NZ618790B2

NZ618790B2 - Treatment of coagulation disease by administration of recombinant vwf

Info

Publication number: NZ618790B2
Application number: NZ618790A
Authority: NZ
Inventors: Bruce Ewenstein; Friedrich Scheiflinger; Tobias M Suiter; Peter Turecek; Wing Yen Wong
Original assignee: Takeda Pharmaceutical Company Limited
Priority date: 2011-06-10
Filing date: 2012-06-11
Publication date: 2015-11-03

Abstract

Disclosed is a use of recombinant Von Willebrand Factor (rVWF) in the manufacture of a medicament for treating Von Willebrand Disease or Hemophilia A in a subject in need thereof, wherein the rVWF is a high molecular weight VWF multimer composition in which at least 20% of the total VWF multimer molecules in the composition are VWF decamers or higher order multimers, and wherein the rVWT has a higher specific activity than plasma derived Von Willebrand Factor. ecules in the composition are VWF decamers or higher order multimers, and wherein the rVWT has a higher specific activity than plasma derived Von Willebrand Factor.

Description

/041957 TREATWEENT QF CQAGHLATEG‘N IBISEASE BY ADRHNISTRATEGN 0F RECGNEBENANF V‘WF BACKGROUND OF THE lNVEN’l‘lﬂN Etltltll} Coagulation diseases, such as von Willehrand Disease (VWD) and Hemophilia, generally result from a deﬁciency in the coagulation cascade. “"von Willehrand Disease” refers to the group of diseases caused by a deficiency of von Willehrand factor. Von Willehrand factor helps blood platelets clump together and stick to the blood vessel wall, which is necessary for normal hlood clotting. Hemophilia A refers to a deficiency of Factor Vlll ty; whereas ophilia l3 relers to a, Factor lX deticiency. Current treatment for these coagulopathies includes a replacement therapy using pharmaceutical preparations comprising the normal ation factor. lﬂtltlZl Replacement therapy in VWD and l-leniophilia A patients involves the repeated administration ofpreparations containing normal coagulation lactors via, intravenous infusion, which can constitute a heavy load on the life of these patients, particularly when venous access is difﬁcult to achieve. it would be advantageous if the frequency of infusions could he reduced. One potentially yiahle therapy is to stabilize Factor Vlli through its association with a second le, such as von Wille’hrand Factor (VWF), with the result that plasma hallllife ot‘Factor \"lll is increased Ellﬁllill VWF is a glycoprotein circulating in plasma as a series of multiiners ranging in size lrorh about Still to 20,038 kl}. The hill length ot‘cDNA ofVWF has been cloned; the propolypeptide corresponds to amino acid residues 23 to 764 of the full length prepro—VWF (Eiheiihooni et al @995) Haemophilia l,,, 77 9G). Multimeric forms ofVWF are composed of 2561} lcl) polypeptide suhuni ts linked together hy disulfide bonds. VW lI es the l platelet adhesion to the suhnendothelium of the d vessel wall., with the larger inultiniers exhibiting ed heniostatic activity. Multimerized VWF binds to the platelet surface glycoprotein Gplhd, through an ction in the Al domain ofVWF, facilitating platelet adhesion. {Either sites on VWF mediate binding to the blood vessel wall. Thus, VVv’l: forms a bridge n the et and the vessel wall that is essential to platelet adhesion and primary hernostasis under conditions of high shear stress Normallyt en dothelial cells e large polymeric forms of VWF and those forms ofV’WF that have a lower molecular weight arise from proteolytic cleavage. The multimers ol‘exeeptionally large lar masses are stored in the Weibel—l’allade hodies ot‘the endothelial cells and liberated upon 2012/041957 stimulation by ts such as thrombin and histamine.

Etlhllél} That F’Vlll pharmacolrinetics are a function of VWF levels is ted by several previous observations. Reduction ofFVlll binding activity in von llebrand Disease (VWD), due to either reduced V‘s/VF protein levels or lowered F‘v’lll binding affinity, results in d steady—state levels of nous F‘v’lll (summarized in Castaman ct at, Disorders of Hemostasis 88(l):94~l 08 (2003‘), and improving survival ofV’WF has been proposed as a viable strategy for improving F‘v’lll stability (Denis et al, Thromb Haemost. 2608 Feb;99(2):27l“8; ’l‘urecek et al, Blood, 2606, 1980 l): ct ltlQZ). Among severe Hemophilia A ts, a correlation between preminlusion VWF levels and the hallllil'e of infused li‘Vlll has been demonstrated by li‘ijnvandraat and colleagues (Fijnvandraat, et al, Br J Haematol. l 9955 Oct;9l(2):474—6). In that study, patients with zoo-300% of e VWF levels were seen to have a F‘v’lll half—lite of l5—29 hours compared to a mean of l 2.5 hours in patients with normal VWF levels. in r study, patients with blood group 0 were demonstrated to have signi ticantly lower VWF levels and shorter F‘Vlll haltllives (l 5.3 hours) compared with those with blood group A if l9.7 hours) (Vlot, et al. 'l‘hromh Haemost. 2800 lan;83(l):65w9). Chemically modiﬁed VWF has been shown to prolong survival of rll‘v'lll ('l'urecel: et al., J. ’l‘hromb. l-laeinost. 2907 Jul 9; 5(2) abstract available at: http/wwwzblachwellpublishing.com/isth2007/abstract,asp?id=6ll898)i As such, co~ stration ofrVWF and rll‘v’lll is a viable strategy for the treatment of coagulation diseases such as von Willebrand Disease and Hemophilia A.

BRIEF t/lARY {3F THE ENVENTEGN ldllllS} Accordingly, the present ion provides methods and combinations for treating coagulation disease by administering recombinant von Willehrand Factor (WW l3) alone or in combination with inant Factor Vlll (rFVlll) to a subject in need thereof, with the result that the in—iriivo haltllife ofli‘actor V E ii is increased.

While} ln one aspect, the present invention provides a, method for treating Von 'Vy’illehrand Disease or lEleinophilia A in a subject in need thereof, the method comprising: administering to the subject recombinant Von Willebrand Factor (rV‘WF) such that Factor V’lll half—life is extended as compared to a t administered plasma derived Von ‘v’v’illebrand Factor, wherein the r‘v’WF is a high molecular weight VWF multimer ition comprising at least 29% WW? decainers or higher order multimers, and wherein the rVVvT has a higher speciﬁc activity than plasma derived Von Willehrand l3actor. {96M} {n thrther ments and in accordance with the above, methods of the invention inciude eo—administering to the subject recombinant Von rand Factor (irVWF) and recombinant Factor VH1 ).

{MEGS} in further embodiments and in accordance with any of the above, the rVWF and rFVE it are stered together in a sin gie composition, {@993 in further embodiments and in accordance with any of the above, the subject is administered between 1,0 iU/hg VWFzRCo and ESQ iU/kg VWF2RCo per dose.

{Witt} in further ments and in ance with any of the above, the subject is administered between 2 iU/kg VWF:RCo and 50 iU/hg V‘v‘szRCo per dose. {9%} t} {n thrther embodiments and in accordance with any of the above, the subject is administered between 5 iU/kg V’WFzRCo and 4t) iEU/kg "\"Vv’FfRCo per dose. {(3912} in further embodiments and in accordance with any of the above, the subject is administered between it} iU/kg VWFRCO and 20 FLT/kg V’WFzRCo per dose.

{WES} in thither embodiments and in accordance with any of the above, the rVWF used in methods of the invention is matured in vitro by treatment with Furin. {thud} in further embodiments and in accordance with any of the above, the rVWF is produced through expression in a Chinese Hamster Ovary {Ci-i0 ceii cuiture). {8015} in further embodiments and in accordance with any of the above, the rFViii and, r‘v’Vt’F are produced through expression in the same ceii cuiture. {time} in further embodiments, and in ance with any of the above, the subject is administered rVWF no more than once every other day.

{M17} in further embodiments and in accordance with any of the above, the subject is administered r‘VWF no more than twice a week. {twig} in further aspects and in accordance with any of the above, the high moiecuiar weight VWF multin'ier composition maintains the at least 20% VWF rs or higher order ers for at ieast 3 hours post-administration. {8019} in further embodiments and in accordance with any of the above, the Factor VIII haif—iife is extended by about 5 hours.

{MEG} in r embodiments and in accordance with any of the above, the Factor Viii i’iaitliite is extended for at least 12 hours.

{M321} in further embodiments and in accordance with any of the above, the Factor Viii haiidife is extended for at least '24 hours. {@223 in further embodiments and in ance with any of the above, the Factor VIII i’iaitliite is extended for at least 36 hours. 39623} hi further embodiments and in accordance with any ot‘the above, the Factor Vii! half-life is extended for at least 48 hours. {(3924} in further embodiments and in ance with any of the above, wherein the Factor Viii haifmiife is extended for at ieast 72 hours. @925} hi thither embodiments and in accordance with any of the above, the ratio ofFVtii procoaguiant activity (1U FVIH:C) to rvwr Ristocetin cofactor activity (1U rVW'FzRCo) administered to the subject is between 2:1 and 1:4, gems; in r embodiments and in accordance with any of the above, the ratio of FVHI procoaguiant activity (1U EVELC) to rVWF Ristocetin cofactor ty (1U rVWRRCo) administered to the subject is between 3:2 and 1:3.

Etch’V} in further embodiments and in accordance with any of the above, the ratio of F‘s/tit procoagniant activity (EU F‘v’iitrC) to rVWF Ristocetin eot’actor activity (EU rVWFiRCo) administered to the subject is between 1:1 and it :2. @028} hi thither embodiments and in ance with any of the above, 23 the ratio of F‘v‘ititi procoaguiant ty (EU F’ViﬂzC) to rV‘v‘i/F Ristocetin cofactor activity (1U rVWFRCo) administered to the subject is about 3:40 {“3293 in further embodiments and in ance with any of the above, the r‘v’Vv’F has a c ty of about ZG—tSG mU/ng 39639} hi further ments and in accordance with any of the above, the high moiecutar weight VWF muttimer composition comprises at least 30% VWF decamers or higher order inuttin'iers, {M331} in further embodiments and in accordance with any of the above, the high moiecuiar weight VWF niuttimer composition comprises at least 48% VWF decanters or higher order ers. {(3632} in further embodiments and in accordance with any of the above, the high moiecniar weight VVV'F muttimer composition comprises at least 50% VWF decanters or higher order muttimers. 39633} hi further embodiments and in accordance with any of the above, the high moiecutar weight VWF muttimer composition comprises at least 60% VWF decamers or higher order inuttin'iers, {M334} in further embodiments and in accordance with any of the above, the high iar weight VWF niuttimer ition comprises at least 78% VWF decanters or higher order muttimers. {(3635} in further s and in accordance with any of the above, the present invention provides a method for treating Henrophitia A or Von Witiehrand Disease in a subject in need thereof, the method comprising: administering to the subject recombinant Von ‘Vv’illebrand Factor (r‘V‘v‘v’F) such that Factor Vlll half—life is extended as compared to a subject stered plasma derived Von Willebrand , wherein: (a) the rVWF has a higher speciﬁc activity than plasma derived Von 'V‘v’illebrand Factor, wherein the specific activity of r\"WF is about 20450 mU/ug; and (b) the F‘v’lll l'ialtllile is at least l5 times higher as compared to F‘v’lll half—life in a subject administered plasma derived von Willebrand Factor.

Eilllfsé} ln further aspects and in accordance with any of the above, the present invention provides a method for treating Hemophilia A or Von Willebrand Disease in a subject in need thereoﬂ the method comprising: administering to the subject recombinant Von Willehrand Factor (r‘V wr) such that Factor Vlll half-life is extended as ed to a subject administered plasma derived Von rand Factor, wherein: (_a) the rVWF is a high molecular weight VWF niultimer ition comprising at least 23% VWF ers or higher order multimcrs, (b) the rVWF has a higher specific activity than plasma derived Von ll’illebrand Factor, wherein the c activity of r\"WF is at least about 2tl~l50 nilj’x’ug; and (c) the F‘Vlll halfmlife is at least l.5 times higher as ed to F‘v’lll half—life in a subject administered plasma derived Von rand Factor. {@8373 in further embodiments and in accordance with any of the above, the level of Factor Vlll procoagulant activity (FVlllzC) in the plasma of the subject 24 hours postnadministration ot‘the r‘v’WF is at least 999/5 of the level ot‘FVllihC activity present in the plasma l hour posts administration {(3638} in r embodiments and in accordance with any of the above, the level of Factor vl ll procoagulant activity (FV’lth) in the plasma of the subject 24 hours post—administration is at least lllll% of the level of FVlllzC activity t in the plasma l hour post~ stration. {(36359} in further embodiments and in ance with any of the above, the level ot‘Faetor Vlll procoagulant activity (F‘v’lll:C) in the plasma of the subject 36 hours post—administration is at least 80% of the level of FVlll2C activity present in the plasma l hour post— administration.

Etllldll} in further ments and in accordance with any of the above, the level of Factor Villl procoagulant activity {FVlllzC} in the plasma of the subject 48 hours post~administratioh is at least 509/2; of the level of F‘v’lllzC activity present in the plasma l hour post— administration.

Eilhcll} in further embodiments and in accordance with any of the above, the higher order rVWF nuiltimers are stable for at least 6 hours post—adniinistration. 2012/041957 39642} in thither embodiments and in accordance with any ot‘the above, the higher order r‘v'WF multimers are stable for at least 12 hours post—administration. {(3643} in r embodiments and in accordance with any of the above, the higher order rVWF ntuitimers are stable for at least 18 hours dministration.

E8944} hi thither embodiments and in accorthtnce with any of the above, the higher order r‘v'WF multimers are stable for at least ‘24 hours post—administration.

S} in further embodiments and in accordance with any of the above, the higher order rV WP“ multimers are stable for at least 36 hours post-administration. {8046} in further embodiments and in accordance with any of the above, the higher order r‘v’Vv’F inultimers are stable for at least 48 hours postsadministration.

Etthdﬁ in further embodiments and in accordance with any of the above, the higher order r‘v’WF niultimers are stable for at least 72 hours post—administration.

{M348} in further aspects and in accordance with any of the above, the present invention provides a method for treating Hemophilia A or Von Willebrand Disease in a subject in need f, the method comprising: stering to the subject recombinant Von Wiliebrand Factor (rVWF). {“3493 in further embodiments and in accordance with any of the above, the method comprises co—administering to the snbj eet recombinant Factor VIII (rFVlli) and recombinant Von Willebrand Factor (id/WEI). ﬁ} in further embodiments and in accordance with any of the above, the rFViii and r‘v’WF are administered togethe in a single composition.

{M351} in further embodiments and in accordance with any of the above, the ratio of rFVHl procoagulant activity (1U rFVillzC) to rVWF Ristocetin cofactor activity {1U rVWF:RCo) stered to the subject is between 2:1 and t :4. {(3652} in further ments and in accordance with any of the above, the ratio ofrFVlH procoagulant activity (E U rFVIll:C) to WW? ltistocetin or activity (it) rV‘v‘v’FiRCo) administered to the subject is between 3:2 and lz3. 39653} in thither embodiments and in accordance with any ot‘the above, the ratio ofrli‘VEEt procoagutant activity (1U rFV1112C) to rVWF Ristocetin cotactor activity (1U I‘VWFIRCO) administered to the subject is between 1:1 and l:2.

{MM} in further embodiments and in accordance with any of the above, the ratio of rFVHl procoagulant activity (1U zC) to rVWF etin cofactor activity {1U rVWF:RCo) administered to the subject is about 3:4. {(3655} in r embodiments and in accordance with any of the above, the rVWF is matured in vitro by treatment with ti‘urin.

ItIIISfi} In r embodiments and in accordance with any ot‘the above, the rVVv’F is expressed in mammaiian ceIi e. {(3657} In r embodiments and in accordance with any of the above, the rFVIII is expressed in mammaIian ceh culture.

ItItISS} In r embodiments and in accordance with any of the above, the manunaiian e comprises CHO cetis.

Ititi59} In further embodiments and in accordance with any of the above, the rFVIII and WWI? are ctr—expressed in the same cuIture. {9963} In further embodiments and in accordance with any of the above, the rFVIII and, r‘v’Vv’F are co-purified.

ItihoI} In further embodiments and in accordance with any of the above, the rFVIII and r‘v’WF are ed separately, ItIIItiZ} In further embodiments and in accordance with any of the above, a rIi'VIII/rVWF con’ipiex is reconstituted prior to administration, gases} In further embodiments and in accordance with any of the above, r‘v'WF is treated with Furin prior to reconstituting the rITVIII/rVWF complex. {sass} In further embodiments and in accordance with any of the above, the reconstituted II/rVWF complex is treated with Furin.

ItIIItiS} In r embodiments and in accordance with any of the above, the Ii‘urin is recombinant Form, {(3666} In further embodiments and in accordance with any of the above, the subject is administered rVWF no more than once daily. @967} In thither embodiments and in accordance with any of the above, the subject is stered rV’WF no more than once every other day. {(3668} In further embodiments and in accordance with any of the above, the t is co— administered rVWF no more than once every third day. {9969} In further embodiments and in accordance with any of the above, the subject is administered rVWF no more than once every fourth day.

WING} In further embodiments and in accordance with any of the above, the subject is administered between I .5 III/kg F‘v’IiirC and ISO III/kg FVIII:C per dose.

KIWI} In further embodiments and in accordance with any of the above, the subject is administered between 10 III/kg F‘v’IiirC and 100 III/kg FVIIIzC per dose.

’Z} In further embodiments and in accordance with any of the above, the subject is administered between 25 ILL/kg FVIII:C and 75 III/kg FVIIIK’L per dose. 396%} in titrther embodiments and in accordance with any ot‘ the above, the subject is stered between 40 lU/kg FVlllzC and 60 Ill/kg FV’lllzC per dose. {(3674} in further embodiments and in ance with any of the above, the level of Factor Vi ll gulant activity (F‘s/”lllzC) in the plasma of the subject 24 hours post—administration is at least 93% of the level of F‘v’ller activity present in the plasma l hour post— administration.

Etlll75} in further embodiments and in accordance with any of the above, the level of Factor Vlll procoagulant activity (FVlll:C) in the plasma of the t 24 hours post—administration is at least ltltl% of the level ofFVlllzC activity present in the plasma l hour postm administration.

Etlh’lti} in further embodiments and in accordance with any of the above, the level of Factor Villl procoagulant ty {FVlllzC} in the plasma ot‘ the subject 36 hours post~administration is at least 809/2; of the level of F‘v’lllzC activity present in the plasma l hour post— administration.

WWW} in further embodiments and in accordance with any of the above, the level of Factor Vlll procoagulant activity (FVlllzC) in the plasma of the subject 48 hours post~administration is at least 59% of the level ot‘FVllihC activity present in the plasma l hour post“ administration. 396%} in titrther embodiments and in accordance with any ot‘the above, the rVVv’F administered to the subject has a BMW VWF multimer composition comprising at least 6 VWF decanters or higher order m ultiiners. {@793 in further embodiments and in ance with any of the above, the rVWF administered to the subject has a lelh/lW VWF multimer composition con'iprising at least 20% VWF decamers or higher order ers. {(3683} in further embodiments and in accordance with any of the above, the rVWF administered to the subject has a BMW VWF multimer composition comprising at least 30% VWF decamers or higher order multimers 39681} in titrther embodiments and in accordance with any ot‘the above, the rVVv’F administered to the subject has a BMW VWF er composition comprising at least t5 VWF decanters or higher order m ultiiners.

{M382} in further embodiments and in accordance with any of the above, the rVWF administered to the subject has a lelh/lW VWF multimer composition con'iprising at least 50% VWF decamers or higher order multimers. {(3683} in r embodiments and in accordance with any of the above, higher order rV WP“ multimers are stable in vitro for at least 3 hours postwadrninistration. 39684} in thither embodiments and in accordance with any ot‘the above, higher order rVWF mnltirners are stable in vitro for at least 6 hours post—administration. {@685} hi further embodiments and in accordance with any of the above, wherein higher order rVWF innltiiners are stable in vitro for at least l2 hours post—administration.

Elllldti} ln thither embodiments and in ance with any of the above, n higher order rVWF nniltiniers are stable in vitro for at least l8 hours post—administration.

Elltl8'7} in further embodiments and in accordance with any of the above, higher order rV WP“ niultiniers are stable in viiro for at least 24 hours post-administration. {9988} in r ments and in accordance with any of the above, higher order r‘v’Vv’lE" inultiiners are stable in vitro for at least 36 hours post—administration.

Ellhlﬁé‘t} in further embodiments and in accordance with any of the above, wherein higher order rVWF nialtiniers are stable in viiro for at least 48 hours post—administration.

{Mild} ln further ments and in ance with any of the above, higher order rVWF inultirners are stable in vitm for at least '72 hours post~adrriinistration.

Etlh‘ﬂ} in further embodiments and in accordance with any of the above, endogenous FV’lll activity is stabilized for at least l2 hours, {him} in further embodiments and in accordance with any of the above, endogenous FVlll activity is stabilized, for at least 24 hours. 39693} in thither embodiments and in accordance with any ot‘the above, endogenous FVlll activity is ized for at least 36 hours, {@694} in further embodiments and in accordance with any of the above, endogenous FVlll activity is stabilized for at least 48 hours. @995} in thither embodiments and in accordance with any of the above, endogenous FVlll activity is stabilized for at least 7‘2 hours. {@696} hi further embodiments and in ance with any of the above, n co~ administered lll activity is stabilized for at least l2 hours. {9997} in further embodiments and in accordance with any of the above, co—administered rli‘Vlll activity is stabilized for at least 24 hours.

Elltl98} in further embodiments and in ance with any of the above, co—administered rFVlll activity is stabilized for at least 36 hours. {($599} ln further embodiments and in accordance with any of the above, era—administered rFVlll activity is stabilized for at least 48. hours, Ellhltltl} in further embodiments and in accordance with any of the above, co—adrninistered rFVlll activity is stabilized for at least 72 hours. ititli Oil in further embodiments and in ance with any ot‘the above, co-administered rFVill activity is stabilized by extending the half—life of the l in vino. {$131112} in further ments and in accordance with any of the above, FVlll hall’llife is extended by about five hours as ed to a patient administered pdli'Vlli.

Etiili 93} in thither embodiments and in ance with any of the above at least l% of the co—administered rF‘Vlii activity is ined for at least 36 hours in a patient administered said rFVlll. {“1134} in further embodiments and in accordance with any of the above, at least 1% of the co~administered rFVlii activity is maintained for at least 48 hours in a patient administered said rF‘v’lll. %195} in further embodiments and in accordance with any of the above, at least 1% of the cc—adrninistered rFVlll activity is ined for at least '72 hours in a, patient stered said rFVlll.

Etiili as} in thither embodiments and in ance with any of the above at least l% of the co—administered rF‘Vlii activity is maintained for at least 90 hours in a patient administered said rFVlll. {“1137} in further embodiments and in accordance with any of the above, at least 1% of the co~administered rFVlli activity is maintained for at least lZO hours in a patient administered said rF‘v’lll. iﬂtimgl in further ments and in accordance with any of the above, at. least l% of the ce—adrninistered rFVlll activity is maintained for at least 168 hours in a patient administered said rFVlll.

BRIEF DESCRIFTEQN QF THE BRA‘WENGS {titling} Figure 1. A schen'iatie of the study design assessing tolerability and safety after single doses of rVV‘v’Fsz‘s/"lll. {$11113} Figure 2. Pharmacokinetic data, (A) provides PK data for rV‘Ji’F/rFVlll and pd‘v’WF/‘pdl?V111. (B) provides data on progressive loss ofhigh molecular weight rVWF upon exposure to ADAMTS l 3. ititli 1i} Figure 3, l’harmacckinetie data showing FVlll PK for r‘s/"WFlil‘FVill and pdFVlli. {(113112} Figure 4. Table summarizing patient demographics for the study {(10113} Figure 5. Pharmacolnnetic data of rVWWrP-‘Vill and pdVWF/pdli'K/lll treatment of Cohort 4A. %114} Figure ti. SDSml’AGE data on VWF mnltimer cleavage by ADAMTS l3. 2012/041957 5911115} Figure 7. Pharmacokinetic data of rVWF/rPV111 and ptiVWR’priPV i ii treatment of Cohort 4A. {(13116} Figure 8. Summary ot‘aoverse events from the study. {(10117} Figure 9, rvwr PK parameters data from the study. {£1111 18} Figure 113. rVWF PK data irom Patient 1. §i Figure 11‘ rVWP PK data from Patient 2. {911123} Figure 12;. rVWF PK data from Patient 3t {“1121} Figure 13. Comparison ofr‘v’Vi’P PK in Patients 1, 2 and 3 comparison of the VWF parameters and. PViii ty. 5911122} Figure 14‘ PV111 activity across aii cohorts in the study.

Eiiu123} Figure 15‘ rVWP PK in patients of Cohort '23 comparison ofVWP ters and FViii activity, {(10124} Figure 16. Summary of cokinetic parameters for Factor Viii procoaguiant activity {PViiizC} for patients in Cohort '2.

Eiiu125} Figure 17‘ rVWP PK in aii patients of Cohort 3 comparison of piasma parameters for VWF and FV111‘ {“1126} Figure 18. Summary ofpharmacokinetic parameters for Factor Viii procoaguiant activity (_FV1112C) for patients in Cohort 3. 5911127} Figure 1553‘ Data from patients in Cohort 4. {911128} Figure 25}. Summary ot‘pharmacokinetic parameters for Factor Viii procoaguiant activity {Pi/iii :C) for patients in Cohort 4A. {(10129} Figure 21. Pharmaeokinetic data of FV’iiFC comparing era—administered rVWP and rFV i ii to Activate Pivotal.

Eiiu13ii} Figure 22‘ Antibody summary for a t receiving co—aciministereti /poF‘i’Hi or rVVv’F/rFVii i.

BE'E‘AILFB PTEGN 0F iHE iNVFﬁN’FiﬂN Overview Eiiiiifii} The present invention provides compositions and s for treating coaguiatiori disease in a subject by stering recombinant yon ‘v‘v’iiie‘branri Pactor (rVWF) aione or in combination with Factor Viii (which can be recombinant or piasma derived), in some aspects, the compositions anti methods of the present invention are used for treating coaguiation diseases such as yon Wiiiebrand Disease (VWD) or Hemophiiia A. 2012/041957 32l in one aspect, rV Wl2 administered to the subject provides increased stability for in viva Factor Vill (F‘s/ill) activity as compared to FVlll stability due to administered plasma derived VWF, allowing for lower doses and/or tirequency oi“ treatment than in traditional treatments for coagulation diseases. increased stability of F‘Vlll activity and levels of l can be measured using methods lrnown in the art and described herein, including standard assays such as one—stage clotting assays, chromogcnic assays, and inimunoassays (see for example Lippi et al., Blood Coagulation & Fibrinolysis, 2009, 260): l~3 and Chandler et al., Am J. Clin. Pathol, 2603, 1293439, each ot’which is hereby orated by reference in its entirety for all purposes and in particular for all teachings related to assays ot‘FVlll level and activity). As shown in FIG. EEG, average FVllE haltllit‘e was increased by rV WEI by 5.2 hours over the halfmlife for ts receiving pdVWF. Fle. '2. and 7 also show increases in F‘Vlll activity in patients administered rVWF as compared to those administered pdVWF: Fit}. 2 shows that average F‘v’lll half—life was increased by 4.7 hours for patients administered rVWF as compared to patients administered pdVWR and the data. lrom Cohort 4A in FlG. 7 shows an se in rv ill half-life of 5.3 hours for ts administered rVWF as compared to patients administered pdVWF in addition, as shown in HG. 2i, the haltllile of plasma PVlll is increased by l2.27 hours in VWD patients receiving rVWF in ation with rFVlll as compared to patients receiving FVlll tie, Advate) alone.

Etltll33l in a further aspect, the administration ot‘r‘V‘r‘y’lI stabilizes endogenous and/or co- administered FVill activity, with the result that the in viva halfnlit‘e and/or activity of FVlll is increased. in embodiments in which rVWF and F‘v’lll are co~adniinistered, the rVWF and F’Vlll can be administered to the subject together in a single composition. in thrther embodin'ients, neither rVWF nor FV l ii are d with a water e polymer. in other embodiments, either the rVWF or F‘v’l ll or both are modified with a water soluble polymer.

As will be appreciated, in en’ibodin’umts in which rVWF is (to—administered with FVlll, the F‘s/ill may be recombinant or plasma derived. } in further s and in accordance with any of the above, the r‘v’WF administered to the subject is a high molecular weight multimer ition comprising decamers or higher order multimers ofrVWF. As discussed above, the use ofi‘V‘v’t’F compositions of the invention provide therapeutic ﬂexibility to dose (or re~dose) with or without l (recombinant or plasma derived). in further embodiments, the rVWF administered to the subject is a high molecular weight VWF multin’ier composition comprising at least 299/5 VWF decamers or higher order multimers. ln specific embodiments, the rvwr‘ administered to subjects is not modified with a water soluble polymer.

WO 71031 2012/041957 Etllll35l hi still further s, the rVWF administered to the subject has a higher specific activity than pd\l’\?i7F. {(33136} in a still lurther aspect, the r‘v’WF alone or in combination with deVlll or r‘F\"lll is administered to the subj ect no more than twice a week.

Elllll '37} hi a yet further aspect, the rVWF is processed with Furin prior to administration to the subject. in n embodiments, the rVW’F is processed with recombinant Furin. lhlllildl in further asp ects, the rVWF of use in the present invention is produced in accordance with methods known in the art and described for example in US 20l 2x/(ltl35l ll), tiled July 8, Bill l and US Patent No. 8,l73,597, issued May 8, 2012, each of which is hereby incorporated by reference in its ty for all purposes and in particular for all teachings related to rVWF compositions and methods for producing these compositions. {(33139} in accordance with any of the above, rVWF alone or in ation with FVlll is used to treat patients with coagulation diseases, such as VWD and Hemophilia A. Patients with VWD have some level ll, but the stability ol’ that F‘v’lll is generally compromise hecause these patients laclr VWF. ent of VWD patients may in some enihodiments e an initial ent with both rVWF and rFVlll tollowed by repeated administrations ofrVWF alone. in other ments, the initial ent may he with r‘v’Vi/F alone while subsequent repeated administrations are with both rVWF and rFVlll, or the initial and subsequent repeat administrations may all include a cowadministration ot‘hoth rVWli‘ and rFVlll, Similarly, Hemophilia A patients (who lack FVlll) may receive an initial treatment ol’hoth rVWF and rFVl ll, and subsequent repeat treatments may comprise the administration of rFV’lll alone or rvwr alone. in other ments, the initial treatment may be rFVlll alone while the subsequent repeat treatments involve co—administration of rVWF and erlll.

Bellnitions Ellhléllll As used herein, “rVWF” refers to recombinant VWF. ll As used herein, “rF‘v’lll” refers to recombinant FVlll, {$31le 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 (n err—recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all. lllllldBl As used herein, ”recombinant ‘V’WF" includes VWF obtained via recombinant DNA technology. in certain embodiments, V WF proteins of the ion can comprise a construct, for example, prepared as in Wt.) Mme/06096 hed on Oct. '23, l986 and US patent application Ser. No. 07,559,599, filed on Jul. '23, l990, in the name of Ginsburg at all, which is incorporated he *ein by reference with respect to the methods of producing recombinant V‘v‘v’l‘. The VW’F in the present in rention can include all potential forms, including the monomeric and multimeric forms. lt should also be understood that the t invention encompasses different forms oft/Wt: to be used in combination. l7or example, the VWF of the present ion may include different multimers, dillerent derivatives and both ically active derivatives and tives not biologically active.

