can be a specific gene or a biologically functional part thereof. In one embodiment, the protein is at least a biologically active part of VWF. A wide variety of vectors can be used for the expression of the VWF and 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., and vectors derived from viral systems such as vaccinia virus, adeno-associated viruses, herpes viruses, retroviruses, etc., using promoters such as CMV, SV40, EF-1, UbC, RSV, ADV, BPV, and β-actin.
In some embodiments of the present invention, the nucleic acid sequence further comprises other sequences suitable for a controlled expression of a protein such as promoter sequences, enhancers, TATA boxes, transcription initiation sites, polylinkers, restriction sites, poly-A-sequences, protein processing sequences, selection markers, and the like which are generally known to a person of ordinary skill in the art.
In certain embodiments, the cell-culture methods of the invention may comprise the use of a microcarrier. In some embodiments, the cell-cultures of the embodiments can be performed in large bioreactors under conditions suitable for providing high volume-specific culture surface areas to achieve high cell densities and protein expression. One means for providing such growth conditions is to use microcarriers for cell-culture in stirred tank bioreactors. The concept of cell-growth on microcarriers was first described by van Wezel (van Wezel, A. L., Nature 216:64-5 (1967)) and allows for cell attachment on the surface of small solid particles suspended in the growth medium. These methods provide for high surface-to-volume ratios and thus allow for efficient nutrient utilization. Furthermore, for expression of secreted proteins in eukaryotic cell lines, the increased surface-to-volume ratio allows for higher levels of secretion and thus higher protein yields in the supernatant of the culture. Finally, these methods allow for the easy scale-up of eukaryotic expression cultures.
The cells expressing VWF can be bound to a spherical or a porous microcarrier during cell culture growth. The microcarrier can be a microcarrier selected from the group of microcarriers based on dextran, collagen, plastic, gelatine and cellulose and others as described in Butler (1988. In: Spier & Griffiths, Animal Cell Biotechnology 3:283-303). It is also possible to grow the cells to a biomass on spherical microcarriers and subculture the cells when they have reached final fermenter biomass and prior to production of the expressed protein on a porous microcarrier or vice versa. Suitable spherical microcarriers can include smooth surface 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).
In certain embodiments, rVWF is expressed in cells cultured in cell culture media that produces high molecular weight rVWF. The terms “cell culture solution,” “cell culture medium or media,” and “cell culture supernatant” refer to aspects of cell culture processes generally well known in the art. In the context of the present invention, a cell culture solution can include cell culture media and cell culture supernatant. The cell culture media are externally added to the cell culture solution, optionally together with supplements, to provide nutrients and other components for culturing the cells expressing VWF. The cell culture supernatant refers to a cell culture solution comprising the nutrients and other components from the cell culture medium as well as products released, metabolized, and/or excreted from the cells during culture. In further embodiments, the media can be animal protein-free and chemically defined. Methods of preparing animal protein-free and chemically defined culture media are known in the art, for example in US 2008/0009040 and US 2007/0212770, which are both incorporated herein for all purposes and in particular for all teachings related to cell culture media. “Protein free” and related terms refers to protein that is from a source exogenous to or other than the cells in the culture, which naturally shed proteins during growth. In another embodiment, the culture medium is polypeptide free. In another embodiment, the culture medium is serum free. In another embodiment the culture medium is animal protein free. In another embodiment the culture medium is animal component free. In another embodiment, the culture medium contains protein, e.g., animal protein from serum such as fetal calf serum. In another embodiment, the culture has recombinant proteins exogenously added. In another embodiment, the proteins are from a certified pathogen free animal. The term “chemically defined” as used herein shall mean, that the medium does not comprise any undefined supplements, such as, for example, extracts of animal components, organs, glands, plants, or yeast. Accordingly, each component of a chemically defined medium is accurately defined. In a preferred embodiment, the media are animal-component free and protein free.
In further embodiments, subsequent to purification from a mammalian cell culture, rFVIII is reconstituted prior to administration. In still further embodiments, the rVWF is treated with furin prior to or subsequent to reconstitution. In further embodiments, the Furin is recombinant furin. In still further embodiments, the rVWF of the invention is not exposed to ADAMTS13, with the result that ultra large (i.e., comprising 10 or more subunits) are present in rVWF compositions of the invention.
In specific aspects, the rVWF used in methods of the present invention is contained in a formulation containing a buffer, a sugar and/or a sugar alcohol (including without limitation trehalose and mannitol), a stabilizer (such as glycine), and a surfactant (such as polysorbate 80). In further embodiments, for formulations containing rFVIII, the formulation may further include sodium, histidine, calcium, and glutathione.
In one aspect, the formulations comprising rVWF is lyophilized prior to administration. Lyophilization is carried out 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)].
Methods of preparing pharmaceutical formulations can include one or more of the following steps: adding a stabilizing agent as described herein to said mixture prior to lyophilizing, adding at least one agent selected from a bulking agent, an osmolarity regulating agent, and a surfactant, each of which as described herein, to said mixture prior to lyophilization. A lyophilized formulation is, in one aspect, at least comprised of one or more of a buffer, a bulking agent, and a stabilizer. In this aspect, the utility of a surfactant is evaluated and selected in cases where aggregation during the lyophilization step or during reconstitution becomes an issue. An appropriate buffering agent is included to maintain the formulation within stable zones of pH during lyophilization.
The standard reconstitution practice for lyophilized material is to add back a volume of pure water or sterile water for injection (WFI) (typically equivalent to the volume removed during lyophilization), although dilute solutions of antibacterial agents are sometimes used in the production of pharmaceuticals for parenteral administration [Chen, Drug Development and Industrial Pharmacy, 18:1311-1354 (1992)]. Accordingly, methods are provided for preparation of reconstituted recombinant VWF compositions comprising the step of adding a diluent to a lyophilized recombinant VWF composition of the invention.
The lyophilized material may be reconstituted as an aqueous solution. A variety of aqueous carriers, e.g., sterile water for injection, water with preservatives for multi dose use, or water with appropriate amounts of surfactants (for example, an aqueous suspension that contains the active compound in admixture with excipients suitable for the manufacture of aqueous suspensions). In various aspects, such excipients are suspending agents, for example and without limitation, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents are a naturally-occurring phosphatide, for example and without limitation, lecithin, or condensation products of an alkylene oxide with fatty acids, for example and without limitation, polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example and without limitation, heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example and without limitation, polyethylene sorbitan monooleate. In various aspects, the aqueous suspensions also contain one or more preservatives, for example and without limitation, ethyl, or n-propyl, p-hydroxybenzoate.
In certain embodiments, compositions of the present invention are liquid formulations for administration with the use of a syringe or other storage vessel. In further embodiments, these liquid formulations are produced from lyophilized material described herein reconstituted as an aqueous solution.
In a further aspect, the compositions of the invention further comprise one or more pharmaceutically acceptable carriers. The phrases “pharmaceutically” or “pharmacologically” acceptable refer to molecular entities and compositions that are stable, inhibit protein degradation such as aggregation and cleavage products, and in addition do not produce allergic, or other adverse reactions when administered using routes well-known in the art, as described below. “Pharmaceutically acceptable carriers” include any and all clinically useful solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like, including those agents disclosed above.

II. Production of Recombinant VWF

The free mature recombinant von Willebrand Factor (rVWF) of the present invention can be produced recombinantly. One skilled in the art recognizes useful methods for expressing a recombinant protein in a host cell. In some instances, 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, prepro-VWF, pro-VWF, and the like.
In certain embodiments, the nucleic acid sequence comprising a sequence coding for VWF can be an expression vector. The vector can be delivered by a virus or can be a plasmid. The nucleic acid sequence coding for the protein can be a specific gene or a biologically functional part thereof. In one embodiment, the protein is at least a biologically active part of VWF. The nucleic acid sequence can further comprise other sequences suitable for a controlled expression of a protein such as promoter sequences, enhancers, TATA boxes, transcription initiation sites, polylinkers, restriction sites, poly-A-sequences, protein processing sequences, selection markers, and the like which are generally known to a person of ordinary skill in the art.
A wide variety of vectors can be used for the expression of the VWF and 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., and vectors derived from viral systems such as vaccinia virus, adeno-associated viruses, herpes viruses, retroviruses, etc., using promoters such as CMV, SV40, EF-1, UbC, RSV, ADV, BPV, and β-actin.
In some aspects, the rVWF used in the methods of the present invention is produced by expression in a mammalian cell culture using methods known in the art. In particular embodiments, the mammalian culture comprises CHO cells. In further embodiments, the rVWF is co-expressed with recombinant Factor VIII (rFVIII) in the same culture. In such embodiments, the rVWF and the rFVIII are purified together (co-purified) or separately using methods known in the art. In other embodiments, the rVWF is expressed in a culture that does not contain rFVIII.
In some embodiments, rVWF is expressed and isolated from a suitable eukaryotic host system. Examples of eukaryotic cells include, without limitation, mammalian cells, such as CHO, COS, HEK 293, BHK, SK-Hep, and HepG2; insect cells, e.g., SF9 cells, SF21 cells, S2 cells, and High Five cells; and yeast cells, e.g., Saccharomyces or Schizosaccharomyces cells. In one embodiment, the VWF can be expressed in yeast cells, insect cells, avian cells, mammalian cells, and the like. For example, in a human cell line, a hamster cell line, or a murine cell line. In one particular embodiment, the cell line is a CHO, BHK, or HEK cell line. Typically, mammalian cells, e.g., CHO cell from a continuous cell line, can be used to express the VWF of the present invention. In certain instances, VWF protein is expressed and isolated from a CHO cell expression system.
VWF can be produced in a cell culture system or according to any cell culture method recognized by those in the art. In some embodiments, the cell cultures can be performed in large bioreactors under conditions suitable for providing high volume-specific culture surface areas to achieve high cell densities and protein expression. One means for providing such growth conditions is to use microcarriers for cell-culture in stirred tank bioreactors. The concept of cell-growth on microcarriers was first described by van Wezel (van Wezel, A. L., Nature, 1967, 216:64-5) and allows for cell attachment on the surface of small solid particles suspended in the growth medium. These methods provide for high surface-to-volume ratios and thus allow for efficient nutrient utilization. Furthermore, for expression of secreted proteins in eukaryotic cell lines, the increased surface-to-volume ratio allows for higher levels of secretion and thus higher protein yields in the supernatant of the culture. Finally, these methods allow for the easy scale-up of eukaryotic expression cultures.
The cells expressing VWF can be bound to a spherical or a porous microcarrier during cell culture growth. The microcarrier can be a microcarrier selected from the group of microcarriers based on dextran, collagen, plastic, gelatine and cellulose and others as described in Butler (1988. In: Spier & Griffiths, Animal Cell Biotechnology 3:283-303). I t is also possible to grow the cells to a biomass on spherical microcarriers and subculture the cells when they have reached final fermenter biomass and prior to production of the expressed protein on a porous microcarrier or vice versa. Suitable spherical microcarriers can include smooth surface 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).
In a further embodiment, the VWF propeptide is cleaved from the non-mature VWF in vitro through exposure of the pro-VWF to furin. In some embodiments, the furin used for propeptide cleavage is recombinant furin.
In certain embodiments, rVWF is expressed in cells cultured in cell culture media that produces high molecular weight rVWF. The terms “cell culture solution,” “cell culture medium or media,” and “cell culture supernatant” refer to aspects of cell culture processes generally well known in the art. In the context of the present invention, a cell culture solution can include cell culture media and cell culture supernatant. The cell culture media are externally added to the cell culture solution, optionally together with supplements, to provide nutrients and other components for culturing the cells expressing VWF. The cell culture supernatant refers to a cell culture solution comprising the nutrients and other components from the cell culture medium as well as products released, metabolized, and/or excreted from the cells during culture. In further embodiments, the media can be animal protein-free and chemically defined. Methods of preparing animal protein-free and chemically defined culture media are known in the art, for example in US 2006/0094104, US 2007/0212770, and US 2008/0009040, which are both incorporated herein for all purposes and in particular for all teachings related to cell culture media. “Protein free” and related terms refers to protein that is from a source exogenous to or other than the cells in the culture, which naturally shed proteins during growth. In another embodiment, the culture medium is polypeptide free. In another embodiment, the culture medium is serum free. In another embodiment the culture medium is animal protein free. In another embodiment the culture medium is animal component free. In another embodiment, the culture medium contains protein, e.g., animal protein from serum such as fetal calf serum. In another embodiment, the culture has recombinant proteins exogenously added. In another embodiment, the proteins are from a certified pathogen free animal. The term “chemically defined” as used herein shall mean, that the medium does not comprise any undefined supplements, such as, for example, extracts of animal components, organs, glands, plants, or yeast. Accordingly, each component of a chemically defined medium is accurately defined. In a preferred embodiment, the media are animal-component free and protein free.
In certain embodiments, the culture of cells expressing VWF can 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 longer. The cell density at which a cell-culture is maintained at for production of a recombinant VWF protein will depend upon the culture-conditions and medium used for protein expression. One of skill in the art will readily be able to determine the optimal cell density for a cell-culture producing an VWF. In one embodiment, the culture is maintained at a cell density of between about 0.5×10⁶and 4×10⁷cells/ml for an extended period of time. In other embodiments, the cell density is maintained at a concentration of between about 1.0×10⁶and about 1.0×10⁷cells/ml for an extended period of time. In other embodiments, the cell density is maintained at a concentration of between about 1.0×10⁶and about 4.0×10⁶cells/ml for an extended period of time. In other embodiments, the cell density is maintained at a concentration of between about 1.0×10⁶and about 4.0×10⁶cells/ml for an extended period of time. In yet other embodiments, the cell density may be maintained at a concentration between about 2.0×10⁶and about 4.0×10⁶, or between about 1.0×10⁶and about 2.5×10⁶, or between about 1.5×10⁶and about 3.5×10⁶, or any other similar range, for an extended period of time. After an appropriate time in cell culture, the rVWF can be isolated from the expression system using methods known in the art.
In a specific embodiment, the cell density of the continuous cell culture for production of rVWF is maintained at a concentration of no more than 2.5×10⁶cells/mL for an extended period. In other specific embodiments, the cell density is maintained at no more than 2.0×10⁶cells/mL, 1.5×10⁶cells/mL, 1.0×10⁶cells/mL, 0.5×10⁶cells/mL, or less. In one embodiment, the cell density is maintained at between 1.5×10⁶cells/mL and 2.5×10⁶cells/mL.
In one embodiment of the cell cultures described above, the cell culture solution comprises a medium supplement comprising copper. Such cell culture solutions are described, for example, in U.S. Pat. Nos. 8,852,888 and 9,409,971, which is hereby incorporated by reference in its entirety for all purposes and in particular for all teachings related to cell culture methods and compositions for producing recombinant VWF.
The polynucleotide and amino acid sequences of prepro-VWF are set out in SEQ ID NO:1 and SEQ ID NO:2, respectively, and are available at GenBank Accession Nos. NM 000552 (Homo sapiens von Willebrand factor (VWF) mRNA) and NP 000543, respectively. The amino acid sequence corresponding to the mature VWF protein is set out in SEQ ID NO: 3 (corresponding to amino acids 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 the sequence of SEQ ID NO:3. See, for example, U.S. Pat. No. 8,597,910, U.S. Patent Publication No. 2016/0129090, as well as FIG. 6 .
One form of useful rVWF has at least the property of in vivo-stabilizing, e.g. binding, of at least one Factor VIII (FVIII) molecule and having optionally a glycosylation pattern which is pharmacologically acceptable. Specific examples thereof include VWF without the A2 domain thus resistant to proteolysis (Lankhof et al., Thromb. Haemost. 77: 1008-1013, 1997), and a VWF fragment from Val 449 to Asn 730 including the glycoprotein 1b-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 a VWF to stabilize at least one FVIII molecule is, in one aspect, carried out in VWF-deficient mammals according to methods known in the state in the art.
The rVWF of the present invention can be produced by any method known in the art. One specific example is disclosed in WO86/06096 published on Oct. 23, 1986 and U.S. patent application Ser. No. 07/559,509, filed on Jul. 23, 1990, which is incorporated herein by reference with respect to the methods of producing recombinant VWF. Thus, methods are known in the art for (i) the production of 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) cultivating the transformed cells, e.g. in a continuous or batchwise manner, (iv) expressing VWF, e.g. constitutively or upon induction, and (v) isolating the VWF, e.g. from the culture medium or by harvesting the transformed cells, in order to (vi) obtain purified rVWF, e.g. via anion exchange chromatography or affinity chromatography. A recombinant VWF is, in one aspect, made in transformed host cells using recombinant DNA techniques well known in the art. For instance, sequences coding for the polypeptide could be excised from DNA using suitable restriction enzymes. Alternatively, the DNA molecule is, in another aspect, synthesized using chemical synthesis techniques, such as the phosphoramidate method. Also, in still another aspect, a combination of these techniques is used.
The invention also provides vectors encoding polypeptides of the invention in an appropriate host. The vector comprises the polynucleotide that encodes the polypeptide operatively linked to appropriate expression control sequences. Methods of effecting this operative linking, either before or after the polynucleotide is inserted into the vector, are well known. Expression control sequences include promoters, activators, enhancers, operators, ribosomal binding sites, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved with the control of transcription or translation. The resulting vector having the polynucleotide therein is used to transform an appropriate host. This transformation may be performed using methods well known in the art.
Any of a large number of available and well-known host cells are used in the practice of this invention. The selection of a particular host is dependent upon a number of factors recognized by the art, including, for example, compatibility with the chosen expression vector, toxicity of the peptides encoded by the DNA molecule, rate of transformation, ease of recovery of the peptides, expression characteristics, bio-safety and costs. A balance of these factors must be struck with the understanding that not all host cells are equally effective for the expression of a particular DNA sequence. Within these general guidelines, useful microbial host cells include, without limitation, bacteria, yeast and other fungi, insects, plants, mammalian (including human) cells in culture, or other hosts known in the art.
Transformed host cells are cultured under conventional fermentation conditions so that the desired compounds are expressed. Such fermentation conditions are well known in the art. Finally, the polypeptides are purified from culture media or the host cells themselves by methods well known in the art.
Depending on the host cell utilized to express a compound of the invention, carbohydrate (oligosaccharide) groups are optionally attached to sites that are 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 they are 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 N-linked and O-linked oligosaccharides and the sugar residues found in each type are different. One type of sugar that is commonly found on both N-linked and O-linked oligosaccharides is N-acetylneuraminic acid (referred to as sialic acid). Sialic acid is usually the terminal residue of both N-linked and O-linked oligosaccharides and, by virtue of its negative charge, in one aspect, confers acidic properties to the glycosylated compound. Such site(s) may be incorporated in the linker of the compounds of this invention and are preferably glycosylated by a cell during recombinant production of the polypeptide compounds (e.g., in mammalian cells such as CHO, BHK, COS). In other aspects, such sites are glycosylated by synthetic or semi-synthetic procedures known in the art.
In some embodiments, sialysation (also referred to as sialylation), can be performed on the column as part of the purification procedures described herein (including the anion exchange, cation exchange, size exclusion, and/or immunoaffinity methods). In some embodiments, the sialylation results in increased stability of the rVWF as compared to rVWF that has not undergone sialylation. In some embodiments, the sialylation results in increased stability of the rVWF in blood circulation (for example, after administration to a subject) as compared to rVWF that has not undergone sialylation. In some embodiments, the increased stability of salivated rVWF results in an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more as compared rVWF that has not undergone sialylation. In some embodiments, the sialylation results in increased half-life for the rVWF as compared to rVWF that has not undergone sialylation. In some embodiments, the sialylation results in increased half-life for the rVWF in blood circulation (for example, after administration to a subject) as compared to rVWF that has not undergone sialylation. 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 as compared rVWF that has not undergone sialylation. In some embodiments, the increased half-life of sialylated rVWF results in rVWF that is stable for 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 24 hours or more in blood circulation (for example, after administration to a subject) as compared to rVWF that has not undergone sialylation. In some embodiments, sialylation increases the number of 2,3 sialylation and/or 2,6 sialylation. In some embodiments, sialylation is increased by the addition of 2,3 sialyltransferase and/or 2,6 sialyltransferase and CMP-NANA (Cytidine-5′-monophospho-N-acetylneuraminic acid sodium salt) as an additional buffer step. In some embodiments, sialylation is increased by the addition of 2,3 sialyltransferase and CMP-NANA (Cytidine-5′-monophospho-N-acetylneuraminic acid sodium salt) as an additional buffer step. In some embodiments, 2,3 sialylation is increased by the addition of 2,3 sialyltransferase and CMP-NANA (Cytidine-5′-monophospho-N-acetylneuraminic acid sodium salt) as an additional buffer step.
In some embodiments, 2,6 sialylation is increased by the addition of 2,6 sialyltransferase and CMP-NANA (Cytidine-5′-monophospho-N-acetylneuraminic acid sodium salt) as an additional buffer step. In some embodiments, 2,3 sialylation and/or 2,6 sialylation are increased by the addition of 2,3 sialyltransferase and/or 2,6 sialyltransferase and CMP-NANA (Cytidine-5′-monophospho-N-acetylneuraminic acid sodium salt) as an additional buffer step. In some embodiments, CMP-NANA is chemically or enzymatic modified to transfer modified sialic acid to potential free position. In some embodiments, sialylation is performed by loading rVWF onto the resin, washing with one or more buffers as described herein to deplete unwanted impurities, apply one or more buffers containing sialyltransferase and CMP-NANA at conditions that allow additional sialylation, and washing with one or more buffers to deplete excess of the sialylation reagents, and eluting with one or more buffers the enhanced rVWF (e.g., the rVWF with increased sialylation). In some embodiments, the sialylation process is performed as part of a cation exchange method, an anion exchange method, a size exclusion method, or an immunoaffinity purification method, as described herein.
Alternatively, the compounds are made by synthetic methods using, for example, 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. Pat. 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 of making individual peptides since it is the most cost-effective method of making small peptides
Fragments, variants and analogs of VWF can be produced according to methods that are well-known in the art. Fragments of a polypeptide can be prepared using, without limitation, enzymatic cleavage (e.g., trypsin, chymotrypsin) and also using recombinant means to generate a polypeptide fragments having a specific amino acid sequence. Polypeptide fragments may be generated comprising a region of the protein having a particular activity, such as a multimerization domain or any other identifiable VWF domain known in the art.
Methods of making polypeptide analogs are also well-known. Amino acid sequence analogs of a polypeptide can be substitutional, insertional, addition or deletion analogs. Deletion analogs, including fragments of a polypeptide, lack one or more residues of the native protein which are not essential for function or immunogenic activity. Insertional analogs involve the addition of, e.g., amino acid(s) at a non-terminal point in the polypeptide. This analog may include, for example and without limitation, insertion of an immunoreactive epitope or simply a single residue. Addition analogs, including fragments of a polypeptide, include the addition of one or more amino acids at either or both termini of a protein and include, for example, fusion proteins. Combinations of the aforementioned analogs are also contemplated.
Substitutional analogs typically exchange one amino acid of the wild-type for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide without the complete loss of other functions or properties. In one aspect, substitutions are conservative substitutions. “Conservative amino acid substitution” is substitution of an amino acid with an amino acid having a side chain or a similar chemical character. Similar amino acids for making conservative substitutions include those having an acidic side chain (glutamic acid, aspartic acid); a basic side chain (arginine, lysine, histidine); a polar amide side chain (glutamine, asparagine); a hydrophobic, aliphatic side chain (leucine, isoleucine, valine, alanine, glycine); an aromatic side chain (phenylalanine, tryptophan, tyrosine); a small side chain (glycine, alanine, serine, threonine, methionine); or an aliphatic hydroxyl side chain (serine, threonine).
In one aspect, analogs are substantially homologous or substantially identical to the recombinant VWF from which they are derived. Analogs include those which retain at least some of the biological activity of the wild-type polypeptide, e.g. blood clotting activity.
Polypeptide variants contemplated include, without limitation, polypeptides chemically modified by such techniques as ubiquitination, glycosylation, including polysialation (or polysialylation), conjugation to therapeutic or diagnostic agents, labeling, covalent polymer attachment such as pegylation (derivatization with polyethylene glycol), introduction of non-hydrolyzable bonds, and insertion or substitution by chemical synthesis of amino acids such as ornithine, which do not normally occur in human proteins. Variants retain the same or essentially the same binding properties of non-modified molecules of the invention. Such chemical modification 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.
Preparing pegylated polypeptide analogs will in one aspect comprise the steps of (a) reacting the polypeptide with polyethylene glycol (such as a reactive ester or aldehyde derivative of PEG) under conditions whereby the binding construct polypeptide becomes attached to one or more PEG groups, and (b) obtaining the reaction product(s). In general, the optimal reaction conditions for the acylation reactions are determined based on known parameters and the desired result. For example, the larger the ratio of PEG:protein, the greater the percentage of poly-pegylated product. In some embodiments, the binding construct has a single PEG moiety at the N-terminus. Polyethylene glycol (PEG) may be attached to the blood clotting factor to, for example, provide a longer half-life in vivo. The PEG group may be of any convenient molecular weight and is linear or branched. The average molecular weight of the PEG ranges from about 2 kiloDalton (“kD”) to about 100 kDa, from about 5 kDa to about 50 kDa, or from about 5 kDa to about 10 kDa. In certain aspects, the PEG groups are attached to the blood clotting factor via acylation or reductive alkylation through a natural or engineered reactive group on the PEG moiety (e.g., an aldehyde, amino, thiol, or ester group) to a reactive group on the blood clotting factor (e.g., an aldehyde, amino, or ester group) or by any other technique known in the art.
Methods for preparing polysialylated polypeptide are described in United States Patent Publication 20060160948, Fernandes et Gregoriadis; Biochim. Biophys. Acta 1341: 26-34, 1997, and Saenko et al., Haemophilia 12:42-51, 2006. Briefly, a solution of colominic acid (CA) containing 0.1 M NaIO₄is stirred in the dark at room temperature to oxidize the CA. The activated CA solution is dialyzed against, e.g., 0.05 M sodium phosphate buffer, pH 7.2 in the dark and this solution was added to a rVWF solution and incubated for 18 h at room temperature in the dark under gentle shaking. Free reagents are optionally be separated from the rVWF-polysialic acid conjugate by, for example, ultrafiltration/diafiltration. Conjugation of rVWF with polysialic acid is achieved using glutaraldehyde as cross-linking reagent (Migneault et al., Biotechniques 37: 790-796, 2004).
It is also contemplated in another aspect that prepro-VWF and pro-VWF polypeptides will provide a therapeutic benefit in the formulations of the present invention. For example, U.S. Pat. No. 7,005,502 describes a pharmaceutical preparation comprising substantial amounts of pro-VWF that induces thrombin generation in vitro. In addition to recombinant, biologically active fragments, variants, or other analogs of the naturally-occurring mature VWF, the present invention contemplates the use of recombinant biologically active fragments, variants, or analogs of the prepro-VWF (set out in SEQ ID NO:2) or pro-VWF polypeptides (amino acid residues 23 to 764 of SEQ ID NO: 2) in the formulations described herein.
Polynucleotides encoding fragments, variants and analogs may be readily generated by a worker of skill to encode biologically active fragments, variants, or analogs of the naturally-occurring molecule that possess the same or similar biological activity to the naturally-occurring molecule. In various aspects, these polynucleotides are prepared using PCR techniques, digestion/ligation of DNA encoding molecule, and the like. Thus, one of skill in the art will be able to generate single base changes in the DNA strand to result in an altered codon and a missense mutation, using any method known in the art, including, but not limited to site-specific mutagenesis. As used herein, the phrase “moderately stringent hybridization conditions” means, for example, hybridization at 42° C. in 50% formamide and washing at 60° C. in 0.1×SSC, 0.1% SDS. It is understood by those of skill in the art that variation in these conditions occurs based on the length and GC nucleotide base content of the sequences to be hybridized. Formulas standard in the art are appropriate for determining 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).
A. VWF Multimers
Assessment of the number and percentage of rVWF multimers can be conducted using methods known in the art, including without limitation methods using electrophoresis and size exclusion chromatography methods to separate VWF multimers by size, for example as discussed by Cumming et al., (J Clin Pathol., 1993 May; 46(5): 470-473, which is hereby incorporated by reference in its entirety for all purposes and in particular for all teachings related to assessment of VWF multimers). Such techniques may further include immunoblotting techniques (such as Western Blot), in which the gel is immunoblotted with a radiolabeled antibody against VWF followed by chemiluminescent detection (see, for example, Wen et al., J. Clin. Lab. Anal., 1993, 7: 317-323, which is hereby incorporated by reference in its entirety for all purposes and in particular for all teachings related to assessment of VWF multimers). Further assays for VWF include VWF:Antigen (VWF:Ag), VWF:Ristocetin Cofactor (VWF:RCof), and VWF:Collagen Binding Activity assay (VWF:CBA), which are often used for diagnosis and classification of Von Willebrand Disease (see, for example, 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 hereby incorporated by reference in their entirety for all purposes and in particular for all teachings related to assays for VWF). In some embodiments, the rVWF obtained using the present methods includes any multimer pattern present in the loading sample of the rVWF. In some embodiments, the rVWF obtained using the present methods includes physiologically occurring multimer patters as well as ultralarge VWF-multimer patterns.
b. VWF Assays
In primary hemostasis VWF serves as a bridge between platelets and specific components of the extracellular matrix, such as collagen. The biological activity of VWF in this process can be measured by different in vitro assays (Turecek et al., Semin Thromb Hemost, 2010, 36: 510-521).
The VWF:Ristocetin Cofactor (VWF:RCof) assay is based on the agglutination of fresh or formalin-fixed platelets induced by the antibiotic ristocetin in the presence of VWF. The degree of platelet agglutination depends on the VWF concentration and can be measured by the turbidimetric method, e.g., by use of 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 the VWF (VWF:RCo) of the present invention is generally described in terms of mU/μg of VWF, as measured using in vitro assays.
In some embodiments, the rVWF purified according to the methods of the present 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, 1 15, 120, 125, 130, 135, 140, 145, 150 or more mU/μg. In some embodiments, rVWF used in the methods described herein has a specific activity of from 20 mU/μg to 150 mU/μg. In some embodiments, the rVWF has a specific activity of from 30 mU/μg to 120 mU/μg. In some embodiments, the rVWF has a specific activity from 40 mU/μg to 90 mU/μg. In some embodiments, the rVWF has a specific activity selected from variations 1 to 133 found in Table 3, below.