Etlhlddl in the context of the present invention, the recombinant VW’F embraces any member of the VWF family irom, 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 ty are r lso embrace as are functional fragments and fusion ns of the V’WF proteins. Furthermore, the VW’F of the invention may turther comprise tags that facilitate purification, detection, or both. The VWF described herein may further be modified with a therapeutic moiety or a moiety le imaging in vino or in viva.

Ellllldfil As used herein, ”plasma—derived VWF {pill/ltd?” es all forms of the protein found in blood including the mature VWF obtained from a mammal having the property of in vivo—stabilizing, cg. binding, of at least one F‘v’lll n’iolecule. {(301465} The term “highly multimeric VW’F” or “high molecular weight "V'WFl’ refers to V‘WF comprising at least ll} subunits, or l2, id, or l6 subunits, to about 28, 2'2, 2-4 or 26 subunits or more, The term “subunit” re ”ers to a, monomer ofVWF. As is known in the art, it is generally dimers of VWF that rize to form the larger order multimers, {see ’l'urecelt et al., Semin. ’l‘hromh l-lernost. , 36(5): 5ll)~52l which is hereby incorporated by reference in its entirety for all purposes and in particular for all teachings regarding multimer analysis ofVVv’F).

Ellllldﬂ As used herein, the term ”factor Vlll" or "FVlll" refers to any form of factor Vlll molecule with the typical teristics of blood coagulation lactor \"lll, r endogenous to a patient, derived from blood plasma, or ed through the use of recombinant DNA techniques, and including all modiﬁed forn'is of factor Vll l. Factor Vlll (F‘v’lll) exists naturally and in eutic preparations as a heterogeneous distribution of polypeptides arising from a single gene product (see, erg, Andersson et al., Proc. Nazi, Acad, Sci. USA, 8329792983 . Commercially available examples of therapeutic preparations ning Factor Vlll include those sold under the trade names of l-llilh/lGF l l; M, ADVAT‘E, and RECOl‘t/TBINATE (available from Baxter Healthcare Corporation, Deerl’ield, lllt, USA.) {(30148} As used herein, “plasma F‘s/Til activity” and “in vivo F‘s/ill activity” are user interchangeably. The in vivo F‘v’lll activity measured using standard assays may he endogenous FV’lll ty, the ty of a therapeutically stered FVlTT trecombinant or plasma derived), or both nous and administered FVlTl activity. Similarly, “plasma F‘s/ill” refers to endogenous F‘v’lll or administered recombinant or plasma derived l. {99149} As used herein “von Willehrand Disease” refers to the group of diseases caused by a ency of von Willehrand factor. Von Willehrand 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 Wi llehrand disease. {(39153} As used herein, the terms "hemophilia," or ”liaemophilia" refer to a, group of disease states hroadly characterized by reduced blood clotting or coagulation. l’lemophilia may refer to Type A, Type B, or Type C hemophilia, or to the composite of all three diseases types.

Type A hemophilia (hemophilia, A) is caused by a reduction or loss of lactor Vlll {FVllll activity and is the most prominent of the ilia subtypes. Type B hemophilia (hemophilia B) results from the loss or reduction of factor lX (FlX) clotting lunction. Type C hemophilia (hemophilia C} is a consequence of the loss or reduction in factor Xi (EXT) clotting activity. ilia A and B are X—linhed diseases, while hemophilia C is autosomal. Common 'ients for hemophilia include both prophylactic and on~demand stration of clotting factors, such as FVlll, FIX, including Be’lnilini’iT/‘w-VH, and EH, as well as FElBAeVll, desmo'pressin, and plasma inlusions.

EllhlSll The terms “isolated,” “purified,” or “biologically pure” re ”er to material that is substantially or essentially free ’l components that normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as rylainide gel ophoresis or high performance liquid cinematography. VWF is the predominant species present in a preparation is substantially purified. The term “purified” in some embodiments denotes that a c acid or protein gives rise to essentially one hand in an electrophoretic gel. in other emlmdiments, it means that the nucleic acid or protein is at least 50% pure, more preferably at least 60%, 65%, 709/23, 75%, 80%, 85%, 93%, 95945, 96%, l 7%, 98%, 99% or more pure. “Purity” or “puriﬁcation” in other embodiments means removing at least one contaminant from the composition to he p d. in this sense, puriﬁcation, does not require that the p untied compound he homogenous, eg, l(3()% pure. 2012/041957 lllllSZ As used herein, ”administerinO" (and all jrammatical e uivalents includes . 33 \ 23‘ q intravenous administration, intramuscular administration, subcutaneous administration, oral administration, stration as a suppository, topical contact, intraperitoneal, intralesional, or intranasal administration, or the implantation of a elease device, eg, a mini-osmotic pump, to a subject. Administration is by any route including parenteral, and transmucosal (6.31, oral, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, egg intravenous, intramuscular, intra—arteriole, intradermal, subcutaneous, eritoneal, intraventricular, and intracranial. {Ether modes of delivery include, but are not d to, the use ol‘liposomal ations, intravenous infusion, transdermal s, etc.

Still} 53} The terms ”therapeutically effective amount or dose” or ”therapeutically sutlicient amount or dose" or ”effective or sufﬁcient amount or dose" refer to a dose that produces therapeutic etl'ects for which it is administered. For e, 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 ms associated with hemophilia. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one sl<il ed in the art using known techniques (see, 61g, Lieberman, Pharmaceutical Dosage Forms (vols. l—3, l992); Lloyd, The Art, Science and iopnz qﬂ’hai‘maceating! mding 0999}; Pickar, Dosage Calculations if l999); and Remington: The Science and Piaizctice ofPharmacy, 2tlthl31dition, 2003, Gennaro, Ed, lyippincott, Williams & Wilkins). {$9154} As used herein, the terms “patient” and “subj ect” are used interchangeably and refer to a manin'ial (preierably human) that has a disease or has the potential of contracting a disease.

Elllll 55} As used , the term "about" denotes an approximate range of plus or minus ltl% from a speciﬁed value. For instance, the language "about 20%" encompasses a range of l 8—2296. {@8156} 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.

Cniniositlons of the invention l The present invention utilizes compositions comprising recombinant von Willebrand Factor (rVWF) for treatment of coagulation disease, such as VWD and llemopliilia A. in some embodiments, the t invention utilizes rVWF in combination with Factor V l ll (F‘v’lll). The co—administered F‘v’lll may be recombinant (rFVlll) or plasma. derived (deVlll). in pre ”erred aspects, the compositions of the present invention stabilize in vino Factor V 5 ll activity (also referred to herein as plasma Factor Vlll activity) such that the in viva half—life of Factor V’lll is extended as compared to that in subjects that have not been administered rVWF or that have been administered pdVWF. Measuring the extent to which rVWF stabilizes FVTll activity tincluding extension of F‘s/ill half—life) can be carried out using methods known in the art. The level of F‘v’lll activity can be n’ieasured by, for ce, one—stage clotting assays, genic assays, and immunoassays (see for example Lippi et al,, Blood Coagulation 6; Fibrinolysis, 2009, 28H): l~3, European Pharmacopoeia (Ph. Burt, 3.sup.rd lid. l99722.7.4), and Chandler et al., Am J. Clin. Pathol, 2093, 12934—39, each of which is hereby incorporated by reference in its entirety for all es and in ular for all ngs related to assays of F‘v’lll level and activity).

Ellh158l in certain embodiments, VW’F proteins of the ion may comprise a construct, for example, prepared as in WC) l986/l)6096 published on Oct. '23, l986 and US patent application Ser. No 07,659,509, filed on Jul. ‘23, l99t), in the name of Ginsburg et al., which is incorporated herein by relerence with respect to the methods of producing recombinant VWF. The VWF useful for the present invention includes all potential forms, including the ric and multimeric forms. One particularly useful form oft/WP are homonmultimers of at least two VWFs. The VWF proteins may be either a biologically active derivative, or when to he used solely as a stabilizer for FVlll the VWF may he of a form not biologically . it should also be tood that the present ion encompasses different forms of VWF to be used in combination. For example, a composition useful for the present invention may include dillerent m ultimers, different derivatives and both biologically active derivatives and derivatives not biologically active. {hill Sill in primary l'iernostasis VWF serves as a bridge between platelets and specilic components of the ellular , such as collagen. The biological activity of V’WF in this process can be measured by different in vitro assays (Turecelr et al Sernin, Thrornb. l-lemost. 28: l49—l60, 2602). The ristocetin cofactor assay is based on the agglutination of fresh or tormalin—fixed platelets induced by the antibiotic ristocetin in the presence ofVWF.

The degree of platelet agglutination depends on the \I’Wli‘ concentration and can be measured by the turbidimetric method, eg. by use of an aggregometer (Weiss et al J. Clin. invest, 52: 27(38~27l6, l973; hilaclarlane et al., Thromb, Diath, llaemorrh. 34: 306—308, l975}. The second method is the en binding assay, which is based on ELISA technology (Brown et Bosak, Tl’iromb, Res. 43: 303—3l l, l986; ro, b. llaernost. 83: l2'7—l 35, 2000), A microtiter plate is coated with type l or ill 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 ELESA reader. As previded herein, the specific Ristecetih (ifet‘actcr activity of the VWE {\I’W’RRCO) of the present invention is generally bed in terms efmU/itg et‘VWF, as measured using in vitro assays. {00} 60} An advantage of the rVWF COHEpOSitiOHS et‘the present invention ever pdVWF is that i’VWF exhibits a higher specific activity than pdV‘iVF. in some embodiments, the i‘v’WF ot themventicn hasaaspeeitic aetivity et at least aheut 20 2'2 5, 25,2 530,, 32.5, 35, 45,0, 42. 5, 45 47.5 50, 52.5,5 57.5 60, 62 .5, 65, 675 70, 5, 77. 5, 80, 82.5, 85,(J; ‘75, 90, 92.5, 95, 97.5, 100, 105, H0, H5, 120, 125, 130, 135, 140, 145, 150 eri'nere mU/ug. hi ene ment, rVWE used in the methods described herein has a specific activity of from 20 mU/ug tc i50 mU/itg. hit r embodiment, the VWF has a specific activity ct 030151(HmL/xug te 120 mUMg hi ahethei enihedinieht, the rV‘Wi has a specific activitv fmm 40 mU/itg to 90 1111.77gig. in yet another embodiment, the :rVWF has a ic attivitv seiet‘ted item vaiiatieiis 1 t0 133 foundiii Tahie i Fabie 1. Exempiaiy embodiments fer the speciﬁc activity of i‘VWF found in the ccmpesitiens and used in the methods ed herein. 37.5 ’ ' E . 3—35 ' _’at74‘—T'\ar. .,i 80—i30 ‘var. 108% 49—h7—0Var Va. 109 Vai.77 Var.110 V31. 7 ea it00 Var.’ilii Var 79 Vai.112 Van 118 - his150 Vai.ii9§ 0/3 Var..0 ”300—140 Vain "EZGE Van 1’21 his120 Var. 122: metie var. "323i ‘v:ar 91 110“ 150 Var, i24i 110—E40 Vat. 30th) '~i. 60110 “01750 Var. ............................................................................................................................................................................................................... —100 V.ar (it . i -922 hit90 v2.22 <25 i222 25o Var 2:25 120 iiiii Vin. 129 ....................................................................................................................................................................................................................... . v.42 2322 -622 , 2252 2:24 ' = . 4225222412 2222 " —:. 6 70 249 252.2 922 2322—: % ' 140—250 Var. = ion {time}; The rVWF ot‘the present invention is highin muitinterie comprising about it) to about 4% subunits. in further embodiments, the niuitimerie rVWF produced using methods of the present ii'ivei'ition comprise about toss, i2—28, 14—26. 16—24, i8~22, 20—21 subunits. in r embodiments, the rVW'F is present in rnuitiniers varying in size from dimers to inuitin'iers of over 43 subui'iits ('> 10 million Daitons). The t innitiiners provide multiple hinding sites that can interact with both piateiet receptors and subendotheiisi niatrix sites of injury, and are the most heniostatieaiiy active form ofVWF. As si'iovvn in the niuitinier anaiysis in PEG. 2 (hottorn panel}, application of/tiiﬂti‘vi’i'SM Wiii eieave the ultra— iarge r‘v’WF muitimers overtime, but during production (generally through expression in cell culture), r‘v’Vv’F compositions of the present invention are generaiiy not exposed to ADAMTS i 3 and retain their highly muitimerie structure.

{SM 62} in one embodiment. a, rVWF ition used in the methods described herein has at distribution of iv WE- oiigorners characterizedin that 959/0 of the ohgorners have n 6 subunits and 20 subunits. in other ments, the a, rVWF composition has a, distribution ofrVWF oiigomers characterized in that 95% of the oiigornei‘s have a range of subunits seieoted 22-0222 variations 458 to 641 found in Tobie 2.

Tobie 2. Exempitny embodiments for the distribution of rvwr oiigornei‘s found in the compositions and used in the methods provided herein. ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ iSuhunits Suhunitsi uhnnits . thnnitsi : ‘ ‘ 4422 .5224 22425 210428 iVar. 596 2.2—5; I: .5225 22422 6 v42. 597i 2—36 6:2 .5226:- 5 2.2 26 20-24 iV-ar. 552s 242.442547122402 522 _____________22v 2—32 v.42 452 -. .. ': :224s : iVar. 62222: 24322 ' t .5225: 435 v.22. 555 224522 .v62.6nii 248510146‘4552232’ 42‘6”» .............it.” 22—26 , 2434 v5: 557 :22.24 viIai; 6225: 2:24:24 222522.465 .5221.i '432 var. 555 22452 .‘var. 222.24: 22.44 2442. 59322 422 .524i :4222 ...... 222222 v52 46s - 22-222.............22viVer. 6226: 2—is ’2 . v2.22. 525 244225 2226 g"var. 222.27 ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------I {\2 >—-A 3" V.ar 473 8-26 Var. 562 2224 Var.

J E;1.21 is ’1 2440 €1,731. ’ ix) 1—“ M2 EVar. " ' ‘ EVa1. E Var. ’ Var.

Var.

Var. ’ Var. ’. 26—38 26—36 2866; 4—12 Var. 581 28454 4—11; 28-32 2.830 {inZil 648 211—311 Var. = Variation {39163} 111 ene embedimenta a rVWF eempesitien ean be characterized aeeerdmg to the percentage ofrVWF molecules that are present in a particular higher erder rVVt’F multimer or larger multimer. Fer example, in one embodiment at least 20% et‘rVWF molecules in a rVWF composition used in the ds described herein are present in an eiignnierie eemplex of at least it 0 subunits. in anetber embodiment, at Eeast 209/2; ef r’VWF molecules in a rVWF composition used in the methods described herein are t in an eiigomerie complex at at least 12 subunits. 111 Vt?tether embodiments, a rVWF eempesitien used in the metbeds presided herein has a ruinin’ial percentage (age, has at least X96) et‘rVWF les present in a partieuEar —Order rV‘WF multimer 0r larger ntuitimer (eg, a 11111111111131 of at ieast ‘1" subunits) aceerdiug to any uue 01121113116115; 134 10 457 found 111 Tahie 3 t0 e 5.

Tahie '3. Exeh’1p1ary eh‘1b011111’1e11is 11.11 the percentage 0171VW157 11101eeu1es that are presem 111 21 13111911 higher order 1V'V1/7F 1111.11t1111er or 1:1.rger multimer 10111111 111 the compositiens 211111 used 111 the 111121110113 {311111111311 hereiu. 1111111111111 Number 111‘ Subunits 1171;1‘17‘171711‘ 11111111111131 .»?- 1am: g 17111 14:75 —731111 1:71 13*" is: 41) Var. 158 ‘1I111. 1713 {13 V3.1. 17.3.1159 V211. 1'7 17.3.1195 M 1’11 1% 17111 222 :Inn 0 .' . 143 Var. 161 ‘1I111. 179 V.:1.1 197 111.215 ‘17a1 233 {13 V3.1. 144 V.ar 162 V3.1. 181) V.ar 198 6 Var.234 2 711% ,. V:11.164 ‘1I111. 182 1711.281) Va1. 218 .236 g 775% ‘11—’31183 Var. 2131 Var. 219 117111.237 1129: —82V111 17111 222 ‘1I111. 11:19 Var. 187 ‘1I111. 2115 V.:1.1 223 Va1. 241 V211.— 8721111111011 7121111: 4. Exempiary embodiments for the percentage 11111711717 11101ee111es that are present 111 (J‘s 21, 11111110111111 higher order 1V‘1VF 11111111111131 61 1a1ge1111u1t1111er found 1111111: £30111pﬂ$1110115 111111 used 111 the 11’1611’10118111‘0‘11111611 herein. iiiuimai Number uf Szughuuits 1112117171711" Muitimer 28 rVWF 111' ‘117111281 Pereeutage 311% 1731.246 Var.264 Var. 282 Var. 3131) ‘1I’a1.318 172111336 % ‘11—’31283 Va1.3{31 Var. 319 .337 22-1711 “17111 232 11711111111211 17111 222 V111. 269 Var. 287 ‘1I111. 3115 V.:1.1 323 / 9 ‘131. 2:12 Va1'1.27) 17.3.1288 V3.1. 386 Va1.3'24 Var. 342 V.111 254 V111. 2'2 V.111 291) V111. 34138 V.111 3‘26 V111 2’73 31111 327 V111 343 :7111. 256 V111 274 V:11. 292 VI111 310 VI111 328 V111. 341:1 V111 275 V111 293 V111. 311 V111. 329 V7111 347 V111 276 V111 348 1111‘ 277 V111 293 V 111'. = ’V111i11‘tion Tahief‘éPumpian 1.1117130611118814 {’01 the tage (11 1VVVF 1110ieeuiesti1111 111e present 111 11 11111110111111 highe1 o1de1 1VWF ei” 01 i111ge11nuitinie1 found in the 1.3011111051110115 11110 115011 in 1111.1 111131110143 1110171111311 . ................................................................................................................................................................................................. 8311113111118 1811111111131 11481111111111.1111111 1V’WF 74411311111111 --- Meieeules 1111‘ 422 1:11 4413 337'V11 V:11. 352 V.111 370 VI.111 388 VI111 406 V:11. 424 V111.442 V.111 353. V111. 374 V.111 389 V211. 4G7 V111. 4‘25 V7111. 443 1VWF Var 31.111 . . ,. .- , 31111409 V111I.427 ,. 11f .9 V111. 356 V211. 374 V111. 392 V111. 411) V111. 4‘28 V7111. 446 Fereeetage 73 ’ 3:11. 358 V111 376 V111. 394 V111 412 V1111430 V111. 448 V111. 413 V111. 43] V7111. 449 V111 7’38 —9{1V111 V111.: 871111.458 841111111331 —393‘111 V111 432 V111 3613 V:11. 38E VI111 399 V:11. 417 V111. 435 V111. 453 V111. 364 V111. 382 V211. 44134) V111. 418 V111. 436 V111. 4:14 V11 4132 V111. 3611’) . , - . VI111. 420 V111. 438 V 111. 456 V111. 367 V111. 385 ' ’ ' ”‘ V 111'. V111iatien {438164} In 11.011011412111111: with the abeve. the 1VWF eompesition administered 10 the subject (with o1witi1out FVEH) genemiiy compi'ises a cant percentage of high moieeuiar weight (BMW) 1VWF 01111111111313. 111 11111he1 embudimentis, the HMVV 1VVVF 11111111111121 1.0111131131111011 111111111131 .51 at least 108/ ~ 80°.41 1VWF decamem 01 h1gher 01131111 1nuiti1ue1s 111 fu1the1 embodiments, the c0111p0sition eemprises abth 10—950’/1:-, 20—900/8, 30—85%, 40—80/0. 50475370,. 61170371. deca111e1$ er 01131111 11111} . 111 1111ihe1 ei'11bedi111er1ts, the E‘iMW 1VWF 11111111111131 composition 60111191111133 at least abeut 10.0. 20%, 30%, 40%, 50%, (10%. 7 %. 80%, 90".8 (41113111111318 13111314331631” 01de1 11111111011115 {843165} ment 01 the mimher and percentage 01 1'VWFim11Etimers can be conducted using 111131110145 known in the 1111. including without 81111111111011 methods using eieet1opheresis and size exclusion tography metheds ‘10 separate VWF muitimers by size. 101 example as discussed by Cumming et a3, (3 C1331 Pathel. 1993 May; 46153: 4713473, which is herehy ineerperated by reference 111 its entirety for all purposes and in particular for all teachings re3ated 111 assessnunt et VWF 1111131111111s,3. Such 111111es may t1irthe1 11131 imntunehletting techniques (such as Western Blot), 111 which the gel is immanehletted with a radielahelled antibody against VW? fellewed by eherniluminescent detectien (see 1111' example ‘3 ’en et a3, (3993), 3. Clin. Lab. Anal” 7: 3 37-323, which is herehy incorporated 31y reference 111 its ty 1131 all purposes and in partieular for all teaehings related to ment et‘VW 3‘ n1ulti1ners).33111131er assays for WW3 include VW3:Antigen (VW313g) VWF:Risteeetin Cetaeter (VWRRCol’), and VWRCollagen Binding Activity assay (‘17’3’17’31’2138/1), which are Often 1.1sed fer sis and classiﬁcatieh of V011 Willehrand Disease. (see for example Fayalere et a3, Patholegy, 39971 29(4): 343 ~45133 which is hereby tnated by 11ee111 its entirety 1111 1133 11u1peses and111 particular 101 2133 t1;11133111105 elated te assays for \I’WF). 1133331163 111 further 111113111131111e11ts, higher order 1VWF 11111311111ers 01’ the invention are stable for about l to about 90 hours post~administration 311 still further embodiments, the higher order rVWF multimers are stable for 113313113 5—833. 113—70, , 2060., 25430, 3G~35 hours pest—~ad111111istratien. 111 vet further ments the higher enler 1VW33n'1ultime1s are stah3e for at least 3 6, l2. 18., 24, 36., 48, 72 heurs pest~administratio11. lh n embodiments the stability 01‘ the 1VW331111.1ltirnersis assessedM 121111) 313333671 311 one embodiment, higher erder 1VWF inultirners used in the compositiens and. 111eth01’ls provided herein have a hall“ life 03’ at least 12 hour pest ad1ninistra.tie11. 111 r “ni'hediment, the higher order 1‘V’WF multiniers have a half life of at least 24 hour post administratien. 311 yet other en’1hedin’1e11tsg the higher order rVWF timers have a, 311133313713 selected from variations 642 to l045 found in Table 11.

Tahie d Exemplary embodiments 1’01 the 31213331111 03 h1ghe1 11113111 rVWF multimeis 111111111111 the cempesitiens and used 111 the metheds provided herein.

Hears Hears at least 3 4:3 X . 11572 atleas13 45 a1 leas1 4 at least 5 at least (1 ' . ~721Var 9513 a1leas1I . . .

, . . Var. 11513 21766 l.‘Va1 953 17111619 171127511 27—611 1:711. 952 17211165111 1627-54 1711.953 1111111111113 172111151 17117.2 34—36 27~48 117111. 9:14 at leastll " . ’52 ~C V" ‘. 3 Var. 854 313—913Var955 .................................................................................................................................................................................................... 611663112 V761. 6:33 678 V7611855 30484 V761. 6472 3674 E71744 ...41..1.22§1-1.2..V64671’66 V416” _____’22:27.24......3.781.2528...... m—LkasLlﬁﬁ V1761.656 66-0 Va1. 858 3066 EV761 _116111126 V7111.§”76 _54 V7111 756 _L26 ' V44” 7:79 1- . 6L 1166L 74 14— V761 _1:12 —-6_680 V—7a18-6} 304488-62V61. 3645 E11766“;.- V44” 762 36-42 V46 i. 4464441178161 ' 7 ‘ 1136 V61. 663 633 V’a1.7644 61l66$139 V61. 664 63i} V761”. 765 ..................................................................................................................................................................................................

V61 8771 33—66 EV61. ........................................................................................................................................................................................................ 3161832 3366 V761 61.11.111.66“1636—'33545V7111. 161661171 V7.61 6:2 Var 773 16—33 V761. 674 314111 44 1.111 84 _6->6 3345 V7211" 6116116180 V761. 67-4— _628-1761.6 33——42 EV761.

V47 776 .

—V47 675 :-46 33129 V761. _6’42 V161878 36-80 EV’ar 880 31761678 8.72 V761. 88G 3684 EV761. 117111.674 V4 746 3678 11.7111 V.61 781 V61. 8823 36—72 EV’61.883 V761 712 V761. 883 3666 EV761. 2—36 V7111. 685 11-36 V7.11 786 1-854 V44 666 Van 7477 1—444 ’0 2.13 3—1.2. V7211”. 7111.2. _843 " - 888 V7a1. 788 ........................................................................................................................................................................................................

V761”. 786 V61.883 V761 884 Va1.785 ........................................................................................................................................................................................................

V761. 786 .1680 ........................................................................................................................................................................................................ 1003 1683 1394 3—2212122221251’22 7112' 222 2222 _54 V211“. 70 _G-42 X—Iar86)}?_€)—39 m739 4548 48911 —"221V22 719 11212 7'20- 227—7 222722—_222 2! -222 223 22.2 2:2 322 “22 740 12214 Var 841 2419 Var 9-42 741 3322 923 V211'. = ‘2’ 11112111011 E11 111111161 2111112322111, 1VWF 03 1186311117111? present 111111111011 111111121511, stability of plasma FVHI, which, as will be appreciated, may include deVl} 01‘ 1"?VIII which has been 3111111111316161'}. ‘10 111,151,12111611‘1 or it 1111137 11113111148 FVE H 1111110g1311011s 10 the 33311131119 or my i11at1‘1‘111 thereaf. For exampie, as Shawn 111 F iii. 22, the haii‘liife 0fpiasma FVIH is increased in VWD patients receiving rVWF in combination with rF‘v’lll as compared to life of F‘v’l it by about l. 5 — 5told as 11111111111ed to the te seen with patients 1eeeivi11g F’Vlll alone. in still 1111111131 embodiments, rVWF increases half—life of F‘Vlll by about it) — 45 l5 4(l 20— 3.525 — 3 it told. ln one embodiment adminisnatwn ot 1‘13?»F increases the stability of plasma FV’lll, as compared to the administration of plasma—derived VWF, by an amount selected liom variations l046 to l089 found in Table 7. in a specific embodiment, administration of a rV WF/FVlll x increases the stability of plasma FVlll, as compared to the administration ofplasmawderived VWF, by an amount selected from variations W46 to l089 found in Table 7 Table '7. Exemplar; embodiments for the increase in halt—life experienced by plasma FVill alter atlininistratmn of rVWF and r\WINS/F1lll complexes as con1pa1ed to after administration of uderived VWF and plasniauderived VWF/F’Vlll complexes.

Increase in increase in increase in increase in plasma FVHE plasma FVIH plasma FVHI plasma FVlll half—life half—life half—life halfmlife Var. ', '1 . at least l.5fold 1046 1 ."0fold 0 fold 441 .1 ~ V3112 1 at 11121512540111 1114s 411-10111 24.5 10111 V3.1: c' ‘. . ' ' at least 30—fold 1049 d 2411111111 ' 3—4. 0 told 11382 Var. ', '1 t, . . atleast 3.5fold 1050 30—fold ' I ’1 ' ' ’ ' 3-3.5 fold 1.122115141110111 1.5 .' ‘ 3.5—5.010111 2111 {321111145 12111 f :3 ' , 3545 111111 3.5-4.0 fold 4- 5. 0 fold 44.5 fold £43341:in in still tiirther e1'nbodi1nents, the increase in F‘v’l ll ife is ined at least ll}. , 30, 4t), 50,, 60, 70, 80, 9‘0, ltltl hours after administration of E'VVVTF. in yet further embodiments. the increase in FVlll 111111—1111: is ined at least 54,25. ll)—l l5. lSmlOS. 21% 95, 25—85, 30—75, 3545, 40—55 hours after administration of 1"VWF. {944174)} in one embodiment. the increase in the mean residence time (MRT) plasma, FVlll is maintained for a time selected from variations lllgtl to l299 found in Table 8, after 2012/041957 administration of rV’WF. in a c embodiment, the increase in the hali‘ilfe of plasma F‘V‘lll is maintained for a. time selected from variations 1090 1:12 1299 found in Table 8, after adniinistratien of a FVWF/F‘v’ill emnpiex. 777777 administration, efrVWF and YVWF/FVIH complexes.

I I Ham's Hours Ham's atleasté Ma: 10911 *2, 2 : , .1196:§ 511—102 EVa: 12159 atleast 12 Va1.l E44 i 60-.96 iV:11 - atleast 18 1—2241198 60-90 at least 24 '- i "i( Var 111:3 Var.il99 511—84 211122215130 20 Vai'=.'2011 60-78 i atleast36 Val. 1148 V31. 12111 60-72 at least 4‘7 _:;__j;__i_9?_§__ . “TEES-.3292... 50:2?___________________ at least 48 ' . 1097' Var. i203 66420 K. : ’ at least 54 222.152.221.29. Var. 1152 V211. 1- at least 66 Var. 1 153 Var. 1206 66-102 ii at least 72 222.152.221.191. : 66-90 at least 84 1.209 66-84 at least 90 ~ 1210 61971 at least 96 atleastliiz '< ' ‘1 ‘ . i ” ‘11— 3 V211,?1 .2 meastnia .. 73.1.1160 Var. 1213 at least 114 Var. 1214 atleastlZOi . 15i h.) -l U] .129 78—96 ' -. {‘0 \3 ON 1.4 M- \i \1 ,, Va. 1225 78-90 V211. 1 2' Var. 1226 78-84 Vain?1. h.) -1 ’73 - Var.122‘7 84—120 Var. IR) \3 O1, 2 28 134-114 V2111 N 00 c) Var. 1229 84-108 ‘= . 2 ’ 2 48—72 Var. ' Var. 1178 48-66 Var. ' 84-96 84-90 ________________ 90-114 911103 W0 2012/171031 12 871121183 54—1118 8111. 1236 90 1112 v11. 1184 817811.123? 9096 “ “ 8711-. ' t 96120 ‘. 1- 1— v11. 1 96—114 , 31171 v11. 1240 96—108 _ Var. 1188 Var. 96102 . 9'11. 102—120 - v11. 192—114 ‘1 Var. ' 11121111 1 v11 1192 v11. 108—120 v11. 1193 108—114: 60-108 Var. , 114—1211 Var. Variation, 588171} in further aspects, the rVWF of the invention shows an increased ton the stability of FVHI as compared to the effect ofpdVWF. For example. as shown in , average FV111 haitliife was increased by rVWF by 5.2 hears over the naEf—iife fer patients receiving pdVWT- . in further embodiments 1VWF increases average FVHI 11111211111. by about 1—15, 21-14, 3 13,—4125ti, 6 113,59, 68iio11rs in sttii tnrtner embodiments, rV’WP ses FVHI hatfniit‘e by about 10%19 about 75% as ed to pdVWF in Vt.t furthei embodiments, rVWF increases FViii fe by about 1098053451 15165841, 1, 255.5%, —500’w, 354°/0 as compared to pdVWP. in certain embodiments, the administration of rVWF increases the 1111111111 of F‘v’iii by an amount selected from ions 1300 to E643 foundin Tahie 9 as compared to administration of piasma—derived \1’\‘1E. in yet further embodiments, the average or percentage increase in FV1H half—life is maintained at least it}, 28, 3t}, 41), 5t}, 6t), 70, 8t), 9t}, EGG hours after administration of rVWF. in yet further embodiments. the increase in FVHI haitllife is maintained at 125 10 115,15 £05, 20— 95, 25~85, 319975, 35—65, 48-55 hours after adntinistra‘rien ot‘rV WEI.

Tabie 9. Exemplary embodiments for the increase in haifntife enced by plasma FVHI atter administration of, rVWF and r\WEE/F8111 complex inereasein increase in Inereasein increase in half life hatt’ Rife 111111“ life haif life atkast 2hr .-"' ' i; .; , 10-90% at least_1 hr 8711-. 13011 —' .- , 1.1;). , v11 1565 Var 1566 v11. 1307 — .- 1: 4090% , 1.1;). ‘ «115% 12 1-” -= a , /',.1j;,," 40-65% Va11313 , ,— 1 40-00% ‘ 1’ - _. : .1 .x. ,0 : .H‘ .xL/f) -_. , 40-50% 10/301570 : : Var.1580 Var 1326 V0182? m, V3111. 591) 5000'% Var. 1594 Var. 1595 Var. 1596 50-55% Vat 1598 55-10094; Var. 1599 -« Var. 1341 - Var. 16136 Var. 16117 Var. 1351} Var. 1353 60-65% Var. 161i) 65—10091; Var. 1616 Var.

W0 2012/171031 ' : Var. 1613 Var.]360 3 .- 57311162] 3-11h1 Var 1361 at Eeast 75% 3 ”' ” 4 '3 , 7 ., '. . ,' Var 1622 atieast 3111‘" ' r- - . . , 711—1 11113 3-9131 Var. 13133 atitast8‘170 ‘- '.". z, . I.’ -j .'"'" '. 116213390170 : 7 at least, 95% ' ‘ a T ‘ - . . 1100270 ' ' ; ,1 Var,1_.68 " i ' 3 - x . Z . . 7.5—}00‘3/0 Var. 1629 Var.1369 ‘ " 1,.‘1 3' ‘ ' " .i ,—..",% 30 Var.137 ? : , 73.1.1376 ’ 3 ' Var.1637 17.31.31” 7:1 i 1.2.3 1 577311638 VE'L" 1311 Val” 13311 " ,oi 7' ' 9111:3031. Var. 1641 Var. 1382 . , 95%~i00% 10070EEV21121 1040072) Viar.

Var. V ariatiori {00172} in still further aspects and in accordance with any of the above, the stabilization of FVHI activity by rVWF as compared to pdVWF can be measured by metrics in addition to FVHEinaif—irte including mean residence time (MRT) and area under some (At1C) exemplary embodiments, rV‘v‘i/F increases MR3" by about 1—15 hours as compared to pdVWF in 1311101631“ embodiments, rVWF increases M RT by about i~25, 2—20. 305, 440, 5—9, 6—8 hours as compared to pdV WF. in stiii further embodiments, rVWF increases F‘Viii MRT by about. 10—80%, 15—65 7'2), 2060/o 2555%3050% 355—45% as compared to pdV‘v’W. In yet tiirther embodiments, the average or percentage se in FViii naEf—iife is maintained at least 10., 20, 30, 40., 50, 60. 70., 80, 90., £00 hours after administration ofrVWF. in certain embodiments, the stration offs/"NF ses the MR? ofil‘Vi it by an amount selected from variations i644 to 1987 found in Tania 10, as compared to administration of plasma— derived VWF. } in yet r embodiments, the increase in MRIE‘ is maintained at least 5-125, i0- llS, 3 5—105, 204955, 25—85, 39—75, 35435, 40—55 hours after administration ef fv’WF. in certain embodiments, the increase in MRI of FVEH caused by administratien efrVWF i s maintained for a time selected from vai‘iatiens 1090 iii 1299 found in Table 8.

Tahie lﬁ. Extii'nplary embodiments for the increase in FVHI mean residence time after administration of TVVVF and V’FVHI complexes, as compared to after aeiniinistiatien of wdeiived VWF and, plasma—derived VWF/FVIH complexes increase in increase in increase in half ﬁfe haif life half iife Var. 1647 .:- at least 7 in“ V7 at least 8 hi Vain 1913 Var. 1914 Var. 191.3 1m~ Var. 1659 , , .‘= _ 40~65% Var. 1917 76.111660 ' '3 .-1 _ 40—60% Var. 1918 Var. 1661 ' ,i. . ‘:.’~, Var. 1919 'anlm -' 2 Var. 1664 ' ' ' " . 45-19094; ' ' y. , .— '5, 45-90% Var, 192.4 ‘ : 45-85% Var. 1925 i . , : 45-75% Var, 192.7 '2. ' a_ . , , , . i. a , . 45-70% Var. 1928 Var, 167i ' v. , an: . ,3 Var.]672 i ; - Var. 1673 i : '3 ~. 52111931 Var. 1675 - a, -. , 50.400175 3331:1676 11.13 ' 3 ' ‘ - -' , Vai'.1935 i x» Vat. 193 6 -23% iii/31:13:51 50-80% 1573:1937 2012/041957 50659/ Var, 1949 % Var. 1941 Var. 1685 z.- , ,, — '_ 1 ,4 , . 3.; 55 11% Va1‘.1945 " ' Var, 16,90 Var. 1691 : : Var. 1954 a ,' = . . Va1‘.1955 76.111698 atieast35% :5 ' ‘ a11eas14995 v, ,; - _, , , 7 Var. 1957 311321314594 " ' I , 1‘: 7211:1701 ' at Eeast 50% ' . -. J, a ', .) Var.1702 211134195595 . . ";.;;. . 311321316994 , 5 ,' 1‘: 3-1;. Var. 1704 atlsast 65% - [L - , , f"; 65-90% Var. 196.7. ' : 654359" Var. 1963 V1111. 1965 Var. 1966 Var.1710 atlsast 95% T ‘ ' , ',.'-' 70-95% x4711191968 Var. 1711 at Eeast 100% 4 '4. 1 , , 1 ', 709094", Var. 1969 75—10094) Var. 1973 75—95% Var. 1974 9 “E 'E E7' > ,_ ',. .4 7211‘. 1726 —12 hr Var. 1727 i ’ ,o . 35—9094;- '-, . 1899 913-10094; Var. 1985 —85%. Var. 1900 90-95% Var. 1986 —_ 95% Var. i815 35—90% Var. i901 )0% Vat Variation Sim} 74} Further exeniptary differences n pdVWF and rVWF are provided in the following table: .......................................................................................................................1........................................................................................................................ pdV‘WF rV‘WF Synthesized in endotheiiai ceiis and a Expressed in CHO eelis ine ,aharvoCVtes Post—translational modification of s E’ropeptide i'einovai mediated in vitro propeptide rernova} occurs intraceiiuiariy: through exposure of the pro—VWF to during passage of the protein to the Goigi recombinant Fnrin and est—Goini coin artments Giyeosyiation ABO blood group Fully glycosyiated ABO blood group s present gyicans absent Consists ofVWF subunits that have been No exposure to ADAMTSB exposed to plasma ADAMTE‘S it 3 E »»> intact VWF subunits —> Uitradarge VWP inuitiniers absent ----> Uitra—iarge 'VWF niers t »»> ts d at TYRmah’iti’t'mﬁ —> Subunit cleavage occurs upon ADAMTS i 3 ex osure Plasmanderived V‘WF concentrates Higher specitie activity than pdVWF contain other proteins incl. ADAMTS 13, E 306111th tutinins {$9175} in some embodiments, the ratio of rFViH proeoaguiant activity (1U rFViiizC) to rV WP“ etin cofactor activity (it) rV‘v‘v’FiRCo) is between 3:1 and "3:5. in i' (J; embodiments, the ratio is between Zzi and 1:4. in still further embodiments, the ratio is between 5:2 and "3:4. in turther embodiments, the ratio i s between 3:2 and i :3. in still further embodiments, the ratio is about lzi , 1:2, 1:3, 1:4, 1:5, 2:1, 2:3, 2:4, 2:5, 3:3 , 3:2, 3:4, or 3:5. in further embodiments, the ratio is between 1:1 and "3:2. in yet thither embodiments, theratioisiiﬂ,1.2:},1.3:t,1.4:i,1.5:},i.6:t,1.7:1,i.8:t,i.9:i, or2:1. in certain embodiments, the ratio ot‘rFViii proeoagnlant activity (1U l‘FVHIZC) to rVWF Ristocetin cot‘actor activity (1U i‘V Wii‘RCb) in a composition 'nseﬁii for a method described herein is selected from variations 1988 to 2140 found in Tabie H.

Tabie 11. Exemplary embodiments for the ratio OfrFViEiEE procoagulant activity (3 U rFV 1112C) to rVWF Rismcetin cofacmr activity (EU rVWF2RCQ) in ccmpcsitians and used in OdS pmvided harem. (1U rFVIH:C) m (1U rFViﬂzC) its} (11} rFVIH:C} :0 (EU x‘FVﬂisc) is} (11} rVWF:RC%) (EU RCo} (EU rVWF:RC%) (EU x'V‘VVF2RCn} Var. Var, Var. Var. 4.1’ . m3.18.5_ 4.3—1.4’ ."7 ‘ 4..,~2.3“g _ 1998 7‘0“ 2065 7103 Var. 203: 8 Var. a I , A , 2087 ''''''''''''''''''''''''''''''1"“ ........ ‘ .......................................... "'_'“:““““ .......................................... ‘ ,......... .......................................... "'_'“:““““ \I': '3:.1 *?1 V' r Ka mm \O- m C" ‘3 mc) av:1 N "J Var. = Variation Ellhliltil in specific aspects, the rV‘vl/TF and/or the F‘Vlll (recombinant or plasma derived) used in accordance with the present invention are not modiﬁed with any conjugation, post“ translation or nt modifications. in particular embodiments, the rVWF and/or F‘y’lll of the present inyention are not modified with a water soluble polymer, including t tion, a polyethylene glycol (PEG), a polypropylene glycol, a polyoxyalltylene, a polysialic acid, hydroxyl ethyl starch, a, arbohydrate moiety, and the like. {“3177} in other aspects, the lI and/or the FVlll (recombinant or plasma derived) used in accordance with the present invention are modified through conjugation, postmtranslation modification, or covalent inodili call on, including niodilicatirnis of the N~ or C— tern’iinal residues as well as modiﬁcations of selected side chains, for example, at free sullhydryl— groups, primary amines, and hydroxyl~groups in one embodiment, a water soluble polymer is linlted to the protein (directly or Via a linlter) by a lysine group or other primary amine. ln one embodiment, the rVWF and/or FVlll proteins of the present invention may be d by conjugation of a water soluhle polymer, including t limitation, a polyethylene glycol (PEG), a opylene , a polyoxyalkylene, a polysialie acid, hydroxyl ethyl starch, a poly—carhohydrate moiety, and the lilte. {(39178} 'Vv’atcr soluble polymers that may he used to modify the rVWF and/or FVlll include linear and branched structures. The conjugated polymers may he e directly to the coagulation ns of the invention, or alternatively may be attached through a linking moiety. Non—limiting examples of n conjugation with water soluble rs can be found in US. Patent Nos, 4,648,835; 4,496,689; 4,30l ,l44; 4,673,4l7; 4,79l,l92, and 337, as well as in Abuchowslri and Davis ”Enzymes as Drugs,” Holcenberg and Roberts, Eds, pp, 367 383, John Wiley and Sons, New York (198l ), and lflernianson t1, Bioconjugate 'l‘echniques 2nd Ed, Academic Press, inc. 2008.

Etllll79l Protein conjugation may be perfoimed by a number of well known techniques in the art, for example, see l-lermanson (1, Bioconjugate ques 2nd Ed, Academic Press, inc. 2008. Examples include linkage through the peptide bond between a carbonyl group on one of either the coagulation protein or waterssoluble r moiety and an amine group of the other, or an ester e etween 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 r 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 ot‘ the polymer by periodate oxidation (Jennings and Lugowski, ,l. lmmunol. l981; l27: llll l—S; Femandes and Gregonradis, Biochim s Acta. l997; l34l; 2634). The generated Schiff Base can be stabilized by specific reduction with NaCNBl—lg to form a secondary amine. An alternative approach is the generation of terminal free amino groups on the polymer by reductive amination with 'Nl-l4Cl alter prior oxidation. Bitunctional reagents can be used for linking two amino or two hydroxyl groups, For example a polymer containing an amino group can be d to an amino group ol’ the coagulation protein with reagents like BS3 {Bis(sulfosuccinimidyl)suberate/l’ierce, Rockford, ill). in addition heterobifunctional cross linking reagents lilre SulldliMCS (Nu-awMaleimidocaproyloxy) srtlltisiicciriii'rriile ester/Pierce) can be used for instance to linlt amine and thiol groups, in other ments, an aldehyde reactive group, such as PEG alkoxide plus diethyl aeetal of hromoacetaldehyde; PEG plus DMSt’Zl and acetic anhydride, and PEG chloride plus the phenoxide ol’zl— hydroxybenzaldehyde, imidyl active esters, activated dithiocarbonate PEG, 2,4,5— trichlorophenylclorolormate and P~nitrophenylcloroformate activated PEG, may be used in the ation ot‘a coagulation protein.

ElltllStll in some aspects, the rVWF used in methods or“ the present invention has been matured in vim) with Furin. in further embodiments, the li‘urin is recombinant Furin.

Etlhlgll in further aspects, the rVWF and/or rFVlll used in the methods of the present invention are ed by expression in a mammalian cell culture using methods ltnown in the art. in particular embodiments, the mammalian culture comprises CHO cells. in further ments, the rVWF and the rFVlll are comexpressed in the same culture, in such embodiments, the r‘v”‘l’v’l7 and the rli‘v’lll are purified together (co-purified) or separately using methods known in the art. in other embodiments, the rV‘iVE and the rFVlll are expressed in different cultures. {(30182} in an exemplary embodiment, the rVWE of the invention comprises rVWF n isolated from a CHE} cell expression system, in a further ment, the propeptide removal is mediated in virro h exposure of the pmvwr to Furin in a, still further embodiment, the Furin used for propeptide removal is recombinant Furin. in as yet r embodiment, tully glycosylated/ABO blood group glycans are absent. {90183} in yet lurther embodiments, the r‘v’WF used in methods and compositions of the present invention by expression in a suitable eukaryotic host system. Examples of otic cells include, Without limitation, mammalian cells, such as CHO, C08, HEK 293, BB K, 333 lflep, and l’lepGZ; insect cells, e.g., SF? cells, SFZl cells, 82 cells, and High Five cells; and yeast cells, e.g., Sacchtrromyces or Sci/i,izostrccizammvces cells. in one embodiment, the VWE can be expressed in yeast cells, insect cells, avian cells, mammalian cells, and the lilre, 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., (Ill-l0 cell from a continuous cell line, can be used to express the ‘i/‘v‘v’lI of the present invention, Etllll 84} in n 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 d 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 VVVF, EtlhlSSl A Wide variety of vectors can be used for the expression of the VWE and can be selected from eulraryotic expression vectors. Examples ol’ vectors for eulraryotic expression include: (i) for expression in yeast, vectors such as pAO, pl’lC, pYES, plle'l‘, using promoters such as AOXl, GAP, GALl, AUGl, etc; (ii) for expression in insect cells, s such as ph’l'l’, pAcS, pllil, prlllEl, pBAC, etc, using ers such as Pl—l, pl {3, MT, AcS, OplEIZ, gpd4, polh, etc, and (iii) for expression in mammalian cells, vectors such as pSVL, pCl‘le, pile/RSV, pcDNA3, , etc, and vectors derived from viral systems such as vaccinia virus, adeno—associated viruses, herpes viruses, retroviiuses, etc., using, promoters such as CMV, S‘Mt}, EF—l, UbC, RSV, ADV, BPV, and li—actin, {noise} in some embodiments of the t invention, the nucleic acid sequence r ses other sequences suitable for a controlled expression of a protein such as promoter sequences, enhancers, TAT/3t, boxes, transcription tion sites, polylinhers, 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. {(33187} ln certain emhodimenta the cell—culture methods ot‘ the invention may comprise the use of a microcarrier. in some embodiments, the cellmcultures of the embodiments can be perlorrned in large hioreactors under conditions suitable for providing high volume~specilic culture surface areas to achieve high cell densities and n expression. One means for providing such growth conditions is to use microcarriers for cellmculture in stirred tanls bioreactors. The t ofcell—growth on niicrocarriers was ﬁrst descrihed by van Wezel (van Wezelﬁ AL, Nature 2l626£lm5 (l967)) and allows for cell attachment on the ce of small solid particles suspended in the growth medium. These methods provide for high surface—to—volume ratios and thus allow for efﬁcient nutrient ation. Furthermore, for expression of sec *eted proteins in eukaryotic cell linesr the increased surtace—to—Volume ratio allows for higher levels of secretion and thus higher protein yields in the supernatant of the cultue. Finally.” these methods allow for the easy scale—up of eulraryotic expression cultures.

Ellhlgdl The cells expressing VWF can be bound to a spherical or a porous microcarrier during cell e growth The microcarrier can be a mieroearrier selected from the group of microcarriers based on dextran, collagen, plastic, gelatine and cellulose and others as described in Butler (l988‘ in: Spier & thsr Animal Cell Biotechnology 3:283~303). lt is also possible to grow the cells to a biomass on cal microcarriers and ture 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 inicrocarriers, such as xlM l, Cytodexm 2, and (CytodexTM 3 (GE. liealthcare) and orous microcarriers such as CytoporeTM l, Cytotuu‘eTM 2, CytolineTM l, and CytolineTM 2 (GE Healthcare). 9} ln certain emhodimenta rVWF is expressed in cells cultured in cell cultu‘e media that produces high molecular weight rVWF. The terms “cell e solution,” “cell culture medium or ” and “cell culture supernatant” refer to aspects of cell e 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 supplementsg to e nutrients and other components for culturing the cells sing "V'WF. The ell culture supen'iatant refers to a cell culture solution comprising the nutrients and other components from the cell culture medium as well as products released, metaholized, and/or excreted from the cells during culture, ln r embodiments the media can he animal protein—tree and chemically defined. s of preparing animal proteinsfree and chemically deﬁned culture media are ltnown in the art, for example in US 260843039049 and US 2t)0'7/02l277€3, which are both orated herein for all purposes and in particular for all teachings related to cell culture media. “Protein tree” 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 an other embodiment, the culture n’iedium is ptide tree. in r embodiment, the culture medium is serum free. ln another embodiment the culture medium is animal n free, in another ment the e 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 r embodiment, the e has recombinant proteins ously added. in another embodiment, the proteins are from a ed pathogen free animal. The term "chemically 'efinet ” as used herein shall mean, that the medium does not comprise any undelii'ied supplements, such as, for example, ext‘acts of animal components, organs, glands, plants, or yeast. Accordingly, each component of a chemically deﬁned medium is accurately deﬁned. in a. pre erred embodiment, the media are animal—componerit free and protein free.

Elltll9lll in certain embodiments, the culture of cells expressing VWF can be maintained for at least about 7 days, or at least about l4 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, ll, l2, l3, l4, l5, l6, l7, l8 months or longer. The cell density at which a cell—culture is maintained at for tion 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 detennine the l cell density for a cell—culture producing an VWF. in one embodiment, the culture is maintained at a, cell density ot‘between about 0.5xlt}6 and 4xltl7 cells/ml for an ed period ol’ time, in other embodiments, the cell density is maintained at a concentration ofbetween about lﬂxl 06 and about l)7 cells/ml for an extended period of time. in other en‘ibodin’ierits, the cell density is maintained at a concentration of between about l .Qxl (36 and about 4.0le6 cells/ml for an extended period of time. in other embodiments, the cell density is maintained at a concentration of between about l .le (ill and about Attlxl ()6 cells/ml for an extended period of time. in yet other embodiments, the cell density may be maintained at a, concentration between about 20x l (l6 and about 4,0xl (36, or between about l .llxl ()6 and about 2.5Xl 0'5, or between about l .le 0'5 and about 3.5X106, or any other similar range, for an extended period ot‘time. Alter an appropriate time in cell culture, the rVWF can be isolated from the expression system using methods ltnown in the art. {(331591} in a specific en’ibodiment, the cell density of the continuous cell culture for tion ofrVWF is maintained at a concentration of no more than 2.5xl {3” cells/mL for an extended period. in other specific embodiments, the cell density is ined at no more than 2.0Xltl6 cells/nth, l,5>;:ltl6 cells/mi, lilitlll6 cells/mL, 0.5Xl06 cells/mL, or less. in one embodiment, the cell density is maintained at between l ,letl6 cells/ml, and 2.5xl ()6 cells/mL.

Etltll :92} ln one specific 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 US ZGlZ/GGSS l l0, tiled July 8, an l, which is hereby incorporated by reference in its entirety for all es and in particular for all teachings d to cell culture s and compositions for producing inant VWF.

Etltll 93} in r ments, subsequent to purification ately or together} from a mammalian cell culture, the rFVlll/rVWl: complex is reconstituted prior to administration. ln still further embodiments, the rVWF is d with Furin prior to or subsequent to econstitution of the ll/rVWF complex. in further ments, the Furin is recombinant Furin.

Eﬁhlé‘tdl in still further embodiments, the rVWF of the invention is not exposed to ADAMTSlS, with the result that ultra large (ie, comprising l0 or more subunits) are present in it"s/"it’ll compositions of the invention. {@0195} ln specific aspects, the rVWF and/or the rFVlll used in methods of the present invention are contained in a tormulation containing a , 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 r embodiments, for torinulations containing rFVlll, the formulation may further include sodium, histidine, calcium, and glutathione.

Etllll 95} ln one aspect, the tormulations comprising rVWF and/or rFVlll are 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, Zl : l9l— 200, (2004) and Chang et al., l’harin Res. l3:243~9 (l 996)}. {@0197} Methods of ing phannaceutical formulations can include one or more of the following steps: adding a stabilizing agent as bed herein to said mixture prior to lyophilizing, adding at. least one agent ed from a bulking agent, an osmolarity regulating agent, and a surtactant, 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 ofa , a hulking agent, and a stabilizer, ln this aspect, the utility of a surfactant is evaluated and selected in cases Where aggre ‘ation during the lyophilization step or during0 ‘econstitution becomes an issue. An appropriate hutt‘ering agent is included to maintain the formulation within stable zones of pl—l during lyopliilization.

Elltll 98} The standard titution practice for lyophilized material is to add hack a volume of pure water or sterile water for ion (Wt-‘1) (typically lent to the volume removed during lyopl'iilization), altl'iough dilute solutions of antibacterial agents are sometimes used in the production of pharmaceuticals for parenteral administration lChen, Drug Development and lndustrial Phannacy, l8rl3l l—l354 ($992)}. Accordingly, methods are provided for preparation of reconstituted recombinant VWF (_with or without recombinant Factor Vlll) itions comprising the step ot‘adding a t to a lyophilized recombinant VWF composition ot‘the invention. {@3199} The lyophilized material may he 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 coi'npound in adn'iixture with excipients suitable for the manufacture of aqueous suspensions). ln s aspects, such excipients are suspending agents, for example and without tion, sodium carhosyn'iethylcellulose, metliylcellulose, hydroij’propylmethylcellulose, sodium alginate, poljyrvinylpyrrolidone, 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 allrylene oxide with fatty acids, for example and without limitation, polyoxyethylcne stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example and without limitation, heptadecaethylmeneoxycetanol, or condensation products of ne oxide with partial esters derived trons fatty acids and a hexitol such as polyoxyetliylene sorhitol eate, or condensation products of ethylene oxide with partial esters ‘lerived from fatty acids and hexitol anliydrides, for example and without limitation, hylene sorhitan monooleate. in various aspects, the aqueous suspensions also contain one or more vatives, for example and without limitation, ethyl, or yl, p—hydroxybenzoate. {tilllelll in certain embodiments, compositions ot‘the present invention are liquid formulations for administration with the use of a syringe or other storage vessel, in further ments, these liquid formulations are produced from lyophilized material described herein reconstituted as an s solution, {tilllel} lo a turther aspect, the compositions of the invention further comprise one or more pharrnaceutically able carriers. The s "phannaceutically" or "phannacologically" acceptable refer to n'iolecular entities and compositions that are stahle, inhibit protein degradation such as aggregation and ge products, and in addition do not produce allergic, or other adverse reactions when administered using routes well—known in the art, as described below. "Pharinaceutically acceptahle carriers” include any and all clinically useful solvents, dispersion media, coatings, cterial and antifungal agents, isotonic and absorption delaying agents and the like, including those agents disclosed ahove.

Administration of com ositions of the invention for s of treatin 5 disease {(33232} ﬁne of the advantages of administering rVWF to subjects to treat ation e is that the higher specific activity of rVWF as compared to pdVWF allows flexibility in the amount ot‘rVWF administered and the n un’iber of times the subject is readosed with r‘v’Vv’lE" (with or without inistered F‘v’lll). in addition, rVWli‘ compositions provide the further lity of re~dosing with rVWF alone after an initial cor—administration ofrVWF and FVl l l, without need for additional dosing with FVill, As will be appreciated and as is discussed in r detail hereinﬁ the co~administered FVlll may be recombinant or plasma derived.

EddZGSl in one , the administration of rVWF in accordance with the invention results in higher plasma l levels and/or activity in the subject than is seen with a subject administered pd‘v’ WF. As discussed above, increases in FVlll levels and activity can be measured using methods standard in the art, thus ng for determination of appropriate dosages for rVWF with or without F‘Vll l. lilllllidl Single or multiple administrations ofi‘VWF (with or t F‘v’lll) are carried out with the dose levels and pattern being selected by the treating physician. For the prevention or ent ot‘diseaseﬁ the riate dosage depends on the type of e to be treated (egg von Willebrand disease), the severity and course of the disease, whether drug is administered for tive or therapeutic purposes, previous therapy, the ts clinical history and response to the drug, and the discretion of the attending pl’iysician. {tilEZtlSl in further embodiments and in accordance with any of the above, treatment of coagulation, diseases such as VWD or Hemophilia A may involve an initial treatment of rvwr alone or in combination with F‘Vlll, followed by one or more repeat doses ofrVWF alone, rVWF plus FVlll together, or FVlll alone. The nature of the initial and then the subsequent repeat administrations will depend in part on the disease being treated. For example, patients with VWD have some level of FVlll, but the stability of that FVlll is generally compromised because these patients lack ‘v’Vv’lE". ent ot‘VWl) patients may thus in some embodiments involve an initial treatment with both rV‘v‘v’F and rF‘Vlll followed by repeated administrations ofr‘VWF alone, ln other embodiments, the initial treatment may be with rVWF alone while subsequent repeated administrations are with both rVWF and rFVlll. in still other en’ibodin'ients, the initial and subsequent repeat administrations may all include a co—administration of both rVWF and rFVlll. Similarly, Hemophilia A patients (who lack F‘s/lit) may receive an initial ent ot‘hoth rVWF and rFVHi, and subsequent repeat treatments may comprise the administration of rFVHl alone or rVWF a1one. in other embodiments, the initial treatn'ient may he rFVHi atone whi1e the subsequent repeat ents invotve eo—administration ofrVWF and rF‘VIli.

E00206} 1n thither aspects, r‘VWF is administered to a subject in doses ranging 110111 0.5 lU/kg 200 ithg in some embodiments, rVWF is administered in doses ranging from 1— 190, 5—180, 10—170, 15—160,20—150,25—140,30—130,35—120,40—110,45—100,50—90,,55—80, or 60—70 iii/kg, in further embodiments and in accordance with any of the above TF (with or without FVIH) is administered to a subj eet at doses of between about 1 IU/ttg to about 150 iii/kg rV WEI. in stiii ﬁrrther embodiments, the rVWF and r17V111 is administered at doses of n 1.5 lU/kg to 150 iU/hg, 2 iU/kg to 50 lU/kg, 5 iU/hg to 40 lU/kg, 10 1U/hg to 20 H.1/kg,_10 ELI/kg to 100 , 25 ELI/kg to 75 iii/kg, and 40 iii/kg to '75 iii/kg, 1n stit1 further embodiments, rVWF is administered at ‘2, 5, 7.5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 iU/hg. As will be appreciated and as is discussed further herein, appropriate dosages of rvwr (or rVWF together with F'VHI) may be ascertained through use of estab1ished assays for determining htood 1eve1 dosages in conjunction with appropriate dose—response data, in one embodiment, rVWF is administered to a t in a dose selected from variations 2141 to 2338 in Table 12, Tabie 12, iﬁlyxemplary embodiments for the dosage ot‘rVWti‘ administered to a suhj eet according to the methods ed herein, About 3 Var. 2144 About 4 Var. 2145 o About 20 Var, 2149 About 35 Var. 2152 About 40 Var. 2153 About 50 About 55 About 65 , About 70 Var. 2159 ---------------------------------------------------------------------------------1—-------------------------- --------------------------- --------------------------- --------------------------- ---------------------------.

About 75 Var. 2160 1—40 Var. 2210 75—10 Var. 2260 30—50 Var. a" Abtsnt 85 Var. 2 E62 10-175 Var: 2’26? Aboutaa Vain2163 10-150 Var.2263 .2312 2313 .2315 -.; Va,r.2316 i Var, 2317 Var. 2169 Var. 2318 ' ' ' .21'' Vat.44.,A”V7”: [\1 ‘LN N C) t IJJ h.) H \J J About 1-45 Var. 2174 ' Abctit 155 Var. 2176 About 160 Var. 2177 76.112178 .. 'L, U i Var.2280 Var.‘ .. Var. 23:30 Var. 2331 Var.2283 Var.‘ Abtsnt 195 Var. 2184 - w 25-200 Vamsggs ..........................................

Var. 2334 Var, 2189 -, 05-i00 Var. ZEQQ Var. = Variation %20’7} in stiii r embodiments, rVWF is stered at a, dnse such that it increases half—lite of piasma FVHI by ahent 1.0 n 4.5., 1.5 n 4.0., 2.0 — 3.5:, 2.5 — 3.0 Enid. in stiii further inients, the dose and/er frequency of rVWF administration is such that the increase in FVHI haifniit‘e is maintained at least 10:, 20, 30,5 40., 50, 6:05, 70, 80, 90:, 100 hours after administration ofrVWF. in yet tiirther ernhedinients, the dose and/er treatieney of rVWF administration is such that the increase in FVEH haifmiife is maintained at least $125, 1043 15, "35—105, 20—95.“, 2535, 30~75, 35—65., 40—55 hours after administration eirVWF, in one embodiment, r‘si’W’F is administered at a dese such that it increases the half-hie of plasma FViEiEi by a value seieeted from variatiens ”30-46 tn 1089 tbund in, Tahie 7.

} As discussed above, the rV‘WF 0f the in rention shnws an sed effect an the stahiiity 0f FVHI as eeinpared tn the effect ofpdVWF in certain aspects, rVWF is administered at a dose and/or with a frequency such that it increases average F‘v’lll half—life by about l—l 5, 2—14, 3—13, 4—12, 5—1 l, 6—H), 5—9, 6—8 hours. in still further embodiments, r‘v’WF is administered at a dose and/or lrequei'icy such that it increases FVlll half—li e by about lilo/ti to about 75% as compared to pdVWF. in yet further embodiments, rVWF increases FVlll half—life by about 10—80%, l5—65%, 20—60945, 25—55945, Bil—50%, 35—45596 as compared to "Wl‘. ln yet further embodiments, the average or percentage increase in FVlll half—life is maintained at least 10, 2t), 30, 40, 5t}, 60, 70, 80, 90, ltlﬁ hours after administration . in yet further ments, the se in F‘s/ill halt—life is maintained at least 5—125, lO—l l5, l5—105, 20—95, 25—85, 30—75, 35—65, 48—55 hours after administration ofrVWF. As will be appreciated, the increase in F‘V‘lll stability, half—life and/or activity can be assessed using methods known in the art, including t tion coagulation assays. in other en‘ibodinients, adn'iinistration ol‘rVWF increases the half—life of F‘s/ill by an amount ed from variations l 3th to l643 ”ound in Table 9, as compared to administiation ofplasma—derived VWF.