TABLE 3

Exemplary embodiments for the specific
activity of rVWF found in the compositions
and used in the methods provided herein.

	(mU/μg)

	20	Var. 1
	22.5	Var. 2
	25	Var. 3
	27.5	Var. 4
	30	Var. 5
	32.5	Var. 6
	35	Var. 7
	37.5	Var. 8
	40	Var. 9
	42.5	Var. 10
	45	Var. 11
	47.5	Var. 12
	50	Var. 13
	52.5	Var. 14
	55	Var. 15
	57.5	Var. 16
	60	Var. 17
	62.5	Var. 18
	65	Var. 19
	67.5	Var. 20
	70	Var. 21
	72.5	Var. 22
	75	Var. 23
	77.5	Var. 24
	80	Var. 25
	82.5	Var. 26
	85	Var. 27
	87.5	Var. 28
	90	Var. 29
	92.5	Var. 30
	95	Var. 31
	97.5	Var. 32
	100	Var. 33
	105	Var. 34
	110	Var. 35
	115	Var. 36
	120	Var. 37
	125	Var. 38
	130	Var. 39
	135	Var. 40
	140	Var. 41
	145	Var. 42
	150	Var. 43
	20-150	Var. 44
	20-140	Var. 45
	20-130	Var. 46
	20-120	Var. 47
	20-110	Var. 48
	20-100	Var. 49
	20-90	Var. 50
	20-80	Var. 51
	20-70	Var. 52
	20-60	Var. 53
	20-50	Var. 54
	20-40	Var. 55
	30-150	Var. 56
	30-140	Var. 57
	30-130	Var. 58
	30-120	Var. 59
	30-110	Var. 60
	30-100	Var. 61
	30-90	Var. 62
	30-80	Var. 63
	30-70	Var. 64
	30-60	Var. 65
	30-50	Var. 66
	30-40	Var. 67
	40-150	Var. 68
	40-140	Var. 69
	40-130	Var. 70
	40-120	Var. 71
	40-110	Var. 72
	40-100	Var. 73
	40-90	Var. 74
	40-80	Var. 75
	40-70	Var. 76
	40-60	Var. 77
	40-50	Var. 78
	50-150	Var. 79
	50-140	Var. 80
	50-130	Var. 81
	50-120	Var. 82
	50-110	Var. 83
	50-100	Var. 84
	50-90	Var. 85
	50-80	Var. 86
	50-70	Var. 87
	50-60	Var. 88
	60-150	Var. 89
	60-140	Var. 90
	60-130	Var. 91
	60-120	Var. 92
	60-110	Var. 93
	60-100	Var. 94
	60-90	Var. 95
	60-80	Var. 96
	60-70	Var. 97
	70-150	Var. 98
	70-140	Var. 99
	70-130	Var. 100
	70-120	Var. 101
	70-110	Var. 102
	70-100	Var. 103
	70-90	Var. 104
	70-80	Var. 105
	80-150	Var. 106
	80-140	Var. 107
	80-130	Var. 108
	80-120	Var. 109
	80-110	Var. 110
	80-100	Var. 111
	80-90	Var. 112
	90-150	Var. 113
	90-140	Var. 114
	90-130	Var. 115
	90-120	Var. 116
	90-110	Var. 117
	90-100	Var. 118
	100-150	Var. 119
	100-140	Var. 120
	100-130	Var. 121
	100-120	Var. 122
	100-110	Var. 123
	110-150	Var. 124
	110-140	Var. 125
	110-130	Var. 126
	110-120	Var. 127
	120-150	Var. 128
	120-140	Var. 129
	120-130	Var. 130
	130-150	Var. 131
	130-140	Var. 132
	140-150	Var. 133

	Var. = Variation

The rVWF of the present invention is highly multimeric comprising about to about 40 subunits. In further embodiments, the multimeric rVWF produced using methods of the present invention comprise about 10-30, 12-28, 14-26, 16-24, 18-22, 20-21 subunits. In some embodiments, the rVWF is present in multimers varying in size from dimers to multimers of over 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 ultralarge multimers (ULMs). Generally, high and ultralarge multimers are considered to be hemostatically most effective (see, for example, Turecek, P., Hämostaseologie, (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 methods described. In some embodiments, when anion exchange and/or cation exchanger methods are employed, the pH, conductivity, and/or counterion concentration of the buffers in the one or more wash step(s) or the gradient buffers can be manipulated to obtain the desired multimer pattern. In some embodiments, then size exclusion chromatography methods are employed, the collection criteria can be employed to obtain the desired multimer pattern. In some embodiments, the described multimer pattern comprises ultralarge multimers. In some embodiments, the ultralarge 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 ultralarge multimers are between about 10,000 kDa and 20,000 kDa. In some embodiments, the ultralarge multimers are between about 11,000 kDa and 20,000 kDa. In some embodiments, the ultralarge multimers are between about 12,000 kDa and 20,000 kDa. In some embodiments, the ultralarge multimers are between about 13,000 kDa and kDa. In some embodiments, the ultralarge multimers are between about 14,000 kDa and 20,000 kDa. In some embodiments, the ultralarge multimers are between about kDa and 20,000 kDa. In some embodiments, the ultralarge multimers are between about 16,000 kDa and 20,000 kDa. In some embodiments, the ultralarge multimers are between about 17,000 kDa and 20,000 kDa. In some embodiments, the ultralarge multimers are between about 18,000 kDa and 20,000 kDa. In some embodiments, the ultralarge multimers are between about 19,000 kDa and 20,000 kDa. In some embodiments, the rVWF obtained using the present methods includes any multimer pattern present in the loading sample of the rVWF. In some embodiments, the rVWF obtained using the present methods includes physiolocical occurring multimer patters as well as ultra large VWF-multimer patterns.
In some embodiments, the rVWF composition prepared by the purification method 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, the rVWF composition has a distribution of rVWF oligomers characterized in that 95% of the oligomers have a range of subunits selected from variations 458 to 641 found in 4.

TABLE 4

Exemplary embodiments for
the distribution of rVWF
oligomers found in the
compositions and used in
the methods provided herein.

	Subunits

	2-40	Var. 458
	2-38	Var. 459
	2-36	Var. 460
	2-34	Var. 461
	2-32	Var. 462
	2-30	Var. 463
	2-28	Var. 464
	2-26	Var. 465
	2-24	Var. 466
	2-22	Var. 467
	2-20	Var. 468
	2-18	Var. 469
	2-16	Var. 470
	2-14	Var. 471
	2-12	Var. 472
	2-10	Var. 473
	2-8	Var. 474
	4-40	Var. 475
	4-38	Var. 476
	4-36	Var. 477
	4-34	Var. 478
	4-32	Var. 479
	4-30	Var. 480
	4-28	Var. 481
	4-26	Var. 482
	4-24	Var. 483
	4-22	Var. 484
	4-20	Var. 485
	4-18	Var. 486
	4-16	Var. 487
	4-14	Var. 488
	4-12	Var. 489
	4-10	Var. 490
	4-8	Var. 491
	6-40	Var. 492
	6-38	Var. 493
	6-36	Var. 494
	6-34	Var. 495
	6-32	Var. 496
	6-30	Var. 497
	6-28	Var. 498
	6-26	Var. 499
	6-24	Var. 500
	6-22	Var. 501
	6-20	Var. 502
	6-18	Var. 503
	6-16	Var. 504
	6-14	Var. 505
	6-12	Var. 506
	6-10	Var. 507
	6-8	Var. 508
	8-40	Var. 509
	8-38	Var. 510
	8-36	Var. 511
	8-34	Var. 512
	8-32	Var. 513
	8-30	Var. 514
	8-28	Var. 515
	8-26	Var. 516
	8-24	Var. 517
	8-22	Var. 518
	8-20	Var. 519
	8-18	Var. 520
	8-16	Var. 521
	8-14	Var. 522
	8-12	Var. 523
	8-10	Var. 524
	10-40	Var. 525
	10-38	Var. 526
	10-36	Var. 527
	10-34	Var. 528
	10-32	Var. 529
	10-30	Var. 530
	10-28	Var. 531
	10-26	Var. 532
	10-24	Var. 533
	10-22	Var. 534
	10-20	Var. 535
	10-18	Var. 536
	10-16	Var. 537
	10-14	Var. 538
	10-12	Var. 539
	12-40	Var. 540
	12-38	Var. 541
	12-36	Var. 542
	12-34	Var. 543
	12-32	Var. 544
	12-30	Var. 545
	12-28	Var. 546
	12-26	Var. 547
	12-24	Var. 548
	12-22	Var. 549
	12-20	Var. 550
	12-18	Var. 551
	12-16	Var. 552
	12-14	Var. 553
	14-40	Var. 554
	14-38	Var. 555
	14-36	Var. 556
	14-34	Var. 557
	14-32	Var. 558
	14-30	Var. 559
	14-28	Var. 560
	14-26	Var. 561
	14-24	Var. 562
	14-22	Var. 563
	14-20	Var. 564
	14-18	Var. 565
	14-16	Var. 566
	16-40	Var. 567
	16-38	Var. 568
	16-36	Var. 569
	16-34	Var. 570
	16-32	Var. 571
	16-30	Var. 572
	16-28	Var. 573
	16-26	Var. 574
	16-24	Var. 575
	16-22	Var. 576
	16-20	Var. 577
	16-18	Var. 578
	18-40	Var. 579
	18-38	Var. 580
	18-36	Var. 581
	18-34	Var. 582
	18-32	Var. 583
	18-30	Var. 584
	18-28	Var. 585
	18-26	Var. 586
	18-24	Var. 587
	18-22	Var. 588
	18-20	Var. 589
	20-40	Var. 590
	20-38	Var. 591
	20-36	Var. 592
	20-34	Var. 593
	20-32	Var. 594
	20-30	Var. 595
	20-28	Var. 596
	20-26	Var. 597
	20-24	Var. 598
	20-22	Var. 599
	22-40	Var. 600
	22-38	Var. 601
	22-36	Var. 602
	22-34	Var. 603
	22-32	Var. 604
	22-30	Var. 605
	22-28	Var. 606
	22-26	Var. 607
	22-24	Var. 608
	24-40	Var. 609
	24-38	Var. 610
	24-36	Var. 611
	24-34	Var. 612
	24-32	Var. 613
	24-30	Var. 614
	24-28	Var. 615
	24-26	Var. 616
	26-40	Var. 617
	26-38	Var. 618
	26-36	Var. 619
	26-34	Var. 620
	26-32	Var. 621
	26-30	Var. 622
	26-28	Var. 623
	28-40	Var. 624
	28-38	Var. 625
	28-36	Var. 626
	28-34	Var. 627
	28-32	Var. 628
	28-30	Var. 629
	30-40	Var. 630
	30-38	Var. 631
	30-36	Var. 632
	30-34	Var. 633
	30-32	Var. 634
	32-40	Var. 635
	32-38	Var. 636
	32-36	Var. 637
	32-34	Var. 638
	34-40	Var. 639
	36-38	Var. 640
	38-40	Var. 641

	Var. = Variation

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

TABLE 5

Exemplary embodiments for the percentage of rVWF molecules that are present
in a particular higher order rVWF multimer or larger multimer found in the compositions
and used in the methods provided herein.

Minimal Number of Subunits in rVWF Multimer

	6	8	10	12	14	16

Minimal	10%	Var. 134	Var. 152	Var. 170	Var. 188	Var. 206	Var. 224
	15%	Var. 135	Var. 153	Var. 171	Var. 189	Var. 207	Var. 225
	20%	Var. 136	Var. 154	Var. 172	Var. 190	Var. 208	Var. 226
	25%	Var. 137	Var. 155	Var. 173	Var. 191	Var. 209	Var. 227
	30%	Var. 138	Var. 156	Var. 174	Var. 192	Var. 210	Var. 228
	35%	Var. 139	Var. 157	Var. 175	Var. 193	Var. 211	Var. 229
	40%	Var. 140	Var. 158	Var. 176	Var. 194	Var. 212	Var. 230
	45%	Var. 141	Var. 159	Var. 177	Var. 195	Var. 213	Var. 231
	50%	Var. 142	Var. 160	Var. 178	Var. 196	Var. 214	Var. 232
	55%	Var. 143	Var. 161	Var. 179	Var. 197	Var. 215	Var. 233
	60%	Var. 144	Var. 162	Var. 180	Var. 198	Var. 216	Var. 234
	65%	Var. 145	Var. 163	Var. 181	Var. 199	Var. 217	Var. 235
	70%	Var. 146	Var. 164	Var. 182	Var. 200	Var. 218	Var. 236
	75%	Var. 147	Var. 165	Var. 183	Var. 201	Var. 219	Var. 237
	80%	Var. 148	Var. 166	Var. 184	Var. 202	Var. 220	Var. 238
	85%	Var. 149	Var. 167	Var. 185	Var. 203	Var. 221	Var. 239
	90%	Var. 150	Var. 168	Var. 186	Var. 204	Var. 222	Var. 240
	95%	Var. 151	Var. 169	Var. 187	Var. 205	Var. 223	Var. 241

Var. = Variation
indicates data missing or illegible when filed

TABLE 6

Exemplary embodiments for the percentage of rVWF molecules that are present in a
particular higher order rVWF multimer or larger multimer found in the compositions
and used in the methods provided herein.

Minimal Number of Subunits in rVWF Multimer

	18	20	22	24	26	28

Minimal	10%	Var. 242	Var. 260	Var. 278	Var. 296	Var. 314	Var. 332
Percentage	15%	Var. 243	Var. 261	Var. 279	Var. 297	Var. 315	Var. 333
of rVVVF	20%	Var. 244	Var. 262	Var. 280	Var. 298	Var. 316	Var. 334
Molecules	25%	Var. 245	Var. 263	Var. 281	Var. 299	Var. 317	Var. 335
	30%	Var. 246	Var. 264	Var. 282	Var. 300	Var. 318	Var. 336
	35%	Var. 247	Var. 265	Var. 283	Var. 301	Var. 319	Var. 337
	40%	Var. 248	Var. 266	Var. 284	Var. 302	Var. 320	Var. 338
	45%	Var. 249	Var. 267	Var. 285	Var. 303	Var. 321	Var. 339
	50%	Var. 250	Var. 268	Var. 286	Var. 304	Var. 322	Var. 340
	55%	Var. 251	Var. 269	Var. 287	Var. 305	Var. 323	Var. 341
	60%	Var. 252	Var. 270	Var. 288	Var. 306	Var. 324	Var. 342
	65%	Var. 253	Var. 271	Var. 289	Var. 307	Var. 325	Var. 343
	70%	Var. 254	Var. 272	Var. 290	Var. 308	Var. 326	Var. 344
	75%	Var. 255	Var. 273	Var. 291	Var. 309	Var. 327	Var. 345
	80%	Var. 256	Var. 274	Var. 292	Var. 310	Var. 328	Var. 346
	85%	Var. 257	Var. 275	Var. 293	Var. 311	Var. 329	Var. 347
	90%	Var. 258	Var. 276	Var. 294	Var. 312	Var. 330	Var. 348
	95%	Var. 259	Var. 277	Var. 295	Var. 313	Var. 331	Var. 349

Var. = Variation

TABLE 7

Exemplary embodiments for the percentage of rVWF
molecules that are present in a particular higher order rVWF
multimer or larger multimer found in the compositions
and used in the methods provided herein.

Minimal Number of Subunits in rVWF Multimer

	30	32	34	36	38	40

Minimal	10%	Var. 350	Var. 368	Var. 386	Var. 404	Var. 422	Var. 440
Percentage	15%	Var. 351	Var. 369	Var. 387	Var. 405	Var. 423	Var. 441
of rVWF	20%	Var. 352	Var. 370	Var. 388	Var. 406	Var. 424	Var. 442
Molecules	25%	Var. 353	Var. 371	Var. 389	Var. 407	Var. 425	Var. 443
	30%	Var. 354	Var. 372	Var. 390	Var. 408	Var. 426	Var. 444
	35%	Var. 355	Var. 373	Var. 391	Var. 409	Var. 427	Var. 445
	40%	Var. 356	Var. 374	Var. 392	Var. 410	Var. 428	Var. 446
	45%	Var. 357	Var. 375	Var. 393	Var. 411	Var. 429	Var. 447
	50%	Var. 358	Var. 376	Var. 394	Var. 412	Var. 430	Var. 448
	55%	Var. 359	Var. 377	Var. 395	Var. 413	Var. 431	Var. 449
	60%	Var. 360	Var. 378	Var. 396	Var. 414	Var. 432	Var. 450
	65%	Var. 361	Var. 379	Var. 397	Var. 415	Var. 433	Var. 451
	70%	Var. 362	Var. 380	Var. 398	Var. 416	Var. 434	Var. 452
	75%	Var. 363	Var. 381	Var. 399	Var. 417	Var. 435	Var. 453
	80%	Var. 364	Var. 382	Var. 400	Var. 418	Var. 436	Var. 454
	85%	Var. 365	Var. 383	Var. 401	Var. 419	Var. 437	Var. 455
	90%	Var. 366	Var. 384	Var. 402	Var. 420	Var. 438	Var. 456
	95%	Var. 367	Var. 385	Var. 403	Var. 421	Var. 439	Var. 457

Var. = Variation

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 order multimers. In further embodiments, the composition comprises about 10-95%, 20-90%, 30-85%, 40-80%, 50-75%, 60-70% decamers or higher order 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 order multimers.
Assessment of the number and percentage of rVWF multimers can be conducted using methods known in the art, including without limitation methods using electrophoresis and size exclusion chromatography methods to separate rVWF multimers by size, for example as discussed by Cumming et al, (J Clin Pathol. 1993 May; 46(5): 470-473, which is hereby incorporated by reference in its entirety for all purposes and in particular for all teachings related to assessment of rVWF multimers). Such techniques may further include immunoblotting techniques (such as Western Blot), in which the gel is immunoblotted with a radiolabeled antibody against VWF followed by chemiluminescent detection (see for example Wen et al., (1993), J. Clin. Lab. Anal., 7: 317-323, which is hereby incorporated by reference in its entirety for all purposes and in particular for all teachings related to assessment of rVWF multimers). Further assays for VWF include VWF:Antigen (VWF:Ag), VWF:Ristocetin Cofactor (VWF:RCof), and VWF:Collagen Binding Activity assay (VWF:CBA), which are often used for diagnosis and classification of Von Willebrand Disease. (see for example Favaloro et al., Pathology, 1997, 29(4): 341-456, which is hereby incorporated by reference in its entirety for all purposes and in particular for all teachings related to assays for VWF).
In some embodiments, the ratio of rFVIII procoagulant activity (IU rFVIII:C) to rVWF Ristocetin cofactor activity (IU rVWF:RCo) for the rVWF prepared according to the methods of the present invention is between 3:1 and 1:5. In further embodiments, the ratio is between 2:1 and 1:4. In still further embodiments, the ratio is between 5:2 and 1:4. In further embodiments, the ratio is between 3:2 and 1:3. In still 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 between 1:1 and 1:2. In yet 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 a composition useful for a method described herein is selected from variations 1988 to 2140 found in Table 8.

TABLE 8

Exemplary embodiments for
the ratio of rFVIII procoagulant
activity (IU rFVIII: C) to
rVWF Ristocetin cofactor
activity (IU rVWF: RCo)
in compositions and used in
methods provided herein.