WWW} As discussed herein, the stabilization of FV’lll activity by rVWF as compared to pdVWF can be measured by metrics in addition to FVlll halt—life, ing mean nce time (MR7?) and area under curve (AUG). in exemplary embodiments, rVVv’F is administered at a dose and/or frequency such that it. increases MRT by about l—l5 hours as compared to pdVWF. in further embodiments, r‘y’Vv’li‘ increases l‘le'l' by about 1—25, 2—20, 3—l 5, 4—10, 5— 9, 6—8 hours as compared to pdVWF in still further embodiments, rVWF increases FVlll MRT by about /5, l5—65‘V5, 20—60%, 25—55%, 30—58%, 35—45% as compared to pet‘s/WT. in yet further ments, the average or percentage increase in F‘v’lll half—life is maintained at least l0, 2t}, 3t}, 40, 5t}, 6t), '70, 80, 90, l (til hours alter administration ot‘rVWF. in yet r ments, the increase in MRI is maintained at least 5—l '25, lil—l l5, 15— lil5, 20—95, 25—85, 30—75, 35—65, 43—55 hours alter administration ot‘r‘VWF. in other embodiments, administration ot‘rVWli‘ increases the MRT oi“ FVill by an amount selected from variations lSGO to lit-43 found in Table 9, as compared to administration of plasma— derived VWF . lﬂlllltll in further aspects, the doses ofrVWF administered to patients are comparable to doses used in administration oi’pdVWF/deVlll. {(30211} Compositions of rVWF with or without F‘v’lll can be contained in pharmaceutical formulations, as described herein, Such, lormulations can be adn'iinistered , topically, transdernially, parenterally, by tion spray, vaginally, rectally, or by intracranial injection. The term parenteral as used herein includes subcutaneous injections, ii'itravenous, intramuscular, intracisternal injection, or infusion techniques. stration by intravenmts, errnal, intramusclar, intramammary, intr‘aperitoneal, intratliecal, retrobulbar, intrapulmonary injection and or surgical implantation at a particular site is plated as well. Generally, compositions are essentially free of ns, as well, as othe ties that could be harmful to the ent. £33212} 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 inyentiye compound is administered as a onetime dose. Those of ordinary skill in the art will readily optimize effective dosages and stration regimens as determined by good medical practice and the clinical ion of the individual patient.

The route of administration can be, but is not d to, by intravenous, intraper‘itoneal, subcutaneous, or uscular administration. The frequency of dosing depends on the pharrnacolrinetic parameters of the agents and the route of administration. The optimal ceutical formulation is ined by one skilled in the art depending upon the route of admini stration and desired dosage. See for example, Remington’s Pharmaceutical Sciences, lStli Ed, l99tl, Maclr: Publishing Co, Easton, Pa. l 8042 pages ilélBS—l7l2, 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 e, and rate of in vivo clearance of the administered . Depending on the route of stration, a suitable dose is calculated according to body weight, body surtace a "ea or organ size. Appropriate dosages may be ascertained througl'i use of established assays for 'etermining blood level dosages in conjunction with appropriate dose~response data. The tinal dosage regimen is determined by the attending physician, considering various factors which modify the action of drugs, eg. the drug's specific activity, the severity of the damage and the responsiveness of the patient, the age, condition, body weight, sex and die. of the patient, the severity of any infection, time of administration and other clinical factors. By way of example, a typical dose ofa recombinant VWF of the present invention is approximately 5t} U/kg, equal to Stilt) rig/kg. As s are conducted, further information will emerge regarding the appropriate dosage levels and duration of treatment for various diseases and conditions, } in some embodiments, r‘VWF ‘s administered to a subject alone. in some embodiments, rVWF is administered to a subject in combination with one or more coagulation factors. in further embodiments, the coagulation factor administered with rVWF is F‘s/ill. in still further embodiments rVWF is stered prior to, subsequent to, or simultaneously with a coagulation factor such as FVlll. ln certain embodiments, rVWF and 2012/041957 F‘Vlll are stered together in a single composition. As will be appreciated, the F‘Vlll that is couadministered with rVWF can be either recombinant FVlll or plasma d. {(33214} in, further embodiments, rVWF (with or without FVlll) is administered to a subject no more than once daily. in further embodiments, rV‘v‘v’F (with or without FVlll) is adn’iinistered to a. subject: no more than once every other day, no more than once every third day, no more than once every fourth day, no more than once every ﬁfth day, no more than once a weelr, no more than once every two weeks, no more than once a month. in still lurther embodiments, rVWF {with or without ll‘V’l E l) is administered to a subject no more than twice a day. 598215} in thither embodiments, rVWF and F‘v’lll are administered er to a subject in an initial dose, and then subsequent re—dosing is conducted with rVWF alone. in other embodiments, re~dosing is conducted with both rVVv’F and FVlll, {$0216} in still further embodiments, rvwr (with or without rFVlll) is administered at a dose such that plasma F‘v’ill activity is stabilized for about l ii to about 90 hours. in further embodiments, plasma, P‘Vlll activity is stabilized for at least l2, ‘24, 36, 48 or 72 hours. As will be appreciated, the stabilized plasma FVlll activity maybe that ot‘endogenons FVlll, con administered F‘v’lll (plasma—derived or inant} or a ation of both endogenous and conadininistered FVlll. 598217} in some embodiments, rVVv’lI and FVlll are administered together at a dose such that extension of in viva) half—life of plasma FVlll activity is ized for at least ll, 24, 36, 48 or 72 hours. in tuither embodiments, the plasma F‘v’lil activity is stabilized for about l0 to about 90 hours. in still further embodiments, the increase in half—life ofplasina FVlli is maintained for at least 24, 36, 48, '72, 93, 120, or left hours in a patient. The (so—administered l can be rFVlll or deV’lll. in some embodiments, plasma F‘Vlll activity is stabilized for a time selected from variations M90 to l299 found in Table 8, after co~administration of WW? and F‘v’lll. {33218} in preferred s, the present invention provides methods for treating ation disease, including hemophilia and von Willehrand Disease (VWDl {$9219} As used herein, the terms "hemophilia" or philia" refer to a group of e states broadly characterized by reduced blood clotting or coagulation. l’len’iophilia may refer to 'l‘ype A, "type B, or Type C hemophilia, or to the composite of all three diseases types.

Type A l'ieinophilia (hemophilia A) is caused by a reduction, or loss of factor Vii}, (F‘v’ll 3) activity and is the most prominent of the hemophilia subtypes. 'l‘ype B ilia (hemophilia l3) results from the loss or reduction ot‘i‘actor lX (FlX) clotting function. Type C ilia (hemophilia (I?) is a consequence of the loss or reduction in factor Xi (Ext) 2012/041957 clotting activity. Hemophilia A and B are Xslinlted diseases, while hemophilia C is autosornal. Common treatments for hemophilia e both prophylactic and onmdemand administration of clotting factors, such as FVlll, FIX“, including Bebulin®—Vl:h and FXl, as well as FElBAuVH, pressin, and plasma infusions.

Eiltl22tll As used herein “von Willebrand Disease” reters to the group of es caused by a deficiency of von \I‘v’illehrand factor. Von 'Vy’illehrand factor helps blood platelets clump together and stick to the blood vessel wall, which is necessary for normal blood clotting.

There are several types ot‘Ven Willehrand e. The following table summarizes the characteristics of dillereiit types oi‘VWD: \§\\\ \\\\\\\\\\\\\§\\\ Quantitative forms ofVWD Partial quantitative deficiencies of VWF VWF plasma levels that are . m to 30/o oi normal approximately (500/o to 80m oi patients lly complete deﬁciency oVWF Approximately l‘}/a to<9/'o ofpatients Qualitative forms ot‘VWD Decreased V’Wl-tiepndent et on Selective deﬁciency oiHMWV’Ml multimers Platelet binding litnctions ofVWF are impaired Gain of function inutation with increased VWF binding to platelets Loss of lelh/lWK-VWF inultiiners from plasma, but not from platelets Loss of both VWF and platelets through a clearance ism Decreased V’WEdependent platelet adhesion without seleCllVe deficiency oflllVlW-- VWF niultiniers Markedly decreased binding afﬁnity for factor Vlll Adapted from Sadler8 J "l‘hromh llaemost 26%; 4i, 2163 {Mini} Methods for treating coagulation e include administering rVWF or a combination of r’V‘WF rFVlll to suhj eets in need thereof in accordance with any of the methods of administration described herein and known in the art. Such subjects may be suffering from any coagulation e, including without limitation von Willehrand Disease 01 hemophilia. As will be appreciated any the of von \‘villehrand Dise eincluding any ol the types listed in the above table, can be d in accordance with any of the s and compositions described herein. lililZ22}l 11 some emwbodimtnts, r‘v’WF {with or without rFVlllf)is adininistered to a subjec such that the level of Factor Vlll gulant activity (F'VlllzC) in the plasma of the subject 24, 3o, 48 or more hours post—adininistration is at least 913% of the level ot‘FVlllf activity present in the plasma l hour post-administration. in further embodiments the level of the C in the plasma of the subject 24, 36, 48 or more hours post~administration is at least between 5t)“/0 and ltl{3% of the level oflWlll: 1: activity present in the plasma l hou1 post- administration. in still linther embodiments the level oi the FVllla‘CLin the plasma of the subject 24, 36, 48 or more hours post—administration is at least 50%, 55%(1the, 65%, 7(30A1; 75%, 80%, 850’/1, 9.0w, 95“71>, 9 1%,, 97%, 98%, 99%, 100% ofthe level ofFVlll:C activity t in the plasma l hour dministration. stration of rVWF {M3223}in one aspect, the present disclosure provides method for treating Von Willebrand Disease (VWD) or Hemophilia, A in a, subject in need thereof, which includes administering a composition of recombinant Von rand Factor (1V Wli) such thatlFactor VlllttVll13l (J‘s stability is sed, as compared to FVlll hall—life in a subject administered a composition of plasma derived Von Willebrand Factor deV WEI). in one embodiment, the composition of rVWT- administered to the subject is a composition ol’high molecular weight rVWF multimers. in one embodiment the composition ol rV‘t’vF administered to the subiect has a higher specific activity than a composition ofpdVWF. in yet another embodiment, the ll} composition ofrVWF administered to the subject is a composition of high molecular weight rVWF multiniers with a higher specific activity than a composition of pdVWVF. in one embodiment, FVlll stability is characterized by the haltlife of F‘Vlll. in another further embodiment, the method is for treating any type ot‘VWD in a, specific ment, the method is for treating Type 3 VWl). {99224} in one embodiment, the method, comprises administering a composition of rVWT- such thatl‘Vl ll stability1s extended by at least 199/1, 2130/”(gill/13;,"a in, 4 in, 6 in, or by an amount selected from variations l3th to l 643 found in Table 9, as compared to FVlll stability in a subject administered a, composition ot‘pd‘VWF, in one embodiment, the composition ot 1VWFadministered to the subject is a composition of high molecular weight rVWF multimers. in one embodiment, the composition ol‘rVW F stered to the t has a higher speciﬁc activity than a composition ofpthWF. in yet another embodiment, the composition of rVWF administered to the subject is a composition of high molecular weight rVWF multimers with a higher specific activity than a composition of pdVWF. in one embodiment, FVlll ity is characterized by the hall‘lii‘e oi‘FVlll. in another ment, F‘s/ill stability is characterized by mean residence time (MRI) of FVlll. in a tlirther embodiment, the method is for treating any type of VWD. in a specific embodiment, the method is for treating ’l'ype 3 V‘s/VD. lilllZZSl ln one embodiment, the method comprises administering a composition ofi‘VWF such that FVlll stability is extended by at least l(l% as compared to lWlll stability in a subject stered a, ition ot‘pdVWF, wherein the composition of rVWF ll) administered to the subject is a composition of high lar weight rVWli‘ multimers having a l percentage of rVWF molecules present in a particular higher—order rV‘yl/TF i'nultin'ier or larger ier according to any one of variations hill to 457 found in Table 3 to "l‘able 5. ln one embodiment, the composition of rVWF administered to the subject has a higher specific ty than a, composition ot‘pdVWltl, ln one ment, l stability is characterized by the half life of FVlll. in another embodiment, FVlll stability is characterized by mean residence time (MRT) of F‘v’lll. ln a further embodiment, the method is for treating any type of VWD. in a specilic ment, the method is for treating ’l'ype 3 VWDG lllllilltil in one embodiment, the method comprises administering a composition OfI‘V‘lVF such that FVlll stability is extended by at least 20% as compared to FVlll stability in a subject administered a composition ofpdVWF, wherein the composition l? administered to the subject is a composition of high molecular weight rVWF multimers having a mii'iimal percentage otif‘lﬂlv’lj molecules present in a particular high er~order rVVv’l: multimer or larger multimer according to any one of variations l 34 to 457 found in Table 3 to Table 5, in one embodiment, the composition otif‘lu’lli’lj administered to the subject has a higher specific activity than a composition of pdVWF. in one embodiment, F‘v’lll stability is characterized by the half life ol‘FVlll, in another ment, F‘v’lll stability is characterized by mean residence time (ls/l RT} ot‘lllv’lll. in a further embodiment, the method is for treating any type ofVWD. in a specific ment, the method is for treating Type 3 VWD. {$0227} ln one embodiment, the method ses administering a composition of rVWF such that FVlll stability is extended by at least 309/5 as compared to FVlll stability in a subject administered a composition of pdV’WF, wherein the ition of i‘VWF administered to the subject is a composition of high molecular weight rVWF multiniers DJ (J; haying a minimal percentage of r‘V'Wl3 molecules present in a particular highersorder r‘V'Wl3 2012/041957 muitimer or iarger mtiitiiner according to any one of variations "334 to 457 found in Tahie 3 to Tahie 5. in one embodiment, the composition of rVWF administered to the subject has a higher speciﬁc activity than a ition of pdVWF. in one embodiment, FViii ity is characterized by the haif life of F‘s/iii. in another embodiment, F‘v’iii stabiiity is characterized by mean residence time ) ot‘FVHi, in a tiirther embodiment, the method is for treating any type of V’WD. in a specific embodiment, the method is for treating 'i‘ype 3 VWD. {“3228} in one embodiment, the method comprises stering a composition ot‘rV WEI, wherein the combination ofrVWF speciﬁc activity in the composition and increase in FVHI it) stabitity, as compared to i stabiiity in a subject administered a composition ot‘pd‘v’WF, is seiected from variations 2339 to 4868 in e 13 to Tahie 19. in one ment, the composition ot‘r‘VWF administered to the subject is a composition of high nioieciiiar weight rVWF rnuitiniers. in one embodiment, F‘v’iii stabiiity is characterized by the half life of FViii. in another en'ihodin'ient, FVEH stability is characterized by mean residence time (MR?) of FVEH. in a further embodiment, the method is for treating any type of VWD, in a c embodimentt the method is for treating Type 3 VWD.

'E‘ahte 13* Exemplary embodiments for the combination ot‘r‘v’Vw’F specific activity in a composition used herein and increase in FVHI stahitity achieved, as compared to FVIH stahiiity in a subject administered a composition ofpdVWF. _____________________i_€!§E§§E§§§§E§Piliiiiiﬁiﬁtttﬁ_____________________________________________________________________ atieasti atteast)‘; atieastii atteast-é atieastS atieasto t‘? atieastS ' ' hr hr at toast atieast , . at ieast ', ', ' . a ':'. '. z, . Var, i so x'.2 .3) ‘:_ , '. , 2 2 to, 2665 atieast ' . . . '. . . Var.

E 70 i<'r " ,» ' ‘ ‘ ’W '1“ i’i” 2620 i666 atieast '- ‘ . '. It, '. . Var. Var. :80 i .-'. W ’1' A. i I»). 2621 256" atieast 1,. Var. ‘ . 1,. Var. i 7 M} i M 23392 2438 ’, i i 7' i 2668 atieast ' Var. 7, ' '. . '. Var. % -‘ 2393 2439 2485 ' ‘ . 2523 2659 ' ' at toast , Var. Var. . . Var. Var. 125 ' 3440 2486 t ,, 2624 23670 atieast V . '. . Var. Var.

ESQ ,1 . if 2441 2487 ,. c.’ .37? 2625 257i t, t, . Var. Var. . . Var. i t 2442 248s <,2 t’ t : 2672 2394 E 29~99 E 2353 23a99 Var. Va1. Var V".1 E 243-84) 2354 2400 A446 2/492 E V211. Var Va1 E 20-370 2355 24012447 H7- Var. V34. Va1. ‘ ', Va1. 29—69 2356 2402 2448 2-494 ,1 ,-' 2678 Var. Va1. Var. V9.1. Var.

E 29—59 E 2357 2493 2449 2495 2679 «<‘ E Ia:i" V31.

E 29449 E 2358 2494 2680 V211. Var. V9.1 ‘ Va1 E 40-154} 2359 2405 2451 2497 2681 Var. Var. Va1. aV1. 44164275 2360 2406 2452 26‘‘82 Var. V91. V91. 40—10%} 2364 2407' 2453 V211. Va'L" V31 49439 2408 2454 2500 40-§0 E 49—79 E 2372 2418 2464 Var. Va: 7 E 79—159 E 2373 2419 Var Vat 2465VcI E 79—125 E 2374 2429 2469 Va1. 4 794499 E 2375 2421 ":3 265'E 4 V211. Var. V7211. Var.

E 79—99 E 2376 2422 2499 2652 E Var. Var. Va1. Var.

E 78—88 ' 2423 2469 2653 E 994425 E 994499 E........................

E 89~99 52331 , : E“ e . g 2658 2794 E ' A ,«_, ‘.' . i 2659 2705 E '-u ‘ 0‘. .t, z E ‘Van E 98—iﬁﬂ E vz.‘ A ,1, . ‘.u i Iiiiillllll 2660 2706 Vat Variatimt 'E‘abie Mt Exemplary embodiments for the cembinaticn efrVWF specific ty in a cempesiticn used herein and increase in FVHI stability achieved, as compared to FVIH stability in a t administered a ition efpdVWF. iacmsmmmtﬁwre____________________________________________________________________ LS hr L7 hr L6 hr 1—5 h!” 14 hr L3 hr L2 h?“ Vat : Y Vat.

§§E atiea§t20 E 2707 a c, E ', h, , 2891 E : Var.

V ﬁttieast 3i} 2892 . . E . , . Var. Var. t atteast4c Elv ‘ ‘..< E t‘E z! 2893 2939 Var. Var. atiezast 50 2, 8 94 2940 Var. Var. Var. ‘c- ‘ atieasst (3&1 2849 2895 2941 ,1 : ' Var. Vat. Var. ziticast’m ’ 23850 2896 2942, Var. Var. atieast 8i] 2851 2897 2943 Var. Var. Var. atiezast 90 2852, 2,898 2944 atieasst Var. Var. Var. ‘o ‘.

Eice 2853 2899 2945 ,1 ziticast Var. Vat. Var. 125 2854 2900 2946 at Eeast Var. Var Var. 335% 2855 2901 2947 Vet. 2906 Vat. 2907 E 40—150 E 48—EZS 2012/041957 E 492199 2 E Var, E 49—99 2868 Var.

MM“) 2869 , E Vat.

E 49_79 2962 E . van E 49259 2871 2917 2963 ‘vat. V91‘.

E 49n59 2872 918 2964 Var. \/91. ‘vat.

E 69—159 2873 2919 2995 Var. V3.1: Var.

E 60~i25 2874' 2929 2966 Var. Var. Vat“.

E 69—199 2875 292: 2967 ' Var. V91‘.

Eq9999 2876 2922 2968 Var. ‘vat.

E 69—89 2877 2969 V29‘. Vat. Var.

E 69~79 2878 2924 2970 E Var. Var. Vat.

E 89_99 2887 2933 2979 Var. Var. Vat“.

E 99—159 2888 2934 2989 Var. ’ E 99n125 2889 '22? E van “ E 99—199 2899 V91: Variation Tabie 15. Exemplaiy embodiments {01" the cambmatimt 0f 1VWF ic activity in a, campositicn used herein and increase in F‘VEH stability achieved” as compared to FVEH stability in a, subj act administered, 9, wmposition 9f pdVWF increased Stability ' 2—31}: 3‘8 151 ...............................................................................................................................................................................................EV/ 331': E VBLT. V(if ittieast 29E1'3975 E' '7 " 3 3259 3395 E3397 at ieasst 3th E Var. .....................................................................................1........................ .................. ........................ ....................... ....................... .......................

I E3076 . I 3214 3260 E Var. Var.

E atkmstdﬁ E3077 3215 Var. Var.

E atieastﬁﬁ E 3078 3216 Var. Var.

E atieastﬁﬁ E 3079 3217 Var. Var. Var.

Eznieas¢70 E3080 3218 3264 Var. Var. Var.

E atieastﬁﬂ E 3081 3219 3265 E Var. Var. Var.

E atiezast 90 308?. 3220 3266 a: ieasst Var. Var. Var.

Eiﬁﬂ E 3083 3221 3267 at ieast Var.

E125 E 3084 FEEEEQE""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" EESG Var. ‘0 ‘. 3276 3322 . ..

Var. Var. - E 29~59 «Av-1*: 34! I E 29_49 . .

E Var. Var. 1 E 40—150 3279 3325 ‘ V31‘. ‘0 ‘.

E 4un125 3280 3326 , ... , Var.

E 40—100 Var. Var. '< E 40~§0 3236 3282 -, E Var. V 3 .13.: 3 4.3 ’7, .: Var. 3438Aq)‘ Var. 3289 Var. 329E) EVM. Vm Vm. Vm 130380 3107 3337 ' V211 V211 Va1. V3.1: Var. 733-325 3] if) 3238 .3294 3:340 . : g V211. V211. Var. Var 1 711411111 3111 '249 3295 ‘ V211“. V211. V31“. 3,341 '0‘. 731—93} 3112 3:250 3332 . _.

V211. V31”. V131. V211“. - 711—811 3113 3251 3297 3343 ' V231. V311 Var. Var. 8334,53} 3] 14 3252 .3298 3:343 V211. V211. Var. Var 1 311-125 3115 3253 3299 3345 ' L V211“. V211. V31‘. 'o‘. 831—3310 3116 35253 3.300 3336 I V211. Var. . V211“. 8114111 3117 3235 3347 : V211 V211. V211. 913-1511 4 3348 Va1 31721132111011 T211339. 16. pl211y enﬁmdinmnts for the cam’bination 0f rV‘WF specific activity 111 a 2:011’111031'13011 used 1113123111 and increase 111 FVHI stability achieved, 218 ed 1:0 FVHE stability in :3. 311111 2:01 stered a composition «of pd‘v’WF 11111133331313 Stabiﬁty (Hams) 1 ---------------------------------------------------------------------------------------------------------------------------------------------- 4—8 111» : V211“. 2-: '. Var. 41111431 211 3443 ., '. 3 3:58] V211. 1'1 " . . Var. . V211“. 211113351311 3444 " _ f 3582 :. 3674 iV211 E V 411123314115:3'445 at 1eas1 1111:'3449 2133132131: 12-5 21132351 1511 21141511 12/171031 ' E 3454 E Vaa'.

E 26-125 E 3455 .3639 3685 E E van VaI. Val“.

E 20—100 E 3456 3640 3686 , VaI.

E 26—96 E 3457 364‘; 3687 .‘ . _ V2112 Val“. Var. .

E 20—80 E 3453 3642 3688 ’ Vaa'. Var. Var. Var.

E 20~70 E 3459 3 597 3 689 Van". Var. Vai'. Var, E 20-60 E 3460 3:398 3644 3690 VaI. VIII. Val“.

E 26—56 E 3461 3 599 3 645 3 691 : V31‘ —40 Var.

Var, 3699 36:34 3700 Var. VIII. ‘van E 66~150 3 609 3 655 3 ”701 Var. Var. ‘va: E ﬁﬂ-EZS 3 6} 0 .3 656 " Var. VctI.

E 60-100 3 61 1 Var. 3;”aI.

E 66—96 3 612 '3658 Van ‘v231‘. 3613 3705 E Var. 30490 3435 V213*. = Variation 717231318 17. Exemplary embodiments for the 3.301331383218013 ofrV WEI Specific activity in a eempesitierl used herein and increase in FVIH stabiiity achieved. as eempared te 1387111 stabiiity in a subject stered a. compesitiori 01’ 1311871337157. increased Stabiiit‘ (ﬁgure) increased Smbiiit‘ (Percent) ”/8 2110/0 333/0 43%: at least Var. Var. 2E} 38 ii I. ., 39113 I. 3 V’ .r 3 '. I“, Var, 4041 Var, 4087 Var. 41,33 312 38123858313904.3950 7 .3 :7, 7- V ‘3 . Val”. 4088 Var. 4134 211318331 - .. . . 48 3-3 - . . g,» . . . Var 4089 Var. 413.5 ratieast V V E Var. 40911 Var. 41363 at: Beast : . .. . .. 611 3‘1 V . . Var 4091 Var 4137 V ' at least E Var. 4092 Var. 4138 at 122331 .. . .. . E 88 I3 .- . .: g.) f . . .. 4001 . Var. 4093 Var. 413:9 V V at least . . . . E 91} 7 ' " V' '2 ' _ _ . '. 4002 z . Var. 4094 Var. 4140 attieast 7 7 . .. . .. 1118 ’7 V V - I' . ., 1 . f Var. 4095 Var. 4141 V V V at Beast E Var, 4096 Var. 4142 331 Va Var. V 15113821 .3867 3 ’ . . . V: Vern-1097 Var.4143 7 V V . 7731 1" . ., Van-40,99 Var.4145 Var. V V 21111111 3824 3870 ' 3 ’V ,‘ u Var-4100 Var.4146 Var Val. ‘. 2119i} 3825 .3871 ‘V 3n; 7 . .L . Vern-11111 Vain/4147 Var. 3872 ’ _' 'V ’4. 3'7 Var.4102 48 Var ' ' E 333 '“ ~, , V394”? Var. 4149 Var. 41.50 Var 4014 . Var. 4152 Var.4015 Vi '2 r .1 . 1'7 Var.4153 WO 71031 V7211: 41116 Var. 41118 Vet. 4154 Var. 4017 Var, 41119 V7312 415:3 .........................................................................................................................................................................................................................

V7211 4018 V7211“. 4110 Var. 4156 48-813 3835 3881 1 , . -1 V7211 1 . 413 711 3’ ’I 3882 ' "I 3‘ ’ ..

-V73.1: Var, V73.1: Var. 411 611 3837 3883 3929 3975 —V7211 V7321 V721r.V7211“. 865113838 3884 3930 397 ' 1:5 ’ . 3 5:1 . .

V781“. : . . . 611-125 3840 3886 3932 3978 611—3111} 3841 3887 3933 3979 ' , J Var. 4117 37314163 V7211 ‘7 “ V7211 V7321“. 618911 384?. 3934 3980 ' _. Var, 4118 V731: 4164 Var. V731‘. Var. V7211‘. {3114311 3843 3889 3935 3981 V7211“: 4119 V7211: 4165 Var. 4 166 Var. 4167 V731 4168 V7 211‘. 4169 Var. 41711 Var. 4171 V:11". 9896 39425 : : _______________________________ V7321“. ’ ’" ’ ( " " - 2. 3897 '3; V7211“. 4127 .' 7’ Var, V73.1: Var. V73.11 811-1138 38:32 3898 394-4 3990 Var. 4174 V7211 . V7211 811-9 3899 3945 3991 V73.1: Var, V73.1: Var. 911—151} 3854 39011 3946 3992 V;§215r.5V721r. V7321“. 98125 V78at '3) 1 3947 3993 V7211“. 4131 V73.1: Var. V73.11 91} 11111 38:36 3902 3948 3994 V7211“.V7 211121111011 Tame 18. ary embodiments fer the 3.3011111812111011 01‘1‘VW 1I Specific activity in a itien used herein and increase in FV’111 stabﬂity achieved? as eempared 10 FV111 312113111137 11'; a subject administered a eempesitien WF. increased SdabiiitV Wanem) 3119331 1 511% 11851198 111w4118/81111m311‘341 11821134; 1561134; 1,5411% 154311378 2012/041957 at 163st at Beam 311 Var. 41811 ................................................................................................................................................................................................................... at least Var. 4181 4366 4412 4458 4504 Var. Var. V3,1: Var. Var. 4229 4368 4414 4460 45116 Var. Var. Var. Var. 11111 Var. 4186 4232 4416 4462 4508 at Hezasﬁ Var. V3,1: Var. Var. 11111 Var. 4187' 4233 at least 76.1: Var. 125 Var. 4188 4418 4464 4510 at 183181 Var. Var. Var. V111‘. 1511 Varl 4189 373 4419 4465 4511 Var. Var. Var. Var. 211—1 51} Var. 4190 4374 4 4466 4512 ' " z Var, '.

Var. 419 1 4437 Va}: Var. 4192 Var. 4195 Var. 4196 .......................................................................................................................................................................................................................

Var. 4198 ‘ 411—1511 Var. 4199 4245 4383 4475 Var. Var. Var. V211“. . 4246 4384 4430 4476 ' ,‘ 7- 41141111 Var. 4201 4385 4431 4477 ‘ 1’ ~ , 1’ V211“. Var. Var. Var. V V 4248 4386 4432 4478 - V V31‘. Var. Var. 411-811 V '. ,- ,1 "= 4387 4433 4479 Var. Var. V3,1: Var. Var. 411—7 4250 4388 4434 4480 4526 , Var. V211‘. Var. Var. '2 . Var. ________________________________________________________________________________________________________________________________________________________________________e1 Var. Var. Var. 4299 1 68—99 Var. 421 U 9". - .L' ,1 4440 4486 4532 68—89 Var. 4211 4257 ' ’ ' 4441 4487 4533 {33‘79 Var. 4212 1’ a, -’. .f 4442 4488 78-151} Var. 4213 4259 '- , 4397 4443 4489 4535 711—125 Var. 4214 4260 " 4398 4444 4490 4536 Var. '2 . Var, Var, Vain 78—188 Var. 4215 4261 ” ' .: 4445 4491. 4537 73499 Var. 4216 4262 ' . ', 4446 4492 4538 Var. 4217 ' 3 Var. 4218 Var. 4220 . . , , a , . Var.