	(IU rFVIII: C)
	to (IU
	rVIVF: RCo)

	4:1	Var. 1988
	3:1	Var. 1989
	2:1	Var. 1990
	3:2	Var. 1991
	4:3	Var. 1992
	1:1	Var. 1993
	5:6	Var. 1994
	4:5	Var. 1995
	3:4	Var. 1996
	2:3	Var. 1997
	3:5	Var. 1998
	1:2	Var. 1999
	2:5	Var. 2000
	1:3	Var. 2001
	1:4	Var. 2002
	1:5	Var. 2003
	1:6	Var. 2004
	4:1-1:6	Var. 2005
	4:1-1:5	Var. 2006
	4:1-1:4	Var. 2007
	4:1-1:3	Var. 2008
	4:1-2:5	Var. 2009
	4:1-1:2	Var. 2010
	4:1-3:5	Var. 2011
	4:1-2:3	Var. 2012
	4:1-3:4	Var. 2013
	4:1-4:5	Var. 2014
	4:1-5:6	Var. 2015
	4:1-1:1	Var. 2016
	4:1-4:3	Var. 2017
	4:1-3:2	Var. 2018
	4:1-2:1	Var. 2019
	4:1-3:1	Var. 2020
	3:1-1:6	Var. 2021
	3:1-1:5	Var. 2022
	3:1-1:4	Var. 2023
	3:1-1:3	Var. 2024
	3:1-2:5	Var. 2025
	3:1-1:2	Var. 2026
	3:1-3:5	Var. 2027
	3:1-2:3	Var. 2028
	3:1-3:4	Var. 2029
	3:1-4:5	Var. 2030
	3:1-5:6	Var. 2031
	3:1-1:1	Var. 2032
	3:1-4:3	Var. 2033
	3:1-3:2	Var. 2034
	3:1-2:1	Var. 2035
	2:1-1:6	Var. 2036
	2:1-1:5	Var. 2037
	2:1-1:4	Var. 2038
	2:1-1:3	Var. 2039
	2:1-2:5	Var. 2040
	2:1-1:2	Var. 2041
	2:1-3:5	Var. 2042
	2:1-2:3	Var. 2043
	2:1-3:4	Var. 2044
	2:1-4:5	Var. 2045
	2:1-5:6	Var. 2046
	2:1-1:1	Var. 2047
	2:1-4:3	Var. 2048
	2:1-3:2	Var. 2049
	3:2-1:6	Var. 2050
	3:2-1:5	Var. 2051
	3:2-1:4	Var. 2052
	3:2-1:3	Var. 2053
	3:2-2:5	Var. 2054
	3:2-1:2	Var. 2055
	3:2-3:5	Var. 2056
	3:2-2:3	Var. 2057
	3:2-3:4	Var. 2058
	3:2-4:5	Var. 2059
	3:2-5:6	Var. 2060
	3:2-1:1	Var. 2061
	3:2-4:3	Var. 2062
	4:3-1:6	Var. 2063
	4:3-1:5	Var. 2064
	4:3-1:4	Var. 2065
	4:3-1:3	Var. 2066
	4:3-2:5	Var. 2067
	4:3-1:2	Var. 2068
	4:3-3:5	Var. 2069
	4:3-2:3	Var. 2070
	4:3-3:4	Var. 2071
	4:3-4:5	Var. 2072
	4:3-5:6	Var. 2073
	4:3-1:1	Var. 2074
	1:1-1:6	Var. 2075
	1:1-1:5	Var. 2076
	1:1-1:4	Var. 2077
	1:1-1:3	Var. 2078
	1:1-2:5	Var. 2079
	1:1-1:2	Var. 2080
	1:1-3:5	Var. 2081
	1:1-2:3	Var. 2082
	1:1-3:4	Var. 2083
	1:1-4:5	Var. 2084
	1:1-5:6	Var. 2085
	5:6-1:6	Var. 2086
	5:6-1:5	Var. 2087
	5:6-1:4	Var. 2088
	5:6-1:3	Var. 2089
	5:6-2:5	Var. 2090
	5:6-1:2	Var. 2091
	5:6-3:5	Var. 2092
	5:6-2:3	Var. 2093
	5:6-3:4	Var. 2094
	5:6-4:5	Var. 2095
	4:5-1:6	Var. 2096
	4:5-1:5	Var. 2097
	4:5-1:4	Var. 2098
	4:5-1:3	Var. 2099
	4:5-2:5	Var. 2100
	4:5-1:2	Var. 2101
	4:5-3:5	Var. 2102
	4:5-2:3	Var. 2103
	4:5-3:4	Var. 2104
	3:4-1:6	Var. 2105
	3:4-1:5	Var. 2106
	3:4-1:4	Var. 2107
	3:4-1:3	Var. 2108
	3:4-2:5	Var. 2109
	3:4-1:2	Var. 2110
	3:4-3:5	Var. 2111
	3:4-2:3	Var. 2112
	2:3-1:6	Var. 2113
	2:3-1:5	Var. 2114
	2:3-1:4	Var. 2115
	2:3-1:3	Var. 2116
	2:3-2:5	Var. 2117
	2:3-1:2	Var. 2118
	2:3-3:5	Var. 2119
	3:5-1:6	Var. 2120
	3:5-1:5	Var. 2121
	3:5-1:4	Var. 2122
	3:5-1:3	Var. 2123
	3:5-2:5	Var. 2124
	3:5-1:2	Var. 2125
	1:2-1:6	Var. 2126
	1:2-1:5	Var. 2127
	1:2-1:4	Var. 2128
	1:2-1:3	Var. 2129
	1:2-2:5	Var. 2130
	2:5-1:6	Var. 2131
	2:5-1:5	Var. 2132
	2:5-1:4	Var. 2133
	2:5-1:3	Var. 2134
	1:3-1:6	Var. 2135
	1:3-1:5	Var. 2136
	1:3-1:4	Var. 2137
	1:4-1:6	Var. 2138
	1:4-1:5	Var. 2139
	1:5-1:6	Var. 2140

	Var. = Variation

In further embodiments, higher order rVWF multimers of the invention are stable for about 1 to about 90 hours post-administration. In still further embodiments, the higher order rVWF multimers are stable for about 5-80, 10-70, 15-60, 20-50, 25-40, 30-35 hours post-administration. In yet further embodiments, the higher order rVWF multimers are stable for at least 3, 6, 12, 18, 24, 36, 48, 72 hours post-administration. In certain embodiments the stability of the rVWF multimers is assessed in vitro.
In one embodiment, higher order rVWF multimers used in the compositions and methods provided herein have a half-life of at least 12 hour post administration. In another embodiment, the higher order rVWF multimers have a half-life of at least 24 hour post administration. In yet other embodiments, the higher order rVWF multimers have a half-life selected from variations 642 to 1045 found in Table 9.

TABLE 9

Exemplary embodiments
for the half-life of higher
order rVWF multimers
found in the compositions
prepared by the methods
provided herein.

Hours

at least 1	Var. 642
at least 2	Var. 643
at least 3	Var. 644
at least 4	Var. 645
at least 5	Var. 646
at least 6	Var. 647
at least 7	Var. 648
at least 8	Var. 649
at least 9	Var. 650
at least 10	Var. 651
at least 11	Var. 652
at least 12	Var. 653
at least 14	Var. 654
at least 16	Var. 655
at least 18	Var. 656
at least 20	Var. 657
at least 22	Var. 658
at least 24	Var. 659
at least 27	Var. 660
at least 30	Var. 661
at least 33	Var. 662
at least 36	Var. 663
at least 39	Var. 664
at least 42	Var. 665
at least 45	Var. 666
at least 48	Var. 667
at least 54	Var. 668
at least 60	Var. 669
at least 66	Var. 670
at least 72	Var. 671
at least 78	Var. 672
at least 84	Var. 673
at least 90	Var. 674
2-90	Var. 675
2-84	Var. 676
2-78	Var. 677
2-72	Var. 678
2-66	Var. 679
2-60	Var. 680
2-54	Var. 681
2-48	Var. 682
2-45	Var. 683
2-42	Var. 684
2-39	Var. 685
2-36	Var. 686
2-33	Var. 687
2-30	Var. 688
2-27	Var. 689
2-24	Var. 690
2-22	Var. 691
2-20	Var. 692
2-18	Var. 693
2-16	Var. 694
2-14	Var. 695
2-12	Var. 696
2-10	Var. 697
2-8	Var. 698
2-6	Var. 699
2-4	Var. 700
3-90	Var. 701
3-84	Var. 702
3-78	Var. 703
3-72	Var. 704
3-66	Var. 705
3-60	Var. 706
3-54	Var. 707
3-48	Var. 708
3-45	Var. 709
3-42	Var. 710
3-39	Var. 711
3-36	Var. 712
3-33	Var. 713
3-30	Var. 714
3-27	Var. 715
3-24	Var. 716
3-22	Var. 717
3-20	Var. 718
3-18	Var. 719
3-16	Var. 720
3-14	Var. 721
3-12	Var. 722
3-10	Var. 723
3-8	Var. 724
3-6	Var. 725
3-4	Var. 726
4-90	Var. 727
4-84	Var. 728
4-78	Var. 729
4-72	Var. 730
4-66	Var. 731
4-60	Var. 732
4-54	Var. 733
4-48	Var. 734
4-45	Var. 735
4-42	Var. 736
4-39	Var. 737
4-36	Var. 738
4-33	Var. 739
4-30	Var. 740
4-27	Var. 741
4-24	Var. 742
4-22	Var. 743
4-20	Var. 744
4-18	Var. 745
4-16	Var. 746
4-14	Var. 747
4-12	Var. 748
4-10	Var. 749
4-8	Var. 750
4-6	Var. 751
6-90	Var. 752
6-84	Var. 753
6-78	Var. 754
6-72	Var. 755
6-66	Var. 756
6-60	Var. 757
6-54	Var. 758
6-48	Var. 759
6-45	Var. 760
6-42	Var. 761
6-39	Var. 762
6-36	Var. 763
6-33	Var. 764
6-30	Var. 765
6-27	Var. 766
6-24	Var. 767
6-22	Var. 768
6-20	Var. 769
6-18	Var. 770
6-16	Var. 771
6-14	Var. 772
6-12	Var. 773
6-10	Var. 774
6-8	Var. 775
8-90	Var. 776
8-84	Var. 777
8-78	Var. 778
8-72	Var. 779
8-66	Var. 780
8-60	Var. 781
8-54	Var. 782
8-48	Var. 783
8-45	Var. 784
8-42	Var. 785
8-39	Var. 786
8-36	Var. 787
8-33	Var. 788
8-30	Var. 789
8-27	Var. 790
8-24	Var. 791
8-22	Var. 792
8-20	Var. 793
8-18	Var. 794
8-16	Var. 795
8-14	Var. 796
8-12	Var. 797
8-10	Var. 798
10-90	Var. 799
10-84	Var. 800
10-78	Var. 801
10-72	Var. 802
10-66	Var. 803
10-60	Var. 804
10-54	Var. 805
10-48	Var. 806
10-45	Var. 807
10-42	Var. 808
10-39	Var. 809
10-36	Var. 810
10-33	Var. 811
10-30	Var. 812
10-27	Var. 813
10-24	Var. 814
10-22	Var. 815
10-20	Var. 816
10-18	Var. 817
10-16	Var. 818
10-14	Var. 819
10-12	Var. 820
12-90	Var. 821
12-84	Var. 822
12-78	Var. 823
12-72	Var. 824
12-66	Var. 825
12-60	Var. 826
12-54	Var. 827
12-48	Var. 828
12-45	Var. 829
12-42	Var. 830
12-39	Var. 831
12-36	Var. 832
12-33	Var. 833
12-30	Var. 834
12-27	Var. 835
12-24	Var. 836
12-22	Var. 837
12-20	Var. 838
12-18	Var. 839
12-16	Var. 840
12-14	Var. 841
14-90	Var. 842
14-84	Var. 843
14-78	Var. 844
14-72	Var. 845
14-66	Var. 846
14-60	Var. 847
14-54	Var. 848
14-48	Var. 849
14-45	Var. 850
14-42	Var. 851
14-39	Var. 852
14-36	Var. 853
14-33	Var. 854
14-30	Var. 855
14-27	Var. 856
14-24	Var. 857
14-22	Var. 858
14-20	Var. 859
14-18	Var. 860
14-16	Var. 861
16-90	Var. 862
16-84	Var. 863
16-78	Var. 864
16-72	Var. 865
16-66	Var. 866
16-60	Var. 867
16-54	Var. 868
16-48	Var. 869
16-45	Var. 870
16-42	Var. 871
16-39	Var. 872
16-36	Var. 873
16-33	Var. 874
16-30	Var. 875
16-27	Var. 876
16-24	Var. 877
16-22	Var. 878
16-20	Var. 879
16-18	Var. 880
18-90	Var. 881
18-84	Var. 882
18-78	Var. 883
18-72	Var. 884
18-66	Var. 885
18-60	Var. 886
18-54	Var. 887
18-48	Var. 888
18-45	Var. 889
18-42	Var. 890
18-39	Var. 891
18-36	Var. 892
18-33	Var. 893
18-30	Var. 894
18-27	Var. 895
18-24	Var. 896
18-22	Var. 897
18-20	Var. 898
20-90	Var. 899
20-84	Var. 900
20-78	Var. 901
20-72	Var. 902
20-66	Var. 903
20-60	Var. 904
20-54	Var. 905
20-48	Var. 906
20-45	Var. 907
20-42	Var. 908
20-39	Var. 909
20-36	Var. 910
20-33	Var. 911
20-30	Var. 912
20-27	Var. 913
20-24	Var. 914
20-22	Var. 915
22-90	Var. 916
22-84	Var. 917
22-78	Var. 918
22-72	Var. 919
22-66	Var. 920
22-60	Var. 921
22-54	Var. 922
22-48	Var. 923
22-45	Var. 924
22-42	Var. 925
22-39	Var. 926
22-36	Var. 927
22-33	Var. 928
22-30	Var. 929
22-27	Var. 930
22-24	Var. 931
24-90	Var. 932
24-84	Var. 933
24-78	Var. 934
24-72	Var. 935
24-66	Var. 936
24-60	Var. 937
24-54	Var. 938
24-48	Var. 939
24-45	Var. 940
24-42	Var. 941
24-39	Var. 942
24-36	Var. 943
24-33	Var. 944
24-30	Var. 945
24-27	Var. 946
27-90	Var. 947
27-84	Var. 948
27-78	Var. 949
27-72	Var. 950
27-66	Var. 951
27-60	Var. 952
27-54	Var. 953
27-48	Var. 954
30-90	Var. 955
30-84	Var. 956
30-78	Var. 957
30-72	Var. 958
30-66	Var. 959
30-60	Var. 960
30-54	Var. 961
30-48	Var. 962
30-45	Var. 963
30-42	Var. 964
30-39	Var. 965
30-36	Var. 966
30-33	Var. 967
33-90	Var. 968
33-84	Var. 969
33-78	Var. 970
33-72	Var. 971
33-66	Var. 972
33-60	Var. 973
33-54	Var. 974
33-48	Var. 975
33-45	Var. 976
33-42	Var. 977
33-29	Var. 978
33-36	Var. 979
36-90	Var. 980
36-84	Var. 981
36-78	Var. 982
36-72	Var. 983
36-66	Var. 984
36-60	Var. 985
36-54	Var. 986
36-48	Var. 987
36-45	Var. 988
36-42	Var. 989
36-39	Var. 990
39-90	Var. 991
39-84	Var. 992
39-78	Var. 993
39-72	Var. 994
39-66	Var. 995
39-60	Var. 996
39-54	Var. 997
39-48	Var. 998
39-45	Var. 999
39-42	Var. 1000
42-90	Var. 1001
42-84	Var. 1002
42-78	Var. 1003
42-72	Var. 1004
42-66	Var. 1005
42-60	Var. 1006
42-54	Var. 1007
42-48	Var. 1008
42-45	Var. 1009
45-90	Var. 1010
45-84	Var. 1011
45-78	Var. 1012
45-72	Var. 1013
45-66	Var. 1014
45-60	Var. 1015
45-54	Var. 1016
45-48	Var. 1017
48-90	Var. 1018
48-84	Var. 1019
48-78	Var. 1020
48-72	Var. 1021
48-66	Var. 1022
48-60	Var. 1023
48-54	Var. 1024
54-90	Var. 1025
54-84	Var. 1026
54-78	Var. 1027
54-72	Var. 1028
54-66	Var. 1029
54-60	Var. 1030
60-90	Var. 1031
60-84	Var. 1032
60-78	Var. 1033
60-72	Var. 1034
60-66	Var. 1035
66-90	Var. 1036
66-84	Var. 1037
66-78	Var. 1038
66-72	Var. 1039
72-90	Var. 1040
72-84	Var. 1041
72-78	Var. 1042
78-90	Var. 1043
78-84	Var. 1044
84-90	Var. 1045

Var. = Variation

In some embodiments, the pro-VWF and/or purified rVWF purified in accordance with the present invention is not modified with any conjugation, post-translation or covalent modifications. In particular embodiments, the pro-VWF and/or purified rVWF of the present invention is not modified with a water soluble polymer, including without limitation, a polyethylene glycol (PEG), a polypropylene glycol, a polyoxyalkylene, a polysialic acid, hydroxyl ethyl starch, a poly-carbohydrate moiety, and the like.
In some embodiments, the pro-VWF and/or purified rVWF purified in accordance with the present invention is modified through conjugation, post-translation modification, or covalent modification, including modifications of the N- or C-terminal residues as well as modifications of selected side chains, for example, at free sulfhydryl-groups, primary amines, and hydroxyl-groups. In one embodiment, a water soluble polymer is linked to the protein (directly or via a linker) by a lysine group or other primary amine. In some embodiments, the pro-VWF and/or purified rVWF of the present invention may be modified by conjugation of a water soluble polymer, including without limitation, a polyethylene glycol (PEG), a polypropylene glycol, a polyoxyalkylene, a polysialic acid, hydroxyl ethyl starch, a poly-carbohydrate moiety, and the like.
Water soluble polymers that may be used to modify the pro-VWF and/or purified rVWF include linear and branched structures. The conjugated polymers may be attached directly to the coagulation proteins 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 in 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.
Protein conjugation may be performed by a number of well-known techniques in the art, for example, see Hermanson G., Bioconjugate Techniques 2nd Ed., Academic Press, Inc. 2008. Examples include linkage through the peptide bond between a carboxyl group on one of either the coagulation protein or water-soluble polymer moiety and an amine group of the other, or an ester linkage between a carboxyl group of one and a hydroxyl group of the other. Another linkage by which a coagulation protein of the invention could be conjugated to a water-soluble polymer compound is via a Schiff base, between a free amino group on the polymer moiety being reacted 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; Fernandes and Gregonradis, Biochim Biophys Acta. 1997; 1341; 26-34). The generated Schiff Base can be stabilized by specific reduction with NaCNBH₃to form a secondary amine. An alternative approach is the generation of terminal free amino groups on the polymer by reductive amination with NH₄Cl after prior oxidation. Bifunctional reagents can be used for linking two amino or two hydroxyl groups. For example, a polymer containing an amino group can be coupled to an amino group of the coagulation protein with reagents like BS3 (Bis(sulfosuccinimidyl)suberate/Pierce, Rockford, Ill.). In addition, heterobifunctional cross linking reagents like Sulfo-EMCS (N-ε-Maleimidocaproyloxy) sulfosuccinimide ester/Pierce) can be used for instance to link amine and thiol groups. In other embodiments, an aldehyde reactive group, such as PEG alkoxide plus diethyl acetal of bromoacetaldehyde; PEG plus DMSO and acetic anhydride, and PEG chloride plus the phenoxide of 4-hydroxybenzaldehyde, succinimidyl active esters, activated dithiocarbonate PE. G., 2,4,5-trichlorophenylcloroformate and P-nitrophenylcloroformate activated PE.G., may be used in the conjugation of a coagulation protein.
Another method for measuring the biological activity of VWF is the collagen binding assay, which is based on ELISA technology (Brown and Bosak, Thromb. Res., 1986, 43:303-311; Favaloro, Thromb. Haemost., 2000, 83 127-135). A microtiter plate is coated with type I or III collagen. Then the VWF is bound to the collagen surface and subsequently detected with an enzyme-labeled polyclonal antibody. The last step is a substrate reaction, which can be photometrically monitored with an ELISA reader.
Immunological assays of von Willebrand factors (VWF:Ag) are immunoassays that measure the concentration of the VWF protein in plasma. They give no indication as to VWF function. A number of methods exist for measuring VWF:Ag and these include both enzyme-linked immunosorbent assay (ELISA) or automated latex immunoassays (LIA.) Many laboratories now use a fully automated latex immunoassay. Historically laboratories used a variety of techniques including Laurell electroimmunoassay ‘Laurell Rockets’ but these are rarely used in most labs today.

III. Kits

As an additional aspect, the invention includes kits which comprise one or more lyophilized compositions packaged in a manner which facilitates their use for administration to subjects. In one embodiment, such a kit includes pharmaceutical formulation described herein (e.g., a composition comprising a therapeutic protein or peptide), packaged in a container such as a sealed bottle or vessel, with a label affixed to the container or included in the package that describes use of the compound or composition in practicing the method. In one embodiment, the pharmaceutical formulation is packaged in the container such that the amount of headspace in the container (e.g., the amount of air between the liquid formulation and the top of the container) is very small. Preferably, the amount of headspace is negligible (e.g., almost none). In one embodiment, the kit contains a first container having a therapeutic protein or peptide composition and a second container having 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 administering the pharmaceutical formulation according to a specific route of administration. Preferably, the kit contains a label that describes use of the pharmaceutical formulations.

IV. rVWF for Methods of Pretreating Subjects with VWD Undergoing Surgery

One of the advantages of administering rVWF to subjects with severe VWD to pretreat for surgery is that the higher specific activity of rVWF as compared to pdVWF allows flexibility in the amount of rVWF administered and the number of times the subject is re-dosed. As will be appreciated and as is discussed in further detail herein, the co-administered FVIII may be recombinant or plasma derived
Single or multiple administrations of rVWF are carried out with the dose levels and pattern being selected by the treating physician. For the prevention or treatment of disease, the appropriate dosage depends on the type of disease to be treated (e.g., von Willebrand disease), the severity and course of the disease, whether drug is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the drug, and the discretion of the attending physician.
In some aspects, rVWF is administered prior to a surgical procedure to a subject at a range from 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 between 12 hours and 24 hours, e.g., 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 12 hours and 24 hours, 14 hours and 24 hours, 16 and 24 hours, 18 hours and 24 hours, or 20 hours and 24 hours prior to the surgical procedure. In some aspects, Factor VIII (FVIII) is not administered with the rVWF prior to the surgical procedure.
In some embodiments, rVWF is administered to the subject at a range of IU/kg, e.g., 5-90, 5-50, 10-90, 15-90, 20-90, 30-90, 40-90, 50-90, 60-90, 70-90, 80-5-80, 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 1 hour prior to surgery. In other embodiments, rVWF is administered at a dose of 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, 40-140, 30, 130, 20-120, 10-110, 70-100, or 70-90 IU/kg after the surgery. In some cases, the surgical procedure is selected from a group consisting of major surgery, minor surgery, and oral surgery.
In some embodiments, the subject is administered 35-60 IU/kg rVWF between 12 hours and 24 hours prior to major surgery. In other embodiments, the subject is administered 15-90 IU/kg rVWF 1 hour prior to major surgery. In another embodiment, the subject is administered 150-220 IU/kg rVWF after major surgery. In some instances, the subject undergoing major surgery is administered a total dosage of 220-320 IU/kg.
In some embodiments, the subject is administered 50-60 IU/kg rVWF between 12 hours and 24 hours prior to minor surgery. In other embodiments, the subject is administered 5-50 IU/kg rVWF 1 hour prior to minor surgery. In another embodiment, the subject is administered 70-150 IU/kg rVWF after minor surgery. In some instances, the subject undergoing minor surgery is administered a total dosage of 100-220 IU/kg.
In some embodiments, the subject is administered 20-40 IU/kg rVWF between 12 hours and 24 hours prior to oral surgery. In other embodiments, the subject is administered 20-50 IU/kg rVWF 1 hour prior to oral surgery. In another embodiment, the subject is administered 10-50 IU/kg rVWF during oral surgery. In another embodiment, the subject is administered 20-50 IU/kg rVWF after oral surgery. In some instances, the subject undergoing oral surgery is administered a total dosage of 70-190 IU/kg.
Compositions of rVWF can be contained in pharmaceutical formulations, 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 injections, intravenous, intramuscular, intracisternal injection, or infusion techniques. Administration by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonary injection and or surgical implantation at a particular site is contemplated as well. Generally, compositions are essentially free of pyrogens, as well as other impurities that could be harmful to the recipient.
In one aspect, formulations of the invention are administered by an initial bolus followed by a continuous infusion to maintain therapeutic circulating levels of drug product. As another example, the inventive compound is administered as a one-time dose. Those of ordinary skill in the art will readily optimize effective dosages and administration regimens as determined by good medical practice and the clinical condition of the individual patient. The route of administration can be, but is not limited to, by intravenous, intraperitoneal, subcutaneous, or intramuscular administration. The frequency of dosing depends on the pharmacokinetic parameters of the agents and the route of administration. The optimal pharmaceutical formulation is determined by one skilled in the art depending upon the route of administration and desired dosage. See for example, Remington's Pharmaceutical Sciences, 18th Ed., 1990, Mack Publishing Co., Easton, Pa. 18042 pages 1435-1712, the disclosure of which is hereby incorporated by reference in its entirety for all purposes and in particular for all teachings related to formulations, routes of administration and dosages for pharmaceutical products. Such formulations influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the administered agents. Depending on the route of administration, a suitable dose is calculated according to body weight, body surface area or organ size. Appropriate dosages may be ascertained through use of established assays for determining blood level dosages in conjunction with appropriate dose-response data. The final dosage regimen is determined by the attending physician, considering various factors which modify the action of drugs, e.g. the drug's specific activity, the severity of the damage and the responsiveness of the patient, the age, condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. By way of example, a typical dose of a recombinant VWF of the present invention is approximately 50 IU/kg, equal to 500 μg/kg. As studies are conducted, further information will emerge regarding the appropriate dosage levels and duration of treatment for various diseases and conditions.
The practice of the present invention may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry, and immunology, which are within the skill of the art. Such conventional techniques include polymer array synthesis, hybridization, ligation, and detection of hybridization using a label. Specific illustrations of suitable techniques can be had by reference to the example herein below. However, other equivalent conventional procedures can, of course, 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, N.Y. and Berg et al. (2002) Biochemistry, 5th Ed., W. H. Freeman Pub., Highly stabilized York, N.Y., all of which are herein incorporated in their entirety by reference for all purposes.
Note that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a polymerase” refers to one agent or mixtures of such agents, and reference to “the method” includes reference to equivalent steps and methods known to those skilled in the art, and so forth.
Note that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a polymerase” refers to one agent or mixtures of such agents, and reference to “the method” includes reference to equivalent steps and methods known to those skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing devices, compositions, formulations and methodologies which are described in the publication and which might be used in connection with the presently described invention.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.
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 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 invention.
Although the present invention is described primarily with reference to specific embodiments, it is also envisioned that other embodiments will become apparent to those skilled in the art upon reading the present disclosure, and it is intended that such embodiments be contained within the present inventive method.
a. Lyophilized VWF Formulations
The present method also provides formulations of rVWF for use in the treatment methods provided herein. In some embodiments, the rVWF composition is used for the production of a pharmaceutical composition. In some embodiments, the rVWF can be formulated into a lyophilized formulation.
In some embodiments, the formulations comprising a VWF polypeptide of the invention are lyophilized after purification and prior to administration to a subject. Lyophilization is carried out 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)).
A lyophilization cycle is, in one aspect, composed of three steps: freezing, primary drying, and secondary drying (A. P. Mackenzie, Phil Trans R Soc London, Ser B, Biol 278:167 (1977)). In the freezing step, the solution is cooled to initiate ice formation. Furthermore, this step induces the crystallization of the bulking agent. The ice sublimes in the primary drying stage, which is conducted by reducing chamber pressure below the vapor pressure of the ice, using a vacuum and introducing heat to promote sublimation. Finally, adsorbed or bound water is removed at the secondary drying stage under reduced chamber pressure and at an elevated shelf temperature. The process produces a material known as a lyophilized cake. Thereafter the cake can be reconstituted with either sterile water or suitable diluent for injection.
The lyophilization cycle not only determines the final physical state of excipients but also affects other parameters such as reconstitution time, appearance, stability and final moisture content. The composition structure in the frozen state proceeds through several transitions (e.g., glass transitions, wettings, and crystallizations) that occur at specific temperatures and the structure may 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 a solute and can be determined by differential scanning calorimetry (DSC). Tg and Tg′ are an important parameter that must be taken into account when designing the lyophilization cycle. For example, Tg′ is important for primary drying. Furthermore, in the dried state, the glass transition temperature provides information on the storage temperature of the final product.
b. Pharmaceutical Formulations and Excipients in General
Excipients are additives that either impart or enhance the stability and delivery of a drug product (e.g., protein). Regardless of the reason for their inclusion, excipients are an integral component of a formulation and therefore need to be safe and well tolerated by patients. For protein drugs, the choice of excipients is particularly important because they can affect both efficacy and immunogenicity of the drug. Hence, protein formulations need to be developed with appropriate selection of excipients that afford suitable stability, safety, and marketability.
A lyophilized formulation is, in one aspect, at least comprised of one or more of a buffer, a bulking agent, and a stabilizer. In this aspect, the utility of a surfactant is evaluated and selected in cases where aggregation during the lyophilization step or during reconstitution becomes an issue. An appropriate buffering agent is included to maintain the formulation within stable zones of pH during lyophilization. A comparison of the excipient components contemplated for liquid and lyophilized protein formulations is provided in Table 10.

TABLE 1

Excipient components of lyophilized protein formulations

Excipient component	Function in lyophilized formulation

Buffer	Maintain pH of formulation during
	lyophilization and upon reconstitution
Tonicity agent/stabilizer	Stabilizers include cryo and
	lyoprotectants
	Examples include Polyols, sugars and
	polymers
	Cryoprotectants protect proteins from
	freezing stresses
	Lyoprotectants stabilize proteins in the
	freeze-dried state
Bulking agent	Used to enhance product elegance and
	to prevent blowout
	Provides structural strength to the lyo
	cake
	Examples include mannitol and glycine
Surfactant	Employed if aggregation during the
	lyophilization process is an issue
	May serve to reduce reconstitution
	times
	Examples include polysorbate 20 and
	80
Anti-oxidant	Usually not employed, molecular
	reactions in the lyo cake are greatly
	retarded
Metal ions/chelating agent	May be included if a specific metal ion
	is included only as a co-factor or where
	the metal is required for protease
	activity
	Chelating agents are generally not
	needed in lyo formulations
Preservative	For multi-dose formulations only
	Provides protection against microbial
	growth in formulation
	Is usually included in the reconstitution
	diluent (e.g. bWFI)

The principal challenge in developing formulations for proteins is stabilizing the product against the stresses of manufacturing, shipping and storage. The role of formulation excipients is to provide stabilization against these stresses. Excipients are also be employed to reduce viscosity of high concentration protein formulations in order to enable their delivery and enhance patient convenience. In general, excipients can be classified on the basis of the mechanisms by which they stabilize proteins against various chemical and physical stresses. Some excipients are used to alleviate the effects of a specific stress or to regulate a particular susceptibility of a specific protein. Other excipients have more general effects on the physical and covalent stabilities of proteins. The excipients described herein are organized either by their chemical type or their functional role in formulations. Brief descriptions of the modes of stabilization are provided when discussing each excipient type.
Given the teachings and guidance provided herein, those skilled in the art will know what amount or range of excipient can be included in any particular formulation to achieve a biopharmaceutical formulation of the invention that promotes retention in stability of the biopharmaceutical (e.g., a protein). For example, the amount and type of a salt to be included in a biopharmaceutical formulation of the invention is 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.
By way of example, inclusion of about 5% sorbitol can achieve isotonicity while about 9% of a sucrose excipient is needed to achieve isotonicity. Selection of the amount or range of concentrations of one or more excipients that can be included within a biopharmaceutical formulation of the invention has been exemplified above by reference to salts, polyols and sugars. However, those skilled in the art will understand that the considerations described herein and further exemplified by reference to specific excipients are equally applicable to all types and combinations of excipients including, for example, salts, amino acids, other tonicity agents, surfactants, stabilizers, bulking agents, cryoprotectants, lyoprotectants, anti-oxidants, metal ions, chelating agents and/or preservatives.
Further, where a particular excipient is reported in molar concentration, those skilled in the art will recognize that the equivalent percent (%) w/v (e.g., (grams of substance in a solution sample/mL of solution)×100%) of solution is also contemplated.
Of course, a person having ordinary skill in the art would recognize that the concentrations of the excipients described herein share an interdependency within a particular formulation. By way of example, the concentration of a bulking agent may be lowered where, e.g., there is a high protein concentration or where, e.g., there is a high stabilizing agent concentration. In addition, a person having ordinary skill in the art would recognize that, in order to maintain the isotonicity of a particular formulation in which there is no bulking agent, the concentration of a stabilizing agent would be adjusted accordingly (e.g., a “tonicifying” amount of stabilizer would be used). 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.
c. Pharmaceutical Buffers and Buffering Agents
The stability of a pharmacologically active protein formulation is usually observed to be maximal in a narrow pH range. This pH range of optimal stability needs to be identified early during pre-formulation studies. Several approaches, such as accelerated stability studies and calorimetric screening studies, are useful in this endeavor (Remmele R. L. Jr., et al., Biochemistry, 38(16): 5241-7 (1999)). Once a formulation is finalized, the protein must be manufactured and maintained throughout its shelf-life. Hence, buffering agents are almost always employed to control pH in the formulation.
The buffer capacity of the buffering species is maximal at a pH equal to the pKa and decreases as pH increases or decreases away from this value. Ninety percent of the buffering capacity exists within one pH unit of its pKa. Buffer capacity also increases proportionally with increasing buffer concentration.
Several factors need to be considered when choosing a buffer. First and foremost, the buffer species and its concentration need to be defined based on its pKa and the desired formulation pH. Equally important is 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 be considered is the sensation of stinging and irritation 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 that are administered via the subcutaneous (SC) or intramuscular (IM) routes, where the drug solution remains at the site for a relatively longer period of time than when administered by the IV route where the formulation gets diluted rapidly into the blood upon administration. For formulations that are administered by direct IV infusion, the total amount of buffer (and any other formulation component) needs to be monitored. One has to be particularly careful about potassium ions administered in the form of the potassium phosphate buffer, which can induce cardiovascular effects in a patient (Hollander-Rodriguez J C, et al., Am. Fam. Physician., 73(2): 283-90 (2006)).
Buffers for lyophilized formulations need additional consideration. Some buffers like sodium phosphate can crystallize out of the protein amorphous phase during freezing resulting in shifts in pH. Other common buffers such as acetate and imidazole may sublime or evaporate during the lyophilization process, thereby shifting the pH of formulation during lyophilization or after reconstitution.
The buffer system present in the compositions is selected to be physiologically compatible and to maintain a 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.
The pH buffering compound may be present in any amount suitable to maintain the pH of the formulation at a predetermined level. In one embodiment, the pH buffering concentration is between 0.1 mM and 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, 100, 200, or 500 mM.
Exemplary pH buffering agents used to buffer the formulation as set out 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, including, but not limited to aspartate, histidine, and glycine. In one embodiment of the present invention, the buffering agent is citrate.
d. Pharmaceutical Stabilizers and Bulking Agents
In one aspect of the present pharmaceutical formulations, a stabilizer (or a combination of stabilizers) is added to prevent or reduce storage-induced aggregation and chemical degradation. A hazy or turbid solution upon reconstitution indicates that the protein has precipitated or at least aggregated. The term “stabilizer” means an excipient capable of preventing aggregation or physical degradation, including chemical degradation (for example, autolysis, deamidation, oxidation, etc.) in an aqueous state. Stabilizers contemplated 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, including polysaccharides such as dextran, starch, hydroxyethyl starch, cyclodextrins, N-methyl pyrollidene, cellulose and hyaluronic acid, sodium chloride, (Carpenter et al., Develop. Biol. Standard 74:225, (1991)). In the present formulations, the stabilizer is incorporated in 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, 100, 200, 500, 700, 900, or 1000 mM. In one embodiment of the present invention, mannitol and trehalose are used as stabilizing agents.
If desired, the formulations also include appropriate amounts of bulking and osmolality regulating agents. Bulking agents include, for example and without limitation, mannitol, glycine, sucrose, polymers such as dextran, polyvinylpyrolidone, carboxymethylcellulose, lactose, sorbitol, trehalose, or xylitol. In one embodiment, the bulking agent is mannitol. The bulking agent is incorporated in a concentration of about 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.
e. Pharmaceutical Surfactants
Proteins have a high propensity to interact with surfaces making them susceptible to adsorption and denaturation at air-liquid, vial-liquid, and liquid-liquid (silicone oil) interfaces. This degradation pathway has been observed to be inversely dependent on protein concentration and results in either the formation of soluble and insoluble protein aggregates or the loss of protein from solution via adsorption to surfaces. In addition to container surface adsorption, surface-induced degradation is exacerbated with physical agitation, as would be experienced during shipping and handling of the product.
Surfactants are commonly used in protein formulations to prevent surface-induced degradation. Surfactants are amphipathic molecules with the capability of out-competing proteins for interfacial positions. Hydrophobic portions of the surfactant molecules occupy interfacial positions (e.g., air/liquid), while hydrophilic portions of the molecules remain oriented towards the bulk solvent. At sufficient concentrations (typically around the detergent's critical micellar concentration), a surface layer of surfactant molecules serves to prevent protein molecules from adsorbing at the interface. Thereby, surface-induced degradation is minimized. Surfactants contemplated herein include, without limitation, fatty acid esters of sorbitan polyethoxylates, e.g., polysorbate and polysorbate 80. The two differ only in the length of the aliphatic chain that imparts hydrophobic character to the molecules, C-12 and C-18, respectively. Accordingly, polysorbate-80 is more surface-active and has a lower critical micellar concentration than polysorbate-20.
Detergents can also affect the thermodynamic conformational stability of proteins. Here again, the effects of a given detergent excipient will be protein specific. For example, polysorbates have been shown to reduce the stability of some proteins and increase the stability of others. Detergent destabilization of proteins can be rationalized in terms of the hydrophobic tails of the detergent molecules that can engage in specific binding with partially or wholly unfolded protein states. These types of interactions could cause a shift in the conformational equilibrium towards the more expanded protein states (e.g. increasing the exposure of hydrophobic portions of the protein molecule in complement to binding polysorbate). Alternatively, if the protein native state exhibits some hydrophobic surfaces, detergent binding to the native state may stabilize that conformation.
Another aspect of polysorbates is that they are inherently susceptible to oxidative degradation. Often, as raw materials, they contain sufficient quantities of peroxides to cause oxidation of protein residue side-chains, especially methionine. The potential for oxidative damage arising from the addition of stabilizer emphasizes the point that the lowest effective concentrations of excipients should be used in formulations. For surfactants, the effective concentration for a given protein will depend on the mechanism of stabilization.
Surfactants are also added in appropriate amounts to prevent surface related aggregation phenomenon during freezing and drying (Chang, B, J. Pharm. Sci. (1996)). Thus, exemplary surfactants include, without limitation, anionic, cationic, nonionic, zwitterionic, and amphoteric surfactants including surfactants derived from naturally-occurring amino acids. Anionic surfactants include, but are not limited to, sodium lauryl sulfate, dioctyl sodium sulfo succinate and dioctyl sodium sulfonate, chenodeoxycholic acid, N-lauroylsarcosine sodium salt, lithium dodecyl sulfate, 1-octanesulfonic acid sodium salt, sodium cholate hydrate, sodium deoxycholate, and glycodeoxycholic acid sodium 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. Non-ionic 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 dioleyl phosphatidyl choline (DOPC), dimyristoylphosphatidyl glycerol (DMPG), dimyristoylphosphatidyl choline (DMPC), and (dioleyl phosphatidyl glycerol) DOPG; sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. Compositions comprising these surfactants, either individually or as a mixture in different ratios, are therefore further provided. In one embodiment of the present invention, the surfactant is TWEEN-80. In the present formulations, the surfactant is incorporated in a concentration of about 0.01 to about 0.5 g/L. In formulations provided, the surfactant concentration is 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.
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 protein's surface. Alternatively, salts can stabilize the denatured state by binding to peptide groups along the protein backbone (—CONH—). Salts can also stabilize the protein native conformation by shielding repulsive electrostatic interactions between residues within a protein molecule. Salts in protein formulations can also shield attractive electrostatic interactions between protein molecules that can lead to protein aggregation and insolubility. In formulations provided, the salt concentration is between 0.1, 1, 10, 20, 30, 40, 50, 80, 100, 120, 150, 200, 300, and 500 mM.
g. Other Common Excipient Components: Pharmaceutical Amino Acids
Amino acids have found versatile use in protein formulations as buffers, bulking agents, stabilizers and antioxidants. Thus, in one aspect histidine and glutamic acid are employed to buffer protein formulations in the pH range of 5.5-6.5 and 4.0-5.5 respectively. The imidazole group of histidine has a pKa=6.0 and the carboxyl group of glutamic acid side chain has a pKa of 4.3 which makes these amino acids suitable for buffering in their respective pH ranges. Glutamic acid is particularly useful in such cases. Histidine is commonly found in marketed protein formulations, and this amino acid provides an alternative to citrate, a buffer known to sting upon injection. Interestingly, histidine has also been reported to have a stabilizing effect, with respect to aggregation when used at high concentrations in both liquid and lyophilized presentations (Chen B, et al., Pharm Res., 20(12): 1952-60 (2003)). Histidine was also observed by others to reduce the viscosity of a high protein concentration formulation. However, in the same study, the authors observed increased aggregation and discoloration in histidine containing formulations during freeze-thaw studies of the antibody in stainless steel containers. Another note of caution with 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; it has been observed to be effective against a number of oxidative stresses (Lam X M, et al., J Pharm ScL, 86(11): 1250-5 (1997)).
In various aspects, formulations are provided which include one or more of the amino acids glycine, proline, serine, arginine and alanine have been shown to stabilize proteins by the mechanism of preferential exclusion. Glycine is also a commonly used bulking agent in lyophilized formulations. Arginine has been shown to be an effective agent in inhibiting aggregation and has been used in both liquid and lyophilized formulations.
In formulations provided, the amino acid concentration is between 0.1, 1, 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.
h. Other Common Excipient Components: Pharmaceutical Antioxidants
Oxidation of protein residues arises from a number of different sources. Beyond the addition of specific antioxidants, the prevention of oxidative protein damage involves the careful control of a number of factors throughout the manufacturing process and storage of the product such as atmospheric oxygen, temperature, light exposure, and chemical contamination. The invention therefore contemplates the use of the pharmaceutical antioxidants including, without limitation, reducing agents, oxygen/free-radical scavengers, or chelating agents. Antioxidants in therapeutic protein formulations are, in one aspect, water-soluble and remain active throughout the product shelf-life. Reducing agents and oxygen/free-radical scavengers work by ablating active oxygen species in solution. Chelating agents such as EDTA are effective by binding trace metal contaminants that promote free-radical formation. For example, EDTA was utilized in the liquid formulation of acidic fibroblast growth factor to inhibit the metal ion catalyzed oxidation of cysteine residues.
In addition to the effectiveness of various excipients to prevent protein oxidation, the potential for the antioxidants themselves to induce other covalent or physical changes to the protein is of concern. For example, reducing agents can cause disruption of intramolecular disulfide linkages, which can lead to disulfide shuffling. In the presence of transition metal ions, ascorbic acid and EDTA have been shown to promote methionine oxidation in a number of proteins and peptides (Akers M J, and Defelippis M R. 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 J. R., /. 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 levels of light and temperature induced methionine-oxidation in rhuMab HER2; however, the formation of a thiosulfate-protein adduct was also reported in this study (Lam X M, Yang J Y, et al., J Pharm Sci. 86(11): 1250-5 (1997)). Selection of an appropriate antioxidant is made according to the specific stresses and sensitivities of the protein. Antioxidants contemplated in certain aspects include, without limitation, reducing agents and oxygen/free-radical scavengers, EDTA, and sodium thiosulfate.
i. Other Common Excipient Components: Pharmaceutical Metal Ions
In general, transition metal ions are undesired in protein formulations because they can catalyze physical and chemical degradation reactions in proteins. However, specific metal ions are included in formulations when they are co-factors to proteins and in suspension formulations of proteins where they form coordination complexes (e.g., zinc suspension of insulin). Recently, the use of magnesium ions (10-120 mM) has been proposed to inhibit the isomerization of aspartic acid to isoaspartic acid (WO 2004039337).
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⁺²ions (up to 100 mM) increased the stability of the enzyme through a specific binding site (Chen B, et al., /Pharm Sci., 88(4): 477-82 (1999)). In fact, removal of calcium ions from the solution with EGTA caused an increase in deamidation and aggregation. However, this effect was observed only with Ca⁺²ions; other divalent cations Mg⁺², Mn⁺²and Zn⁺²were observed to destabilize rhDNase. Similar effects were observed in Factor VIII. Ca⁺²and Sr⁺²ions stabilized the protein while others like Mg⁺², Mn⁺²and Zn⁺², Cu⁺²and Fe⁺²destabilized the enzyme (Fatouros, A., et al., Int. J. Pharm., 155, 121-131 (1997). In a separate study with Factor VIII, a significant increase in aggregation rate was observed in the presence of Al⁺³ions (Derrick T S, et al., I. Pharm. Sci., 93(10): 2549-57 (2004)). The authors note that other excipients like buffer salts are often contaminated with Al⁺³ions and illustrate the need to use excipients of appropriate quality in formulated products.
j. Other Common Excipient Components: Pharmaceutical Preservatives
Preservatives are necessary when developing multi-use parenteral formulations that involve more than one extraction from the same container. Their primary function is to inhibit microbial growth and ensure product sterility throughout the shelf-life or term of use of the drug product. Commonly used preservatives include, without limitation, benzyl alcohol, phenol and m-cresol. Although preservatives have a long history of use, the development of protein formulations that includes preservatives can be challenging. Preservatives almost always have a destabilizing effect (aggregation) on proteins, and this has become a major factor in limiting their use in multi-dose protein formulations (Roy S, et al., J Pharm ScL, 94(2): 382-96 (2005)).
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 enabling patient convenience, and increased marketability. A good example is that of human growth hormone (hGH) where the development of preserved formulations has led to commercialization of more convenient, multi-use injection pen presentations. At least four such pen devices containing preserved formulations of hGH are currently available on the market. Norditropin® (liquid, Novo Nordisk), Nutropin AQ® (liquid, Genentech) & Genotropin (lyophilized—dual chamber cartridge, Pharmacia & Upjohn) contain phenol while Somatrope® (Eli Lilly) is formulated with m-cresol.
Several aspects need to be considered during the formulation development of preserved dosage forms. The effective preservative concentration in the drug product must be optimized. This requires testing a given preservative in the dosage form with concentration ranges that confer anti-microbial effectiveness without compromising protein stability. For example, three preservatives were successfully screened in the development of a liquid formulation for interleukin-1 receptor (Type I), using differential scanning calorimetry (DSC). The preservatives were rank ordered based on their impact on stability at concentrations commonly used in marketed products (Remmele R L Jr., et al., Pharm Res., 15(2): 200-8 (1998)).
Development of liquid formulations containing preservatives are more challenging than lyophilized formulations. Freeze-dried products can be lyophilized without the preservative and reconstituted with a preservative containing diluent at the time of use. This shortens the time for which a preservative is in contact with the protein significantly minimizing the associated stability risks. With liquid formulations, preservative effectiveness and stability have to be maintained over the entire product shelf-life (−18-24 months). An important point to note is that preservative effectiveness has to be demonstrated in the final formulation containing the active drug and all excipient components.
Some preservatives can cause injection site reactions, which is another factor that needs consideration when choosing a preservative. In clinical trials that focused on the evaluation of preservatives and buffers in Norditropin, pain perception was observed to be lower in formulations containing phenol and benzyl alcohol as compared to a formulation containing m-cresol (Kappelgaard A. M., Horm Res. 62 Suppl 3:98-103 (2004)). Interestingly, among the commonly used preservative, benzyl alcohol possesses anesthetic properties (Minogue S C, and Sun D A., AnesthAnalg., 100(3): 683-6 (2005)). In various aspects the use of preservatives provide a benefit that outweighs any side effects.
k. Methods of Preparation of Pharmaceutical Formulations
The present invention further contemplates methods for the preparation of pharmaceutical formulations.
The present methods further comprise one or more of the following steps: adding a stabilizing agent as described herein to said mixture prior to lyophilizing, adding at least one agent selected from a bulking agent, an osmolality regulating agent, and a surfactant, each of which as described herein, to said mixture prior to lyophilization.
The standard reconstitution practice for lyophilized material is to add back a volume of pure water or sterile water for injection (WFI) (typically equivalent to the volume removed during lyophilization), although dilute solutions of antibacterial agents are sometimes used in the production of pharmaceuticals for parenteral administration (Chen, Drug Development and Industrial Pharmacy, 18:1311-1354 (1992)). Accordingly, methods are provided for preparation of reconstituted rVWF compositions comprising the step of adding a diluent to a lyophilized rVWF composition of the invention.
The lyophilized material may be reconstituted as an aqueous solution. A variety of aqueous carriers, e.g., sterile water for injection, water with preservatives for multi dose use, or water with appropriate amounts of surfactants (for example, an aqueous suspension that contains the active compound in admixture with excipients suitable for the manufacture of aqueous suspensions). In various aspects, such excipients are suspending agents, for example and without limitation, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents are a naturally-occurring phosphatide, for example and without limitation, lecithin, or condensation products of an alkylene oxide with fatty acids, for example and without limitation, polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example and without limitation, heptadecaethyl-eneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example and without limitation, polyethylene sorbitan monooleate. In various aspects, the aqueous suspensions also contain one or more preservatives, for example and without limitation, ethyl, or n-propyl, p-hydroxybenzoate.
l. Exemplary rVWF Formulation for Administration
In some embodiments, the present methods provide for an enhanced formulation that allows a final product with high potency (high rVWF concentration and enhanced long term stability) in order to reduce the volume for the 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.
In some embodiments, the formulation for administration comprises one or more zwitterionic compounds, including for example, amino acids like Histidine, Glycine, Arginine. In some embodiments, the formulation for administration comprises a component with amphipathic characteristic having a minimum of one hydrophobic and one hydrophilic group, including for example polysorbate 80, octylpyranosid, dipeptides, and/or amphipathic peptides. In some embodiments, the formulation for administration comprises a non reducing sugar or sugar alcohol or disaccharides, including for example, sorbitol, mannitol, sucrose, or trehalose. In some embodiments, the formulation for administration comprises a nontoxic water soluble salt, including for example, sodium chloride, that results in a physiological osmolality. In some embodiments, the formulation for administration comprises a pH in a range from 6.0 to 8.0. In some embodiments, the formulation for administration comprises 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 comprises one or more bivalent cations that stabilize rVWF, including for example, Ca2+, Mg2+, Zn2+, Mn2+ and/or combinations thereof. In some embodiments, the formulation for administration comprises about 1 mM to about 50 mM Glycine, about 1 mM to about 50 mM Histidine, about zero 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 with a pH of about 7.0 and having a physiological osmolarity at the time point of administration.
In some embodiments, the formulation for administration can be freeze dried. In some embodiments, the formulation for administration is stable and can be stored in liquid state at about 2° C. to about 8° C., as well as at about 18° C. to about 25° C. In some embodiments, the formulation for administration is stable and can be stored in liquid state at about 2° C. to about 8° C. In some embodiments, the formulation for administration is stable and can be stored in liquid state at about 18° C. to about 25° C.
m. Administration/Dosing
To administer compositions to human or test animals, in one aspect, the compositions comprises one or more pharmaceutically acceptable carriers. The phrases “pharmaceutically” or “pharmacologically” acceptable refer to molecular entities and compositions that are stable, inhibit protein degradation such as aggregation and cleavage products, and in addition do not produce allergic, or other adverse reactions when administered using routes well-known in the art, as described below. “Pharmaceutically acceptable carriers” include any and all clinically useful solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like, including those agents disclosed above.
The pharmaceutical formulations are administered orally, topically, transdermally, parenterally, by inhalation spray, vaginally, rectally, or by intracranial injection. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intracisternal injection, or infusion techniques. Administration by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, retrobulbar, and/or intrapulmonary injection at a particular site is contemplated as well. Generally, compositions are essentially free of pyrogens, as well as other impurities that could be harmful to the recipient.
According to the present invention, rVWF is administered in the absence of Factor VIII (FVIII). In some embodiments, FVIII is not administered.
In some embodiments, the rVWF is administered prior to the surgical procedure, as discussed herein. In some embodiments, the rVWF is administered at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, or at least 24 hours, prior to the surgical procedure. In some embodiments, the surgical procedure is a minor surgical procedure. In some embodiments, the surgical procedure is a major surgical procedure. In some embodiments, FVIII is not administered.
In some embodiments, 35-60 IU/kg rVWF is administered at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, or at least 24 hours, prior to the surgical procedure. In some embodiments, 50-60 IU/kg rVWF is administered at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, or at least 24 hours, prior to the surgical procedure. In some embodiments, 20-40 IU/kg rVWF is administered at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, or at least 24 hours, prior to the surgical procedure. In some embodiments, the surgical procedure is a major surgical procedure. In some embodiments, the surgical procedure is a minor surgical procedure. In some embodiments, the surgical procedure is an oral surgical procedure. In some embodiments, FVIII is not administered.
In some embodiments, about 50-60 IU/kg rVWF is administered between 12 hours and 24 hours prior to the surgical procedure. In some embodiments, 50-60 IU/kg rVWF is administered between 12 hours and 24 hours prior to the surgical procedure and the surgical procedure is a minor surgical procedure. In some embodiments, about 55-60 IU/kg rVWF is administered between 12 hours and 24 hours prior to the surgical procedure and the surgical procedure is a minor surgical procedure. In some embodiments, about 50-55 IU/kg rVWF is administered between 12 hours and 24 hours prior to the surgical procedure and the surgical procedure is a minor surgical procedure. In some embodiments, about 50 IU/kg, about 52 IU/kg, about 54 IU/kg, about 56 IU/kg, about 58 IU/kg, or about 60 IU/kg rVWF is administered between 12 hours and 24 hours prior to the surgical procedure and the surgical procedure is a minor surgical procedure. In some embodiments, FVIII is not administered.