Var, 4221 2’7 i ' . .I. L) i . 4497 ' i V . . i . Var. 913451} Var. 4222 ’ 7‘ ‘ ' - a 4498 Var. 4223 Var. 4224 V31‘. Variation T3189. 19. Exemplary embodiments for the combination ofr‘v’Vv’F speciﬁc activity in a composition used herein, and increase in, FVHI siabiiiiy achieved, as compared to FVHE srahiiity in a. t administered a composition «ofpdVWF. increased Stabilit‘ nt} 152“ 9.5.8.22.....i..%.1’::€§f§f§{éz........21§:§1§.%2........é§:§§f{2........§.€3:£€332§.........a?8:59.12?..........1 g) at 1933:1128 Var.454 a. , . 39 Var.4685 Var,4731 . 77 (mi?! :m 4640 , Var.4733 . 4644 Var. 4690 Var. 4829 WVWVaV‘VVVa/ai30 _____________________________________________Via Var. 4693 Var. 4739 Var. 4785 Var. 4831 E Var, 4741 Var. 4787 Var. 4833 Var. 4696 Var. 4742 Var. 4788 Var. 4834 V 4697 Var, 4744 Var. 4790 Var. 4836 Var. 4699 Var. 4745 Var. 4791 Var. 4837 WO 71031 Var 4701 Var 4747 Var. 4793 ar 4839 Var. 4712, Var. 4758 Var. 4804 Var. 4850 Var. 4805 Var. 4851 3233... ...‘.‘.’:i3.4.‘.'.v..ﬂ§9... .Xitizfiﬁﬁé... “ ______________________________________________ Var. 4715 Var. 4761 Var. 4807 Var. 4853 ‘ 16 Var.4808 7211‘. 4854 Var 4717 Var 4763 Var. 4809 Var 4855 ............................................................................................................................... .4 Var. 4718 Var 4764 Va} 4810 Var. 4856 7t}ﬁll w ‘ 44. 444.47 70125 4 7th iﬁtl 4 4 -, _‘v44 4747 70-90 w . -_V44 7t)8t) ~ 47 444 4’44 44:5 8% 35% r 44.4 4442 l i .99 3 9% * 4444:4444 Var.I:V’ariatieii {$4229} in one embodiment, the rnethed comprises administering a eempesitien ot‘rV/ WEI, wherein the eernpesitinn ef rV’WF administered to the subject is a eonipesition ot‘high melecniar weight rVWE mnitintersin whieh at ieast .39"/0 0t rVWPimelecuiesin the (J‘s eeinpesition are present in a innitnner of at least 10 ts, and wherein the combination of rV’WF speciﬁc activity in the composition and increase in FVEH stability, as compared to F‘s/”Hi stabiiity in a subject administered a eempesitien of pdV’WF, is selected from variations 339 to 4868 in Tahiti 13 tn Tahiti 159. in one entbodintmit, the eernpesiticn F administered to the subject is a composition ef high molecular weight rV"WF nntitiniers. in it} one embodiment, FVEH stability is terized by the i’iait‘iife ot‘F‘ViH. in another embediment, FV’ililE stability is characterized by mean residence time (MR'F) et‘ FVIll. in a titrther einbedinient. the niethnd is for treating any type ofVWD. in a specitie embodiment, the method is far treating ’l'ype 3 V’WD.

Etltl23ﬂl ln one embodiment, the method comprises administering a composition ol‘r‘v’WF, wherein the composition of rvwr administered to the t is a composition of high molecular weight it'll/WV? multimers in which at least 50% ofrVWF molecules in the composition are present in a multimer of at least l0 ts, and wherein the combination of rVWF ic actiyity in the composition and increase in FVlll stability, as compared to F‘Vlll stability in a t administered a composition ofpthWF, is selected from variations 2339 to 4868 in Table T3 to Table 19 ln one embodiment, the composition ofrVWF administered to the subject is a composition h molecular weight rVWF multimers. iln one embodiment, FVlll stability is terized by the half life of FVlll. In another ll) embodiment, FVlll stability is characterized by mean nce time (MR7?) of l. ln a further embodiment, the method is for treating any type of VWD. In a specific embodiment, the method is for ng Type 3 VWT). {$02M} ln one embodiment, the method comprises administering a composition of rVWF, wherein the composition of r‘VWF administered to the subject is a, composition of high molecular weight rVWF multimers in which at least 70% of rVWF molecules in the composition are present in a multiiner of at least ll) ts, and wherein the combination of rV WT: specitic activity in the ition and increase in FVlll stability, as compared to FVlll stability in a subject administered a composition ofpdVWF, is selected from variations 2339 to 4868 in Table l3 to Table 1‘). ln one embodiment, the composition ofrVWl" administered to the subject is a composition ofhigh molecular weight rVWF multimers. ln one embodiment, FVlll stability is terized by the i’iait‘lit‘e of F‘Vlll. in another embodiment, F‘s/ill stability is characterized by mean residence time (MRT) of F‘Vlll. ln a turther embodiment, the method is for treating any type of VWT). ln a specific en'ibodiment, the method is for treating Type 3 VWD. {(33232} ln one embodiment, the method comprises administering a composition F, wherein the composition ot‘r‘i/ Wl2 administered to the subject is a composition of high molecular weight rVWF inultimers having a minimal percentage ofrVWF les present in a particular hi ghersorder r‘vPVt’F multimer or larger multimer according to any one of variations l3l-‘l to 457 found in Table 3 to Table ‘5, and wherein the combination ofr‘v’WF specific actiyity in the composition and increase in FVlll ity, as compared to FVlll stability in a subject administered a composition of pdVWF, is selected from variations 339 to 4868 in Table lit to Table l9, ln one €1’l’ll30t‘lll‘tlfﬁil, the composition ot‘r‘VWF administered to the subject is a composition of high molecular weight rVWF multimers. In one embodiment, FVlll ity is characterized by the i’iait‘life ot‘FVlll. in another DJ (J; embodiment, ll stability is characterized by mean residence time (l‘vlll'l') of FVﬁt in a thither embodiment, the method is for ng any type of VWI). in a specific embodiment, the method is for treating ’l'ype 3 VWD. {@3233} in one embodiment, the method comprises administering a dosage of a rVWF composition containing from l {l lU/ltg to 40 lid/kg r’V’WFzRCo activity, n the combination ot‘r‘v’WF speciﬁc ty in the composition and increase in FVlll stability, as compared to F‘v’lll stability in a subject administered a composition of pdVWF, is selected from variations 2339 to 4868 in Table l3 to Table l9. in a, c embodiment, the composition contains from 20 lid/kg to 30 ill/kg rV‘iNFillCo activity. in one embodiment, the composition ofrVWF administered to the subject is a composition of high molecular ll) weight rVWP multimers. in one embodiment, FVlll ity is characterized by the half life of FVlll. in another embodiment, FVlll stability is characterized by mean residence time (h/lRT) ot‘FVlll. in a thrther embodiment, the method is for treating any type of VWD. in a ic embodiment, the method is for treating Type 3 VWD. lllllilfléll ln one embodiment, the method ses administering a dosage of a r‘VWF composition containing from 25 lU/kg to 75 ill/kg rVWRRCo activity, wherein the combination of rVWl? specific actiyity in the composition and increase in FVlll stability, as ed to F‘Vlll stability in a subject administered a composition ot‘pdVWF, is selected from yariations 2339 to 4868 in Table 13 to Table 19. in a speciﬁc embodiment, the composition contains from 46 ill/kg to oil ill/leg rVVv’lhRCo activity. in one embodiment, the composition ofrVWF administered to the subject is a composition ofhigh molecular weight rVWF multin'iers, in one en'ibodiment, F‘v’lll stability is characterized by the halt‘lit‘e of F‘s/ill. in r embodiment, F‘v’lll ity is characterized by mean nce time (MRT) of F‘y’lll. in a further en'ibodin'ient, the method is for treating any type oft/7WD. in a speciﬁc embodiment, the method is for treating 'l‘ype 3 "V'WD. {@3235} in one embodiment, the method comprises administering a dosage of a rVWF composition containing from 7:3 ill/kg to l25 ill/kg rVi’v’lhRCo activity, wherein the combination ofr‘v’WF speciﬁc activity in the composition and increase in FVlll stability, as compared to FVlll stability in a subject administered a composition v’Wii‘, is selected from variations 2339 to 4868 in Table l3 to Table l9. in a, speciﬁc ment, the composition contains trom 75 ill/lag to lOG lU/hg rRCo activity, ln one embodiment, the composition of rVWF administered to the subject is a ition of high molecular weight rVW F multimers. in one embodiment, FVlll stability is characterized by the hall’lil’e of FVlll. in another embodiment, FVlll stability is characterized by mean residence time (h/lRT) ot‘FVlll, lit a. further n'ient, the method is for treating any type oft/7WD. in a DJ (J; speciﬁc embodiment, the method is for treating ’l'ype 3 VWD.

E98236} En one embodiment, the method comprises administering a composition oErVWF, wherein the subject is administered a dose ofrVWF selected from variations 214E to 2338 in TahEe E2, and wherein the comhination ot‘rVWF specitic activity in the ition and se in F‘VHE stability, as compared to FVEEE stabilityin a subject administered a ition oEpdVWF, is selected from Variations 2339 to 4868 in EahEe E3 to EaEiEe E9, En one ment, the composition of r‘V7WE administered to the sahj cct is a ition ot high moiecuiar weight rVVVT mnitimers, in one embodiment, FVEEE stability is characterized by the haEt‘Eife of FVEEE. in another embodiment, FVEEE stability is characterized by mean residence time (MEET) ot‘FVEEE, in a further ment, the method is for treating any type E0 ot‘VWEZ). En a specific embodiment, the method is for treating Type 3 ‘V”‘E‘7V7D.

EtEhZS’EE En one embodiment, the method comprises stering a composition of rVWF, wherein the dose ofrVWF and increase in FVEEE stahihty, as compared to FVEEE stahiiity in a, snhject administered a composition ofpdVWE, is seEected from variations 4869 to 8003 in EahEe 29 to EahEe 27. in oneembodiment, the composition ot rVWF administered to the subject is a composition of high moEecuiar weight rVWE maitimers. En one embodiment, the composition ofrVWF administered to the subject has a higher specific activity than a composition ot pd‘v’WF En yet another embodiment, the composition otrVWFEadniinistered to the subject is a composition of high moiecuiar weight rVWF muEtimers with a higher specitic activity than a composition ofpdVWF. En one embodiment, FVEEE stabiEity is characterized by the t‘e of EVEEE. in r embodiment, FVEEE stabiiity is characterized by mean residence time (EVERT) of FVE E E. En a further embodiment, the method is for treating any type of V‘WD. in a specific embodiment, the method is for treating Type 3 V‘ND, E‘ahEe 2t} Exemplary embodiments ”or the combination of rVWF dosage and increase in FVEEE stahiEity achieved, as compared to EVEEE stabiiity in a subject administered a ition ot‘ pd‘V/WF Ericreased StahEEEtV (Hours) at toast 1 at Eeast 2 at Eeast 3; t toast 4 at toast 5 at Eeast 6 at 7 .III F, Va1. 4927 {3! yearns 4 : V’a15156 ,9 ‘ a; . g i 37,4 0 at E 2 ER Var. 4873 Va, is s vmm "= '24 * 3:“ i bi) Var. 4875 Var. 493.2 :1 Var. 4876 Var. 4933 WO 71031 ,.4994 , _ “ ' . - 24995 79h: , . .

V31 4939 ’- '- - - .- ,1 _ .. 7&5167 18418.9E25 : , 1:85 2E525_814884 “ “ ' '. .

. . . EL‘}?-81 V3114885 V81 4942 *- '- ' .. ,, -. ., i211. 51,8 7211‘. 5227 19 2 9517' E5 Va: 4944 “ ' 2 . . . 1 - 55—11 .I. 981—. 4888 V81 4945 HE18 188 9'8 4955 9.9.1956 129.4959 ' ‘= E25—288 Van'.4902 '25150 981. 4983 Var. 4908 09 ‘~ .V . ‘. 188 4967 I.

Emil-209 V81 49% V81" 4973 E81913:175 V211. 4917' V81 49/4 1E25—15i} V111 492'.

E151}209M211 4923 E159288 1 Ems—288 ' 1/88.492598~.<-<" " I . 128.5210 Var. = V 8.1183011 Tabie 21., Exemplary embodiments fer the combinatinn OfrV WEI (image and increase in FVHEE stability achieved, as compared to FVEH stability in a. subject administered a eompnsition of 'pdVWF Inc:eased Stabilii (Ham‘s) atEeastShrEighr M773: EV/31‘ 2Q ty) EVar.

Bosage rV‘WszCn (EU/kg 77.777677 -_m5563 Var.5566 Var. 7 -5 . .. 3 —..7 V37. 5518 Var. 5575 Var, 5632 I’, Var. 5519 Var. 5576 Var. 5633 Var, 5521 Var. 5578 Var. 5635 - I . . - ,7, Var. 5636 Var 5524 Var. 5581 Var. 5638 277' .29 Var 558:: ' —72 7476 7r 7647 Var.5649 _ y! n," a Va.5650 "’ Var.5594 Var, 5651 -- M.: ' : ' _’a1.5602 .75151159 --V11 E'.’ ‘ ’ 3 -—‘11: ’2 1 4. .5490 125—175 5751' ‘ ' 1 551115515 1751-. 5605 175151162 125-151} Var. 7E ' ' . .’"' Var.5549 Va15606 Vzar.5663 1511121111 31751. " 1 ~. ., . «. . .“ .5 57111256111 '~ E E 1111 :. ’- V111. 5608 “17.11 51165 175121.111 V111. ’< , . 1 1 . 5 ., 1111. 5609 V 211‘. Variation T211115 22 ary embediments for the combination ofWW? dosage and. increase in EE'VEEE stability achieved, as compared to F‘VEII stability in a subject administered 21 composition of pd‘ﬂVF. increased Stahiiitv (H0111s) a W E activity) 15.6010 Var. 601 {1 Var. 6012 Dusage :RC Var.{1013 FVWF Var 6014 (RU/kg Var. {1016 Var 6017 1 Val“. {3019 Var 6020 ‘,I! _‘v . , V1115. £3022 2.55 5 5111111023 1711 ' 7 .::1 111161121 -v1.1 5717 ,1 . .7 1111111115 '11 I ‘ . . 1.1021 —751 ‘1". '.,E .,. Va: 6023 5125 Wu. 55112 ~ E 1151.160311 1111155133 7' , . "E . .. 11111151331 1 5/111 5910 : V51 6033 Vat, 60.36 V'ar. 6037 --Var 7697 -\Im5757 7’77 m5775 VaI. 6047', Var 6048 -w.77647 -75 V 7' '7 '. . 4 ', . , VaI. 6050 vat 9951 jﬁﬁnlﬁﬁ Var. .7' 10639125 VarI. i --Vang; a . . , Var. ,, .-: , -. Vat 606i 3254159 Va.57 ' ' 'I. 77. . . .- .' V23I.6062 -Va:5777 5 -7 I . 153-290 Var .723“ "7.9 ' "' ’ '. . ’2. . I: Van 5950:45VaI. Vat 6064 1759295} Var. 5723 Var. 5780 Var. 5837 7 5' . Van. 59517:5VaI. . '. Var. 6065 Var. Variation Fabie 23 Exemplaw embodiments n or the combination of IVWE- dosage and increase in EV1H stability acmeved, as ed to EVE} stability in a subject admiﬂisiered a, composition of pdV’WF increased Stahiii: ' ('Hmars 37-497 57-777 77779 929.6966 'I 3EVar. ar ., Va6408 1 EVaa 6295::757:9 9.352 Vat 6499 IV'WFERK 9 Va16296 Var.. - Var. 6297. Var ., Va9411 5 Vat 641? (EU/kg " ‘ Van. 62995 Var,. - Var.6300:Va: .7 ,I Var.9414 EVai 6391799 1151.513 V7311 5159 V31: -2115 175151175 1.1125133175151911 -25~151} 7 Var. 6134 V31. 6191 12-51 -Var _")0 Var 6084 V31. 614E!» 3731.316 98 V31. i ' .1 H; '. _ .. 21.1". (1436 ‘ ' ' ‘ 1151,5427 V211. 6429 V31" 6430 -5i0:=:\7;11 6093 V31. 6151) ' ‘. 1 “~13 , I‘]< 3—25 p -. «31“: “1251,5440 252115 ' ' 441 —11%} ;’75 -75—175 3 ‘ 2.. . . ,. . 3‘3 .31515451 75—1513 ' ~. - Q 17515452 V211. £1460 7 ‘v 31‘ 6461 -125 -- ' WO 71031 1755293 Var. 612:3 V21I.6179 VaI. 6236 Var. 6293E VaI 63503Var 6407 Var 6464 Var. Variation Tabie 24. Exemplmy embodiments for the atien of IVWF dosage and ineIease in FV111 stabiEity achieved, as: compared I0 FVHI stabiiity in a subject administered a composition of pdVWF increased Smhiiit ' (Hours) 54m E5513? 671” .._-'~ :. 4 , , . 3 VIII. 6636 Var. 6693 Val“ 6807 activity) Var, 5523; ‘ ’ -. g, 5‘ .V33' 6694 ’7 VaI. 6809 :RCo 3211‘ 6810 IVWF (EU/kg -_525 .7536480 -W6538 -5—209 m 6482 v I, — .

I. . ,~ . , . ,3 VaI.6825 ' ‘ VaI,6826 54m; V’i. 'r ' ,1 ~. x: --—31mm: 550 '~ 2 I) i‘ 'r -. 5 )i '3 VaI. 6>331 Val“ 6832 .5729-E Var . .6730EVa: Var 673 EV/31‘. " ---— 511-75 V2117. 6506 V211".6563 V211'.66'211 ~2111} 7 V-a1.6564 V7211. 66‘21 .. . . . 2 2 . . -_5175.6526508 _I256565 Var 6622 ' ’ E ' I. I. ,5 - 16221 7 I. 1 2 V21 6512 V211 " .. V52 6854 ' 7 E ' V211 6855 Va1.6856 Var 6857 2. ~ 2 1 '. ,7." .’I 32 151121111 Vat 6520 V211. 6577 V7111 6634 V211. 66911: 6748 V211. 6862 175472111} V211. 6521 V7211. 65 78 V7211. 6635 Va1. 6692+ 6749 2. . 2 V7 a1. 6863 V7111. ion T211116 25 ary 6111116161111191113 101 1116: combination 01 1VVVF dosage and1116516121616: 111 FV1115111111111y achieved as (36111111211611 10 FV111 6112111111137111 a t 216111111216161'261 21 COlﬂpOSitiOi’l of pdVWF. 11161eased 6112111111117 ('11611115 / 1’81cent) 211 162151 at 12:21:11 ad 112.2151 211 162151 at 12:21:11 73-8111 1.11% 11—11% 411% 511% 19—56% activity)” _"2116864 1’52 _7211.6978V1721170—5 1252,7149 1’62: 7206 Dusage YVVV’FzRCQ 117126;, 687—2‘11} _7a169311 7211‘. 76141. 1I.52 71611 _’52I.7, —S-5 —122.2 6874 v22 6988 122.2 7045 Var 7102 V V52 7103 _52I7160 — V2117. 71147 12721671114 V211.",2 161 V2111721‘ ~57 .. 11.17 7048 V7211 7105. Var. 7162 VI'>1'. 7219 —5Si} 552 6935 Var 754912.22 7106 552 .7162 2—25 V2117. 711511 12721671117 111.7051 V211 7108 V2117. 6882. V7211. 6939 V211 6996 V2117115j 1272167110 V7211. 6883 V7a1 6940 V7a1.€199/ 7-054V7211' 71721137111 V7211. 71: 14 V7211. 7115 1 . . V117116 7L317 525 1. V7.31 7117 V7211 7174 1111 7404 '41 7061 111I 115 Va 7705 1E11:1511V71 — 2111 1.1193 111I V731.1 6894 V7211. 7008 V71. _ V7211 7122 V7211. 7179 E11—511 .116952 41.”. t: , .2111711137 V211'7124 11.1 7068 V211 7125 '25-11111 V7211. 6899 V7111. 6956 V7.31 7013 V7211. 71370 31.1 6900 V7211 6957 V7211. 7014 111. 711,7 1 V7211. 7185 V7111. 7242 .E25475 711 7186 14117241 E50—2111) V7211. 6902 V7:11.6959 V7111. 7016 V7211. 7117.3 1272117130 V7517I 187 V7211. 7244 E511150 V7211. 6903 V7211. 7017 V7211. 7074 V211. 7131 V7211. 7188 V7211. 7245 1111 II 111172.1.1 V7211. 119115 V11 71119 V7211 7113 1211.71.10 V7211.

E75—21111Va11111133 70771212117111.7134 11... Va 1112 VIII .2111 14:11.11 1111 7E5-1110 V7111 6910 16967 11721127024 V711117081 8: E101121111 V211 6968\1717025‘1/21 71182 V7211 7139 71173117084 V117141 V72:.r 71185 V7211 7,142 E125—1‘7S V7:11.7144 V7211'7201 —1E25150 V7.21 7145 V7211. 7202 V7211“. 71721112111111.1177] 717111116 26. 1911311111111ij embodiments ”1111 e combination of 1V7WF dosage 111121 1111311311813 111 FV111 1112111111152 3,1:1'1iovo11, as 6011111211611 10 FV’HI 5112111111157 111 21 i 3.1111111115113169. a, composition of pdV’WF V7:11. 7492 V7211 7,493 —A.1 . . - —2772 _’a77329 _’a7‘.7 6.11 74273 Var. 7500 V76 7444 V6 756 72217377 F677759 -767: —7275 V71117389 Var. 75613 7276Va1‘ 7333 V‘1.1 7447 Va1.7504 V‘.11 727177 ‘27 “6I371— V672 7762 622.1. 7679 ’ Var. 7392 ' 2‘-.‘.7 7449 Var. 7563 V2‘-.7‘. 7620 ............................................................................................................................................................................................................................ 727'.Va1‘ 7336 V217. 7393 aV'r. 7450 V217. 7564 V'a7‘. 7621 ‘2 7776 7'6. 7451 7712172122 7".78 Var. 7.333: Var 7395 V217. 7452 V2171 762.3 ......................................................................................................................................................................................................................................

Var 7'339 V217. 7396 a..r 7453 V'a.7‘. 7624 776 7.776 7762 7367 7771 2:27 16.172127 —125 V177 7398 V217. 7455 . V2171 7626 Var. 7399 Var 7456 Var 7513 . V23717627 —§76 7‘6 777' 7624 _7267V76 v2.1 7462 —76_7‘677269 7‘6 7774 7‘67 763: 1.1. 7747 776 7464 776 7577 Var. 741.15 “ V217 7406 V7.71 7463 . 7. .

Var 740, V2:.r 7464 V217. /635 Var. 76.37 V2112, [[638 V:77". 6407 , . .1 . . V211”. 1,641 .67 66—11.1 776 757.7 6:666 7—1171 7767472 6.11.7217 66766 _67766 76.7664 4 ‘ ‘ ' ‘ ‘ Var. 7474 ‘ ..................................................................................................................................................................................................................................

Var. 7473_ V'a.7‘. 7646 Var 7420 V217.'/'477 V21717648 ......................................................................................................................................................................................................................................

V217.7421a..7 747'8 a7. 7649 76 7721 7596 11013-150 7'..122 V277. /48.3 Var 7547') ‘ ..... Var. 7654 ................................................................................................................................................................................................ +111012513 Var 7484 Var a7 7598 Var. 7655 1726—266 7762 77726 75772 7‘62 7766 1127175 7 5 V3127—V27/ 86 Var ”.7600 W0T2012/171031 73 I . 15 '0 .151121111 '73 -7 11.2"”? ' 1. ;, Va. 7651, " * 21111 . : ._ V211: Variation Fabio 27 aw embodiments 1‘ or the combination of 1‘]WE- dosage and increase 111 FV1H stability 1111111111111, as compared to EVE} stability 111 a subject admimisiered a composition of pdVWF increased Stahiﬁitv (Percent) 211511110 2113111/ 2111—5111/11 3311—4111:, .11 11.521111 11211. 7662 1111191110 .1111 71.147 ty} 115—1511 111171163: " " E ' ‘ FV‘VVFIRCQ . '7/80 Var.

(EU/kg V9176??? 5475 -151} 1’11 77:16 1111 71112 ‘ 5 1869E V211,,“ : 513-2131} 3131777130 V31. 51} 1511 . -511—11111 ’31.?702 _1317739 -V’31”816 V31“ E-V,’V31 77133 V31. 771313 " =7517S1121111 1'11 -Var7774"II -—m1131.7775 1132 V31 \7311311011 {11112381111 0116 6011101111116111 the 1116111011 1301111111363 3111111111st61111g 3 03111011 of 117W?- 11111616111 the 00111003111011 WF 30111111131616(1 t0 the subject18 3 03111011 01 high 111016011131 weight 1VWF t1mers111 11hich 31 16331 30"/o 01 1‘11’W1 1110160111611111 the ’J‘: 1.101111303111011 310 111636111111 31111111111161 of 3116331 113 subunits, 31111 1111010111 the dose- 011V“? and i1101‘e33e 111 17V111 31313111131, 33 60111113161110 FVEEE 1ity 111 3 311111 e6t administe1‘ed. 21 00011303111011 11fpdV’WF, is 361001.611. 110111 1101115 4869 1.0 8003 111 1311116 213 to T211118 27, 111 0116 embmiiment, the 00111110310011 of 1‘11’WF 311111101816161110 the subject has 3 11ig11e1‘ speciﬁc activity 111311 3 composition 011311173131?” 111 0116 0111110111111611’1, FVIH stabiiity is characterized 11} by the 1131111176 01’ F1711}. 111 3110171161“ embodiment, FV111 stability 13 1:1131'30161‘12611113’ 1116311 631361106 111116 (MR7?) of FVIH. 111 3 further ment, the 111681011 11; for 1163111111 31131131116 1713. 111 3 speciﬁc 61.111101111116113 the 1011 13 1’01“ 116311113; Type 3 VW13. 5118239} 111 0116 embodiment, the method 001111111363 administering 3 composition 01’1‘V‘WFD wherein the 60111003111011 01’1‘VW F 3.1111111113161611 10 the subject is 3 00111003111011 011111311 1110161311131 weight WW1.111111111116111111 which at 16381 50% 0f YVWE 11101613u1es111 the 00111003111011 3.16 1311650111 in 3 1111111111101 01 at 10331 10 311111111113 31111 wherein the 11056 01137111? 31111 111616336 111 F‘VEEI st311i1ity, 33 compared t0 17V111 ity in a subj 601 31111111113161611 3 1.3011113051111011 01131117131335, is 501661.011 110111 variations 4869 to 80133 111 T311110 213 10 131110 27. 1111 011e embodiment, the 00101105111011 01’ 1311/11/13 administered t0 the 311131601 has 3 h1gh6r 3p66iﬁe ...... by the half life ofFViltl. ln another embodiment, F‘y’lll stability is characterized by mean residence time (MRI) of F‘Vlll. in a further embodiment, the method is for treating any type of VWt). in a specific embodiment, the method is for treating Type 3 VW D. {(30243} in one embodiment, the method comprises administering a composition of rVWF, wherein the composition ofrVWF administered to the subject is a composition of high molecular weight rVWF multimers in which at least 7 % of rVWF molecules in the composition are present in a iner of at least ill subunits, and wherein the dose ofrVW? and increase in F‘s/ill stability, as compared to F‘Vttl stability in a subject administered a composition ofpdVWF, is selected trom variations 4869 to 8003 in Table 2% to Table 27, in l0 one embodiment, the composition ofrVWF administered to the subject has a higher speciﬁc activity than a composition F. in one embodiment, F‘v’lll ity is characterized by the l’ialt‘life of F‘Vlll. in another embodiment, FVill stability is characterized by mean ce time (MR7?) of l. in a further embodiment, the method is for treating any type oily/7WD. in a c embodiment, the method is for treating Type 3 VWD.

Ebedi} in one embodiment, the method comprises administering a composition of rVWF, wherein the composition ol‘r‘v’WF administered to the subject is a composition of high molecular weight r‘y’Vv’li‘ multimers having a minimal percentage Vv’F molecules present in a particular higher—order rVWF er or larger er according to any one of variations 134 to 457 found in Table 3 to Table 5, and wherein the dose ofrVWF and se in FVlll stability, as compared to FVlll stability in a subject administered a ition ot‘pdVWF, is selected from variations 4869 to 8383 in Table 2b to Table 27. in one embodiment, the composition of rVW’F administered to the subject has a higher speciﬁc activity than a composition ofpdVWF. in one embodiment, FVlll stability is characterized by the half life of F‘s/lit. ln another ment, F‘s/ill stability is characterized by mean residence time (MET) of FVill. in a further embodiment, the method is for treating any type of VWt). in a specific embodiment, the method is for treating Type 3 VWD. {90242} in one embodiment, the method comprises administering a ition of rVWlT, wherein the it"s/"NF in the composition has a specific ty ot‘from 40 mil/gig to 60 mU/pg, and wherein the dose ot‘rVVVF and increase in FREE stability, as compared to FVlll stability in a subject administered a composition of pdVWF, is selected tron'i variations 4869 to 8803 in Table Ell to Table 27. in one embodiment, the composition ofrVWF stered to the subject is a composition of high molecular weight rVWF multimers. in one embodiment, i stability is characterized by the half life of F‘v’lli. in another embodiment, F‘s/ill stability is characterized by mean residence time (M RT) ot‘FVlll. in a further embodiment, the method is for treating any type of VWD. in a speciﬁc embodiment, the method is for ng Type 3 VWD. {(33243} in one embodiment, the method comprises administering a composition ot‘rV‘tlv’F, wherein the rVWF in the composition has a specific activity of at least 60 mid/pg, and wherein the dose oti:"tl”tlv’lj and increase in FViil stability, as compared to EVE} stability in a subject administered a composition of pdV’WF, is selected from variations 4869 to 8093 in Table 28 to Table 27, in one embodiment, the composition ofrVWF administered to the subject is a composition of high molecular weight rVWF mnltimers. in one embodiment, FVlll stability is characterized by the halflite of FVlll. in another embodiment, FVIH ll) stability is characterized by mean nce time (MR’I‘) of F‘Vlll. in a thither embodiment, the method is for ng any type of VWD. in a specific embodiment, the method is for treating Type 3 VWI). {(30244} in one embodiment, the method comprises administering a composition of rVWF, wherein the rVWF in the composition has a speciﬁc activity of at least 83 trill/pg, and n the dose of rVWF and increase in F‘s/ill stability, as compared to F‘v’lll stability in a t administered a composition ofpdVWF, is selected from variations 4869 to 8003 in e 28 to Table 27. in one embodiment, the composition ofrVWF administered to the subject is a, composition of high molecular weight rVWF mnltimers. in one embodiment, li‘Vlll stability is characterized by the half life of F‘Vlll. in another embodiment, F‘v’lll stability is characterized by mean nce time (MET) lt in a further embodiment, the method is for treating any type ot‘VWD, in a speciﬁc embodiment“, the method is for treating Type 3 VWD. £33245} in one embodii'rierin the method comprises administering a composition of rVVv’R wherein the rVW’F in the composition has a ic activity selected from variations l to 133 found in Table la and wherein the dose F and increase in FVlll ity as compared to FVl ll stability in a t administered a composition of pd‘s/"NF, is selected from variations 4869 to 8003 in Table 28 to Table 27. in one embodiment, the composition of r‘v’Vy’F administered to the subject is a composition ot‘high molecular weight rVWF erst in one embodiment, F‘v’lll stability is characterized by the halt‘lit‘e of F‘v’lll. in another embodiment FVlli ity is characterized by mean residence time (MRT) of F'Vlll. in a further embodiment, the method is for treating any type ofVWD. in a specific en'ibodin'ient, the method is for treating Type 3 VWD, i} in one embodiment, the method comprises administering a composition of rvwr, wherein the dose ofrVWF and c activity ol‘rVWF in the composition is selected troni DJ '4‘; variations 8394 to 10625 in Table 28 to Table 34. in one embodiment, the composition of 1878717 ste1ed t0 the suhj ect is a cernposition ot high meiecniar weight rV’WF iiiuitiniers. in one enihediiiient, the composition cf i‘V‘WF administered to the subject has a higher speciﬁc activity than a een’iposition et‘pdVWF. in yet another embodiment, the cempeSitien of 1VWFFadrniniStered to the Subjectis a composition of high iiai weight rVWF i'riuitiiners with a higher speciﬁc activity than a composition of pdVWF. in one ment, FVIH stability iS terized by the half iife of F‘s/"Hi. in another embodiment, FVIH stability is characterized by mean residence time (MRT) ot‘FViii, in a further embodiment, the methed is for treating any type ei‘VWiZ). in a specific embodiment, the method is for treating Type 3 VWD i0 Tahie 281 Exemplary embodiments for the combinatien ot‘rV WEI desage and W WEI speciﬁc activity useful in the methods described . at teas: 70 at Eeast 80 76.1: 8289 Var. 8346 activity} Var 8290 Var 8347 Van 8291 ‘17>1 8348 Dosage rVWF:RCe 8:182:81 Vzir.8296 :3323 (EU/kg : Var. 8013. Vet 8070 7- ‘- '- . '. '. ‘. '.