In some embodiments, about 35-60 IU/kg rVWF is administered between 12 hours and 24 hours prior to the surgical procedure. In some embodiments, about 35-IU/kg rVWF is administered between 12 hours and 24 hours prior to the surgical procedure and the surgical procedure is a major surgical procedure. In some embodiments, about 30-60 IU/kg rVWF is administered between 12 hours and 24 hours prior to the surgical procedure and the surgical procedure is a major surgical procedure. In some embodiments, about 40-60 IU/kg rVWF is administered between 12 hours and 24 hours prior to the surgical procedure and the surgical procedure is a major surgical procedure. In some embodiments, about 45-60 IU/kg rVWF is administered between 12 hours and 24 hours prior to the surgical procedure and the surgical procedure is a major surgical procedure. In some embodiments, about 50-60 IU/kg rVWF is administered between 12 hours and 24 hours prior to the surgical procedure and the surgical procedure is a major surgical procedure. In some embodiments, about 35 IU/kg, about 40 IU/kg, about 45 IU/kg, about 50 IU/kg, about 55 IU/kg, or about 60 IU/kg rVWF is administered between 12 hours and 24 hours prior to the surgical procedure and the surgical procedure is a major surgical procedure. In some embodiments, FVIII is not administered.
In some embodiments, about 20-40 IU/kg rVWF between 12 hours and 24 hours prior to said surgical procedure and said surgical procedure is an oral surgical procedure. In some embodiments, about 20 IU/kg rVWF between 12 hours and 24 hours prior to said surgical procedure and said surgical procedure is an oral surgical procedure. In some embodiments, about 25 IU/kg rVWF between 12 hours and 24 hours prior to said surgical procedure and said surgical procedure is an oral surgical procedure. In some embodiments, about 30 IU/kg rVWF between 12 hours and 24 hours prior to said surgical procedure and said surgical procedure is an oral surgical procedure. In some embodiments, about 35 IU/kg rVWF between 12 hours and 24 hours prior to said surgical procedure and said surgical procedure is an oral surgical procedure. In some embodiments, about 40 IU/kg rVWF between 12 hours and 24 hours prior to said surgical procedure and said surgical procedure is an oral surgical procedure. In some embodiments, FVIII is not administered.
In some embodiments, the method comprises a second pre-treatment step of administering rVWF 1 hour prior to the surgical procedure. In some embodiments, about 5-50 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is a minor surgical procedure. In some embodiments, about 5 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is a minor surgical procedure. In some embodiments, about 10 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is a minor surgical procedure. In some embodiments, about 15 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is a minor surgical procedure. In some embodiments, about 20 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is a minor surgical procedure. In some embodiments, about 25 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is a minor surgical procedure. In some embodiments, about 30 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is a minor surgical procedure. In some embodiments, about 35 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is a minor surgical procedure. In some embodiments, about 40 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is a minor surgical procedure. In some embodiments, about 45 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is a minor surgical procedure. In some embodiments, about 50 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is a minor surgical procedure. In some embodiments, FVIII is not administered.
In some embodiments, about 15-90 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is a major surgical procedure. In some embodiments, about 15 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is a major surgical procedure. In some embodiments, about 20 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is a major surgical procedure. In some embodiments, about 25 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is a major surgical procedure. In some embodiments, about 30 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is a major surgical procedure. In some embodiments, about 35 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is a major surgical procedure. In some embodiments, about 40 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is a major surgical procedure. In some embodiments, about 45 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is a major surgical procedure. In some embodiments, about 50 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is a major surgical procedure. In some embodiments, about 55 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is a major surgical procedure. In some embodiments, about 60 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is a major surgical procedure. In some embodiments, about 65 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is a major surgical procedure. In some embodiments, about 70 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is a major surgical procedure. In some embodiments, about 75 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is a major surgical procedure. In some embodiments, about 80 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is a major surgical procedure. In some embodiments, about 85 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is a major surgical procedure. In some embodiments, about 90 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is a major surgical procedure. In some embodiments, FVIII is not administered.
In some embodiments, about 20-50 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is an oral surgical procedure. In some embodiments, about 25-50 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is an oral surgical procedure. In some embodiments, about 30-50 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is an oral surgical procedure. In some embodiments, about 25-40 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is an oral surgical procedure. In some embodiments, about 40-50 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is an oral surgical procedure. In some embodiments, about 20 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is an oral surgical procedure. In some embodiments, about 25 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is an oral surgical procedure. In some embodiments, about 30 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is an oral surgical procedure. In some embodiments, about 35 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is an oral surgical procedure. In some embodiments, about 40 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is an oral surgical procedure. In some embodiments, about 45 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is an oral surgical procedure. In some embodiments, about 50 IU/kg rVWF is administered 1 hour prior to the surgical procedure when the surgical procedure is an oral surgical procedure. In some embodiments, FVIII is not administered.
In some embodiments, the method comprises administration of rVWF during the surgical procedure. In some embodiments, about 10-50 IU/kg rVWF is administered during said surgical procedure and said surgical procedure is an oral surgical procedure. In some embodiments, about 20-50 IU/kg rVWF is administered during said surgical procedure and said surgical procedure is an oral surgical procedure. In some embodiments, about 30-50 IU/kg rVWF is administered during said surgical procedure and said surgical procedure is an oral surgical procedure. In some embodiments, about 40-50 IU/kg rVWF is administered during said surgical procedure and said surgical procedure is an oral surgical procedure. In some embodiments, about 20-40 IU/kg rVWF is administered during said surgical procedure and said surgical procedure is an oral surgical procedure. In some embodiments, about 30-40 IU/kg rVWF is administered during said surgical procedure and said surgical procedure is an oral surgical procedure. In some embodiments, 10 IU/kg rVWF is administered during said surgical procedure and said surgical procedure is an oral surgical procedure. In some embodiments, about 15 IU/kg rVWF is administered during said surgical procedure and said surgical procedure is an oral surgical procedure. In some embodiments, about 20 IU/kg rVWF is administered during said surgical procedure and said surgical procedure is an oral surgical procedure. In some embodiments, about 25 IU/kg rVWF is administered during said surgical procedure and said surgical procedure is an oral surgical procedure. In some embodiments, about 30 IU/kg rVWF is administered during said surgical procedure and said surgical procedure is an oral surgical procedure. In some embodiments, about 35 IU/kg rVWF is administered during said surgical procedure and said surgical procedure is an oral surgical procedure. In some embodiments, about 40 IU/kg rVWF is administered during said surgical procedure and said surgical procedure is an oral surgical procedure. In some embodiments, about 45 IU/kg rVWF is administered during said surgical procedure and said surgical procedure is an oral surgical procedure. In some embodiments, about 50 IU/kg rVWF is administered during said surgical procedure and said surgical procedure is an oral surgical procedure. In some embodiments, FVIII is not administered.
In some embodiments, about 70-220 IU/kg rVWF is administered after the surgical procedure. In some embodiments, about 90-220 IU/kg rVWF is administered after the surgical procedure. In some embodiments, about 110-220 IU/kg rVWF is administered after the surgical procedure. In some embodiments, about 120-220 IU/kg rVWF is administered after the surgical procedure. In some embodiments, about 140-220 IU/kg rVWF is administered after the surgical procedure. In some embodiments, about 150-200 IU/kg rVWF is administered after the surgical procedure. In some embodiments, about 160-220 IU/kg rVWF is administered after the surgical procedure. In some embodiments, about 180-220 IU/kg rVWF is administered after the surgical procedure. In some embodiments, about 180-200 IU/kg rVWF is administered after the surgical procedure. In some embodiments, about 180-190 IU/kg rVWF is administered after the surgical procedure. In some embodiments, about 190-220 IU/kg rVWF is administered after the surgical procedure. In some embodiments, about 190-210 IU/kg rVWF is administered after the surgical procedure. In some embodiments, about 200-220 IU/kg rVWF is administered after the surgical procedure. In some embodiments, about 210-220 IU/kg rVWF is administered after the surgical procedure. In some embodiments, about IU/kg rVWF is administered after the surgical procedure. In some embodiments, about IU/kg rVWF is administered after the surgical procedure. In some embodiments, about 100 IU/kg rVWF is administered after the surgical procedure. In some embodiments, about 110 IU/kg rVWF is administered after the surgical procedure. In some embodiments, about 120 IU/kg rVWF is administered after the surgical procedure. In some embodiments, about 130 IU/kg rVWF is administered after the surgical procedure. In some embodiments, about 140 IU/kg rVWF is administered after the surgical procedure. In some embodiments, about 150 IU/kg rVWF is administered after the surgical procedure. In some embodiments, about 160 IU/kg rVWF is administered after the surgical procedure. In some embodiments, about 170 IU/kg rVWF is administered after the surgical procedure. In some embodiments, about 180 IU/kg rVWF is administered after the surgical procedure. In some embodiments, about 190 IU/kg rVWF is administered after the surgical procedure. In some embodiments, about 200 IU/kg rVWF is administered after the surgical procedure. In some embodiments, about 210 IU/kg rVWF is administered after the surgical procedure. In some embodiments, about 220 IU/kg rVWF is administered after the surgical procedure. In some embodiments, FVIII is not administered.
In some embodiments, about 70-150 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a minor surgical procedure. In some embodiments, about 80-150 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a minor surgical procedure. In some embodiments, about IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a minor surgical procedure. In some embodiments, about 100-150 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a minor surgical procedure. In some embodiments, about 110-150 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a minor surgical procedure. In some embodiments, about 120-150 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a minor surgical procedure. In some embodiments, about 130-150 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a minor surgical procedure. In some embodiments, about 100-140 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a minor surgical procedure. In some embodiments, about 90-140 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a minor surgical procedure. In some embodiments, about 140-150 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a minor surgical procedure. In some embodiments, about 70 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a minor surgical procedure. In some embodiments, about IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a minor surgical procedure. In some embodiments, about 90 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a minor surgical procedure. In some embodiments, about 100 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a minor surgical procedure. In some embodiments, about 110 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a minor surgical procedure. In some embodiments, about 120 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a minor surgical procedure. In some embodiments, about 130 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a minor surgical procedure. In some embodiments, about 140 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a minor surgical procedure. In some embodiments, about 150 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a minor surgical procedure. In some embodiments, when the surgical procedure is a minor surgical procedure, the pre-treatment comprises administering at least two doses of rVWF prior to the surgical procedure. In some embodiments, when the surgical procedure is a minor surgical procedure, the pre-treatment comprises administering at least two doses of rVWF prior to the surgical procedure, wherein the first dose is larger than the second dose. In some embodiments, FVIII is not administered.
In some embodiments, about 150-220 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a major surgical procedure. In some embodiments, about 160-220 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a major surgical procedure. In some embodiments, about 170-220 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a major surgical procedure. In some embodiments, about 180-220 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a major surgical procedure. In some embodiments, about 180-210 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a major surgical procedure. In some embodiments, about 190-220 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a major surgical procedure. In some embodiments, about 190-210 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a major surgical procedure. In some embodiments, about 200-220 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a major surgical procedure. In some embodiments, about 150 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a major surgical procedure. In some embodiments, about 160 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a major surgical procedure. In some embodiments, about 170 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a major surgical procedure. In some embodiments, about 180 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a major surgical procedure. In some embodiments, about 190 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a major surgical procedure. In some embodiments, about 200 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a major surgical procedure. In some embodiments, about 210 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a major surgical procedure. In some embodiments, about 220 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is a major surgical procedure. In some embodiments, when the surgical procedure is a major surgical procedure, the pre-treatment comprises administering at least two approximately equal doses of rVWF prior to the surgical procedure. In some embodiments, FVIII is not administered.
In some embodiments, about 20-50 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is an oral surgical procedure. In some embodiments, about 25-50 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is an oral surgical procedure. In some embodiments, about 30-50 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is an oral surgical procedure. In some embodiments, about 25-40 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is an oral surgical procedure. In some embodiments, about 30-40 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is an oral surgical procedure. In some embodiments, about 40-50 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is an oral surgical procedure. In some embodiments, about 45-50 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is an oral surgical procedure. In some embodiments, about 20 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is an oral surgical procedure. In some embodiments, about 25 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is an oral surgical procedure. In some embodiments, about 30 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is an oral surgical procedure. In some embodiments, about 35 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is an oral surgical procedure. In some embodiments, about 40 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is an oral surgical procedure. In some embodiments, about 45 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is an oral surgical procedure. In some embodiments, about 50 IU/kg rVWF is administered after the surgical procedure when the surgical procedure is an oral surgical procedure. In some embodiments, the surgical procedure is an oral surgical procedure and the pre-treatment comprises administering at least two approximately equal doses of rVWF prior to the surgical procedure. In some embodiments, FVIII is not administered.
In some embodiments, a total dosage of about 100-220 IU/kg rVWF is administered when the surgical procedure is a minor surgical procedure. In some embodiments, a total dosage of about 110-220 IU/kg rVWF is administered when the surgical procedure is a minor surgical procedure. In some embodiments, a total dosage of about 120-220 IU/kg rVWF is administered when the surgical procedure is a minor surgical procedure. In some embodiments, a total dosage of about 130-220 IU/kg rVWF is administered when the surgical procedure is a minor surgical procedure. In some embodiments, a total dosage of about 140-220 IU/kg rVWF is administered when the surgical procedure is a minor surgical procedure. In some embodiments, a total dosage of about 150-220 IU/kg rVWF is administered when the surgical procedure is a minor surgical procedure. In some embodiments, a total dosage of about 160-220 IU/kg rVWF is administered when the surgical procedure is a minor surgical procedure. In some embodiments, a total dosage of about 170-220 IU/kg rVWF is administered when the surgical procedure is a minor surgical procedure. In some embodiments, a total dosage of about 180-220 IU/kg rVWF is administered when the surgical procedure is a minor surgical procedure. In some embodiments, a total dosage of about 190-220 IU/kg rVWF is administered when the surgical procedure is a minor surgical procedure. In some embodiments, a total dosage of about 180-210 IU/kg rVWF is administered when the surgical procedure is a minor surgical procedure. In some embodiments, a total dosage of about 190-210 IU/kg rVWF is administered when the surgical procedure is a minor surgical procedure. In some embodiments, a total dosage of about 200-220 IU/kg rVWF is administered when the surgical procedure is a minor surgical procedure. In some embodiments, a total dosage of about 210-220 IU/kg rVWF is administered when the surgical procedure is a minor surgical procedure. In some embodiments, a total dosage of about 100 IU/kg rVWF is administered when the surgical procedure is a minor surgical procedure. In some embodiments, a total dosage of about 110 IU/kg rVWF is administered when the surgical procedure is a minor surgical procedure. In some embodiments, a total dosage of about 120 IU/kg rVWF is administered when the surgical procedure is a minor surgical procedure. In some embodiments, a total dosage of about 130 IU/kg rVWF is administered when the surgical procedure is a minor surgical procedure. In some embodiments, a total dosage of about 140 IU/kg rVWF is administered when the surgical procedure is a minor surgical procedure. In some embodiments, a total dosage of about 150 IU/kg rVWF is administered when the surgical procedure is a minor surgical procedure. In some embodiments, a total dosage of about 160 IU/kg rVWF is administered when the surgical procedure is a minor surgical procedure. In some embodiments, a total dosage of about 170 IU/kg rVWF is administered when the surgical procedure is a minor surgical procedure. In some embodiments, a total dosage of about 180 IU/kg rVWF is administered when the surgical procedure is a minor surgical procedure. In some embodiments, a total dosage of about 190 IU/kg rVWF is administered when the surgical procedure is a minor surgical procedure. In some embodiments, a total dosage of about 200 IU/kg rVWF is administered when the surgical procedure is a minor surgical procedure. In some embodiments, a total dosage of about 210 IU/kg rVWF is administered when the surgical procedure is a minor surgical procedure. In some embodiments, a total dosage of about 220 IU/kg rVWF is administered when the surgical procedure is a minor surgical procedure. In some embodiments, FVIII is not administered.
In some embodiments, a total dosage of about 220-320 IU/kg rVWF is administered when the surgical procedure is a major surgical procedure. In some embodiments, a total dosage of about 230-320 IU/kg rVWF is administered when the surgical procedure is a major surgical procedure. In some embodiments, a total dosage of about 240-320 IU/kg rVWF is administered when the surgical procedure is a major surgical procedure. In some embodiments, a total dosage of about 250-320 IU/kg rVWF is administered when the surgical procedure is a major surgical procedure. In some embodiments, a total dosage of about 260-320 IU/kg rVWF is administered when the surgical procedure is a major surgical procedure. In some embodiments, a total dosage of about 270-320 IU/kg rVWF is administered when the surgical procedure is a major surgical procedure. In some embodiments, a total dosage of about 280-320 IU/kg rVWF is administered when the surgical procedure is a major surgical procedure. In some embodiments, a total dosage of about 280-310 IU/kg rVWF is administered when the surgical procedure is a major surgical procedure. In some embodiments, a total dosage of about 290-310 IU/kg rVWF is administered when the surgical procedure is a major surgical procedure. In some embodiments, a total dosage of about 290-320 IU/kg rVWF is administered when the surgical procedure is a major surgical procedure. In some embodiments, a total dosage of about 300-320 IU/kg rVWF is administered when the surgical procedure is a major surgical procedure. In some embodiments, a total dosage of about 300-310 IU/kg rVWF is administered when the surgical procedure is a major surgical procedure. In some embodiments, a total dosage of about 220 IU/kg rVWF is administered when the surgical procedure is a major surgical procedure. In some embodiments, a total dosage of about 230 IU/kg rVWF is administered when the surgical procedure is a major surgical procedure. In some embodiments, a total dosage of about 240 IU/kg rVWF is administered when the surgical procedure is a major surgical procedure. In some embodiments, a total dosage of about 250 IU/kg rVWF is administered when the surgical procedure is a major surgical procedure. In some embodiments, a total dosage of about 260 IU/kg rVWF is administered when the surgical procedure is a major surgical procedure. In some embodiments, a total dosage of about 270 IU/kg rVWF is administered when the surgical procedure is a major surgical procedure. In some embodiments, a total dosage of about 280 IU/kg rVWF is administered when the surgical procedure is a major surgical procedure. In some embodiments, a total dosage of about 290 IU/kg rVWF is administered when the surgical procedure is a major surgical procedure. In some embodiments, a total dosage of about 300 IU/kg rVWF is administered when the surgical procedure is a major surgical procedure. In some embodiments, a total dosage of about 310 IU/kg rVWF is administered when the surgical procedure is a major surgical procedure. In some embodiments, a total dosage of about 320 IU/kg rVWF is administered when the surgical procedure is a major surgical procedure. In some embodiments, FVIII is not administered.
In some embodiments, a total dosage of about 70-190 IU/kg rVWF is administered when the surgical procedure is an oral surgical procedure. In some embodiments, a total dosage of about 80-190 IU/kg rVWF is administered when the surgical procedure is an oral surgical procedure. In some embodiments, a total dosage of 9 about 0-190 IU/kg rVWF is administered when the surgical procedure is an oral surgical procedure. In some embodiments, a total dosage of about 100-190 IU/kg rVWF is administered when the surgical procedure is an oral surgical procedure. In some embodiments, a total dosage of about 110-190 IU/kg rVWF is administered when the surgical procedure is an oral surgical procedure. In some embodiments, a total dosage of about 120-190 IU/kg rVWF is administered when the surgical procedure is an oral surgical procedure. In some embodiments, a total dosage of about 130-190 IU/kg rVWF is administered when the surgical procedure is an oral surgical procedure. In some embodiments, a total dosage of about 140-190 IU/kg rVWF is administered when the surgical procedure is an oral surgical procedure. In some embodiments, a total dosage of about 150-190 IU/kg rVWF is administered when the surgical procedure is an oral surgical procedure. In some embodiments, a total dosage of about 160-190 IU/kg rVWF is administered when the surgical procedure is an oral surgical procedure. In some embodiments, a total dosage of about 170-190 IU/kg rVWF is administered when the surgical procedure is an oral surgical procedure. In some embodiments, a total dosage of about 180-190 IU/kg rVWF is administered when the surgical procedure is an oral surgical procedure. In some embodiments, a total dosage of about 70 IU/kg rVWF is administered when the surgical procedure is an oral surgical procedure. In some embodiments, a total dosage of about 80 IU/kg rVWF is administered when the surgical procedure is an oral surgical procedure. In some embodiments, a total dosage of about 90 IU/kg rVWF is administered when the surgical procedure is an oral surgical procedure. In some embodiments, a total dosage of about 100 IU/kg rVWF is administered when the surgical procedure is an oral surgical procedure. In some embodiments, a total dosage of about 110 IU/kg rVWF is administered when the surgical procedure is an oral surgical procedure. In some embodiments, a total dosage of about 120 IU/kg rVWF is administered when the surgical procedure is an oral surgical procedure. In some embodiments, a total dosage of about 130 IU/kg rVWF is administered when the surgical procedure is an oral surgical procedure. In some embodiments, a total dosage of about 140 IU/kg rVWF is administered when the surgical procedure is an oral surgical procedure. In some embodiments, a total dosage of about 150 IU/kg rVWF is administered when the surgical procedure is an oral surgical procedure. In some embodiments, a total dosage of about 160 IU/kg rVWF is administered when the surgical procedure is an oral surgical procedure. In some embodiments, a total dosage of about 170 IU/kg rVWF is administered when the surgical procedure is an oral surgical procedure. In some embodiments, a total dosage of about 180 IU/kg rVWF is administered when the surgical procedure is an oral surgical procedure. In some embodiments, a total dosage of about 190 IU/kg rVWF is administered when the surgical procedure is an oral surgical procedure. In some embodiments, FVIII is not administered.
In some embodiments, when the surgical procedure is a major surgical procedure, the pre-treatment comprises administering at least two approximately equal doses of rVWF prior to the surgical procedure. In some embodiments, the dosage is a dosage as listed above. In some embodiments, FVIII is not administered. In some embodiments, when the surgical procedure is a minor surgical procedure, the pre-treatment comprises administering at least two doses of rVWF prior to the surgical procedure. In some embodiments, when the surgical procedure is a minor surgical procedure, the pre-treatment comprises administering at least two doses of rVWF prior to the surgical procedure, wherein the first dose is larger than the second dose. In some embodiments, the dosage is a dosage as listed above. In some embodiments, FVIII is not administered. In some embodiments, the surgical procedure is an oral surgical procedure and the pre-treatment comprises administering at least two approximately equal doses of rVWF prior to the surgical procedure. In some embodiments, the dosage is a dosage as listed above. In some embodiments, FVIII is not administered.
Generally, Type 1 VWD is indicated by <30 IU/dL VWF:RCo, <30 IU/dL VWF:Ag, low or normal FVIII, and >0.5-0.7 IU/dLVWF:RCo/VWF:Ag Ratio. Type 2A VWD is indicated by <30 IU/dL VWF:RCo, <30-200 IU/dL VWF:Ag, low or normal FVIII, and <0.5-0.7 IU/dLVWF:RCo/VWF:Ag Ratio. Type 2B VWD is indicated by <30-200 IU/dL VWF:RCo, <30 IU/dL VWF:Ag, low or normal FVIII, and usually <0.5-0.7 IU/dLVWF:RCo/VWF:Ag Ratio. Type 2M VWD is indicated by <30 IU/dL VWF:RCo, <30-200 IU/dL VWF:Ag, low or normal FVIII, and <0.5-0.7 IU/dLVWF:RCo/VWF:Ag Ratio. Type 2N VWD is indicated by 30-2000 IU/dL VWF:RCo, 30-200 IU/dL VWF:Ag, very low FVIII, and >0.5-0.7 IU/dLVWF:RCo/VWF:Ag Ratio. Type 3 VWD is indicated by <3 IU/dL VWF:RCo, <3 IU/dL VWF:Ag, extremely low (<10 IU/dL) FVIII, and the VWF:RCo/VWF:Ag Ratio is not applicable. Normal is indicated by 50-200 IU/dL VWF:RCo, 50-200 IU/dL VWF:Ag, normal FVIII, and >0.5-0.7 IU/dLVWF:RCo/VWF:Ag Ratio. 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.