Var. 8014 Var 807i 711,989 8711 8388 263 Van 8301 ‘1ar7 838 7 3’ E2525 V’ai 81118 ‘7 87818828 -~..1 87111 8821 871118878 " ‘1 1111 11.11112 —5 85 8711 8888 “ 7 "= 87111 883 8 871118887 “ ‘= Elli-200 Var 80313.1“. 8088 \zar. 8034 . .Q .'1 Var. 8035 V": . c‘ ( Var. 8036 \/VIC. ( 49 . ,. . , .. 7594544 . ’- . .. 4744,4400 . 7 '~. 4 "v.4.~. ‘4/448450 8444347 8744,8046 4744284443 ' . . :4“ 7.

V744 444747 V44. 3404 ' 42448332 .. , 944 4048 ' 4.4 4:435 . 7 4244.83.33 4744.43.99 77518K} V44 4444: :4 4444 _. - 4 V444. 8050 ' '41. 8107 ‘: 44244—2944 4244,8054 4744.44.08 Var. 8339 Var. 8396 V44. 8340 'V44. 8397 Var. 8341 V34 83 98 74254544 737 ' . ,. 4 ,. 4 . ,. :. . . :4592944 944 4058 . . i ,. . .. 4444,8343 Var 4400 .VI‘44 44.454 “717:“ -. 7.4: ’ V448060‘Ja48117 " (4.238 Var. V 44444447044 Tabie 29. Exemplmy embodiments for the 4.7044473444314044 0f 4VWF desage 2444414‘47‘4‘471? speciﬁc activity useful in the methods described .

S ecific Activitv (mil/:13 444784457 244183444 4447434454 347454434 9% 137} 12.8 - . 4 4. 28—125 28—188 ‘ ‘ ’ ‘ ‘ ' 7 ' 44.4584 744.8745 4444444344 , . .. . . . . . 4 K724418746 . 7 4744,4405 Var.8462 " ' . '~. 3 I. 47448747 424.44% 74.4 1 " . ’v . . _ . 4 748 Damage 2444244 Var. 8409 Var. 8466 8/244 8410 V44. 8467 ' (442/444; " V444 847.7 ~ V34: 817? k) DC U} 72.5158 Var. 8414 7 . b) (X) U! 06 ‘J1 7251735} V244. 8415 1'4 00 Ki! {.4\ ' . \C‘ 444444 ' ’ _. I 44744 .44 444424 4444 " . 4444424 42444444 ’- “34» 2—4;: 2 7 -4 , *-,:.i. 7.4-.

I5. 5.

V5: 8427 V55 5454 “ 7 7 V55 8428 :7:55. 84575 " V55. 8429 55 84576 ins-255 ) i561 Emu-3:5 57511. 5432 .

V555490 5551 8457 V55. 5494 E25155 57115 8438 5‘51. 8495 5115 8439 5751254913 536%550 515%“30 Isa—75 V55 5794 3575—203 ‘57at“.— ‘5’ n Tame 35}. Exemplary embodimems for the eombinatiem of r‘v’WF dosage and r‘v’WF speciﬁc activity useful in the methods described herein.

Seciﬁc Aetivitv m T/ Desege 25555 2959 87119916 8711.8874 " 1 8818 87111 87‘11:1. 9975 :19$188848885 ' .8948. . 2 , ‘. 8949 8711 8883 “ 28959 E29199 8711:1. 9994 " .9951 _V311‘ 88.18 Var 8895 :559~299 8311. 99.49 711,; E59159 817111. 9941 :11. 9999 .

E59199 ' 871188411 8711. 81119 E79125 87111. 8847 94 E75480 Var 8905 E199—299 . . 878798906 1991199 199—125 E.125299 1E25- 375 V 8711 8:112 ‘7 28968 87118913 “ 28879 8711.1: 9914 " .9971 1E75—299 , . 1 . - 7.9 E87111 .9929 V793. 9143 Var. 9299 8211'.— ‘8’ 9119311011 Tabie 311 Exemplary embodimsms for the 1.201187111911011 (81“17‘11/‘87E1/7EI (image and 17V WEI speciﬁc activity useful in the s described herein. 4%—5i} . . . .1 : . . . . .. 7211‘. 9486 728.1: 9543 .

Var. 9202 Var. 9259 '8 71 . 1‘ . . .... . . V3.11 9487 Var. 97544 8719.92.93 1.9431 V111: 9499 8711.1. 9545 W0 2012/171031 Var. 592114 79.1.9211' Var, 9489 Var. 954s 115-511 Var. 92112 V111. 94911 V111. 9547 9925 V921 9263 E115111 ‘17>1: 9264 V111. 9549 EL1595 E95125 *- 11.511 V111. 92111 111 V111. 9211 1 ' ,. ,/ 51-19% V912 9272 V91 9229 V91 9223 V91 9217 I V211‘. 9505 ‘1" 211‘. 9562 V19- 9222 V11 9279 911195113 - V111. 95117 V111. 9564 1111' Var. 9282 ‘1‘17211 9283 1-. 995 Eiﬁ~HEW Var. 92:3:0 V211 328: 111.9115 —11925 -. 9. 9119 Esmmu V111. 9229 911 -. ., 1 1 1129129524 V291.95111 E5111511 V111. 92411 V111 9297 '1 . 11211129525 Var 95122 E51111111 V111. 9241 1 ’1 1 .- V11>1~9526 1121119593 E“is—21111 9111192114 1791292111 19193112 E 2 "1711119303 — 1E911—2911 . ., '1911175 V111. 92511 V111 9307 EM225 ' 21.1” 9593 12.592911 ...................................................... 125-175 V912 9322 V912 9322 V111. 92515 V111: 9313 LENS—20E} ‘1/111 9257 Var 9314 WO 71031 Var. Variation T211119. 32. Exemplary 606111131113 for the combination Vv’F dosage and, YVWF 111613151111: activity useful in the methods describsd herein. "1111151 611 V 1 activity) Var. 9602 V9.1. 9659 Var. 9603 V9.1. 9660 Bssage 'WF2RC‘0 ’ .: 1. 3 1V Var. 9667 7211'. 9664 (EU/kg Var. 9608 Var. 9665 ‘ ‘ V111. 9609 V111'. 9666 Var. 9898 I a 1 171129675 “ 1 .' - 1 1 —Va1.9619 1711:9676 " ', , ' E 1 :2, 1711,9677 v, , Var. 99115 Var, 0171 '- .f .: : '23», Var. 99116 —Var.%22 ' .. Var.

Var-9623 ” C . "a —511 1:. : .' . 1 1711:9909 ‘25 : » : :111 ' : : a . . . . ,, 1131,9911 —Va1.9628 1711:9615 " ', v “711.9629 "1" " U ‘ Var.963‘2 Var.9689 7t: '_ '1 ~. 1131:9917 E25490 Var.9633 Var. . . , 1711:9918 63.6 Va ' 1 1 , Var. 91337 Var. " ' . . 1' 3511121111 Var.9638 695 " '1 ' v " . 1721191123 1711,9539 V" " ' ' ' v . ', 1721191124 .13 1131,9698 '- .f g x, ,1 .f V31199‘46 Var.964‘2 \x’ar.9699 t" ' _ '~..: ~ 171129928 $1.970691. 9707 {EYES—20% 15125375 1." . _. 9853 '11.<7 7 . '~..:1 9938 315331299 11. , V7111.9939 31533—2339 ” ’ ' E 175299 V2313*. = Variation 391338 33. Exemplary embodiments 33313139 cambination afr‘V/VV'F dosage and rV‘V’V'F speciﬁc activiiy 318131111 111317133 IlthhOdS 71de 116193111. 7311‘99 73-83 8%- SE} V7111. 9942 V111. 9999 3211.1 10056 V111. 13333319227 activity) V7111.199339 V7111.133957V11.1 1333111 3111139228 . 313229 . 39.273 Engage 931719233139 1 17 1. 7231.101}: .

Va: 10175 37131130232 193395 V7111. V7111. 19178 19235 13V Var 9949 ' (EU/kg V “ '7. -—2 . . 1339318 . . . . . 1 V711 91352 V111. 39999 V111. V111. 19189 31111119237 -251533 V711.1. 9953 V7111. 3991 9 V711.1 13391.17 \7’131.10238 -..:1¥317.111.9954 V7111-.199:11 V7111.133968 V7111. 3333 25 V7111. 39182 V7111.392;39E V3 19969 V711.1333271 V7111. 313383 V7111.392139E 19979 V711. 193 27 Va. 393 84 31711392131: V11.9957 V711 19178 V7111 39185 V7111. 39242 ’ "——713110129 372131102435 3171111133139 3171111119187 3117111149244 3171119131 217-5183018221 11927353 ............................................................................................................................................................................................

Var.1‘10 89 Var. 1024(3E -53533 V711 913312 V111. 39919 V111. 133978 V:{T.10i:ﬁ3 V111. 19199 31111119247 -—V7111. 931.13 V7111.19929 V7111.1339.7 V2317. 10191 V231“. 10248 V119964V111113973V11133928V7111331 “”99“ V31” V331‘.9965 Var.1=’)022 Var. 10079 Var. H 136 Var. 10193 V2317. 1.. Var,9966 1002. $10080 Var. 10137 Var.10194 Var.

-WV1111. 113332 73 37131 303 95 371312 -V7111.1933;11~1.19338211119139 V7111. 19198 Var.’ -1912933 V7.11 191326 V7.11 193383 31711119149 31711199197 V7111. -—1.:V711 91372 V111. 39929 V111. 133988 V111.1331 43 V111.19299 V111. . -—1—”V11. 9973 19939 V7111.133987 V111.13331311 311111333258 V7111. 3 97.59 -2533333 71.119999 31711193137 1 1729 1111. 193335 ‘V7111~.193392 V7111.19149 V7111.19298 V7111.19265§ —50721-1195979 V31.10EJ3'6 EJ93 71.732.10E50 VarLE0207 71.7312 0E2€1IE -—1721119980 Var. 111037 Var. 111094 ' 7 Var 1112117, Var. 11.1265 -":I11.11931 038 V221.111152V.11.11_12119 V211.11_1266 511-11111 172211111139 V221.111153 1_12113 V211.11_1267 71/410040 71/ 10097 17211. 10154 0211 V211.10268 Va1.10041 "1100.98 V211. 1EJ15S ll\ I . . Var.10269 E Var 11.11142 Var. 101199 1721110213 VI211.1112711 171111211143 V3.1. 11111111 V'2.1.1111 57 V211.111214 VI211.111271E 75—125 V7211.9987 V7111". E0044 V;1.1'.LEOE01 7' E I’ 711721100215 Var. E0272___E 75—100 1721119988 V7111" E0045 Var LEOE02 E17111. LEO S9 Var. E02E6 Var. E027 "—199.29? ""73215791399"79311'11447 wig-177719193"- "93-2—7141 2 "9379—92—17"- "177.27.171'92 ,:1 -111112175 1721119990 11047 172111111114 ' " V211.10218 V'212.1(127I5E -1111121511 VI'I.2,1.9991 Var.10048 172211111115 V221.111162 V211.11_1276E 11111—125 Var. 11.11149 Var. 1111116 Var. 111163 V211.111277 -1W\7211. 1111151721. 10109 V7; 1016113 172111022 VI211.111231.1 151121.111 V21 9996 VI111 1211153 17.21 1111111 V'I.>.1.111167 921110224 872110281 299 141.9997 1411101154 121111111 1 V.11'.LE0168 V212. 1112.25 V52. 1112ng 727017099 172110075 ‘IEEEJE? 71191727 WEEQW‘ E71E0282 ____________________E25:22:11..

V211*. = Variaﬁon 7172113141 34. ExemEﬂary embodiments 1701' 11113 61311111111211.1011 1317117171717 dosage and 117171717 speciﬁc 211111191157 115131111 4: 111611110615 {1638011de 116113111.

.S.11.25E£5..2§3.113111:..1'1111111121 ____________________________ .___________________________ 80—125 SEE—1008090 90-150 90-125 90400 VI211 111455 Var. 111512 Var. 111569 activity) V'211. 111399 VI211. 111456 VI211 111513 V211. 1115711 9.5211111 V212. 1114.57 17222111514 Var. ' ' 7 ' ' ' ' 1 1 02 ______________ 1:21 :13 2 .22". .10345 VarﬁE0402 V731'EI0ES9 71.7'211 .9731}. m E 27371717117212.1313 V711, V21 111346 V211. 11.14113 V.211 11.14611 Var. Var. 1.2.1 V2:11112911 V2:r.111347 V211.11_14114 V211.11_1461 V51.1- V511 V7211.1EJ29E Var.11J.":4.8 V211I.1EJ405 EV’aI.

ELIE/14g 7211'. 10293. 7211'. 102150 0407 -L 2:5200 . -.L V211. 2721110294 :721' 10.351 0408 V7211 10465 Var. 10522 Var. 1' 2. 2~5 150 231. 10295 3.1. 10352 Var. 10409 Var. 10466 V211". 10523 V211. E q Var. E0437 Var.

........................................................................................................................................................................................... Var LE 0297 E17111'.LE0.3S4 Var. E0411 Var E0468 VaE'.E ,. V2.2 111293 V2.2 1113.55 Var111412 V2.1 01469 Va: 1115 --V21 111299-—V-V21 111357 V.211 11.14711 Var. 111527E V2.11 1 11512115262 I: --“‘Var. 22201 551 .5 211'. 0.10.5 1110360 Va1'.10417 Var 10474 -57150 ‘17211'.E0.304 ‘17211'10361 ‘17211'10418 V7211“ 10475 ‘V7 1' -—VI'I.>.1 1113115 V'.>.1.111362 V'211. 111419 VI211. 111476 VI211. 1 71173710306 71173740363 Var. E0420 Var E077 .......................................................................................................................................................................................... 71273210307 712732.103 E VarﬁE0421 Var E0471 -&V2171273210308 712732403115 VarﬁE0422 Var E0IE/9 V7211'.E. . 19 V211.10366 V211.1(1423 V211.11141311 153,7 V211~.111594 --V221.1113111 V221. 111367 V211.11_1424 V211. 11.1431 V51.1153 8 V5.1.111595 V61111036.55 Var. 10482 41 . . . Va1. 10425 V2117. 10.5 .. 1 1 "1721110369 "17211: 104213 V211”. 10483 Vat. 10540 Var. 10597 -1{1~1111} 'V71>11.18313 'V7a1. 18378 'V7a1. 111427 Va1. 111484 Va1~.111541 V'a1.1115981 -—'V7a1. 18314 'V7a1. 18371 'V7a1. 111428 Va1~ 111485 Va1~.111542 V'a1,111599 Va1, 3. 17111.111.6811 ................................................................... , . . 1731. 111544 V111. 1116111 -25121111 V111.111274 i8411 V111. V1111.111488 Va1. ~. 111682 -— ' Va11. 111218 V7a11. 111275 111111114891 . 18882 251-1013 '2 . 31,1 . 10376 Var. 1.0433 V2117. 1.0490 10604 —75 11 Va;1.111.3:77 Var. 111434 1491 . . 1 2558 71.118321 711 1837. Va1 18435 Va1 18492 7 .1061‘161 -—Va. 18322 V7a1 18379 Va1 111492 Va1.111558 Va1. 11115117 -—V791.1 18323 Va1~ 111494 Va1~.111551 V7a1.111688 V111, 111495 Va1, 111552 V7111: 111689 75151.1 Va11.111228 V7a11.111285 17111111442 11111111499 Va1.‘1 --Va;1. 1113/9.5— Var. 111443 Va1. 1115811 V117. - —.11111 - 721.1. 10.131 7211 1038.55 1173110415 -11.111 175 Va. 18322 V7a1 5 Va1 1115112 Va1. ‘1' -1119111511 V7>1. 18323 V7a1 1838911.1 18398 V7a1.111447 Va1~ 111584 Va1. 1 1 1 7~ ' 1 . 37 1 1251511 V7111.111458m V1111.i11588 : - 1V11.111294 V111. 11.1451 V'a1. 1115155 V111. 111622 ___-151112811 V7111. 111238 V7a1.1 111295 11111. 111589 Va1. 18566 Va1.18822 V211*. = 11711111111011 {0112117} in one embodiment, the inethed 1301111111865 administering a een1pesitien ot‘rV W13, wherein the eempesitien 01’1VWF administered to the subject is 711. compesitien ethigh nieieenitti VVeight YVW1 muitime‘rsin W 111611 at 1e85‘1 30°/6 et 17‘117’1371n11'11e01116s1n the 'J‘: 1.101111388111011 are 11121386111111 11111161 of 811611.81 10 subunits, 81111177111311.1111 the dese- ef 1VW? and specific activity of 1VW13 in the 611n1pesitien is ed 1mm variations 8004 to 10625 in T511316 28 to T221116 341. 111 one embediment, the eenipesitien of TVW13 administered to the 8111.11 eet has 11.1'1ig1'1e1” speciﬁc activity than a composition 111’pd‘V’WF. 111, one embodiment, 19-"51’111 stahiiity is characterized by the 1181f iife 6f 19-"51’111. 111 another embodiment, F‘s/"111 stabiiity is eharaetetizeti 11y 11168.11, residence time (M RT) 0117117111. 111 21. further 6111151111111119111, the methed is for treating any type 01’VW1). in a ie embodiment. the method is: 1191‘ treating Type 3 VWD. 48} in ene ment, the method comprises stering 21 composition 0111/11/17 n the compesitien of 17VWF administered t1) the subject is 11 een1pesitien 01’ high 1110161311181 weight 1‘V7’V‘V7’131.n1111timeisin which at ieest 50% of YVW1 1110116130165;111 the eempesitien are present in a. mtiitimet‘ of at 1east 1t) ts, and wherein the dese 191’ 1‘17’VV’F WO 71031 and specific activity ot‘r‘y’Wb in the composition is selected from variations 8004 to liloZS inrs Table 28 to Table 34. in one embodiment, the composition of rvwr administered to the subject has a higher specific activity than a sition ot‘pdVWF. in one embodiment, FVTTl stability is characterized by the half life of FVTTl. in another embodiment, F‘y’lll stability is characterized by mean residence time (MRT) of FVTH. in a tiirther ernbodimenn the method is for treating any type of VWD. in a specific embodiment, the method is for treating Type 3 VWD. {“3249} in one embodiment, the method comprises administering a composition ot‘r‘i/ WEI, wherein the composition ofrVWF administered to the subject is a composition of high ll) molecular weight rVWl“ mnltiiners in which at least 70% ot‘r‘t/Wb molecules in the1 composition are present in a multimer of at least ll) subunits, and wherein the dose of rVWF and specific activity ofrVWF in the composition, is selected from variations 8004 to ”H3625 in Table 28 to Table 34. in one embodiment, the composition of r‘v‘wr administered to the subject has a higher specific activity than a composition ol‘pdVWF. in one embodiment, F‘v‘lll stability is characterized by the half life of F‘v‘lll. in another embodiment, FVllT stability is characterized by mean residence time (MET) oflTVTllc Tn a r embodiment, the method is for treating any type of‘v’Vy’l). in a ic embodiment, the method is for ng Type 3 VWD, EtitlZSﬂ} in one ment, the method comprises administering a composition ofr‘tPVt’F, wherein the composition ’WF stered to the subject is a composition of high molecular weight rVWF mnitirners having a minimal percentage ot‘rVWF molecules present in a particular higher—order rVWF multimer or larger mnltimer ing to any one of variations 134 to 457’ found in Table 3 to Table 5, and wherein the dose ol‘rVWF and c activity of rvwr in the composition is selected from variations 8004 to l 0625 in Tabie 28 to Tabie '34. in one einbodirnenn the composition ofrVWF administered to the subject has a higher specific activity than a composition of pd‘v'Vi/F. in one embodiment, FVTTT stability is characterized by the halfliie of FVTTT. in another embodiment, FVllT stability is characterized by mean residence time (MRT) of EVE”. in a thither embodiment, the method is for treating any type ofVWD. Tn a specich embodiment? the method is for ng Type 3 VWT). {(30251} in one embodiment, the method comprises administering a composition of rVWF, wherein F‘v’lll in the subject is stabilized for at least l8 hour-s post~administration, and wherein the dose of r‘t/WF and speciﬁc actiyity ofrVWF in the ition is selected from variations 8384 to 10625 in Table 2% to Table 34, hi one dinient, the cornposition of DJ (J; rV WP“ administered to the subject has a higher speciﬁc ty than a composition of pdVW13 . 1n one embodiment, 1iV111 stability is characterized by the halflife 11. 1n another embodiment, F‘v’111 stability is characterized by mean nce time (MRT) of 17117111. in a titrther embodiment, the method is for treating any type oi“ VW1). in a, specili c embodiment, the method is for treating Type 3 VWD.

E10252} 1n one embodiment, the method comprises administering a composition of rVWF, wherein F‘v’lil in the subject is stabilized for at least '24 hours post—administration, and wherein the dose ofrVWF and speciﬁc ty ofrVWF in the composition is seiected from variations 81194 to 10625 in ’1‘ab1e 28 to Table 34. hi one embodiment, the composition ot‘ rVWT- administered to the subject has a higher speciﬁc activity than a composition of it) pdVW13 . 1n one embodiment, 1iV111 stability is characterized by the halflife Gibb/111. 1n another ment, F‘v’111 stability is characterized by mean residence time (MRT) of 17117111. in a titrther embodiment, the method is for treating any type oi“ VW1). in a, specili c ment, the method is for ng Type 3 VWD. 1311253} 1n one embodiment, the method comprises administering a composition of rVWF, wherein F‘v’lil in the subject is stabilized for at least 30 hours post—administration, and wherein the dose ofrVWF and speciﬁc activity ofrVWF in the composition is seiected from variations 81194 to 10625 in ’1‘ab1e 28 to Table 34. hi one embodiment, the composition ot‘ rVWT- administered to the subject has a higher c activity than a composition of pdVW13 . 1n one embodiment, 1iV111 stability is characterized by the halflife Gibb/111. 1n another ment, F‘v’lll stability is characterized by mean residence time (MRT) of 17117111. in a, further embodin'ient, the method is for treating any type ot‘VWD. in a, c embodiment, the method is for treating Type 3 VWD. stration of rFVTi 11 133254} 1n one aspect, the present disclosure provides method for treating Von Wiliebrand Disease (VWD) or Hemophilia A in a subject in need thereof, which includes administering a composition of recombinant Von Willebrand Factor (rVVvT) and recombinant FV111 (rFV111) such that 17actor V111 (F‘v’ll1) stability is sed, as compared to 13V11l halt—life in a subject administered a composition ofplasma d Von Wiilebrand Factor ‘r‘s’F), in one embodiment, the composition ofrVWan?V111 stered to the subject is a composition of high molecular weight rV’W'F multimei‘s. 1n one embodiment, the composition of rVWF/rFVill administered to the subject has a higher VWF specitic activity than a composition of pdVWF. 1n yet another embodiment, the composition of rVWT-V’rFVll1 administered to the subject is a composition of high molecular weight r‘VWF ers with a higher V’WF specific activity than a composition ofpdVWF. 1n one embodiment, FV111 stability is characterized by the halt‘lit‘e of F‘s/ill. in another embodiment, F‘s/ill stability is terized by mean residence time (MRI) of F’Vlll. in a further embodiment, the method is for treating any type of VWD. lit a specilic embodiment, the method is for treating Type 3 VWD. } in one embodiment, the method comprises administering a composition of rVWF/rFVlll such that FVlll stability is extended by at least lil%, 209/2), 30%, '2 hr, 4 hr, 6 hr, or by an amount selected from variations 8th to loll-3 found in Table 9, as compared to l stability in a subject administered a composition of pdV WEI/'FVEH. in one embodiment, the composition ofrVW17/1"lelll administered to the subject is a composition of ll) high molecular weight rVWli‘ rnnltimers. in one embodiment, the composition of rVWF/rFVlll administered to the t has a higher VWF specific activity than a composition WF/FVlll, in yet another embodiment, the composition ot‘r‘v’WF/rFVlll administered to the snbj ect is a composition of high molecular weight rVWF multimers with a higher speciﬁc activity than a composition of pdVWF, ln one embodiment, FVlll stability is characterized by the half life of . in r embodiment, F‘Vlll ity is characterized by mean residence time (MRT) of FVlll. in a thither ment, the method is for treating any type of VWD. in a ic embodiment, the method is for ng Type 3 VWD, 598256} in one embodiment, the method comprises administering a composition of rVVv’F/'rFVlll such that FVlll stability is extended by at least l0% as compared to FVlll stability in a subject administered a composition ol‘pdVWF/FVHL wherein the composition of FVVVF/I‘FVl ll administered to the subject is a composition of high molecular weight rVWF mnltimers having a minimal percentage ot‘rVWF molecules present in a particular high er~ order rVW'F multimei‘ or larger mnltimer according to any one of variations l34 to 457 found in Table 3 to Table 5, in one embodiment, the composition ot‘rVWF/rFVlll administered to the snbj ect has a higher VWF specitie activity than a ition of pdV‘v‘y’F/F‘t/ill. in one embodiment, FVlll stability is characterized by the hall‘life ofFVlll, in another ment, FVlll stability is characterized by mean residence time (MR7?) of F‘v’lll. lo a further embodiment, the method is for treating any type ol‘VWD In a specific embodiment, the method is for treating Type 3 VWit). {(30257} in one embodiment, the method comprises administering a composition of rVVv’F/rFVlll such that FVlll stability is extended by at least 23% as compared to FVlii ity in a t administered a composition of pdVVv’F/F‘Jlll, wherein the composition oltVWR’rF‘y’l ll, adn'iiriistered to the sttbj ect is a composition of high molecular weight r‘v’W F DJ (J; mnltimers haying a minimal tage ofr‘v’Wl? molecules present in a particular highers order r‘y’Vy’lI mer or larger mnltimer according to any one ot‘yariations l34 to 457 found in Table 3 to Table 5. in one embodiment, the composition of rVVv’F/rFVlll stered to the subject has a higher VWF’ speciﬁc activity than a composition ofpdVVv’F/FVlli. in one embodiment, F‘s/ill stability is characterized by the half life of F‘s/ill. in another en'ibodin'ient, FVlll stability is characterized by mean residence time (MET) of FVlll. in a r embodiment, the method is for treating any type of VWD. in a speciﬁc ment, the method is for ng Type 3 VWD. {“3258} in one ment, the method comprises administering a ition of rV‘yt’T/rFVlll such that EVHI stability is extended by at least 30% as compared to F‘v’lll ll) stability in a subject administered a composition ot‘pdVWF/FVEll, wherein the composition of i'\l"\7t7F/rF\/’lil administered to the snbj eet is a ition of high molecular weight rvwr multin'iers haying a, minin'ial percentage of rVWF molecules present in a ular higher— order rVWF multimer or larger mnitimer according to any one of variations 134 to 457 found in Table 3 to Table 5. in one embodiment, the composition of /rFVlli administered to the subject has a higher specific activity than a composition of pdVWF/E-‘Vill. in one embodiment, FVlll ity is characterized by the half life of FVlll. in another embodiment, F‘V‘lll stability is characterized by mean nce time (h’lR'l’) of FVill, in a titrther embodiment, the method is for treating any type ofVWD. in a specific embodiment, the method is for treating ’l'ype 3 \I’Wii). ﬂl in one embodiment, the method comprises administering a composition of r‘y’WF/rFVlll, wherein the combination of r‘VWF specific activity in the composition and increase in F‘s/ill stability, as compared to F‘s/ill stability in a stibject administered a con’iposition oi pdVWR/FVlll, is selected from Variations 2339 to 4868 in Table 13 to Table 19. in one embodiment, the composition of r‘yf‘tKI’F/R'FVlll administered to the subject is a composition of high molecular weight rVWF multimers. in one embodiment, FVlll stability is terized by the half life of F‘Vlll. in another ment, F‘y’lll stability is characterized by mean residence time (MRT) ot‘FVlll, in a further embodiment, the method is for treating any type ofVWli). in a specific embodiment, the method is for treating ’l'ype 3 VWD. {(33269} in one embodiment, the method comprises adn’iinistering a composition of rVWF/rFVlll, wherein the composition of rV‘y‘y’F/rFVlli stered to the snbj eet is a con’iposition of high n’iolecnlar weight rVWF imiltimers in which at least 30% ot‘rVWF molecules in the composition are present in a mnltimer of at least ll) ts, and wherein the combination oi‘rVWF specific activity in the composition and increase in F‘y’ill stability, DJ (J; as compared to F‘V‘lll stability in a subject administered a composition of pd‘t/"NF/FVHI, is selected from variations 2339 to 4868 in 'l‘abie E3 to 'l‘abie l9. in one embodiment, the composition of rVVv’F/‘rli'Vlli administered to the subject is a composition of high molecular weight rVWF multimers, in one embodiment, FVlll stability is characterized by the it‘e of F‘s/ill. in another embodiment, F‘v’lll stability is characterized by mean residence time (MRT) of FVlii. in a further ment, the method is for treating any type oft/7WD. in a speciﬁc embodiment, the method is for treating "l‘ype 3 "V'WD. {$9261} in one embodiment, the method comprises administering a composition of rV ‘Vlll, wherein the composition ot‘r‘yI’V‘tI’iV/rF‘s/lll administered to the subject is a ition of high lar weight rVWF multimers in which at least 50% of I‘VWF it) molecules in the composition are present in a multimer ot‘ at least it) subunits, and wherein the ation ofrVWF specific activity in the ition and increase in F‘v’lll stability, as compared to FVlll stability in a subject administered a ition ot’pd‘w’WF/FVHI, is selected from variations 339 to 4868 in Table 13 to Table 19. in one embodiment, the composition of rV‘v’v’F/rli‘y’lli administered to the subject is a composition ot~ high molecular weight rVWF multimers. in one embodiment, F‘y’lll stability is characterized by the half life of FVlll. in another ment, FVlll stability is characterized by mean nce time (MR’E‘) Ol‘i:\/'iii. in a r embodiment, the method is for ng any type of VWl). in a speciﬁc embodiment, the method is for treating Type 3 VWD. 598262} in one embodiment, the method comprises administering a composition of r‘v’W’E’rFVlll, wherein the composition ot‘rVWF/rFVlll administered to the subject is a composition of high molecular weight rVWF multimers in which at least 70% ot‘rVWF molecules in the composition are present in a multimer of at least it) subunits, and n the combination ot‘r‘VWF speci tic activity in the composition and increase in FVlil stability, as compared to FV’lll stability in a subject administered a composition of pdV’Wli'le-‘Vilil, is ed from ions 2339 to 4868 in Table 13 to Tahie 159. in one embodiment, the composition ot‘r‘yI’V‘tI’iV/rF‘s/lll administered to the subject is a composition of high molecular weight rVWF mnltimers. in one embodiment, FVlll stability is characterized by the half life of FVill, in another embodiment, F‘Viil stability is characterized by mean residence time (MET) ofFVlllt in a further embodiment, the method is for treating any type ot‘VWﬂ in a specili c ment, the method is for treating Type 3 VWD. {(30263} in one embodiment, the method comprises administering a composition of rV‘t‘v’F/rFViil, wherein the composition of rV‘v’v’Fx’rF‘y’lii adn'iinistered to the subject is a composition of high molecular weight rVWF multimers having a minimal percentage of r‘v’WF molecules present in a particular higher—order rVWF multimer or larger multimer DJ (J; ing to any one of variations 134 to 457 found in Table 3 to Table 5, and wherein the WO 71031 combination ofrV WEI specific activity in the composition and increase in FVlll stability, as compared to F’Vlll stability in a subject administered a composition of pdVWF/FVlll, is selected from variations 2339 to 4868 in Table 13 to Table 159. in one embodiment, the composition of rVWF/rFVlll administered to the subject is a composition of high molecular weight rVW F mnltimers. in one embodiment, FVlll stability is characterized by the hall’lil’e of FVlll. in another embodiment, FVlll stability is characterized by mean residence time (MET) oflTVlll, in a farther embodiment, the method is for ng any type ofVWD. ln a c embodiment, the method is for treating Type 3 VWD. {911264} 1n one embodiment, the method comprises administering a dosage ofa rVWF/rFVlll ll) ition containing from 10 ill/kg to 4G lll/kg WWFdlCo activity, wherein the ation of rVWF specific activity in the composition and increase in F‘s/"ill stability, as compared to F‘v’iil stability in a subject administered a ition of pdV‘v’v’E’FVlll, is selected from variations 2339 to 4868 in Table 13 to Table 19. ln a specific embodiment, the ition contains from 20 lU/lrg to 30 ill/lag rVVv’FRt’jo activity. in one embodiment, the composition of i'VWFI'rF‘v’lll administered to the subject is a composition of high molecular weight rVVvT multimers, in one embodiment, FVlll stability is characterized by the halt‘life of FVlll. in another embodiment, FVlll stability is characterized by mean nce time (MR1) of FVlll, in a further embodiment, the method is for treating any type ot‘VWlZ). in a speciﬁc ment, the method is for treating Type 3 VWD. {$11265} in one embodiment, the method comprises administering a dosage of a /rF‘v’lll composition containing from 25 lU/lrg to 75 ill/lag Co activity, wherein the combination of rvwr specific activity in the composition and increase in li'Vlill stability, as compared to FVlll stability in a subject administered a composition ofpdVWF/FVlll, is selected from variations 2339 to 4868 in Table 13 to Table 19. in a specific embodiment, the composition ns li'on'i 4t} lU/hg to so ill/kg r‘v’WFrRCo activity, ln one embodiment, the composition of t‘v’hVF/l‘li‘lilll administered to the subject is a composition of high molecular weight rVVVF multimers. in one embodiment, FVlll stability is characterized by the half life ofli‘Vlll. in another embodiment, F‘v’lll stability is terized by mean residence time (_MR1“) of FVlll. in a further embodiment, the method is tor ng any type of VWl). in a specific embodiment, the method is for treating Type 3 VW D. {(10266} ln one embodiment, the method comprises administering a dosage of a rVWF/rFVlll composition containing from 75 ill/kg to 125 lU/lrg TV‘WFZRCO activity, wherein the combination of rVWF ic activity in the composition and se in F‘s/"ill stability, as compared to F‘v’iil stability in a t administered a composition of pdV‘v’v’E’FVlll, is DJ (J; selected from variations 2339 to 4868 in Table 13 to Table 19. in a ic embodiment, the composition contains from '75 lit/leg to tilt) Ell/kg YVWFiRCO activity. in one embodiment, the composition of rVWF/il’hdll administered to the subject is a composition of high molecular weight rVWF n'iultimers, in one ment, FVlll stability is characterized by the half life of ll. in another ment, F‘Vlll stability is characterized by mean residence time (MRT) ot‘FVlll, in a further embodiment, the method is for treating any type ofVWD. in a specific embodiment, the method is for treating Type 3 VWD. lilll267l in one embodiment, the method comprises administering a composition of rV WF/rli‘Vlll, wherein the subject is administered a dose ot‘rV WP“ selected from variations lell to 2338 in Table l2, and wherein the combination ofrVWF specific activity in the ll) composition and increase in F‘s/ill stability, as compared to li‘V’lll stability in a subject administered a composition of FVlll, is selected from variations 2339 to 4868 in Table B to Table it). in one embodiment, the composition, ofrV‘v‘v’F/erlll administered to the subject is a composition of high molecular weight rvwr multimers. in one embodiment, F‘y’lli stability is characterized by the halflite of F‘y’lli. in r en'ibodin'ient, FVlll stability is characterized by mean residence time (MR?) of F‘Vlll. in a further ment, the method is for treating any type ofVWD. in a ic embodiment, the method is for ng Type 3 VWt). {90268} in one embodiment, the method comprises administering a composition of r‘v’Vy’F/rFVill, wherein the dose i‘it’r“\,/"\lt7lI and increase in FVlll stability, as ed to FVlll stability in a t stered a composition ofpd‘v’WF/F‘y’lll, is selected from variations 4869 to 8003 in Table 2t} to Table 27, in one embodiment, the composition of rVWF/rFVlll administered to the subject is a composition of high molecular weight rVWF multimers. in one embodiment, the composition of rVVv’F/rFVlll administered to the subject has a higher speciﬁc actiyity than a composition ofpthWF. in yet another embodiment, the composition ot‘rVWF/rFVlll administered to the subject is a composition of high molecular weight ’F multimers with a higher specific activity than a ition of pdV‘iNF/EIV l l l. in one embodiment, FVlll stability is characterized by the half life ol‘FVlll, in another embodiment, FVlll stability is characterized by mean residence time (MR7?) of F‘s/ill. in a further embodiment, the method is for treating any type ol‘VWD in a specific embodiment, the method is for treating Type 3 VWD. } in one embodiment, the method comprises administering a composition of rVWF/rFVlll, n the composition of rVVv’F/rFVlH adn'iinistered to the subject is a composition of high molecular weight rVWF multimers in which at least 30% of rVWF molecules in the ition are present in a multimer of at least ll) subunits, and wherein DJ (J; the dose ot‘rV WP“ and increase in li‘Vlll stability, as compared to FVlll stability in a subject administered a composition ofpdV WED/F‘Vlll, is selected from variations 4869 to 8063 in Table 23 to Table 27. in one embodiment, the composition of i"\i"t‘ti’F’/"r¥‘\"lll administered to the subject has a higher speciiic activity than a. composition of pd‘t’Vt’F,l’FVlll, in one embodiment, l stability is characterized by the half life of F‘s/ill. in another embodiment, FVlll stability is terized by mean residence time (MET) of FVlll. in a further embodiment, the method is for treating any type of VWD. in a, speciﬁc embodiment, the method is for ng Type 3 VWD. {“3279} in one embodiment, the method comprises administering a composition of rV‘t‘yT/rFVlll, wherein the composition of FVlll administered to the snbj ect is a, ll) ition of high molecular weight r‘v’WF multimers in which at least 50% ofWW? molecules in the ition are present in a er of at least it) ts, and wherein the dose ol’rVWF and increase in F‘y’lll stability, as compared to FVlll stability in a subject stered a composition of pthWR/FVlll, is selected from variations 4869 to 8003 in Table 29 to Table 27. in one embodiment, the composition /rFVlll administered to the subject has a higher specific activity than a composition of pdVWF/E-‘Vlll. in one embodiment, FVlll stability is characterized by the half life of FVlll. in another embodiment, FVllE stability is terized by mean residence time (h’lR'l’) of FVill, in a titrther embodiment, the method is for treating any type ofVWD. in a speciﬁc embodiment, the method is for treating ’l'ype 3 \I’WD.