V. Surgical Procedures

The surgical procedure according to the methods of the present invention can be a major surgical procedure or a minor surgical procedure.
Generally, major surgery includes any invasive operative procedure in which a more extensive resection is performed, e.g., a body cavity is entered, organs are removed, or normal anatomy is altered. Generally, if a mesenchymal barrier is opened (for example, pleural cavity, peritoneum, meninges), the surgery is considered major. In some embodiments, a major surgery is one in which there is an expected blood loss of greater than 500 mL, significant fluid shifts and typically, at least one night in hospital. Exemplary major surgical procedures include but are not limited to bariatric surgeries/gastric bypass, septal myotomy, pancreatectomy, thoracic aortic dissection repair, esophagectomy, bladder cystectomy, coronary revascularization, spinal osteomyelitis surgery, surgical ventricular restoration, craniectomy, laparoscopic surgery (except cholecystectomy and tubal ligation), open resection of organs, large joint replacements, mastectomy with reconstruction, and/or spine, thoracic, vascular, and/or intracranial surgery. Further examples of major surgeries include but are not limited to maxillary or mandibular osteotomy, laryngectomy, resection of large benign or malignant mass and/or lymph node dissection requiring overnight stay in hospital (with or without reconstructive surgery), mastectomy with immediate tissue reconstruction (with or without lymph node biopsy or axillary dissection), laparoscopic or open repair or resection (of, for example, stomach, small bowel, colon, liver, pancreas, spleen, adrenals or liver), open cholecystectomy, large incisional, epigastric or ventral hernia repairs, hysteroscopic resection or ablation, hysterectomy and/or adnexal surgery, laparoscopy for extensive endometriosis, abdominal or transvaginal pelvic floor surgery, intracranial surgery, spinal laminectomy and/or fusion, knee replacement, hip replacement, shoulder replacement, elbow joint replacement, hardware removal or revision for infection or failure, amputation, spinal laminectomy and/or fusion, free flap reconstruction (plastic surgery), panniculectomy, mediastinoscopy, lung resection, esophagus resection, mediastinal mass resection (thoracoscopic or open), hiatal hernia repair (thoracoscopic or open), bladder tumor resection (transurethral or open), prostate tumor (transurethral or open), resection of kidney resection (laparoscopic or open), ureteral resection (laparoscopic or open), resection of testis (transscrotal or abdominal), amputation, peripheral arterial bypass surgery, aortic aneurysm repair (endovascular or open), and/or carotid endarterectomy.
Minor surgery is any invasive operative procedure in which only skin or mucus membranes and connective tissue is resected e.g. vascular cutdown for catheter placement, implanting pumps in subcutaneous tissue. A minor surgical procedure typically includes any procedure that can be safely performed in an outpatient setting, without the use of general anesthesia or the need for respiratory assistance. In some embodiments, a minor surgery is one in which there is an expected blood loss of less than 500 mL, minimal fluid shifts and is typically done on an ambulatory basis (day surgery/same day discharge). Such outpatient surgical procedures can include but are not limited to cataract surgery, breast surgery without reconstruction, laparoscopic cholecystectomy and tubal ligation, and most cutaneous, superficial, endoscopic and arthroscopic procedures. Further examples of minor surgeries include but are not limited to tooth extraction, tonsillectomy, adenoidectomy, septoplasty, turbinectomy, rhinoplasty, pharyngeal biopsy, laryngeal biopsy, minor excision by laser or other means, middle ear surgery, mastoidectomy, cochlear implantation, endoscopic sinus surgery, small resections of benign and malignant masses (done on an ambulatory basis; i.e., mandibular tori, brachial cleft cyst, small tongue cancer), thyroidectomy, breast lumpectomy (with or without lymph node biopsy or axillary dissection), mastectomy (with or without lymph node biopsy or axillary dissection), inguinal hernia repair (laparoscopic or open approach), umbilical hernia repair (laparoscopic or open approach), laparoscopic cholecystectomy, hemorrhoidectomy, dilation, curettage, diagnostic hysteroscopy, laparoscopy, endometrial ablation by thermal balloon, tubal ligation, laparoscopy—limited endometriosis, transvaginal tape insertion, discectomy, cataract extraction, most ophthalmological procedures, arthroscopic surgery (including ACL repair), routine hardware removal (not for infection), tendon surgery, bunionectomy, discectomy, carpal tunnel release, Dupuytren's contracture release, major tendone surgery, minor tendon surgery, small rotational flaps and skin grafts, basal cell carcinoma resection, lipoma excision, reduction mammoplasty and other surgery for benign breast disease, cosmetic breast surgery, bronchoscopy, cystoscopy, ureteroscopy, renoscopy for stone, renoscopy for stricture, renoscopy for biopsy, hydrocele excision, varicocele excision, vasectomy, circumcision, and/or varicose vein excision.
Oral surgical procedures include, but are not limited to, various dental procedures and oral surgeries, including for example tooth extractions.
In some embodiments, the surgical procedure is a major surgical procedure. In some embodiments, the surgical procedure is a minor surgical procedure. In some embodiments, the surgical procedure is an oral surgical procedure.

EXAMPLES

Example 1: Hemostatic Efficacy and Safety of rVWF

This study evaluated the hemostatic efficacy and safety of rVWF with or without ADVATE (antihemophilic factor [recombinant]), Baxalta US Inc., Westlake Village, CA (rFVIII) in patients with severe VWD undergoing elective surgery.

Methods

Phase 3, open-label, uncontrolled, nonrandomized study at 14 sites in 10 countries (NCT02283268) in patients ≥18 y of age who had severe VWD and were scheduled to undergo elective surgery. Patients were monitored for 14 d after surgery.

Treatment

12-24 h before surgery, rVWF 40-60 IU/kg rVWF:RCo was given intravenously to allow endogenous FVIII:C levels to increase to ≥30 IU/dL (minor/oral surgery) or ≥60 IU/dL (major surgery). FVIII:C levels were assessed within 3 h of initiation of surgery. If target FVIII:C levels were achieved, rVWF alone was administered 1 h before surgery to achieve the peak levels described in Table 2. If target FVIII:C levels were achieved, rVWF alone was administered 1 h before surgery to achieve the peak levels described in Table 2. Intraoperative and postoperative dosing were individualized to maintain target trough levels according to pharmacokinetic (PK) and pharmacodynamic (PD) results, as well as the intensity and duration of the hemostatic challenge.

TABLE 2

VWF: RCo and FVIII: C Target Levels: Recommendations for the
Prevention of Excessive Bleeding During and After Surgery

	VWF: RCo	FVIII: C
	Target Peak	Target Peak
Type of	Plasma Level	Plasma Level*	Calculation of rVWF Dose
Surgery	(IU/dL)	(IU/dL)	(IU VWF: RCo Required)†

Minor/Oral	50-60	40-50	ΔVWF: RCo × BW (kg)/IR‡
Major	100	80-100	ΔVWF: RCo × BW (kg)/IR‡

BW = body weight; FVIII: C = factor VIII activity; IR = incremental recovery; rVWF = recombinant von Willebrand factor; VWF: RCo = von Willebrand factor ristocetin cofactor activity.
*Additional rFVIII may be required to attain the recommended FVIII: C target peak plasma levels.
†Administered within 1 h before surgery.
‡If the IR was not available, assume an IR of 2.0 IU/dL per IU/kg.
ΔVWF: RCo = target peak plasma VWF: RCo − baseline plasma VWF: RCo.

Assessment

Overall hemostatic efficacy (primary outcome) was assessed by the investigator at 24 h after the last perioperative infusion or at study completion, whichever occurred earlier (Table 3). Intraoperative hemostatic efficacy was assessed by the surgeon (Table 3), along with intraoperative actual versus predicted blood loss. Safety evaluations included adverse events (AEs) and antibodies to rVWF, rFVIII, Chinese hamster ovary (CHO) proteins, murine immunoglobulin G (IgG), and rFurin.

TABLE 3

Overall* and Intraoperative† Hemostatic Efficacy Rating Scale

Rating	Assessment

Excellent	Hemostasis achieved with rVWF with or without rFVIII was
	as good or better than that expected for the type of surgical
	procedure performed in a hemostatically normal subject
Good	Hemostasis achieved with rVWF with or without rFVIII was
	probably as good as that expected for the type of surgical
	procedure performed in a hemostatically normal subject
Moderate	Hemostasis with rVWF with or without rFVIII was clearly
	less than optimal for the type of procedure performed but was
	maintained without the need to change the rVWF concentrate
None	Patient experienced uncontrolled bleeding that was the result
	of inadequate therapeutic response despite proper dosing,
	necessitating a change of rVWF concentrate

rFVIII = recombinant factor VIII; rVWF = recombinant von Willebrand factor.
*As assessed by the investigator.
†As assessed by the surgeon.

Statistics

Descriptive analyses included point estimates and 90% CIs for the number of patients with hemostatic efficacy rated “excellent/good” using a Clopper Pearson test. PK/PD and safety were summarized using descriptive statistics.

Results

Patients

TABLE 4

Baseline Demographics and Clinical Characteristics

		Parameter	N = 15

Sex, n (%)

Male	7	(46.7)
Female	8	(53.3)
Median age (range), y	40	(20-70)
Median weight (range), kg	73.5	(52.0-127.2)
Median BMI (range), kg/m2	25.6	(17.1-38.0)

VWD type, n (%)

1	3	(20.0)
2A	2	(13.3)
2B	1	(6.7)
2M	1	(6.7)
3	8	(53.3)

Surgery classification, n (%)

Major	10	(66.7)
Minor	4	(26.7)
Oral	1	(6.7)

Mean (SD) FVIII: C, IU/dL

		All VWD types (n = 11)	20.6	(23.7)
		Type 3 VWD (n = 5)	1.8	(1.1)

Mean (SD) VWF: RCo, IU/dL

	All VWD types (n = 11)	9.7	(11.0)
	Type 3 VWD (n = 5)	<8	(0.0)

BMI = body mass index; FVIII: C = factor VIII activity; VWD = von Willebrand disease; VWF: RCo = von Willebrand factor ristocetin cofactor activity.

Overall hemostatic efficacy was rated as “excellent” or “good” for all 15 patients (90% CI: 81.9-100.0) (FIG. 1 ).

Efficacy

Overall hemostatic efficacy was rated as “excellent” or “good” for all 15 patients (90% CI: 81.9-100.0) (FIG. 1 ).
Intraoperative hemostatic efficacy was rated “excellent” or “good” for all 15 patients (90% CI: 81.9-100.0) (FIG. 2 ). Among the 8 patients with type 3 VWD, overall and intraoperative hemostatic efficacy were both rated “excellent” for 7 patients and “good” for 1 patient. Mean±SD intraoperative actual blood loss relative to predicted blood loss was 70%±45% and was rated “excellent” for 13 patients and “good” for 2 patients.

Exposure

Patients received a total of 104 infusions of rVWF to prevent or treat surgical bleeding; the median overall surgical dose of rVWF was 220.4 IU/kg (range, 63.8-648.4 IU/kg) (Table 4). 93 (89.4%) infusions of rVWF alone: 15 (12-24 h before surgery), 12 (1 h before surgery), and 66 (postoperatively). 11 (10.6%) infusions of rVWF with rFVIII: 3 (1 h before surgery), 1 (intraoperatively), and 7 (postoperatively). 5 patients received the 11 infusions of rVWF with rFVIII, and 6 of the 7 postoperative infusions of rVWF with rFVIII were in 1 patient.
Of the 10 patients undergoing major surgery, 7 (70%) did not require coadministration of rFVIII.

TABLE 5

Median rVWF Exposure Overall and by Surgery Classification

Surgery Classification

	Minor (n = 4)	Major (n = 10)	Oral (n = 1)	Overall (N = 15)

Median total number of	3	(2-4)	7.5	(4-15)	5	6	(2-15)
infusions* (range)
Median exposure (range),	3	(2-4)	6.5	(4-15)	4	6	(2-15)

d
Median dose 12-24 h	57.2	(55.0-59.9)	49.3	(37.4-57.6)	36.1 55.0	(36.1-59.9)
before surgery (range),
IU/kg

Median dose 1 h before

39.3

(8.0-46.4)

37.6

(15.7-82.7)

18.1

35.8

(8.0-82.7)

surgery (range), IU/kg
Median intraoperative	0	0	18.1	18.1
dose (range), IU/kg

Median postoperative	79.3	(42.8-115.9)	214.8	(47.7-533.3)	36.1	189.8	(36.1-533.3)
dose (range), IU/kg
Median total surgical dose	119.9	(63.8-217.3)	307.6	(125.2-648.4)	108.4	220.4	(63.8-648.4)

(range), IU/kg

rVWF = recombinant von Willebrand factor.
*Total number of preoperative priming infusions, preoperative initial loading doses, preoperative supplemental loading doses, intraoperative doses, and postoperative doses.

Safety

6 patients reported 12 treatment-emergent AEs; none considered related to treatment. patients had serious AEs (diverticulitis and deep vein thrombosis [DVT]; each occurred in 1 patient); neither event was considered related to factor replacement treatment.
The serious DVT occurred on postoperative day 8 (initially reported as a nonserious DVT on postoperative day 4). The event was asymptomatic and observed after routine duplex scan. The event was assessed as unlikely related to rVWF and not related to rFVIII or the study procedures; causally associated with the patient's major surgery (total hip replacement) and ongoing history of obesity. Postoperative levels of FVIII:C never exceeded 150 IU/dL. The event resulted in placement of caval filter and subsequently resolved without sequelae. No severe allergic reactions; neutralizing antibodies to rFVIII, CHO proteins, murine IgG, or rFurin.
One patient with VWD type 3 who had an intraoperative transfusion of packed red blood cells during major total knee replacement surgery tested positive for binding antibodies to VWF on postoperative day 7.
The PK parameters for VWF:RCo for the patients who underwent PK analysis (n=11) are shown in FIG. 4 . Mean concentrations of VWF:RCo, VWF:Ag, and VWF collagen binding activity (VWF:CB) reached peak levels by 30 min and gradually declined over a period of 72 h post-infusion (FIG. 4 ).
Administration of rVWF alone resulted in substantial, rapid stabilization of endogenous FVIII:C levels 6-12 h after infusion, with peak FVIII:C levels reached by 24 h among all patients assessed (n=11; FIG. 5A), as well as in the subset of patients with type 3 VWD (n=5; FIG. 5B). Overall, patients achieved mean FVIII:C >60 IU/dL by 6 h postinfusion (FIG. 5A), and patients with higher baseline FVIII:C (e.g., with type 1 or type 2 VWD) were able to achieve target levels more rapidly. Despite having mean FVIII:C levels <2 IU/dL at baseline, administration of rVWF alone allowed patients with type 3 VWD to achieve target VWF:RCo and FVIII:C levels quickly, with FVIII:C >60 IU/dL achieved by 12 h post-infusion (FIG. 5B).

Conclusions

In this surgery study, overall and intraoperative hemostatic efficacies were rated as “excellent” or “good” for all 15 patients. For major surgeries, overall hemostatic efficacy was “excellent” in 7 patients and “good” in 3 patients, and intraoperative efficacy was “excellent” in 8 patients and “good” in 2 patients. Nearly 90% of infusions to achieve intraoperative and postoperative hemostasis were with rVWF alone; 70% of major surgeries were managed with rVWF alone. rVWF targets the primary dysfunction of VWD and allows physicians to focus on achieving optimal efficacy without concern for FVIII accumulation. These data support the safe and effective use of rVWF in major and minor surgeries.

Example 2: Recombinant Von Willebrand Factor in Subjects with Severe Von Willebrand Disease Undergoing Surgery

This example provides the study results from a study examining treatment of subjects with severe von Willebrand Disease (VWD) undergoing surgery.

Outcome Measures:

Primary Outcome Measures:

Overall Hemostatic Efficacy as Assessed by the Investigator (Hemophilia Physician) [Time Frame: 24 hours after last peri-operative infusion or at completion of Day 14 (±2 days) visit, whichever occurs earlier]. Hemostatic efficacy was rated on a scale of excellent—good—moderate—none.

- Excellent: Intra-, and postoperative hemostasis achieved with rVWF with or without ADVATE was as good or better than that expected for the type of surgical procedure performed in a hemostatically normal subject.
- Good: Intra-, and postoperative hemostasis achieved with rVWF with or without ADVATE was probably as good as that expected for the type of surgical procedure performed in a hemostatically normal subject.
- Moderate: Intra-, and postoperative hemostasis with rVWF with or without ADVATE was clearly less than optimal for the type of procedure performed but was maintained without the need to change the rVWF concentrate.
- None: Participant experienced uncontrolled bleeding that was the result of inadequate therapeutic response despite proper dosing, necessitating a change of rVWF concentrate.

Secondary Outcome Measures:

#1: Intraoperative Actual Versus Predicted Blood Loss as Assessed by the Operating Surgeon [Time Frame: Day 0 (at completion of surgery)]. The predicted blood loss was estimated preoperatively by the operating surgeon based on a hemostatically normal individual of the same sex, age, stature and co-morbidities as the participant. The actual blood loss was assessed consisting of the estimated blood loss, including into swabs, towels and suction during the procedure, per the anesthesiologist's record.
#2: Intraoperative Actual Blood Loss Relative to Predicted Blood Loss [Time Frame: Day 0 (at completion of surgery)]. Actual blood loss relative to predicted blood loss was calculated as [Actual Blood loss (mL)] divided by [Predicted Blood Loss (mL) multiplied by 100.
#3: Intraoperative Actual Versus Predicted Blood Loss Score as Assessed by the Operating Surgeon [Time Frame: Day 0 (at completion of surgery)]

- Hemostatic efficacy was rated on a scale of excellent—good—moderate—none.
- Excellent: Intraoperative blood loss was less than or equal to the maximum blood loss expected for the type of procedure performed in a hemostatically normal subject (<100%).
- Good: Intraoperative blood loss was up to 50% more than the maximum expected blood loss for the type of procedure performed in a hemostatically normal subject (101-150%) Moderate: Intraoperative blood loss was more than 50% of the maximum expected blood loss for the type of procedure performed in a hemostatically normal subject (>150%).
- None: Uncontrolled hemorrhage that was the result of inadequate therapeutic response despite proper dosing, necessitating a change of clotting factor replacement regimen.

#4: Intraoperative Hemostatic Efficacy Score as Assessed by the Operating Surgeon [Time Frame: Day 0 (at completion of surgery)]

- Hemostatic efficacy was rated on a scale of excellent—good—moderate—none.
- Excellent: Intraoperative hemostasis achieved with rVWF with or without ADVATE was as good or better than that expected for the type of surgical procedure performed in a hemostatically normal subject.
- Good: Intraoperative hemostasis achieved with rVWF with or without ADVATE was probably as good as that expected for the type of surgical procedure performed in a hemostatically normal subject.
- Moderate: Intraoperative hemostasis with rVWF with or without ADVATE was clearly less than optimal for the type of procedure performed but was maintained without the need to change the rVWF concentrate.
- None: Participant experienced uncontrolled bleeding that was the result of inadequate therapeutic response despite proper dosing, necessitating a change of rVWF concentrate.

#5: Daily Intra- and Postoperative Weight-adjusted Dose of rVWF With or Without ADVATE [Time Frame: Daily, from day of surgery through postoperative Day 14 (±2 days)]
#6: Occurrence of Adverse Events [Time Frame: From first infusion of investigational product through study completion (i.e., 14 (±2) days post-surgery)]. Treatment emergent adverse events (TEAEs) and treatment emergent serious adverse events (TESAEs) were evaluated.
#7: Occurrence of Thrombotic Events [Time Frame: From first infusion of investigational product through study completion (i.e., 14 (±2) days post-surgery)]. Treatment emergent adverse events (TEAEs) and treatment emergent serious adverse events (TESAEs) were evaluated for thrombotic events.
#8: Occurrence of Severe Allergic Reactions (e.g., Anaphylaxis) [Time Frame: From first infusion of investigational product through study completion (i.e., 14 (±2) days post-surgery)]. Treatment emergent adverse events (TEAEs) and treatment emergent serious adverse events (TESAEs) were evaluated for severe allergic reactions.
#9: Number of Participants Who Developed Inhibitory and Total Binding Antibodies to Von Willebrand Factor (VWF) and Inhibitory Antibodies to Factor VIII (FVIII) [Time Frame: Testing occurred throughout the study at screening, prior PK infusion, pre-surgery, post-surgery in case of excessive bleeding or unexplained bleeding, at postoperative day 7 and at study completion visit (ie. 14 (±2) days post-surgery)]. Participants were treated with recombinant van Willebrand Factor (rVWF) with or without ADVATE.
#10: Number of Participants Who Developed Antibodies to Chinese Hamster Ovary (CHO) Proteins, Mouse Immunoglobulin G (IgG) or Recombinant Furin (rFurin) [Time Frame: Testing occurred throughout the study at screening, prior PK infusion, pre-surgery, post-surgery in case of excessive bleeding or unexplained bleeding, at postoperative day 7 and at study completion visit (ie. 14 (±2) days post-surgery).] Participants were treated with recombinant van Willebrand Factor (rVWF) with or without ADVATE.
#11: Pharmacokinetics: Area Under the Plasma Concentration Versus Time Curve From 0 to 72 Hours Post-infusion (AUC 0-72 h/Dose) [Time Frame: PK measurements were done within 30 minutes pre-infusion, and post infusion at 30 (±5) minutes, 60 (±5) minutes, 6 (±1) hours, 12 (±1) hours, 24 (±2) hours, 48 (±2) hours and 72 (±2) hours.] This assessment was only required for subjects undergoing major surgery. Subjects received a PK infusion at a dose of 50±5 IU/kg rVWF:RCo within 42 days prior to surgery. The area under the plasma concentration/time curve from 0 to 72 hours post-infusion was computed using the linear trapezoidal rule. For the calculation of AUC(0-72h) the levels at 72 hours was linearly interpolated/extrapolated from the 2 nearest sampling time points. PK analysis was performed for the following analytes: VWF Ristocetin Cofactor Activity (VWF:RCo), VWF Antigen Activity (VWF:Ag), VWF Collagen Binding Activity (VWF:CB), VWF Activity Measured INNOVANCE VWF Ac Assay (VWF:Ac), FVIII Coagulation Activity (FVIII:C)
#12: Pharmacokinetics: Area Under the Plasma Concentration Versus Time Curve From Time 0 to Infinity (AUC 0-∞/Dose) [Time Frame: PK measurements were done within 30 minutes pre-infusion, and post infusion at 30 (±5) minutes, 60 (±5) minutes, 6 (±1) hours, 12 (±1) hours, 24 (±2) hours, 48 (±2) hours and 72 (±2) hours.] This assessment was only required for subjects undergoing major surgery. Subjects received a PK infusion at a dose of 50±5 IU/kg rVWF:RCo within 42 days prior to surgery. The area under the plasma concentration/time curve from time 0 to infinity and the area under the first moment curve from time 0 to infinity was calculated as the sum of AUC or AUMC from time 0 to the time of last quantifiable concentration plus a tail area correction calculated as Ct/λz and Ct/λz(t+1/λz), respectively, where Ct was the last quantifiable concentration, t was the time of last quantifiable concentration and λz was the terminal or disposition rate constant. PK analysis was performed for the following analytes: VWF Ristocetin Cofactor Activity (VWF:RCo), VWF Antigen Activity (VWF:Ag), VWF Collagen Binding Activity (VWF:CB), VWF Activity Measured INNOVANCE VWF Ac Assay (VWF:Ac), FVIII Coagulation Activity (FVIII:C)
#13: Pharmacokinetics: Mean Residence Time (MRT) [Time Frame: PK measurements were done within 30 minutes pre-infusion, and post infusion at 30 (±5) minutes, 60 (±5) minutes, 6 (±1) hours, 12 (±1) hours, 24 (±2) hours, 48 (±2) hours and 72 (±2) hours.] This assessment was only required for subjects undergoing major surgery. Subjects received a PK infusion at a dose of 50±5 IU/kg rVWF:RCo within 42 days prior to surgery. Mean residence time was calculated as area under the first moment curve from time 0 to infinity divided by the area under the curve time 0 to infinity minus T/2 where T was the duration of the infusion. PK analysis was performed for the following analytes: VWF Ristocetin Cofactor Activity (VWF:RCo), VWF Antigen Activity (VWF:Ag), VWF Collagen Binding Activity (VWF:CB), VWF Activity Measured INNOVANCE VWF Ac Assay (VWF:Ac)
#14: Pharmacokinetics: Clearance (CL) [Time Frame: PK measurements were done within 30 minutes pre-infusion, and post infusion at 30 (±5) minutes, 60 (±5) minutes, 6 (±1) hours, 12 (±1) hours, 24 (±2) hours, 48 (±2) hours and 72 (±2) hours.] This assessment was only required for subjects undergoing major surgery. Subjects received a PK infusion at a dose of 50±5 IU/kg rVWF:RCo within 42 days prior to surgery. Clearance was calculated as dose (IU/kg) divided by the area under the curve time 0 to infinity. PK analysis was performed for the following analytes: VWF Ristocetin Cofactor Activity (VWF:RCo), VWF Antigen Activity (VWF:Ag), VWF Collagen Binding Activity (VWF:CB), VWF Activity Measured INNOVANCE VWF Ac Assay (VWF:Ac)
#15: Pharmacokinetics: Incremental Recovery (IR) [Time Frame: PK measurements were done within 30 minutes pre-infusion, and post infusion at 30 (±5) minutes, 60 (±5) minutes, 6 (±1) hours, 12 (±1) hours, 24 (±2) hours, 48 (±2) hours and 72 (±2) hours.] This assessment was only required for subjects undergoing major surgery. Subjects received a PK infusion at a dose of 50±5 IU/kg rVWF:RCo within 42 days prior to surgery. Incremental recovery was calculated as (Cmax minus Cpreinfusion) divided by the dose (IU/kg) where kg refers to the body weight at the time of dosing and Cmax was the observed maximum concentration before correction for pre-infusion values. PK analysis was performed for the following analytes: VWF Ristocetin Cofactor Activity (VWF:RCo), VWF Antigen Activity (VWF:Ag), VWF Collagen Binding Activity (VWF:CB), VWF Activity Measured INNOVANCE VWF Ac Assay (VWF:Ac)
#16: Pharmacokinetics: Elimination Phase Half-life (T1/2) [Time Frame: PK measurements were done within 30 minutes pre-infusion, and post infusion at 30 (±5) minutes, 60 (±5) minutes, 6 (±1) hours, 12 (±1) hours, 24 (±2) hours, 48 (±2) hours and 72 (±2) hours.] This assessment is only required for subjects undergoing major surgery. Subjects received a PK infusion at a dose of 50±5 IU/kg rVWF:RCo within λz days prior to surgery. Terminal or disposition half-life (T1/2) was calculated as 1n2/λz where)\,z was the terminal elimination rate constant as calculated in WinNonlin NCA using at least three quantifiable concentrations. PK analysis was performed for the following analytes: VWF Ristocetin Cofactor Activity (VWF:RCo), VWF Antigen Activity (VWF:Ag), VWF Collagen Binding Activity (VWF:CB), VWF Activity Measured INNOVANCE VWF Ac Assay (VWF:Ac)
#17: Pharmacokinetics: Volume of Distribution at Steady State (Vss) [Time Frame: PK measurements were done within 30 minutes pre-infusion, and post infusion at 30 (±5) minutes, 60 (±5) minutes, 6 (±1) hours, 12 (±1) hours, 24 (±2) hours, 48 (±2) hours and 72 (±2) hours.] This assessment is only required for subjects undergoing major surgery. Subjects received a PK infusion at a dose of 50±5 IU/kg rVWF:RCo within 42 days prior to surgery. Vss was calculated as the clearance multiplied with the mean residence time. PK analysis was performed for the following analytes: VWF Ristocetin Cofactor Activity (VWF:RCo), VWF Antigen Activity (VWF:Ag), VWF Collagen Binding Activity (VWF:CB), VWF Activity Measured INNOVANCE VWF Ac Assay (VWF:Ac)

Eligibility Criteria:

Ages Eligible for Study: 18 Years and older; Sexes Eligible for Study: All

Inclusion Criteria:

Diagnosis of severe von Willebrand disease (VWD) as listed below and elective surgical procedure planned:

- 1. Type 1 (Von Willebrand factor: Ristocetin cofactor activity (VWF:RCo)<20 IU/dL), or
- 2. Type 2A (as verified by multimer pattern), Type 2B (as diagnosed by genotype), Type 2N (FVIII:C<10% and historically documented genetics), Type 2M, or
- 3. Type 3 (Von Willebrand factor antigen (VWF:Ag)<3 IU/dL)

VWD with a history of requiring substitution therapy with von Willebrand factor (VWF) concentrate to control bleeding.
If type 3 VWD (VWF Antigen/VWF:Ag≤3 IU/dL), participant has a medical history of at least 20 exposure days to VWF/FVIII coagulation factor concentrates (including cryoprecipitate or fresh frozen plasma).
If type 1 or type 2 VWD, participant has a medical history of 5 exposure days or a past major surgery requiring VWF/FVIII coagulation factor concentrates (including cryoprecipitate or fresh frozen plasma).
Participant was at least 18 years of age.
If female of childbearing potential, participant presents with a negative pregnancy test.
If applicable, participant agrees to employ adequate birth control measures for the duration of the study.
Participant is willing and able to comply with the requirements of the protocol.