EilllZ’Li'll in one embodiment, the method comprises administering a composition of r‘v’VVF/rﬂ’lll, wherein the composition ot‘rVWF/rlTVlll administered to the subject is a composition of high lar weight rVWF multimers in which at least 70% of rVWF molecules in the composition are t in a. multimer of at least l ll subunits, and n the dose of rvwr and increase in FVlll stability, as compared to F‘s/ill stability in a subject administered a. composition of pii\"Vt"l7,l’F\/'lll, is selected iron't variations 4869 to 8803 in 'l‘able 28 to Table 27. in one embodiment, the composition of rV‘v‘v’F/rFVlll administered to the subject. has a higher speciﬁc activity than a composition ofpd‘v’WF/FVIH. in one embodiment, FVlll stability is characterized by the halt‘life of FVlll. in another embodiment, FVlll stability is characterized by mean residence time (MRT) ot‘FVlll, in a further embodiment, the method is for treating any type oft/7WD. in a, speoilic embodiment, the method is for treating 'l‘ype 3 VWD.

E33272} in one ment, the method comprises administering a composition of rVWF/rFVlll, wherein the composition of rVVv’F,/i‘l9"y’ l ll administered to the subject is a composition of high molecular weight rVWF multimers having a minimal percentage of DJ (J; rV Wli molecules present in a particular higher-order rVWF er or larger multimer ing to any one of variations 134 to 457 found in Table 3 to Table 5, and wherein the dose ofrVWF and increase in FV’HT stability, as ed to F‘Vlll stability in a subject administered a composition of pdVWF/FVlli, is selected iron'i variations 4869 to 8803 in Table 20 to Table 27. in one embodiment, the composition of r‘v’Vy’F/rFVlll administered to the subject has a higher specitie activity than a composition ot‘pdVWF/FVlll. in one embodiment, ll stability is characterized by the half life of F‘s/"ill. in another embodiment, FVlll stability is characterized by mean residence time (MRT) ot‘FVlll, in a r embodiment, the method is for treating any type oft/Wt). in a specific embodiment, the method is for treating Type 3 VWD, it) 598273} in one ment, the method comprises administering a composition of rVWF/rFVlll, wherein the rV‘WF in the ition has a speciﬁc activity of from 40 mil/tag to so mU/ug, and wherein the dose ol‘rVWF and increase in FViil stability, as compared to rv ill stability in a subject administered a composition of pdVWF/T‘Vlll, is selected irom ions 4869 to 8003 in Table 26} to Table 27. in one embodiment, the composition of rVVv’F/‘rlé‘v’lll administered to the subject is a composition of high lar weight rVWF multimers, in one embodiment, FVlll ity is characterized by the halflife of F‘s/lit. in another embodiment, F‘v’lll stability is characterized by mean residence time (MRT) of FVlll. in a further embodiment, the method is for treating any type ot‘VWD in a specitic embodiment, the method is for treating Type 3 ‘v’Vv’D.

EilllZMl in one embodiment, the method comprises administering a composition of r‘v’WF/rltl‘y’lll, wherein the rVWF in the composition has a specitie activity of at least 60 mU/ug, and wherein the dose ofrVWF and increase in F‘s/ill stability, as compared to F‘Vlll stability in a subject administered a composition of pdVWF/FVlll, is selected from variations 4869 to 8003 in Table Ztl to Table 27. in one embodiment, the composition of i"\i"t‘ti’F’/"r¥"\"lll administered to the subject is a composition of high molecular weight rVWF niultimers. in one embodiment, FVlll stability is characterized by the life of FVlll. in another embodiment, FVlll stability is characterized by mean residence time (MET) of FVlll. in a timber ment, the method is for treating any type of VWT). in a specitic embodiment, the method is for treating Type 3 VWD. {(33275} in one embodiment, the method comprises administering a composition of rVWF/rFVlll, n the r‘v’ WT in the composition has a specific ty of at least 80 mU/ug, and n the dose ol‘rVWF and increase in FVlil stability, as compared to FVlll stability in a subject administered a composition of F‘v’lll, is selected from variations 4869 to 8003 in Table 26} to Table 27. in one embodiment, the composition ofr‘v’h‘v’F/rltl‘v’lll DJ (J; administered to the subject is a composition ot‘high molecular weight rVWF multimers. in one embodiment, FViEE stability is characterized by the half life of FVEEE. in another embodiment, F‘s/"iii stabiiity is characterized by mean residence time (MR?) of F‘s/"iii. in a further enibodin'ient, the method is for treating any type ot‘VWD. in a specific embodiment, the method is for treating ",E‘ype 3 VWD.

E38276} in one embodiment, the method comprises administering a composition of rVWF/rFVHi, wherein the i‘V’WF in the composition has a speciﬁc activity selected from variations 1 to 133 found in Table 1, and wherein the dose ot‘rVWF and increase in F‘v’lii stability, as ed to F‘Vill stability in a subject administered a composition of pdVWF/FVHL is selected from variations 4869 to 8003 in Tabie 25} to Tabie 27‘ in one it) embodiment, the ition ofi‘VWF/rilVill administered to the snbj ect is a composition of high moiecuiar weight r‘v7W'F mnltimers. in one embodiment, i stability is characterized by the l'ialt‘lii‘e of F‘Viii. in another embodiment, FVlii ity is characterized by mean esidence time (MR7?) of F‘s/iii. in a further embodiment, the method is for treating any type ot‘VWD. in a speciﬁc embodiment, the method is for treating Type 3 VWD.

WWW} in one embodiment, the method comprises administering a ition of r‘v’Vv’F/rFViH, wherein the dose ofrVWF and speciﬁc activity ofrVWF in the composition is selected from variations 8904 to M3625 in ’E‘ahie 28 to 'i'ahie 34%. in one ment, the ition of rVWF/rFVlll administered to the subject is a composition ot‘high molecular weight rV Wt2 muitimers. in one embodiment, the composition ofrVWF/rFWH administered to the subject has a higher specific activity than a composition of pd‘v’WF/FVHI. in yet another ment, the composition /rFViii administered to the subject is a composition of high molecniar weight rVWF muitimers with a higher VWF speciﬁc activity than a composition of pd‘v’Vv’F/FVHI, in one en'ibodiment, FViii stability is characterized by the bait life of FVlll. in another ment, F‘s/"iii stability is terized by mean residence time (MET) of FVEH. in a further en'ibodiment, the method is for treating any type of VWt). in a specific ment, the method is for treating 'fi‘ype 3 V‘t’v’i}. } in one embodiment, the method ses administering a composition of r‘v’Vt’F/rFViii, wherein the composition of ETVEii administered to the subject is a composition of high moiecnlar weight rVWF multimers in which at ieast 30% of rVWF molecules in the composition are present in a muttimer of at least it) subunits, and wherein the dose of r‘v’ WF and specific activity of rV‘v‘v’F in the composition is seiected from variations 8004 to lt3625 in Tahiti 28 to Tabie 34. in one embodiment, the composition of i‘VWF/rFViii administered to the subject has a higher VWF specific activity than a composition ot‘pdVWF/FVHL in one embodiment, F‘v’iii stability is characterized by the DJ (J; haltiife of FVHI. in another embodiment, FViii stability is terized by mean residence 2012/041957 time (MR3? ot‘FVlii. in a further embodiment, the method is for treating any type of‘v W i).1111 in a specific embodiment, the method is for treating ",l‘ype 3 VWD. {(33279} in one embodiment, the method comprises administering a ition of rVWF/rFVlli, wherein the composition of rV‘tI‘i/TF/rFVlll administered to the subject is a composition of high molecular weight rVWF i'nuitimers in which at least 50% ofrVWF moiecules in the composition are present in a multimer of at least it) subunits, and wherein the dose ot‘i‘VWF and, specific actiyity ot‘rVWF in the composition is seiected from variations 8394 to 10625 in Table 28 to Table 34. in one embodiment, the composition of rV‘t‘yT,/’1"FVHI administered to the subject has a higher VWT- specific activity than a it) composition of pdV‘v‘y’F/FVHI. in one embodiment, F‘v’lii ity is characterized by the half life of F‘Vlll. in r embodiment, F‘s/Eli stability is characterized by mean residence time (MRT) of F‘v’iii. in a tiirther embodiment, the method is for treating any type ot‘VWD. in a specific embodiment, the method is for ng Type 3 VWD.

Eilti’ZSti} in one embodiment, the method comprises administering a composition of i‘V‘WF/rFViii, wherein the composition of rVVv’Fx’i‘F-‘y’l ll administered to the t is a composition of high molecular weight YVWF muitimers in which at least 70% of rVWF molecules in the composition are present in a muitimer of at least it) subunits, and wherein the dose of rVWT- and specific activity ofrVWF in the composition is selected from variations 8304 to M3625 in 'i'ahle 28 to Tania 34. in one embodiment, the composition of rV‘t’v'F/rFViH administered to the subject has a higher VWF speciﬁc ty than a composition ot‘pdVWF/FVHL in one embodiment, F‘v’iii stability is characterized by the half life of H. in another embodiment, F‘y’iii stability is characterized by mean residence time (MELT) ot‘FVlii, in a r embodiment, the method is for treating any type ofVWD, in a specific ment, the method is for treating ",l‘ype 3 VWD. {(33281} in one embodiment, the method comprises administering a composition of rV ‘Vlli, wherein the composition ot‘i'\/’XR7'i9‘//'ri7\/'lll administered to the subject is a ition of high molecular weight rVWF innitimers having a minimal percentage of ’F moiecules present in a particuiar hi ghersorder rVWF mnitimer or larger muitimer according to any one of variations 134 to 4557 found in Tabie 3 to Tabie 5., and n the dose F and speciﬁc activity oi‘rVWF in the composition is selected from variations 8004 to 10625 in Tahie 28 to Tabie '34. in one embodiment, the composition of rVVv’F/rFViii administered to the subject has a, higher VWF specific actiyity than a, composition of pdVWF/FVlll. in one ment, F’Vlii stability is characterized by the l'ialt‘life of FVlll. in r embodiment, FViii stability is characterized by mean residence time (l‘v’lR’l‘) ot‘ll‘ﬂll. in a further embodiment, the method is for treating any type otW/"ND. in a ic embodiment, the method is for ng Type 3 VWD. {(33282} in one embodiment, the method comprises administering a composition of rVWF/rFVlll, wherein F‘s/ill in the subject is stabilized for at least 18 hours post— administratiom and wherein the dose oi‘rVWF and specific ty ot‘rVW F in the composition is selected from variations 8904 to 10625 in "Fable 28 to "Fable 34. in one embodiment, the composition ofrVW l administered to the subject has a higher VWF speciﬁc activity than a composition WF/F‘v’lll. in one ment, F‘Vlll stability is characterized by the half life ol‘FVlll, in another menti F‘v’lll stability is it) characterized by mean residence time (ls/l RT} ot‘ll‘s/lll. in a further embodiment, the method is for ng any type ofVWD. in a specific embodiment, the method is for treating Type 3 VWD. {(30283} in one embodiment, the method comprises administering a composition of rVWF/rFVllL wherein FVlll in the subject is stabilized for at least 2-4 ho tirs post“ administrationD and wherein the dose ofrywr and specific activity of rVWF in the composition is selected from variations 8004 to 106215 in Table 28 to Table 34‘ ln one embodiment, the composition of rVWF/riiVlll stered to the subject has a higher VWF speciﬁc ty than a composition of F/FVlll. in one embodiment, FVlll stability is characterized by the half life of FVll 5. in another embodiment, F‘Vlll stability is characterized by mean residence time (MRT) of FVlll. in a thither embodiment? the method is for treating any type of VWD. in a speciﬁc ment, the method is for treating Type 3 VWD.

E33284} in one embodiment the method comprises administering a composition of rVWF/rFVlll, wherein F‘Vlll in the subject is stabilized for at least 30 hours post— administration, and wherein the dose ofrVWF and specitic ty of r‘VWF in the composition is selected from variations 8904 to lil625 in Table 28 to ’l‘ahle 34. in one embodiment, the composition ol‘r‘v’WF/rFVlll administered to the subject has a higher VWF c activity than a composition 0fpdV‘yVE/EIVEll. in one embodiment, F‘v’lll stability is characterized by the lialt‘lil‘e of FVlll. in another embodiment, FVlll ity is characterized by mean residence time (MRT) of F‘y’lll. in a thither ernbodirnenn the metl'iod is for treating any type of VWD. in a specific embodiment, the method is for treating Type 3 V‘WD.

EﬁhZSSl in one embodiment, the method comprises administering a composition of rVWF/rFVlll, wherein the ratio ol‘rFVlll procoagnl ant activity (lU rFVlll :C) to rVWF DJ (J; Ristocetin cot-actor activity {it} rywrzatm in the composition is from 4: l—3:2, and wherein the composition of rV‘v‘v’F/rli‘Vlll administered to the subject is a composition of high molecular weight rVWF multimers having a minimal percentage of rVWF molecules present in a particular higher—order r‘v’WF m ultimer or larger mnl timer ing to any one of variations 134 to 457 found in Table 3 to Table 5. in a specific embodiment, the ratio of rF‘v’lll procoagtdant activity (1U rFVlll:C) to rVWF Ristocetin cot‘actor activity (1U i‘VWF2RCo) in the composition is from 31—32, in one embodiment, the composition of rV‘v’v’F/rFVill administered to the subject has a higher VHtVF c ty than a composition ot‘pd‘v’l’VF/F‘v’lll. in one ment, F‘Vlll stability is characterized by the half life of FViil. in r embodiment, F‘v’lll ity is characterized by mean residence it) time (l‘le’l‘) ot‘li‘ﬂll. lit a further embodiment, the method is for treating any type of‘v'Vi/l). in a specific embodiment, the method is for ng "l‘ype 3 VWD. 6} in one ment, the method comprises administering a ition of rVWF/rFVlll, wherein the ratio of rFVlH procoagnlant activity (lU rF‘Vlll :C) to rVWF Ristocetin cotactor activity (it) r‘v’WhrRCo) in the composition is from 2: l —l :2, and wherein the composition of r‘v’V‘y’F/rE-‘Vlll administered to the subject is a composition of high molecular weight rVWF mnltimers having a minimal percentage F molecules present in a particular higher-order rV WEI multimer or larger mnltimer according to any one of variations 134 to 457 found in Table 3 to Table 5. ln a speciﬁc embodiment, the ratio of rli‘V l ll procoagniant activity (1U rFVlth) to it"s/"Jill? Ristocetin cot‘actor activity (ill rVWFRCo) in the composition is from 3:2—2z3, in one embodiment, the composition of rVWF/rFVlll administered to the subject has a higher VWF specitic activity than a ition of pdVWF/FVlli. in one ment, FVlll stability is characterized by the hall’lite ot‘FVlll. in another embodiment, F‘v’lll stability is terized by mean residence time (MRI) of F‘v’lii. in a further embodiment, the method is for treating any type ofvwo. in a specilic embodiment, the method is for treating Type 3 VWD. {“3287} in one embodiment, the method comprises administering a composition of rV‘v‘yT/rFVlll, wherein the ratio ot‘rFVlll procoagnlant activity (lU zC) to rVWF Ristocetin cofactor activity (it) WW FiliCo) in the composition is from 223-126, and wherein the composition of rVWF/rFVlll administered to the subject is a composition of high lar weight rVWF multimers having a minimal percentage otitf‘thv’lj molecules present in a particular higher—order rVWF multimer or larger mnltimer according to any one of variations 134 to 457’ found in Table 3 to Table 5, in a specilic embodiment, the ratio of my lll pi‘ocoagnlant ty (1U rFVlii:C) to rVW’F Ristocetin cofactor activity (1U rVWFRCo) in the composition is from 2:3—l :5, in one embodiment, the ition of DJ (J; rV WF/rli‘Vlll administered to the subject has a higher speciﬁc activity than a composition ot‘ 2012/041957 pdVWF/FVill, in one embodiment, F‘s/ill stability is characterized by the halt‘life of FVlll. in another embodiment, FVlll stability is characterized by mean residence time (MR7?) of FVlll. in a further embodiment, the method is for treating any type oft/7WD. in a. specific embodiment, the method is for treating Type 3 VWD. } in one embodiment, the method ses administering a composition of rVWF/rFVlll, wherein the ratio of rli'Vlll procoagnlant activity {ll} rFV’lllzC) to rVWF etin cofactor activity (lU rVlezRCo) in the composition is selected from variations l988 to 2149 found in Table ll, and wherein the composition ofrVWli/rli‘Vlll administered to the subject is a composition of high molecular weight rVWF multimers having a, minimal it) percentage of rVWli“ molecules present in a particular -order rVW lI multimer or larger multimer according to any one of variations 134 to 457 found in Table 3 to Table 5. in one embodiment, the composition ol‘rVWF/rli‘y’lll administered to the subject has a higher VWP specific activity than a composition of pd\l7\?t7'F/"F'\i’lll. in one embodiment, FVlll stability is characterized by the half life ot‘FVlil, in another embodiment, F‘v’lll ity is characterized by mean residence time (MRI) of F’Vlll. in a further embodiment, the method is for treating any type ot‘VWD. in a specific embodiment, the method is for treating Type 3 VWD. {90289} in one embodiment, the method comprises administering a composition of r‘v’Vt’lEi/rFVill, wherein the ratio ofrli‘Vl ll procoagulant activity (ll) rli‘s/lllif) to rVWli‘ Ristocetin cofactor activity (lU rVWRRCo) in the composition is selected from variations l988 to 2140 found in Table ll. in one embodiment, the composition ot‘W’WF/rltiVlll administered to the subject is a composition of high molecular weight rVWF mnltimers. in one ment, FVill ity is terized by the hall‘lit‘e of FVlll. in r embodiment, F‘s/”ill stability is characterized by mean residence time (MR’l‘) of F‘s/ill. ln a further embodiment, the method is for treating any type D. in a specili c embodiment, the method is for treating Type 3 VWl). {902%} in one embodiment, the method comprises administering a composition of t"lit/r}3Vill, wherein the ratio ot‘rli‘Vlll procoagnlant activity (l U rli‘y’lllf) to rVWli‘ Ristocetin cofactor activity (lU rVWFRCo) in the composition is from 4-: L322, and wherein the ation ol‘rVWF specific activity in the composition and increase in F‘v’lll ity, as compared to li'Vlll stability in a subj ect administered a ition of pdVWR’FVlll, is selected item variations 2339 to 4868 in Table B to Table ii). in a speciﬁc embodiment, the ratio of rFV l ll procoagnlant activity (lU rFVlll2C) to rVWVF Ristocetin cofactor activity (lU rVWF:RCo) in the composition is from 3:l~3:2, in one embodiment, the composition of DJ (J; rV Wli/rli‘Vlll administered to the t is a composition of high molecular weight rVWli‘ llll WO 71031 multimers. in one ment, F‘v’lii stability is terized by the half life ot‘FVlll. in another embodiment, F‘v’lii stability is characterized by mean residence time (MRT) of FVTH. in a r embodin'ient, the method is for treating any type oft/7WD. in a speciﬁc embodiment, the method is for treating Type 3 VWD.

E33291} in one embodiment, the method comprises administering a composition of i‘V‘WF/rFVHl, wherein the ratio of rFVill procoaguiant activity (it; rFV’lllzC) to rVWF Ristocetin cofactor activity (TU rVWF:RCo) in the composition is from 2: lwl :2, and wherein the combination ofrVWF specific activity in the composition and increase in li‘Vlll stability, as compared to FVTH stability in a subject stered a composition of pdVWF/FVHT, is it) selected from variations 2339 to 4868 in Tabte E3 to Table l9. in a speciﬁc embodiment, the ratio of i‘FVl ll procoagulant activity (lU rFVHhC) to rV’WF Ristocetin or activity (TU rVWFRCo) in the composition is from 3:2~2:3, in one embodiment, the composition of rVWF/rFVl’l’l administered to the subject is a composition of high molecular weight rV’WF multimers. in one ment, FVlTl stability is characterized by the half life ot‘FVTTl, in another embodiment, F‘v’lii stability is characterized by mean residence time (MRT) of FVTTl, in a r embodiment, the method is for treating any type ot‘VWD in a speciﬁc embodiment, the method is for treating Type 3 VWD. {90292} in one embodiment, the method comprises administering a composition of r‘v"‘t’t"F/rFVill, wherein the ratio ot‘rli‘Vlll procoagalant activity (l U rli‘tl’lllf) to r‘v’WF Ristocetin cofactor activity (TU rVWF:RCo) in the ition is from 223% :6, and wherein the combination ol‘rVWF ic activity in the composition and ease in F‘v’lil stability, as compared to F‘s/ill stability in a subject administered a composition of pdVWF/FVHT, is selected item variations 2339 to 4868 in Tabte B to Table ii). in a speciﬁc embodiment, the ratio of i‘FVl ll procoagulant activity (lU rFVHhC) to rV’WF Ristocetin cofactor ty (TU rVWFRCo) in the ition is from 2:3~l:5, in one embodiment, the composition of rV ‘Vlll administered to the t is a composition ot‘bigb molecular weight E‘V‘WYF ers. in one embodiment, FVlll stability is characterized by the half life ot‘FVlll, in another embodiment, F‘v’lli stability is characterized by mean residence time (MR’l‘) of FVTTl, in a further embodiment, the method is for treating any type ot‘VWD in a speciﬁc embodiment, the method is for treating Type 3 VWD. {(30293} in one embodiment, the method comprises administering a composition of rFVTTl, wherein the ratio ot‘rFVTll procoagnlant activity ( l U rFVllizC) to rVWF Ristocetin cofactor activity {it} rVWFRCo) in the composition is selected from variations l988 to 2140 found in Table 11, and wherein the combination ot‘r‘VWF speciﬁc activity in DJ (J; the composition and increase in F‘Vlll stability, as compared to F‘v’lii ity in a subject 1012 administered a composition ofpdV WEI/F‘y’iil, is selected from variations 2339 to 4868 in Tabie 13 to Tabie 19. in one embodiment, the composition of ‘vi’F’/"r¥‘\"iil stered to the subject is a composition of high moiecui ar weight r‘VWF multimers, in one embodiment, FVTTT stabiiity is characterized by the half life of FVTTT. in another embodiment, F‘Vill stability is characterized by mean residence time (MRT) of F‘v’lii. in a tiirther embodiment, the method is for treating any type of VWD. in a specific embodiment, the method is for treating Type 3 VWD. } in one embodiment, the method comprises administering a composition of rV‘i‘vT/rFVTTl, wherein the ratio ot‘rFVTlT gniant ty (TU rFVliizC) to rVWF it) itistocetin cofactor activity (it) WWFdiCo) in the ition is from 4: i ~32, and wherein the dose of rVWF and increase in FVTll stabiiity, as compared to F‘s/iii stability in a subject administered a composition of pdV't’JF/FVTTT, is selected 'i variations 4869 to 8803 in Tabie 20 to Tabie 27. in a specific ment, the ratio of rFVili procoaguiant activity (EU rFVilT:C) to r‘v’WF Ristocetin cotactor activity (it) rVWFRCo) in the composition is from 3: '3 -3:2. in one embodiment, the composition of rF‘t/ill administered to the t has a higher ic actiyity than a composition ot‘pdVWF/FVTTT, in one embodiment, F‘v’lli stabiiity is characterized by the half iife ofFViii. in another embodiment, FVTTT stability is characterized by mean residence time (MET) of FVlll. in a further ment, the method is for treating any type of VWit). in a specitic embodiment, the method is for treating Type 3 VWD. {(332595} in one embodiment, the method comprises administering a composition of rVWF/rFViil, wherein the ratio of rFVilT procoagniant activity (TU rF‘Vlii :C) to rVWF Ristocetin coiactor activity (it) rVWFRCo) in the composition is from 2: l —l :2, and wherein the dose of rVWF and increase in FVTll stabiiity, as compared to F‘s/iii ity in a subject administered a composition of pdV't’JF/FVTTT, is selected ii'oii'i variations 4869 to 8803 in 'T‘ahie 28 to Tobie 27. in a speciﬁc embodiment, the ratio of rFViii procoagniant activity (TU rFV1112C) to r‘v’WF Ristocetin cotactor activity (TU rVWRRCo) in the composition is from 322-223. in one embodiment, the composition ‘v’ii‘I/rii‘Vill administered to the subject has a higher ic actiyity than a composition ot‘pdVWT/TVTTT, in one embodiment, FVlll stability is characterized by the hait’lii’e ofFVlll. in another embodiment, F‘s/ill stability is characterized by mean residence time (MRT) of FViil. in a tiirther embodiment, the method is for treating any type of VWT). in a specitic embodiment, the method is for treating Type 3 VWD. {(332596} in one embodiment, the method comprises stering a composition of DJ (J; rV WF/rii‘Viil, wherein the ratio of rli‘y’ili procoagniant activity (TU rFVi E E :C) to rVWF Etistocetin cofactor activity (ll) WW FiltCo) in the composition is from 2:3-l :6, and wherein the dose of i‘VWF and increase in FVlll stability, as compared to F‘s/ill ity in a subject administered a. composition of pdVW’F/FVHI, is selected from ions 4869 to 8803 in 'l‘ahte 20 to 'l‘ahte 27. in a specific embodiment, the ratio of rFVlll procoagulant activity (l'U rFVlll:C) to r‘v’WF Ristocetin cotactor activity (It) rVWFRCo) in the composition is from 234:5. in one embodiment, the composition of r‘V’WlQ-II’rFVlll administered to the subject has a higher specific activity than a, composition ol‘pdV‘leT/FVHL in one embodiment, FVllE stability is characterized by the half life ll. in another embodiment, FVIH stability is characterized by mean residence time (MET) of FVlll. in a it) further ment, the method is for ng any type of VWt). in a specitic embodiment, the method is for treating ’l'ype 3 V‘v‘v’ll). {(332597} in one embodiment, the method comprises administering a composition, of FVlll, wherein the ratio of rFVlll procoagniant activity (lU rF‘Vlll :C) to rVWF Ristocetin cotactor activity (It) rVWFRCo) in, the composition is selected from variations l988 to 2140 found in Table ll, and wherein the dose of rVWF and increase in FVlll stability as compared to F‘v’lll stability in a, subject administered a composition of pd‘v’Wli/li‘Vlll, is selected from variations 4869 to 8603 in Table lit? to Table 27. in one embodiment, the composition ofrVWF/rFVHl stered to the subject has a higher ic activity than a ition OEpdVVVF/FVHI. in one embodiment, F‘v’lll stability is characterized by the halflife of FVIH. in another embodiment, FVlll stability is characterized by mean nce time (MRT) of WV l l l. in a tirrther embodiment, the method is for treating any type of VWD. in a specific embodiment, the method is for treating Type 3 V‘WD, %298} in one embodiment, the method comprises administering a composition of rVWF/rltiVlll, wherein the ratio ot‘rFVlll gul ant activity (1U rFVlll :C) to rVWF Ristocetin cofactor activity {ltl r‘v'WRRtLZo) in the composition is from 4: l—3:2, and wherein the dose of rVWT- and specific activity ofrVWF in the ition is selected from variations 8304 to M3625 in 'i'abie 28 to Table 34. ln a speciﬁc ment, the ratio of rFVlll procoagulant activity (lU rFVl112C) to rVWF Ristocetin cofactor activity (1U rVWFRCo) in, the composition is from 3: l «3:2, in one embodiment, the composition of rVWF/rFVlll administered to the subject has a higher V W13 speciﬁc ty than a composition of pdV‘v‘v’F/F‘v’lll. in one embodiment, FVlil stability is characterized by the half life of F‘Vlll. in another embodiment, F‘s/ill stability is characterized by mean residence time (MRT) of F‘v’lll. ln a tirrther embodiment, the method is for treating any type ot‘VWD.

DJ (J; in a specific embodiment, the method is for treating ’l'ype 3 \I’Wli).

WO 71031 59829.9} in one embodiment, the method comprises administering a composition of rVWF/rFVHi, wherein the ratio of rFViii procoaguiant activity (1U rFV’HizC) to rV‘v‘v’F Ristocetin cot‘actor activity {it} rVWFRt’jo) in the ition is from 2:14 :2, and wi'ierein the dose of r\’ WF and specific activity of rV‘v‘v’F in the composition it ed from variations 8004 to 30625 in Tahie 28 to Tahte 34. in a specitic embodin’ient, the ratio of rF‘v’ iii procoaguiant activity (1U rFViH:C) to rvwr Ristocetin cofactor ty (1U rVWFRCo) in the composition is from 3:2—2z3, in one embodiment, the composition of rV WF/rii‘Viii administered to the subject has a higher VWF specific activity than a composition ofpdVWF/FVHI. in one embodiment, FViii stahiiity is terized by the it) half iife of FVEEE. in another embodiment, F‘v’iii ity is characterized by mean nce time (MRI) of FVIH. in a further emhodiment, the method is for treating any type ofVWD. in a specific embodiment, the method is for ng Type 3 VWD. {timtitti in one emhodiment, the method ses administering a composition of rVWF/rFViii, wherein the ratio ot‘rFViii printoagtiiant ty (3 U rFVtiizC) to rVWF Ristocetin cofactor activity (EU rVWF:RCo) in the composition is from 2:3-1 :6, and n the dose ofrVWF and specific activity Ofl’VYVVTF in the composition is seiected from variations 8394 to 10625 in ’E‘ahie 28 to Table 34. in a specific ment, the ratio of rFViii procoagtdant activity {1U rFViiLC) to rVW? Ristocetin cot‘actor activity (EU r‘v’Vy’FRCo) in the composition is from 2:34 :5. in one embodiment, the composition of rV‘t’v'F/rFVHt administered to the subject has a higher VWF specific activity than a composition ot‘pdVWF/FVHL in one embodiment, t stahiiity is characterized by the haif iife of F‘s/71H. in another emhodiment, FVHI stahiiity is characterized hy mean residence time (MELT) ii, in a further embodiment, the method is for treating any type ofVWD, in a specific ment, the method is for treating Type 3 VWD. {(33331} in one embodiment, the method comprises administering a composition, of rV WF/rF‘V’iii, wherein the ratio of rii‘v’iii procoagniant activity (1U rii‘Vi E E :C) to rVWF Ristocetin coiactor activity (1U rVWRRCo) in the composition is seiected from variations 1988 to 2143 found in ’E‘ahie it, and wherein the dose ofrV WtI and specific activity of rVWF in the composition is selected from variations 8084 to t0625 in Tahie 28 to Tahie 34, in one emhodiment, the composition ot‘rVW F/rF‘VtH administered to the subject has a higher VWF specific activity than a composition of pdVWFz’P-"Viii. in one emhodiment, F‘s/iii stahiiity is characterized by the hait‘iit‘e of F‘v’iit. in another embodiment, FVHI stahiiity is characterized hy mean residence time (MRT) of F‘s/’11}. in a further embodiment, the method is for treating any type of VWD. in a specific ment, the method is for treating Type 3 DJ (J; VWD .

Ellllfltlfll The practice of the present invention may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular y {including inant techniques), cell biology, biochemistry, and immunology, which are within the skill of the art. Such conventional ques include polymer array synthesis, hybridization, ligation, and detection ofhybridization using a label.

Specific illustrations of suitable techniques can be had by nce to the example herein below, However, other equivalent tional procedures can, of course, also be used. Such conventional techniques and descriptions can be found in standard laboratory s such as Genome r's: A Laboratory ila’anaai Series (Vols. l—lV), Using Antibodies: A l0 Laboratory Marina}, Ceiis: Al Laboraz‘oiﬁv , PCR Primer: .41 Laboratory miai, and ilioiecziiar Cloning: A Laboratory Manual ifall from Cold Spring Harbor Laboratory Press), Stryer, l... U995) Biochemistry (4th lid.) Freernan, Highly stabilized York, Gait, “Gli’g‘omicleoririe synthesis: A Practical Approach " l984, lRL Press, London, Nelson and Cox , Leimingw‘, Primripies of'diochemisiijii 3rd Ed, W, H. Freeman Pub, Highly stabilized York, NY. and Berg et al. (2002) Biochemistry, 5th Ed, W. H. Freeman Pub, Highly ized York, NY, all ot‘vvhich are herein incorporated in their entirety by reference for all purposes. {90333} 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, relerence to "a polymerase” refers to one agent or mixtures of such agents, and ‘eference to "the method" includes reference to equivalent steps and methods ltnovvn to those skilled in the art, and so forth.

E38394} Unless delined 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 s. All publications mentioned herein are incorporated herein by reference for the e of bing 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. @9395} Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit ol’ 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, t to any speciﬁcally excluded limit in the stated range. Where the stated lilo range includes one or both ot‘the limits, ranges excluding either both of those included limits are also ed in the invention. {(33336} ln the above description, numerous specific details are set lorth 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 with out one or more of these speciﬁc details. in other instances, well—lmown features and procedures well known to those skilled in the art have not been described in order to avoid ing the invention, {@3337} Although the present ion 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 methods.

EXAMPLES Example 1 {@8338} Study ot‘rVWlhrF‘t/lll co—administration. The immediate tolerability and safety after single doses RrF‘v’lll at 2 lU/hg 7.5 lU/hg 2t) lU/kg and 5t) lU/hg VWFRCO was asstssed as a primary endpoint of the study. Secondary endpoints included PK for znce, "\"Vv’RCB, VVt"F:Ag, F‘v’lll and multimeric composition of the VWF. An additional ary endpoint was a PK comparison with pthVv’Fx’fdeVlll {Cohort 4 {5t} lU/ltg oﬂ. See Flt}. l for a schematic illustration of the study design. lllllfillgl Recombinant human Von lilv’illebrand Factor (rVWF) was expressed in CllQ cells. l’ropeptide l was mediated in vitro through exposure of the pTO~VW lI to recombinant Furin, Fully ylated/ABO blood groups glycans were absent. The recombinant VWF has higher specific activity than plasma—derived VWF (pdVWF) and offers the therapeutic tlexihility of dosing with or t r The rVWF used in this study was not exposed to ADAM’E‘Sl 3, resulting in the presence ofultra~large VWF multimers and intact VWF subunits. ADAMTSB results in subunit cleavage at TYRE605—h/lE'l‘l606. liltiﬁlllll Co PK/VWF niultimer kinetics showed highly r PK. for Vil’llzRCo (VWF activity) between rVWF and Humate l?’ {Flt}. 2A). Humate l?’ is human derived medium purity Factor Vlll concentrates complexed to VWF. A surrogate marker was used for efficacy and dosing recommendations. The data in Flt}. 2A show that rVWF shows similar activity to that ot‘plasma d VWr Progressive loss of high molecular weight r‘i/ Wli‘ was seen upon exposure to A DA l‘vl’l'Ell3 (F iii. 23}, showing that rVWF is present in high molecular weight multimers prior to the ADAMTS l 3 exposure ll)? 598311} l-ligher FVlll levels were observed in rV WEI patients as eornpared to llurnate {Fit}. 3’)” These data show that rvwr stabilizes nous F‘Vlll in viva. There was a difference in effects seen with different ‘v’WFrFVlll ratios (l .3:l vs. ~2,l VWF/FVlll), These different ratios suggest that less rVWF can he used to stabilize FVlll than is needed when using plasnia derived VWF. The study design provided a flexibility ot‘remdosing with rVWF alone (no rFVlll) after the initial dose. {993 12} Ultra~large moleeular weight nniltiiners are present with rVWF — these ultranlarge molecular weight inultirners rapidly disappear following intiision. ADAl‘vl’l‘Sl 3 mediated ge fragments were seen in all subjects The pharniaeolrinetie proﬁle ot‘V‘thRC/o was similar to that ofpdVWF. There was sustained stabilization of endogenous FVlll with rVWli‘ which was comparable to that observed with pdVWF. The adverse drug reaction e with r‘v’WF v as similar to that ol‘pd‘v’lvlv’li/deVlll, and all related adverse events (fills) were mild.

E30313} The overall safety and pharniaeohinetie proﬁle suggests that r‘VWF can he used for the treatment and preventing of hleeding episodes at doses comparable to pdVWF/deVlll, while ing the levels of FVlll as compared to the effect. from the use F.

Without being limited by , it is le that the high proportion ot‘ultra—large ners (deeainers or higher) in the population ofrVWF leads to the enhaneed stabilization of FVlll seen with rVWF as compared to that by plasma derived VWF .

Example 2 Etlhftléll ln—hurnan stud ’ evaluatin . .hannaeoltineties stratin ' and tolerahilitv . safet rVWF in patients with type 3 "V'WD or severe 'l‘ype l V‘WD.

E38315} The pdVWF used in this study was synthesized in endothelial cells and niegaharyoeytes. Post—translational modiﬁcation of tide removal occurred intracellularly during passage of the protein to the Golgi and post—Golgi COl‘l’lpElIllllGl‘l’lSt (l lyeosylation/AB0 blood group glyeans were present. The pd‘v’ Wli‘ consisted of VW lI subunits that had been exposed to plasma ADAMTSBl There were no ultranlarge VWF niultiniers in the pd‘v’Wli‘ population and ts were cleaved at TYRJ(’OS-l‘v'llirl‘lmﬁ. pdVWF concentrates contained other proteins, including ADAMTSl 3 and heinagglutins. {(39316} The rVWF used in this study was expressed in (flit) eells, Propeptide removal was mediated in vitro through exposure of the pro—VWF to recombinant Furin. Fully glyeosylated/ABt’) blood group glyeans were absent. The rVWF was not d to ADAMTS l 3. The ts of the rvwr were intact and large \I’WF niultimers were ltlll present. Subunit cleavage did occur upon AlIFAl‘vl'i‘Sl 3 exposure. The r‘v’WF showed higher speciﬁc activity than F. {(33317} The inclusion criteria tor this study were: 6 'l‘ype 3 V’WD (VWF:Ag S 3 lU/dL) 6 Severe 'i‘ype l VWD (V'Wlthiio S it} ill/dill and FVlllﬂ < “(3 lU/dL a 18 to 60 years of age 9 Previous coagulation factor replacement therapy (2:25 ED) is Nonmbleeding state {@8318} The exclusion criteria for this study were: as Other coagulation disorders 6 History ’F and/or l7Vlll inhibitors 8% Cardiovascular disease a Medical history of thromhoeinholic event in Medical history of other immunological disorders {(39319} The immediate tolera‘oiiity and safety after single doses of rV’WFszVlll at '2 iii/kg, 7.5 ill/kg 2t) ill/kg and 5t} lU/lcg VWFRCo was assessed as a primary endpoint of this study. Secondary nts included PK for VWFERCo, EB, VWRAg, F‘Vill and inultinieric composition of the VWFi An additional secondary nt was a PK comparison with ptl‘t"‘t’y”l7//pdllVlll [Cohort 4 (5t) ill/kg VWt::RCoﬂ. Patient demographics for this study are shown in HG, d. Phannacokinetic analysis of Cohort 4A (Type 3 VWD) VWFRCo/VWF:Ag is shown in PEG. 5, These data show that the total activities ot‘r‘v’WF were comparable to those ofpdVWF. l’rogressive loss of high molecular weight rVWF was seen upon exposure to ADAMTSB (HG, 6). Further pharmacohinetic analysis ot‘Cohort 4A, (Type 3 VW’D) lli“, is shown in FlG. 7. The data in Fit}. 7 ts that rVWF was more effective at stabilizing the in vivo activity of F‘v’iii than pdVWF, resulting in increased F‘Vlii procoagulant ty after treatment with rVWF/rVWF as compared to treatment with pdVWF/‘deVliL {“3329} Ultra—large molecular weight niultirners were t in r‘v’W l: and rapidly disappeared following infusion ADAMTSlS mediated cleavage fragments were seen in all subjects. The pharmacoltinetic proﬁle of VWRRCO was similar to that ofpdVWF. There was ned stahilization of endogenous FV’lll that was contparahle to that ed with pdVWF. The adverse drug reaction proﬁle was similar to that of pdVVt’F/‘pilF‘v’ill i, and all related adverse events were mild. €983le The overall safety and pharmacokinetic proﬁle suggests that rVWF can he used for the treatment and preventing of bleeding episodes at doses comparable to pdVWF/de‘Vlll.

Example 3 {(33322} Clinical stud of rVWF:rFVllltcom :ilex. This study was a pharniacokinetus immediate safety and tolerability studyin congenital yon Willebrand disease Type3. FFG 9 shows the rVWF PK parameters for three patiei'its in the study.

EllllBZBllEG ltl shows Pl»; data tor latient l. There was a good correlation between VWFRCo and VWF:CBA. Activities were measurable up to l2 hours (both were below the limit ot‘quantiticatlon at 24 hours). V‘WFiAg was still measurable at 96 hours. Endogenous FVlll activity increased to a maximum oft). l3 lU/ntL.

E33324} FEG. l l shows Pls: data in t 2. As with Patient l, there was a good correlation hetween io and VWF:CBA Both activities t rere measurable (alheit at the limit of quai'ititication) up to l2 hoursi VWFiAg was still measurable at '72 hours (the 96 hour sample was not tested). Endogenous FVlll activity increased to a maximum of (HS lU/mL for this patient.

EtlllBZSllHG l2 shows PK datain Patient 3 VWF: Elli:o activity was measurable up to 24 hours (albeit at the limit of ﬁcation} while VWF:CBA was below LoQ at 24 hours, VWFEAg was still measurable up to 48 hours. liinrlogenous F‘s/ll} activity increased to a maximum of (3. l3 lU/ntL for this patient.

E303loll75G l3 shows data comparing the l parameters andlWlll activity tor the three patients. There was a good ation see in all three patients for all parameters tested (VWFrRCo, VWFzCB/3t, VWFzAg, and FVlerhr activity). {$0327} F had lth‘“/(.i recovery in all patients. The three patients showed slightly ditt‘irent r‘v’WF PF; For VWF antigen there was a maximum ll (36 Rbml 30 minutes attei application of 2 ill VWFFCo/lrg ed by a steady decline. For F‘Vlll ty, the baseline FVllliactivity increased to imately til lU/inl l5. minutes after ation of rV WF-lrlVll lhereattei ty lurther increased to (3 lot U/ml reached a u at 3%) hours that stayed up to 218 hours followed by a steady decline. The delayed se in FVlll indicates that the secondary rise was induced by rVWF.

Example 4 Etlh328l r‘if WF stud ' F‘Vll PK Assessment and TA assessment. FTG. l4 shows data for FVlll actiyity across all cohorts. FlG. 15 shows data troin Cohort l (2 ill/kg V‘v‘v’FiRCo/l .5 lU/lrg F‘v’lll). There was good correlation for all three patients in this cohort for all parameters tested. llll 59832.53} tilt}. l5 shows data from Cohort 2 (75 ill/kg ‘V’Vt’l72RCti,«’5.8 ill/kg FVEH). Flt}. lo shows the pharmacoltinetics data for this cohort for FVlll2C. {(333%)} HG, , 7 shows data for Cohort 3 (2i) lU/ltg VWFRCo/lizl ill/kg FVlli). These data show a comparison of plasma parameters for VWF and FVlll. shows the pharmacokinetics data for this cohort for FVlllzC. %331} FlG. 19 shows data for Cohort 4 (5t) lU/kg VWRRCo). l dosing was 38.5 lU/kg rFVlll or 25 lU/lsg pdlj‘v’lllt These data show that the parameters for i‘VWF are similar to those for pdVW l3 (Haeniate), but that r‘v’WF is more effective at stabilizing FVill activity, resulting in an se seen in FVlll activity as compared to that seen with pdVWF.

MG. 29 shows a summary of pharmacoltinetics data for this cohort for FVHMZ As can be seen in these data, the median and mean Tm was increased for rVWF/P'Vlll as compared to de/WVF/FVEH, ting that rVWF is more effective at stabilizing in viva FVlli than is pet‘s/WP resulting in sed half—life.

Example 5 E33332} Clinical studi Old'VVVTF effect on rFVlll half—lite in the treatment ofhen'io' hilia A, This study evahiates the immediate tolerahility and safety ot‘rVWli‘ after single doses ot‘ﬁﬁ lU/kg rFVlll (Advate) alone or in combination with rVWF at ll) or 50 lU/lgg von Willehrand Factorﬁistocetin cot‘actor activity (VW’FERCo). This study also evahiates the pharmacokinetics after single doses of 50 lU/hg rFVlll (Advate) alone or in combination with r‘v’WF at ll) or St} ill/leg {VW132Rtifoi).

Etlh333l The tion for the study is ed using the following criteria: lﬂt3334i lattliisiannotate: 9 Severe Hemophilia A (19"! lll:C <l lU/dL) 6 iii to 60 years ol‘age s Previous coagulation lactor replacement therapy {ZlSG ED) % Non—bleeding state {@0335} Exclusion criteria: e Other coagulation ers a History ofVWF and/or FVlll inhibitors 6 Cardiovascular disease a Medical history ofthromhoemholic event a Medical history of other immunological disorders (exceptions) } Subject participation is for l6 weeks with an overall study duration of? months, WO 71031 E98337} The design of the study is a multicenter, uncontrolled, non randomized, open-label clinical study to assess safety, immediate tolerability and pharmacokinetics of rFVlllr‘t/WF in subjects with hereditary severe lunnophilia A (F‘Jlllf <Zl 9/0). {@9338} Each subject (N = l 4) participating in the triple—period PK analysis receives in a non randomized t‘ashion 50 ill/leg rFVlll (Ads/ate) with no, it} lU/ltg V‘WFrltCo, or 50 ill/kg VWFRCo rVWF, and is expected to participate for approximately l6 weeks.

EtlllSS9l The intra—indiyidual pharmacolrinetics are spaced at 5—l4 days apart to ensure a “wash—out” period of at least 5 days and a timely completion of the study. Safety analyses are performed tely once 6 suhj ects have teen dosed with it) lU/KG VWF2RCo rVWF and once 6 subjects have been dosed with St) Eli/KG Co i'V WEI.

Etlhftéltll Subjects should not have ed ciyoprecipitate, fresh frozen plasma or other drugs interfering with VWF or FVlll PK for at least 5 days before either of the infusions, {(39341} Dosage of rVWF is escalated when immediate tolerahility and safety are demonstrated after a minimum of 6 subjects treated with l it ill VW F:RCo rVWF. The e elects of the investigational product on yital signs, hematology, and clinical chemistry parameters will determine short—term safety {@8342} Samples for the determination els ot‘ll‘v’lll activity {FVlll:t3), VWF antigen (VWT-Ag), VWF activity (VWFIRCo), VWRCB, (and VWF er distribution) are taken pre—intusion (within 3t) minutes prior to the start of the intiision) and alter the end of the inlusion at l5 minutes (15 s), 30 s (i5 s), i hour (i5 minutes), 3 hours (ill) minutes), 6 hours tilt) minutes} 9 hours tilﬁ minutes}, 2.4 hours {i7 hours) and 48 hours (i2 hours), 72 hours (i2 hours) 96 hours (i2 hours), and LN) hours (i7 hours) tor at least every 24 hours therealter until FVlll drops lelow <l%). FVlll activity is detemiined using both the genic and one-stage al"l"l‘—hased assay methods performed at the local and central laboratory. {(33343} The effects ol’eaeh intusion on vital signs and other symptoms indicative of an adverse event (Ali), logy, and clinical chemistry parameters are used as indicators of term . Vital signs, clinieal chemistry, and hen'iatology parameters are assessed pre—infusion and until thl hours nfusion. The occurrence ofABS are continuously monitored for up to 2 hours postninfusion (rVWF treated subjects) and at various time points during the follow—up period. {99344} The presence of inhibitors to FVlll and VWF are assessed before investigational product infusion and at the study completion yisit‘ EtlllSL‘lS} it“ at any time a, serious AE related to the investigational t occurs, an independent data. monitoring eon'imittee (Uh/ll?) will advise whether to continue the study. ll '2 598346} Pharmacokinetic primary endpoints for the study include: Alltilg—ce/Dose (area under the plasma concentration/time curve from time t) to infinity); AUCagsa/Dose (area under the plasma eoneentration/time curve lrom time (i to 96 hours); mean residence time (h/lR’l“); nce (CL); 'l‘l/E (elimination phase half—life); Volume of distribution at steady state (Vss) ofVWFRCm VWF:Ag NWFKIB, and FVlll.

Ellﬁ34l’7l Analysis for pharmacokinetic primary nts include AUCMéh/Dose, AUCg- 0JDose, MR1 CL, Tu; and Vss summarized per treatment group (50 lU/lrg rFVlll (Advate) alone, 50 Ell/kg rli‘Vlll (Ad's/ate) premixed with rV W? at ll) lid/kg VWF2RCo, 5i} Ell/kg rFVlll (Advate) premixed with rVWF at 50 lU/lrg VWF2RCo) by median and twonsided 9 % Cls for the median, mean, standard deviation, coefﬁcient ot’yariation and geometric mean.

Descriptive statistics (medians and ranges) are used to summarize VWF2RCo, VWRAg, VWRCB and FVlll levels overtime. {@0348} Safety primary endpoint es: occurrence of treatment related AEs. is of the safety primary endpoint es the number and percentage of subjects who experience a treatment related AE. The number and rate of treatment related AEs are ted. d9l Pliarinacohinetic secondary endpoints include: in Vivo recovery thR) and incremental recovery tilt} ot‘FVlll, ‘v’Vv’EﬁRCo, VWF:Ag and VWF:CB. Analysis of pharmacoliinetic secondary endpoints includes ining lVR and lR of F‘v’lll, VWRRCQ V'WFLAg and VWF1C l3 by subject and summarizing by medians and ranges. liltlSSlll Safety secondary endpoints include: development of inhibitory and total binding WF dies, development of inhibitory antibodies to FVllL development of antibodies to Chinese hamster ovary (CHO) proteins, mouse immunoglobulin G (lgG) and rFurin, occurrence of thrombotic events.

Ellll'BSll Safety secondary endpoints are analyzed by tabulating the number ot‘ ts who experience an SAE and the number of SAEs a in addition, the number of subjects who experi en ee a treatment related AE. and the number and rate of treatment related A Es are subcategorized for thrombotic events, inhibitory and total binding WF dies, inhibitory dies to FVll l, antibodies to Chinese hamster ovary (CllO) nsg antibodies to mouse immunoglobulin G {lgG} and antibodies to rFurin.

EddSSZ} A listing of all AEs are presented by subject identiﬁer, age, sex, preferred term and reported term of the All, duration, tyr seriousness, action talren, outcomeﬁ causality assessment, onset date, stop date and medication or non-drug therapy to treat the AE. An overview table for Abs are provided, presenting the number of AES the number of subjects with AEs and the corresponding percent of subjects in total and by seriousness and relationship to treatment. An atlditionsi summary table ts the totai number of (mild, moderate, severe) ABS by system organ class and pre ”erred term with reiationship t0 treatment.

} No format sample size eaieuistion has been conducted for this study. {@9354} it is understood that the exanipies and embodiments described herein are for illustrative es oniy and that various rrtoditieations or changes in light thereof wiii he suggested to persens skiiied in the art and are to be included within the spirit and purview of this application and, seope of the appended . Ail publications, patents, and. patent appiieatiens cited herein are hereby incorporated by reference in their entirety for ah purposes.

Claims

WHAT IS CLAIMED IS:

1. Use of recombinant Von Willebrand Factor (rVWF) in the manufacture of a medicament for treating Von Willebrand Disease or Hemophilia A in a t in need thereof, wherein the rVWF is a high molecular weight VWF multimer composition in which at least 20% of the total VWF multimer molecules in the composition are VWF decamers or higher order multimers, and wherein the rVWT has a higher specific activity than plasma derived Von Willebrand .

2. The use of claim 1, wherein the ment comprises recombinant Von Willebrand Factor (rVWF) and recombinant Factor VIII (rFVIII).

3. The use according to claim 1 or claim 2, wherein the medicament is formulated to administer between 1.0 IU/kg VWF:RCo and 150 IU/kg VWF:RCo per dose.

4. The use according to claim 1 or claim 2, n the ment is formulated to administer between 2 IU/kg VWF:RCo and 50 IU/kg VWF:RCo per dose.

5. The use according to claim 1 or claim 2, wherein the medicament is formulated to administer between 5 IU/kg VWF:RCo and 40 IU/kg VWF:RCo per dose.

6. The use according to claim 1 or claim 2, wherein the medicament is formulated to administer between 10 IU/kg VWF:RCo and 20 IU/kg VWF:RCo per dose.

7. The use according to any one of claims 1 to 6, wherein the rVWF is matured in vitro by treatment with Furin.

8. The use ing to any one of claims 1-7, wherein the rVWF is produced through expression in a e Hamster Ovary (CHO cell culture). 9. The use according to any one of claims 1-8, wherein the rFVIII and rVWF are produced through expression in the same cell culture. 10. The use according to any one of claims 1-9, wherein the medicament is to be administered no more than once every other day. 11. The use of claim 10, wherein the medicament is to be administered no more than twice a week. 12. The use according to any one of claims 1 to 11, wherein the high lar weight VWF multimer ition maintains the at least 20% of the total VWF multimer molecules as VWF decamers or higher order multimers for at least 3 hours post-administration. 13. The method according to any one of claims 1 to 12, wherein the Factor VIII half-life is extended by at least 5 hours. 14. The method of claim 13, wherein the Factor VIII half-life is extended by at least 12 hours. 15. The method of claim 13, wherein the Factor VIII half-life is extended by at least 24 hours. 16. The method of claim 13, wherein the Factor VIII half-life is extended by at least 36 hours. 17. The method of claim 13, wherein the Factor VIII half-life is extended by at least 48 or 72 hours. 18. The use of any one of claims 2-17, wherein the ratio of FVIII procoagulant activity (IU F VIII:C) to rVWF Ristocetin cofactor activity (IU rVWF:RCo) administered to the subject is to be between 2:1 and 1:4. 19. The use of claim 18, wherein the ratio of FVIII gulant activity (IU F VIII:C) to rVWF Ristocetin cofactor activity (IU rVWF:RCo) administered to the subject is to be between 3:2 and 1:3. 20. The use of claim 18, wherein the ratio of FVIII procoagulant activity (IU F VIII:C) to rVWF Ristocetin cofactor activity (IU rVWF:RCo) administered to the subject is to be between 1:1 and 1:2. 21. The use of claim 18, n the ratio of FVIII procoagulant activity (IU F VIII:C) to rVWF Ristocetin cofactor activity (IU rVWF:RCo) stered to the subject is to be about 3:4. 22. The use of any one of claims 1-21, wherein the rVWF has a specific ty of about 20- 150 mU/g. 23. The use of any one of claims 1-22, in which at least 30% of the total VWF multimer molecules in the high molecular weight VWF multimer ition are VWF decamers or higher order multimers. 24. The use of any one of claims 1-22, in which at least 40% of the total VWF multimer molecules in the high molecular weight VWF multimer composition are VWF decamers or higher order ers. 25. The use of any one of claims 1-22, in which at least 50% of the total VWF er molecules in the high molecular weight VWF multimer composition are VWF rs or higher order multimers. 26. The use of any one of claims 1-22, in which at least 60% of the total VWF multimer molecules in the high molecular weight VWF multimer composition are VWF rs or higher order multimers. 27. The use of any one of claims 1-22, in which at least 70% of the total VWF multimer molecules in the high molecular weight VWF multimer composition are VWF decamers or higher order multimers. 28. The use of any one of claims 1 -27, wherein the medicament is for treating Von Wiliebrand Disease Type 3. 1 /23 mHmW ....... 03> m w 55% 33. £25. ....... 95.5w {1.31%5E»;«6 PEOIOU rmmﬁ-»w m ..... _‘ kﬁmﬁw 025 52?: =5uEs> .OE n Eat a mu»... PMOIOU Amucv Eat? ........ h"“Mm Law'uvv—Ckw® Emom matey—.22 w coaquoucmm 5&3 kmOxGU commas Ema u «

9. 9:2. mama SUBSTITUTE SHEET (RULE 26)