Selected Exclusion Criteria:

Diagnosis of pseudo VWD or another hereditary or acquired coagulation disorder (e.g., qualitative and quantitative platelet disorders or elevated prothrombin time [PT]/international normalized ratio [INR]>1.4).
History or presence of a VWF inhibitor at screening.
History or presence of a factor VIII (FVIII) inhibitor with a titer ≥0.4 BU (Nijmegen-modified Bethesda assay) or ≥0.6 BU (by Bethesda assay).
Known hypersensitivity to any of the components of the study drugs, such as to mouse or hamster proteins.
Medical history of immunological disorders, excluding seasonal allergic rhinitis/conjunctivitis, mild asthma, food allergies or animal allergies.
Medical history of a thromboembolic event.
HIV positive with an absolute CD4 count <200/mm³.
Platelet count <100,000/mL.
Diagnosis of significant liver disease, as evidenced by, but not limited to, any of the following: serum alanine aminotransferase (ALT) 5 times the upper limit of normal; hypoalbuminemia; portal vein hypertension (e.g., presence of otherwise unexplained splenomegaly, history of esophageal varices) or liver cirrhosis classified as Child B or C.
Diagnosis of renal disease, with a serum creatinine level >2.5 mg/dL.
Participant had been treated with an immunomodulatory drug, excluding topical treatment (e.g., ointments, nasal sprays), within 30 days prior to signing the informed consent.
Participant was pregnant or lactating at the time informed content is obtained.
Participant had participated in another clinical study involving an investigational product (IP), other than rVWF with or without ADVATE, or investigational device within 30 days prior to enrollment or was scheduled to participate in another clinical study involving an IP or investigational device during the course of this study. However, eligible patients participating in the rVWF Prophylaxis Study (071301) may be enrolled.
Progressive fatal disease and/or life expectancy of less than 3 months.

Results:

Enrollment was conducted at 14 study sites in 10 countries (USA, Australia, Taiwan, Germany, Russia, Spain, Ukraine, United Kingdom, Italy, Turkey).

TABLE 6

Reporting Groups

	Description

Recombinant Von Willebrand Factor	Surgery participants treated with
(rVWF)	Recombinant von Willebrand Factor
	(rVWF)

TABLE 6

Participant: Overall Study

	Recombinant Von Willebrand Factor (rVWF)

STARTED	15
COMPLETED	14
NOT COMPLETED	1
Withdrawal by Subject	1

TABLE 7

Baseline Measures

	Recombinant Von Willebrand Factor
	(rVWF)

Overall Participants Analyzed	15
[Units: Participants]
Age	40.0
[Units: Years]	(20.0 to 70.0)
Median (Full Range)
Sex: Female, Male
[Units: Participants]
Count of Participants
Female
	8 53.3%
Male
	7 46.7%

TABLE 8

Outcome Measures

1. Primary: Overall Hemostatic Efficacy as Assessed by the Investigator
(Hemophilia Physician) [Time Frame: 24 hours after last pen-operative
infusion or at completion of Day 14 (±2 days) visit, whichever occurred
earlier].

TABLE 9

Primary Outcome #1

Measure	Primary
Type
Measure	Overall Hemostatic Efficacy as Assessed by the Investigator
Title	(Hemophilia Physician)
Measure	Hemostatic efficacy was rated on a scale of excellent -
Description	good - moderate - none.
	Excellent: Intra-, and postoperative hemostasis achieved
	with rVWF with our without ADVATE was as good or
	better than that expected for the type of surgical procedure
	performed in a hemostatically normal subject.
	Good: Intra-, and postoperative hemostasis achieved with
	rVWF with or without ADVATE was probably as good as
	that expected for the type of surgical procedure performed
	in a hemostatically normal subject.
	Moderate: Intra-, and postoperative hemostasis with rVWF
	with or without ADVATE was clearly less than optimal for
	the type of procedure performed but was maintained without
	the need to change the rVWF concentrate.
	None: Participant experienced uncontrolled bleeding that
	was the result of inadequate therapeutic response despite
	proper dosing, necessitating a change of rVWF concentrate.
Time	24 hours after last pen-operative infusion or at completion
Frame	of Day 14 (±2 days) visit, whichever occurs earlier

Population Description Outcome #1

Number of participants with major, minor and oral surgery and number of participant with Von Willebrand Type 1, 2A, 2B, 2M and 3 do sum up to the overall number of participants analyzed. The full analysis data set, including all participants who received investigational product and have at least 1 hemostatic assessment, was used for analysis.

TABLE 10

Reporting Groups Outcome #1

	Description

Recombinant Von Willebrand	Surgery participants treated with
Factor (rVWF)	Recombinant von Willebrand Factor
	(rVWF)
Minor Surgery	All participants who underwent minor
	surgery.
Major Surgery	All participants who underwent major
	surgery.
Oral Surgery	All participants who underwent oral
	surgery.
Von Willebrand Disease Type 1	All participants with von Willebrand
	Disease Type
1.
Von Willebrand Disease Type 2A	All participants with von Willebrand
	Disease Type
2A.
Von Willebrand Disease Type 2B	All participants with von Willebrand
	Disease Type
2B.
Von Willebrand Disease Type 2M	All participants with von Willebrand
	Disease Type
2M.
Von Willebrand Disease Type 3	All participants with von Willebrand
	Disease Type
3.

TABLE 11

Measured Values Outcome #1

Recombinant
Von				Von	Von	Von	Von	Von
Willebrand				Willebrand	Willebrand	Willebrand	Willebrand	Willebrand
Factor	Minor	Major	Oral	Disease	Disease	Disease	Disease	Disease
(rVNVF)	Surgery	Surgery	Surgery	Type	1	Type 2A	Type 2B	Type 2M	Type	3

Participants Analyzed

	15	4	10	1	3	2	1	1	8

Overall
Hemostatic
Efficacy as
Assessed by the
Investigator
(Hemophilia
Physician)
[Units:
Participants]
Count of
Participants
Excellent
	11	73.3%	4	100.0%	7	70.0%	0	0.0%	2	66.7%	1	50.0%	1	100.0%	0	0.0%	7	87.5%
Good
	4	26.7%	0	0.0%	3	30.0%	1	100.0%	1	33.3%	1	50.0%	0	0.0%	1	100.0%	1	12.5%
Moderate
	0	0.0%	0	0.0%	0	0.0%	0	0.0%	0	0.0%	0	0.0%	0	0.0%	0	0.0%	0	0.0%
None
	0	0.0%	0	0.0%	0	0.0%	0	0.0%	0	0.0%	0	0.0%	0	0.0%	0	0.0%	0	0.0%

Secondary Outcome: Outcome #2

2. Secondary: Intraoperative Actual Versus Predicted Blood Loss as Assessed by the Operating Surgeon [Time Frame: Day 0 (at completion of surgery)]

TABLE 12

Primary Outcome #2

Measure Type	Secondary
Measure Title	Intraoperative Actual Versus Predicted Blood Loss as
	Assessed by the Operating Surgeon
Measure	The predicted blood loss was estimated preoperatively by
Description	the operating surgeon based on a hemostatically normal
	individual of the same sex, age, stature and co-morbidities
	as the participant.
	The actual blood loss was assessed consisting of the
	estimated blood loss, including into swabs, towels and
	suction during the procedure, per the anesthesiologist's
	record.
Time Frame	Day 0 (at completion of surgery)

Population Description Outcome #2

For predicted blood loss the number of participants analyzed was 14 as for one participant (included in the major surgery reporting group) the predicted blood loss was not collected. The full analysis data set, including all participants who received investigational product and had at least 1 hemostatic assessment, was used for analysis.

TABLE 13

Reporting Groups Outcome #2

TABLE 26

Measured Values Outcome #2

Recombinant
Von				Von	Von	Von	Von	Von
Willebrand				Willebrand	Willebrand	Willebrand	Willebrand	Willebrand
Factor	Minor	Major	Oral	Disease	Disease	Disease	Disease	Disease
(rVWF)	Surgery	Surgery	Surgery	Type	1	Type 2A	Type 2B	Type 2M	Type	3

Participants Analyzed

	15	4	10	1	3	2	1	1	8

Intra-

operative

Actual

Versus

Predicted

BloodLoss

as Assessed

by the

Operating

Surgeon

[Units: mL]

Mean

(Standard

Deviation)

Actual

blood loss

Participants Analyzed

	15	4	10	1	3	2	1	1	8

Actual	94.3	(177.88)	0.0	(0.00)	127.0	(209.27)	145.0	[1]	115.0	(103.32)	42.5	(53.03)	50.0	[1]	50.0	[1]	110.6	(240.87)
blood
loss
Predicted
blood
loss

Participants Analyzed

	14	4	9	1	3	1	1	1	8

Predicted	106.1	(161.82)	2.5	(5.00)	152.8	(186.33)	100.0	[1]	100.0	(100.00)	10.0	[1]	50.0	[1]	50.0	[1]	134.4	(206.46)
blood
loss

[1] No Standard Deviation Possible as Only One Participant was Analyzed.

No Statistical Analysis Provided for Intraoperative Actual Versus Predicted Blood Loss as Assessed by the Operating Surgeon

Secondary Outcome: Outcome #3

3. Secondary: Intraoperative Actual Blood Loss Relative to Predicted Blood Loss [Time Frame: Day 0 (at completion of surgery)]

TABLE 14

Outcome #3

Measure Type	Secondary
Measure Title	Intraoperative Actual Blood Loss Relative to
	Predicted Blood Loss
Measure Description	Actual blood loss relative to predicted blood loss
	was calculated as [Actual Blood loss (mL)]
	divided by [Predicted Blood Loss (mL)
	multiplied by 100.
Time Frame	Day 0 (at completion of surgery)

Population Description Outcome #3

Number of participants analyzed was 11, as for 3 participants the actual and the predicted blood loss was zero and for 1 participant the predicted blood loss was not collected. Therefore ‘actual blood loss relative to predicted blood loss’ could not be calculated. The full analysis data set was used for the analysis of this outcome measure.

TABLE 15

Reporting Groups Outcome #3

	Description

Recombinant Von Willebrand	Surgery participants treated with
Factor (rVWF)	Recombinant von Willebrand Factor
	(rVWF)
Minor Surgery	All participants who underwent minor
	surgery.
Major Surgery	All participants who underwent major
	surgery.
Oral Surgery	All participants who underwent oral
	surgery.
Von Willebrand Disease	All participants with von Willebrand
Type
1	Disease Type 1.
Von Willebrand Disease	All participants with von Willebrand
Type
2A	Disease Type	2A.
Von Willebrand Disease	All participants with von Willebrand
Type
2B	Disease Type	2B.
Von Willebrand Disease	All participants with von Willebrand
Type
2M	Disease Type	2M.
Von Willebrand Disease	All participants with von Willebrand
Type
3	Disease Type 3.

TABLE 16

Measured Values

Recombinant				Von	Von	Von	Von	Von
Von				Willebrand	Willebrand	Willebrand	Willebrand	Willebrand
Willebrand	Minor	Major	Oral	Disease	Disease	Disease	Disease	Disease
Factor (rVIVF)	Surgery	Surgery	Surgery	Type	1	Type 2A	Type 2B	Type 2M	Type	3

Participants Analyzed

	11	1	9	1	2	1	1	1	6

Intraoperative

69.6

(44.77)

0.0 ^[1]

68.9

(34.48)

145.0 ^[1]

122.5

(31.82)

50.0 ^[1]

100.0 ^[1]

45.0

(38.92)

Actual Blood Loss

Relative to Predicted

Blood Loss

[Units: Percent]

Mean (Standard

Deviation)

^[1] No standard deviation possible as only one participant was analyzed.

No Statistical Analysis Provided for Intraoperative Actual Blood Loss Relative to Predicted Blood Loss

Secondary Outcome: Outcome #4

4. Secondary: Intraoperative Actual Versus Predicted Blood Loss Score as Assessed by the Operating Surgeon [Time Frame: Day 0 (at completion of surgery)]

TABLE 17

Outcome #4

Measure Type	Secondary
Measure Title	Intraoperative Actual Versus Predicted Blood Loss
	Score as Assessed by the Operating Surgeon
Measure Description	Hemostatic efficacy was rated on a scale of
	excellent - good - moderate - none.
	Excellent: Intraoperative blood loss was less than
	or equal to the maximum blood loss expected for the
	type of procedure performed in a hemostatically
	normal subject (≤100%).
	Good: Intraoperative blood loss was up to 50% more
	than the maximum expected blood loss for the type
	of procedure performed in a hemostatically normal
	subject (101-150%) Moderate: Intraoperative blood
	loss was more than 50% of the maximum expected
	blood loss for the type of procedure performed in a
	hemostatically normal subject (>150%).
	None : Uncontrolled hemorrhage that was the result
	of inadequate therapeutic response despite proper
	dosing, necessitating a change of clotting factor
	replacement regimen.
Time Frame	Day 0 (at completion of surgery)

TABLE 18

Population Description Outcome #4

Number of participants with major, minor and oral surgery and number
of participant with Von Willebrand Type 1, 2A, 2B, 2M and 3 do sum up
to the overall number of participants analyzed. The full analysis data set,
including all participants who received investigational product and have at
least 1 hemostatic assessment, was used for analysis.

TABLE 19

Reporting Groups Outcome #4

	Description

Recombinant Von Willebrand Factor	Surgery participants treated with
(rVWF)	Recombinant von Willebrand Factor
	(rVWF)
Minor Surgery	All participants who underwent minor
	surgery.
Major Surgery	All participants who underwent major
	surgery.
Oral Surgery	All participants who underwent oral
	surgery.
Von Willebrand Disease Type 1	All participants with von Willebrand
	Disease Type 1.
Von Willebrand Disease Type 2A	All participants with von Willebrand
	Disease Type 2A.
Von Willebrand Disease Type 2B	All participants with von Willebrand
	Disease Type 2B.
Von Willebrand Disease Type 2M	All participants with von Willebrand
	Disease Type 2M.
Von Willebrand Disease Type 3	All participants with von Willebrand
	Disease Type 3.

TABLE 20

Measured Values Outcome #4

Recombinant				Von	Von	Von	Von	Von
Von				Willebrand	Willebrand	Willebrand	Willebrand	Willebrand
Willebrand	Minor	Major	Oral	Disease	Disease	Disease	Disease	Disease
Factor (rVNVF)	Surgery	Surgery	Surgery	Type 1	Type 2A	Type 2B	Type 2M	Type 3

Participants Analyzed

	15	4	10	1	3	2	1	1	8

Intraoperative
Actual Versus
Predicted Blood
Loss Score as
Assessed by the
Operating Surgeon
[Units: Participants]
Count of Participants
Excellent	13	86.7%	4	100.0%	8	80.0%	1	100.0%	3	100.0%	1	50.0%	1	100.0%	1	100.0%	7	87.5%
Good	2	13.3%	0	0.0%	2	20.0%	0	0.0%	0	0.0%	1	50.0%	0	0.0%	0	0.0%	1	12.5%
Moderate	0	0.0%	0	0.0%	0	0.0%	0	0.0%	0	0.0%	0	0.0%	0	0.0%	0	0.0%	0	0.0%
None	0	0.0%	0	0.0%	0	0.0%	0	0.0%	0	0.0%	0	0.0%	0	0.0%	0	0.0%	0	0.0%

No Statistical Analysis Provided for Intraoperative Actual Versus Predicted Blood Loss Score as Assessed by the Operating Surgeon

Secondary Outcome: Outcome #5

5. Secondary: Intraoperative Hemostatic Efficacy Score as Assessed by the Operating Surgeon [Time Frame: Day 0 (at completion of surgery)]

TABLE 21

Outcome #5

Measure Type	Secondary
Measure Title	Intraoperative Hemostatic Efficacy Score as Assessed
	by the Operating Surgeon
Measure Description	Hemostatic efficacy was rated on a scale of
	excellent - good - moderate - none.
	Excellent: Intraoperative hemostasis achieved with
	rVWF with our without ADVATE was as good or
	better than that expected for the type of surgical
	procedure performed in a hemostatically normal
	subject.
	Good: Intraoperative hemostasis achieved with
	rVWF with or without ADVATE was probably
	as good as that expectedfor the type of surgical
	procedure performed in a hemostatically
	normal subject.
	Moderate: Intraoperative hemostasis with rVWF
	with or without ADVATE was clearly less than
	optimal for the type of procedure performed but
	was maintained without the need to change the
	rVWF concentrate.
	None: Participant experienced uncontrolled bleeding
	that was the result of inadequate therapeutic response
	despite proper dosing, necessitating a change
	of rVWF concentrate.
Time Frame	Day 0 (at completion of surgery)

TABLE 22

Population Description Outcome #5

Number of participants with major, minor and oral surgery and number
of participant with Von Willebrand Type 1, 2A, 2B, 2M and 3 do sum up
to the overall number of participants analyzed. The full analysis data set,
including all participants who received investigational product and have at
least 1 hemostatic assessment, was used for analysis.

TABLE 23

Reporting Groups Outcome #5

TABLE 24

MeasuredValues Outcome #5

Participants Analyzed

	15	4	10	1	3	2	1	1	8

Intraoperative
Hemostatic
Efficacy Score as
Assessed by the
Operating Surgeon
[Units: Participants]
Count of Participants
Excellent	13	86.7%	4	100.0%	8	80.0%	1	100.0%	3	100.0%	1	50.0%	1	100.0%	1	100.0%	7	87.5%
Good	2	13.3%	0	0.0%	2	20.0%	0	0.0%	0	0.0%	1	50.0%	0	0.0%	0	0.0%	1	12.5%
Moderate	0	0.0%	0	0.0%	0	0.0%	0	0.0%	0	0.0%	0	0.0%	0	0.0%	0	0.0%	0	0.0%
None	0	0.0%	0	0.0%	0	0.0%	0	0.0%	0	0.0%	0	0.0%	0	0.0%	0	0.0%	0	0.0%

No Statistical Analysis Provided for Intraoperative Hemostatic Efficacy Score as Assessed by the Operating Surgeon

Secondary Outcome: Outcome #6

6. Secondary: Daily Intra- and Postoperative Weight-adjusted Dose of rVWF With or Without ADVATE [Time Frame: Daily, from day of surgery through postoperative Day 14 (±2 days)]

TABLE 25

Outcome #6

Measure Type	Secondary
Measure Title	Daily Intra- and Postoperative Weight-adjusted
	Dose of rVWF With or Without ADVATE
Measure Description	No text entered.
Time Frame	Daily, from day of surgery through postoperative
	Day 14 (±2 days)

TABLE 25

Population Description Outcome #6

Number of participants analyzed was different for the time points
according to individual treatment. The full analysis data set, including
all participants who received investigational product and have at least 1
hemostatic assessment, was used for analysis.

TABLE 40

Reporting Groups Outcome #6

	Description

Recombinant Von Willebrand	Surgery participants treated with
Factor (rVWF)	Recombinant von Willebrand Factor
	(rVWF)

TABLE 26

Measured Values Outcome #6

	Recombinant Von Willebrand
	Factor (rVVVF)

Participants Analyzed	15
Daily Intra- and Postoperative Weight-
adjusted Dose of rVWF With or
Without ADVATE
[Units: IU/kg]
Median (Inter-Quartile Range)
intraoperative
Participants Analyzed	1
intraoperative	18.1
	(18.1 to 18.1)
postoperative day 1
Participants Analyzed	3
postoperative day 1	23.5
	(16.9 to 47.2)
postoperative day 2
Participants Analyzed	11
postoperative day 2	42.3
	(23.2 to 50.6)
postoperative day 3
Participants Analyzed	12
postoperative day 3	28.6
	(20.6 to 48.9)
postoperative day 4
Participants Analyzed	9
postoperative day 4	33.9
	(23.2 to 44.3)
postoperative day 5
Participants Analyzed	7
postoperative day 5	31.5
	(18.8 to 47.2)
postoperative day 6
Participants Analyzed	5
postoperative day 6	23.2
	(18.8 to 23.6)
postoperative day 7
Participants Analyzed	5
postoperative day 7	23.8
	(23.6 to 50.8)
postoperative day 8
Participants Analyzed	7
postoperative day 8	33.9
	(23.6 to 53.6)
postoperative day 9
Participants Analyzed	3
postoperative day 9	23.6
	(16.3 to 53.6)
postoperative day 10
Participants Analyzed	3
postoperative day 10	23.6
	(16.3 to 34.8)
postoperative day 11
Participants Analyzed	3
postoperative day 11	23.6
	(16.3 to 53.6)
postoperative day 12
Participants Analyzed	4
postoperative day 12	29.3
	(20.1 to 44.2)
postoperative day 13
Participants Analyzed	1
postoperative day 13	16.3
	(16.3 to 16.3)
postoperative day 14
Participants Analyzed	2
postoperative day 14	25.5
	(16.3 to 34.8)
postoperative day 15
Participants Analyzed	1
postoperative day 15	16.3
	(16.3 to 16.3)

No Statistical Analysis Provided for Daily Intra- and Postoperative Weight-Adjusted Dose of rVWF with or without ADVATE

Secondary Outcome: Outcome #7

7. Secondary: Occurrence of Adverse Events [Time Frame: From first infusion of investigational product through study completion (i.e., 14 (±2) days post-surgery)].

TABLE 27

Outcome #7

Measure Type	Secondary
Measure Title	Occurrence of Adverse Events
Measure Description	Treatment emergent adverse events (TEAEs)
	and treatment emergent serious adverse events
	(TESAEs) was evaluated.
Time Frame	From first infusion of investigational product
	through study completion (i.e., 14 (±2) days
	post-surgery)

TABLE 28

Population Description Outcome #7

The safety analysis data set, including all participants who received
any amount of investigational product, was used for analysis of this
outcome measure.

TABLE 29

Reporting Groups Outcome #7

TABLE 30

Measured Values

		Recombinant Von
		Willebrand Factor
		(rVWF)

	Participants Analyzed	15
	Occurrence of Adverse Events
	[Units: Adverse Events]
	Treatment emergent Adverse Events	12
	(TEAEs)
	Severe TEAEs	1
	TEAEs related to rVWF	0
	TEAEs related to ADVATE	0
	TEAEs related to both rVWF	0
	and ADVATE
	Treatment emergent Serious	2
	Adverse Events (TESAEs)
	TESAEs related to rVWF	0
	TESAEs related to ADVATE	0
	TESAEs related to both rVWF and	0
	ADVATE
	TEAEs leading to discontinuation F	0
	of rVW
	TEAEs leading to discontinuation of	0
	ADVATE
	TEAEs leading to discontinuation	0
	of study
	TEAEs leading to death	0
	TEAEs related to study procedure	0
	TESAEs related to study procedure	0

No statistical analysis provided for Occurrence of Adverse Events

Secondary Outcome: Outcome #8

8. Secondary: Occurrence of Thrombotic Events [Time Frame: From first infusion of investigational product through study completion (i.e., 14 (±2) days post-surgery)]

TABLE 31

Outcome #8

	Measure Type	Secondary
	Measure Title	Occurrence of Thrombotic Events
	Measure Description	Treatment emergent adverse
		events (TEAEs) and treatment
		emergent serious adverse events
		(TESAEs) were evaluated for
		thrombotic events.
	Time Frame	From first infusion of investigational
		product through study
		completion (i.e., 14 (±2) days post-surgery)

TABLE 32

Population Description Outcome #8

The safety analysis data set, including all
participants who received any amount of
investigational product, was used for analysis of this outcome measure

TABLE 33

Reporting Groups

TABLE 34

Measured Values Outcome #8

Recombinant Von

Willebrand Factor (rVWF)

Participants Analyzed 15

Occurrence of Thrombotic Events

[Units: Adverse Events]

Thrombotic TEAEs 2

Thrombotic TESAEs 1

No statistical analysis provided for Occurrence of Thrombotic Events

Secondary Outcome: Outcome #9

9. Secondary: Occurrence of Severe Allergic Reactions (e.g., Anaphylaxis) [Time Frame: From first infusion of investigational product through study completion (i.e., 14 (±2) days post-surgery)]

TABLE 35

Outcome #9

	Measure Type	Secondary
	Measure Title	Occurrence of Severe Allergic
		Reactions (e.g., Anaphylaxis)
	Measure Description	Treatment emergent adverse
		events (TEAEs) and treatment
		emergent serious adverse events
		(TESAEs) were evaluated for
		severe allergic reactions.
	Time Frame	From first infusion of investigational
		product through study
		completion (i.e., 14 (±2) days post-surgery)

TABLE 36

Population Description Outcome #9

The safety analysis data set, including all
participants who received any amount of
investigational product, was used for analysis of this outcome measure.

TABLE 37

Reporting Groups Outcome #9

TABLE 38

Measured Values Outcome #9

		Recombinant Von
		Willebrand Factor
		(rVWF)

	Participants Analyzed	15
	Occurrence of Severe Allergic Reactions (e.g.,
	Anaphylaxis)
	[Units: Adverse Events]
	Severe allergic reaction TEAEs	0
	Severe allergic reaction TESAEs	0

No statistical analysis provided for Occurrence of Severe Allergic Reactions (e.g., Anaphylaxis)

Secondary Outcome: Outcome #10

10. Secondary: Number of Participants Who Developed Inhibitory and Total Binding Antibodies to Von Willebrand Factor (VWF) and Inhibitory Antibodies to Factor VIII (FVIII) [Time Frame: Testing occurred throughout the study at screening, prior PK infusion, pre-surgery, post-surgery in case of excessive bleeding or unexplained bleeding, at postoperative day 7 and at study completion visit (ie. 14 (±2) days post-surgery).]

TABLE 39

Population Description Outcome #10

Measure Type	Secondary

Measure Title	Number of Participants Who
	Developed Inhibitory and Total
	Binding Antibodies to Von Willebrand
	Factor (VWF) and Inhibitory
	Antibodies to Factor VIII (FVIII)
Measure	Participants were treated with
Description	recombinant van Willebrand Factor
	(rVWF) with or without ADVATE.
Time Frame	Testing occurred throughout the
	study at screening, prior PK
	infusion, pre-surgery, post-surgery
	in case of excessive bleeding or
	unexplained bleeding, at postoperative day 7 and at study
	completion visit (ie. 14 (±2) days post-surgery).

TABLE 40

Population Description Outcome #10

The safety analysis data set, including all
participants who received any amount of
investigational product, was used for analysis of this outcome measure.

TABLE 41

Reporting Groups Outcome #10

TABLE 55

Measured Values Outcome #10

	Recombinant Von
	Willebrand Factor (rVWF)

Participants Analyzed	15
Number of Participants Who Developed
Inhibitory and Total Binding Antibodies
to Von Willebrand Factor (VWF) and
Inhibitory Antibodies to Factor VIII (FVIII)
[Units: Participants]
Count of Participants
Development of inhibitory antibodies to	0
VWF
Development of total binding antibodies to	1
VWF
Development of inhibitory antibodies to	0
FVIII

No Statistical Analysis Provided for Number of Participants Who Developed Inhibitory and Total Binding Antibodies to Von Willebrand Factor (VWF) and Inhibitory Antibodies to Factor VIII (FVIII)

Secondary Outcome: Outcome #11
11. Secondary: Number of Participants Who Developed Antibodies to Chinese Hamster Ovary (CHO) Proteins, Mouse Immunoglobulin G (IgG) or Recombinant Furin (rFurin) [Time Frame: Testing occurred throughout the study at screening, prior PK infusion, pre-surgery, post-surgery in case of excessive bleeding or unexplained bleeding, at postoperative day 7 and at study completion visit (ie. 14 (±2) days post-surgery).]

TABLE 42

Outcome #11

Measure	Secondary
Type
Measure	Number of Participants Who Developed Antibodies to
Title	Chinese Hamster Ovary (CHO) Proteins, Mouse
	Immunoglobulin G (IgG) or Recombinant Furin
	(rFurin)
Measure	Participants were treated with recombinant von
Description	Willebrand Factor (rVWF) with or without ADVATE.
Time Frame	Testing occurred throughout the study at screening, prior
	PK infusion, pre-surgery, post-surgery in case of excessive
	bleeding or unexplained bleeding, at postoperative day 7
	and at study completion visit (ie. 14 (±2) days post-
	surgery).

TABLE 43

Population Description Outcome #11

	The safety analysis data set, including all participants who received
	any amount of investigational product, was used for analysis of this
	outcome measure.

TABLE 44

Reporting Groups Outcome #11

	Description

Recombinant Von	Surgery participants treated with
Willebrand Factor (rVWF)	Recombinant von Willebrand Factor
	(rVWF)

TABLE 59

Measured Values Outcome #11

	Recombinant Von
	Willebrand Factor (rVWF)

Participants Analyzed	15
Number of Participants Who Developed	0
Antibodies to Chinese Hamster Ovary
(CHO) Proteins, Mouse Immunoglobulin G
(IgG) or Recombinant Furin (rFurin)
[Units: Participants]
Count of Participants

No Statistical Analysis Provided for Number of Participants Who Developed Antibodies to Chinese Hamster Ovary (CHO) Proteins, Mouse Immunoglobulin G (IgG) or Recombinant Furin (rFurin)

Secondary Outcome: Outcome #12
12. Secondary: Pharmacokinetics: Area Under the Plasma Concentration Versus Time Curve From 0 to 72 Hours Post-infusion (AUC 0-72 h/Dose) [Time Frame: PK measurements were done within 30 minutes pre-infusion, and post infusion at 30 (±5) minutes, 60 (±5) minutes, 6 (±1) hours, 12 (±1) hours, 24 (±2) hours, 48 (±2) hours and 72 (±2) hours.]

TABLE 45

Outcome #12

Measure	Secondary
Type
Measure	Pharmacokinetics: Area Under the Plasma Concentration
Title	Versus Time Curve From 0 to 72 Hours Post-infusion
	(AUC 0-72 h/Dose)
Measure	This assessment was only required for subjects undergoing
Description	major surgery. Subjects received a PK infusion at a dose of
	50 ± 5 IU/kg rVWF: RCo within 42 days prior to surgery.
	The area under the plasma concentration/time curve from 0
	to 72 hours post-infusion was computed using the linear
	trapezoidal rule. For the calculation of AUC(0-72 h) the
	levels at 72 hours was linearly interpolated/extrapolated
	from the 2 nearest sampling time points.
	PK analysis was performed for the following analytes:
	VWF Ristocetin Cofactor Activity (VWF: RCo), VWF
	Antigen Activity (VWF: Ag), VWF Collagen Binding
	Activity (VWF: CB), VWF Activity Measured
	INNOVANCE VWF Ac Assay (VWF: Ac),
	FVIII Coagulation Activity (FVIII: C)
Time Frame	PK measurements were done within 30 minutes pre-
	infusion, and post infusion at 30 (±5) minutes, 60 (±5)
	minutes, 6 (±1) hours, 12 (±1) hours, 24 (±2) hours,
	48 (±2) hours and 72 (±2) hours.

TABLE 46

Population Description Outcome #12

	The PK analysis data set, including all participants who underwent
	PK assessment with data collected at the relevant time points,
	was used for analysis of this outcome measure.

TABLE 47

Reporting Groups Outcome #12

TABLE 48

Measured Values Outcome #12

	Recombinant Von
	Willebrand Factor (rVWF)

Participants Analyzed	11
Pharmacokinetics: Area Under the Plasma
Concentration Versus Time Curve From 0 to
72 Hours Post-infusion (AUC 0-72 h/Dose)
[Units: hours*IU/dL]
Geometric Mean (Geometric Coefficient
of Variation)
VWF: RCo
Participants Analyzed	11
VWF: RCo	31.91 (37.5%)
VWF: Ag
Participants Analyzed	11
VWF: Ag	57.08 (25.6%)
VWF: CB
Participants Analyzed	11
VWF: CB	63.91 (29.4%)
VWF: Ac
Participants Analyzed	11
VWF: Ac	54.61 (28.1%)
FVIII: C
Participants Analyzed	5
FVIII: C	67.49 (31.1%)

No Statistical Analysis Provided for Pharmacokinetics: Area Under the Plasma Concentration Versus Time Curve from 0 to 72 Hours Post-Infusion (AUC 0-72 h/Dose)

Secondary Outcome: Outcome #13

13. Secondary: Pharmacokinetics: Area Under the Plasma Concentration Versus Time Curve From Time 0 to Infinity (AUC 0-Go/Dose) [Time Frame: PK measurements were done within 30 minutes pre-infusion, and post infusion at 30 (±5) minutes, 60 (±5) minutes, 6 (±1) hours, 12 (±1) hours, 24 (±2) hours, 48 (±2) hours and 72 (±2) hours.]

TABLE 49

Outcome #13

Measure	Secondary
Type
Measure	Pharmacokinetics: Area Under the Plasma Concentration
Title	Versus Time Curve From Time 0 to Infinity (AUC 0-∞/
	Dose)
Measure	This assessment was only required for subjects undergoing
Description	major surgery. Subjects received a PK infusion at a dose of
	50 ± 5 IU/kg rVWF: RCo within 42 days prior to surgery.
	The area under the plasma concentration/time curve from
	time 0 to infinity and the area under the first moment curve
	from time 0 to infinity was calculated as the sum of AUC
	or AUMC from time 0 to the time of last quantifiable
	concentration plus a tail area correction calculated
	as Ct/λz and Ct/λz(t + 1/λz), respectively, where Ct
	was the last quantifiable concentration, t was the time of
	last quantifiable concentration and λz was the terminal or
	disposition rate constant.
	PK analysis was performed for the following analytes:
	VWF Ristocetin Cofactor Activity (VWF: RCo), VWF
	Antigen Activity (VWF: Ag), VWF Collagen Binding
	Activity (VWF: CB), VWF Activity Measured
	INNOVANCE VWF Ac Assay (VWF: Ac), FVIII
	Coagulation Activity (FVIII: C)
Time Frame	PK measurements were done within 30 minutes pre-
	infusion, and post infusion at 30 (±5) minutes, 60 (±5)
	minutes, 6 (±1) hours, 12 (±1) hours, 24 (±2) hours,
	48 (±2) hours and 72 (±2) hours.

TABLE 50

Population Description Outcome #13

TABLE 51

Reporting Groups Outcome #13

TABLE 52

Measured Values

	Recombinant Von
	Willebrand Factor (rVWF)

Participants Analyzed	11
Pharmacokinetics: Area Under the Plasma
Concentration Versus Time Curve From
Time 0 to Infinity (AUC 0-∞/Dose)
[Units: hours*IU/dL]
Geometric Mean (Geometric Coefficient
of Variation)
VWF: RCo
Participants Analyzed	11
VWF: RCo	34.43 (43.3%)
VWF: Ag
Participants Analyzed	11
VWF: Ag	68.87 (31.5%)
VWF: CB
Participants Analyzed	11
VWF: CB	71.82 (34.1%)
VWF: Ac
Participants Analyzed	11
VWF: Ac	61.90 (32.2%)
FVIII: C
Participants Analyzed	3
FVIII: C	75.00 (30.9%)

No Statistical Analysis Provided for Pharmacokinetics: Area Under the Plasma Concentration Versus Time Curve from Time 0 to Infinity (AUC 0-∞/Dose)

Secondary Outcome: Outcome #14

14. Secondary: Pharmacokinetics: Mean Residence Time (MRT) [Time Frame: PK measurements were done within 30 minutes pre-infusion, and post infusion at 30 (±5) minutes, 60 (±5) minutes, 6 (±1) hours, 12 (±1) hours, 24 (±2) hours, 48 (±2) hours and 72 (±2) hours.]

TABLE 53

Outcome #14

Measure	Secondary
Type
Measure	Pharmacokinetics: Mean Residence Time (MRT)
Title
Measure	This assessment was only required for subjects undergoing
Description	major surgery. Subjects received a PK infusion at a dose of
	50 ± 5 IU/kg rVWF: RCo within 42 days prior to surgery.
	Mean residence time was calculated as area under the first
	moment curve from time 0 to infinity divided by the area
	under the curve time 0 to infinity minus T/2 where T was
	the duration of the infusion.
	PK analysis was performed for the following analytes:
	VWF Ristocetin Cofactor Activity (VWF: RCo), VWF
	Antigen Activity (VWF: Ag), VWF Collagen Binding
	Activity (VWF: CB), VWF Activity Measured
	INNOVANCE VWF Ac Assay (VWF: Ac)
Time Frame	PK measurements were done within 30 minutes pre-
	infusion, and post infusion at 30 (±5) minutes, 60 (±5)
	minutes, 6 (±1) hours, 12 (±1) hours, 24 (±2) hours,
	48 (±2) hours and 72 (±2) hours.

TABLE 54

Population Description Outcome #14

TABLE 55

Reporting Groups Outcome #14

TABLE 56

Measured Values

	Recombinant Von
	Willebrand Factor (rVWF)

Participants Analyzed	11
Pharmacokinetics: Mean Residence Time
(MRT)
[Units: Hours]
Geometric Mean (Geometric Coefficient
of Variation)
VWF: RCo	22.69 (41.3%)
VWF: Ag	37.92 (28.4%)
VWF: CB	29.35 (31.1%)
VWF: Ac	29.75 (28.6%)

No Statistical Analysis Provided for Pharmacokinetics: Mean Residence Time (MRT)

Secondary Outcome: Outcome #15

15. Secondary: Pharmacokinetics: Clearance (CL) [Time Frame: PK measurements were done within 30 minutes pre-infusion, and post infusion at 30 (±5) minutes, 60 (±5) minutes, 6 (±1) hours, 12 (±1) hours, 24 (±2) hours, 48 (±2) hours and 72 (±2) hours.]

TABLE 57

Outcome #15

Measure	Secondary
Type
Measure	Pharmacokinetics: Clearance (CL)
Title
Measure	This assessment was only required for subjects undergoing
Description	major surgery. Subjects received a PK infusion at a dose of
	50 ± 5 IU/kg rVWF: RCo within 42 days prior to surgery.
	Clearance was calculated as dose (IU/kg) divided by the
	area under the curve time 0 to infinity.
	PK analysis was performed for the following analytes:
	VWF Ristocetin Cofactor Activity (VWF: RCo), VWF
	Antigen Activity (VWF: Ag), VWF Collagen Binding
	Activity (VWF: CB), VWF Activity Measured
	INNOVANCE VWF Ac Assay (VWF: Ac)
Time Frame	PK measurements were done within 30 minutes pre-
	infusion, and post infusion at 30 (±5) minutes, 60 (±5)
	minutes, 6 (±1) hours, 12 (±1) hours, 24 (±2) hours,
	48 (±2) hours and 72 (±2) hours.

TABLE 58

Population Description Outcome #15

TABLE 59

Reporting Groups Outcome #15

TABLE 60

Measured Values

	Recombinant Von
	Willebrand Factor (rVWF)

Participants Analyzed	11
Pharmacokinetics: Clearance (CL)
[Units: dL/hour/kg]
Geometric Mean (Geometric Coefficient
of Variation)
VWF: RCo	0.02904 (43.3%)
VWF: Ag	0.01452 (31.5%)
VWF: CB	0.01392 (34.1%)
VWF: Ac	0.01616 (32.2%)

No Statistical Analysis Provided for Pharmacokinetics: Clearance (CL)

Secondary Outcome: Outcome #16

16. Secondary: Pharmacokinetics: Incremental Recovery (IR) [Time Frame: PK measurements were done within 30 minutes pre-infusion, and post infusion at 30 (±5) minutes, 60 (±5) minutes, 6 (±1) hours, 12 (±1) hours, 24 (±2) hours, 48 (±2) hours and 72 (±2) hours.]

TABLE 61

Outcome #16

Measure	Secondary
Type
Measure	Pharmacokinetics: Incremental Recovery (IR)
Title
Measure	This assessment was only required for subjects undergoing
Description	major surgery. Subjects received a PK infusion at a dose of
	50 ± 5 IU/kg rVWF: RCo within 42 days prior to surgery.
	Incremental recovery was calculated as (Cmax minus
	Cpreinfusion) divided by the dose (IU/kg) where kg refers
	to the body weight at the time of dosing and Cmax was
	the observed maximum concentration before correction for
	pre-infusion values.
	PK analysis was performed for the following analytes:
	VWF Ristocetin Cofactor Activity (VWF: RCo), VWF
	Antigen Activity (VWF: Ag), VWF Collagen Binding
	Activity (VWF: CB), VWF Activity Measured
	INNOVANCE VWF Ac Assay (VWF: Ac)
Time Frame	PK measurements were done within 30 minutes pre-
	infusion, and post infusion at 30 (±5) minutes, 60 (±5)
	minutes, 6 (±1) hours, 12 (±1) hours, 24 (±2) hours,
	48 (±2) hours and 72 (±2) hours.

TABLE 77

Population Description Outcome #16

TABLE 62

Reporting Groups Outcome #16

TABLE 63

Measured Values Outcome #16

	Recombinant Von
	Willebrand Factor (rVWF)

Participants Analyzed	11
Pharmacokinetics: Incremental Recovery
(IR)
[Units: IU/dL]
Mean (Standard Deviation)
VWF: RCo	1.961 (0.45445)
VWF: Ag	1.991 (0.38395)
VWF: CB	2.780 (0.56640)
VWF: Ac	2.635 (0.38050)

No Statistical Analysis Provided for Pharmacokinetics: Incremental Recovery (IR)

Secondary Outcome: Outcome #17

17. Secondary: Pharmacokinetics: Elimination Phase Half-life (T1/2) [Time Frame: PK measurements were done within 30 minutes pre-infusion, and post infusion at 30 (±5) minutes, 60 (±5) minutes, 6 (±1) hours, 12 (±1) hours, 24 (±2) hours, 48 (±2) hours and 72 (±2) hours.]

TABLE 64

Outcome #17

Measure	Secondary
Type
Measure	Pharmacokinetics: Elimination Phase Half-life (T½)
Title
Measure	This assessment was only required for subjects undergoing
Description	major surgery. Subjects received a PK infusion at a dose of
	50 ± 5 IU/kg rVWF: RCo within 42 days prior to surgery.
	Terminal or disposition half-life (T½) was calculated
	as ln2/λz where λz was the terminal
	elimination rate constant as calculated in WinNonlin
	NCA using at least three quantifiable concentrations.
	PK analysis was performed for the following analytes:
	VWF Ristocetin Cofactor Activity (VWF: RCo),
	VWF Antigen Activity (VWF: Ag), VWF Collagen
	Binding Activity (VWF: CB), VWF Activity Measured
	INNOVANCE VWF Ac Assay (VWF: Ac)
Time Frame	PK measurements were done within 30 minutes pre-
	infusion, and post infusion at 30 (±5) minutes, 60 (±5)
	minutes, 6 (±1) hours, 12 (±1) hours, 24 (±2) hours,
	48 (±2) hours and 72 (±2) hours.

TABLE 65

Population Description Outcome #17

TABLE 66

Reporting Groups Outcome #17

TABLE 66

Measured Values

	Recombinant Von Willebrand
	Factor (rVWF)

Participants Analyzed	11
Pharmacokinetics: Elimination Phase
Half-life (T½)
[Units: Hours]
Geometric Mean (Geometric Coefficient
of Variation)
VWF: RCo	16.52 (42.7%)
VWF: Ag	26.88 (26.5%)
VWF: CB	21.07 (33.2%)
VVVF: Ac	22.19 (28.5%)

No Statistical Analysis Provided for Pharmacokinetics: Elimination Phase Half-Life (T1/2)

Secondary Outcome: Outcome #18

18. Secondary: Pharmacokinetics: Volume of Distribution at Steady State (Vss) [Time Frame: PK measurements were done within 30 minutes pre-infusion, and post infusion at 30 (±5) minutes, 60 (±5) minutes, 6 (±1) hours, 12 (±1) hours, 24 (±2) hours, 48 (±2) hours and 72 (±2) hours.]

TABLE 67

Outcome #18

Measure Type	Secondary
Measure Title	Pharmacokinetics: Volume of Distribution at Steady State (Vss)
Measure Description	This assessment was only required for subjects undergoing major
	surgery. Subjects received a PK infusion at a dose of 50 ± 5 IU/kg
	rVWF:RCo within 42 days prior to surgery. Vss was calculated as
	the clearance multiplied with the mean residence time.
	PK analysis was performed for the following analytes:
	VWF Ristocetin Cofactor Activity (VWF: RCo), VWF Antigen
	Activity (VWF: Ag), VWF Collagen Binding Activity (VWF: CB),
	VWF Activity Measured INNOVANCE VWF Ac Assay (VWF:Ac)
Time Frame	PK measurements were done within 30 minutes pre-infusion, and
	post infusion at 30 (±5) minutes, 60 (±5) minutes, 6 (±1) hours, 12
	(±1) hours, 24 (±2) hours, 48 (±2) hours and 72 (±2) hours.

TABLE 68

Population Description Outcome #18

The PK analysis data set, including all participants who underwent PK
assessment with data collected at the relevant time points, was used
for analysis of this outcome measure.

TABLE 69

Reporting Groups Outcome #18

		Description

	Recombinant	Surgery participants treated
	Von Willebrand	with Recombinant von Willebrand
	Factor (rVWF)	Factor (rVWF)

TABLE 69

Measured Values Outcome #18

	Recombinant Von
	Willebrand
	Factor (rVWF)

Participants Analyzed	11
Pharmacokinetics: Volume of Distribution at
Steady State (Vss)
[Units: dL/kg]
Geometric Mean (Geometric Coefficient of Variation)
VWF: RCo	0.6591 (28.8%)
VWF: Ag	0.5506 (18.4%)
VWF: CB	0.4086 (24.0%)
VVVF: Ac	0.4806 (21.5%)

No Statistical Analysis Provided for Pharmacokinetics: Volume of Distribution at Steady State (Vss)

The examples set forth above are provided to give those of ordinary 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 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 persons of skill in the art are intended to be within the scope of the following claims. All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.
All headings and section designations are used for clarity and reference purposes only and are not to be considered limiting in any way. For example, those of skill in the art will appreciate the usefulness of combining various aspects from different headings and sections as appropriate according to the spirit and scope of the invention described herein.
All references cited herein are hereby incorporated by reference herein in their entireties 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.
Many modifications and variations of this application can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments and examples described herein are offered by way of example only, and the application is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which the claims are entitled.

Claims

1. A method for pre-treatment of a subject with severe von Willebrand disease (VWD) prior to surgery, wherein said pre-treatment comprises administering 40-60 IU/kg of recombinant Von Willebrand Factor (rVWF) to said subject between 12 hours and 24 hours prior to the surgical procedure and wherein Factor VIII (FVIII) is not administered with the VWF prior to the surgical procedure, wherein the rVWF comprises highly multimeric VWF comprising at least 10 subunits.

2. The method of claim 1, wherein said pre-treatment further comprises administering 5-90 IU/kg rVWF to said subject 1 hour prior to said surgical procedure.

3. The method of claim 1, wherein FVIII is not administered after said surgical procedure.

4. The method of claim 1, wherein said surgical procedure is selected from the group consisting of major surgery, minor surgery, and oral surgery.

5. The method of claim 1, wherein said subject is administered 50-60 IU/kg rVWF between 12 hours and 24 hours prior to said surgical procedure and said surgical procedure is a minor surgical procedure.

6. The method of claim 1, wherein said subject is administered 40-60 IU/kg rVWF between 12 hours and 24 hours prior to said surgical procedure and said surgical procedure is a major surgical procedure.

7. The method of claim 1, wherein said subject is administered about 40 IU/kg rVWF between 12 hours and 24 hours prior to said surgical procedure and said surgical procedure is an oral surgical procedure.

8. The method of claim 1, wherein said subject is administered 5-50 IU/kg rVWF 1 hour prior to the surgical procedure and said surgical procedure is a minor surgical procedure.

9. The method of claim 1, wherein said subject is administered 15-90 IU/kg rVWF 1 hour prior to said surgical procedure and said surgical procedure is a major surgical procedure.

10. The method of claim 1, wherein said subject is administered 20-50 IU/kg rVWF 1 hour prior to said surgical procedure and said surgical procedure is an oral surgical procedure.

11. The method of claim 1, wherein said subject is administered 10-50 IU/kg rVWF during said surgical procedure and said surgical procedure is an oral surgical procedure.

12. The method of claim 1, wherein said subject is administered 70-220 IU/kg rVWF after said surgical procedure.

13. The method of claim 1, wherein said subject is administered 70-150 IU/kg rVWF after said surgical procedure and said surgical procedure is a minor surgical procedure.

14. The method of claim 1, wherein said subject is administered 150-220 IU/kg rVWF after said surgical procedure and said surgical procedure is a major surgical procedure.

15. The method of claim 1, wherein said subject is administered 20-50 IU/kg rVWF after said surgical procedure and said surgical procedure is an oral surgical procedure.

16. The method of claim 1, wherein said subject is administered a total dosage of 100-220 IU/kg rVWF and said surgical procedure is a minor surgical procedure.

17. The method of claim 1, wherein said subject is administered a total dosage of 220-320 IU/kg rVWF and said surgical procedure is a major surgical procedure.

18. The method of claim 1, wherein said subject is administered a total dosage of IU/kg rVWF and said surgical procedure is an oral surgical procedure.

19. The method of claim 1, wherein said surgical procedure is a major surgical procedure and said pre-treatment comprises administering at least two approximately equal doses of rVWF prior to the surgical procedure.

20. The method of claim 1, wherein said surgical procedure is a minor surgical procedure and said pre-treatment comprises administering at least two doses of rVWF prior to the surgical procedure, wherein the first dose is larger than the second dose.

21. The method of claim 1, wherein said surgical procedure is an oral surgical procedure and said pre-treatment comprises administering at least two approximately equal doses of rVWF prior to the surgical procedure.

22. The method of claim 1, wherein the rVWF comprises highly multimeric VWF comprising over 40 subunits.

23. The method of claim 1, wherein the rVWF comprises ultra-large multimers (ULMs), wherein the ULMs are at least 10,000 kDa.

24. The method of claim 1, wherein the rVWF comprises a percentage of high molecular weight (BMW) rVWF multimers, wherein the BMW rVWF multimers comprises at least 10% rVWF decamers or higher order multimers.