LV10193B - Process for an industrial - scale preparation of a standardized human von willebrand factor concentrate of very high purity and suitable for therapeutic use - Google Patents

Abstract

Izgudrojums attiecas uz cilvēka plazmas Villebranda faktora iegūšanas paņēmienu no krioprecipitētasplazmasfrakcijas. Paņēmiens ietvertrīshromatogrāfiskāssadalīšanas stadiju kombināciju. Iegūtajam koncentrātam ir ļoti augsta īpatnējā aktivitāte un paaugstināts multimēru ar lielu molekuiārmasu saturs.The invention relates to a method for obtaining human plasma Villebrand factor from cryoprecipitated plasma fractions. The technique involves a combination of the stages of chromatographic separation. Acquired the concentrate has a very high specific activity and a high content of high molecular weight multimers.

Description

LV 10193

Field of the Tnvention

The invention relates to a process for preparing an industrial-scale, standardized human von VVillebrand factor concentrate of very high purity, very high specific activity, and high content in high molecular \veight multimers, intended in particular for therapeutic use.

Backeround of Related Art von Willebrand factor (vWF) is the largest known molecule circulating in plasma. It exists as a series of large disulfide-linked multimers, the basie subunit of which has a molecular weight of about 260 kilodaltons (KDa). The smallest form of vWF in plasma is a dimer of about 440-500 KDa and the largest forms are multimers of the dimer with molecular vveights reaching up to 20 million daltons. The assembly of subunits which are linked together may be celi specific, the vWF being synthesized and polymerized in the megacaryocytes and endothelial celis.

This factor plays an essential role in hemostasis through two distinet funetions : it transports and stabilizēs factor VIII in the blood stream and, as an adhesive protein, it permits the spreading, the attachment and the aggregation of the blood platelets on the vascular subendothelium thus contributing to the swift healing of injured vessels. A congenital vWF deficiency, or a structural anomaly of this factor, gives rise to von Willebrand disease which initially takes the form of hemorrhages, particularly cutaneous and of the mucous membranes. The clinical forms taken by this disease are very heterogenous and pose major problems in the event of surgery. Treatment of von Willebrand disease is essential in order to correct primary hemostasis (bleeding time) and coagulation (activated cephalin time and F VIII activity) anomalies. - 2 -

The disease is treated by substitute therapy with vWF-enriched human plasma derīvatives (for example, the cryoprecipitated fraction of plasma or the concentrates of Factor Vilī containing a sufficient quantity of vWF).

However, these products are not standardized for the treatment of von Willebrand disease. In addition, the poorly purified fractions of blood plasma especially cryoprecipitate are not free from the risk of virai contamination because they are often not subjected to any efficient virai inactivation step. Furthermore, they lead to an excess of contaminating proteīns which the patient does not need and which can cause immune reactions after multiple injections.

Purified Factor VIII, on the contrary, can be subjected to efficient virus inactivation treatment, but its purification process has been optimized for treating hemophilia A patients and not for vWF deficient-patients. In fact, the recently developed and increasingly effective processes, such as immunoaffinity or ion exchange purification used for preparing Factor VIII, producē concentrates that no longer contain enough vWF to be efficient in the treatment of von Willebrand disease.

It is to meet this need of an efficient way of treating von WiIIebrand disease that the Applicant has developed a new industrial process for purifying vWF while stili obtaining optimum benefit from the isolation of different plasma molecules. In particular, it permits, in one step, to prepare a concentrate of Factor VIII (according to a process described in EP Application 0 359 593) and to recover a separate vWF fraction from the same batch of cryoprecipitate, thus allovving to make an optimal use of human plasma. The vWF fraction thus obtained is purified by two additional - 3 - LV 10193 chromatographic steps which providc a vWF concentrate of very high purity.

The complexity of the vWF molecule makes it very difficult to purify. Small-scale methods, i.e., 5 to 2000 ml for the purposes of analytical study, have already been described (Thorell et al., Thromb. Res. 1984, 35: 431-450), but it has not been possible to adapt these methods for v\VF preparation on an industrial scale. In addition, the concept of making the best possible use of cryoprecipitate by producing vWF in addition to FVIII was not considered. vWF has been purified by differential solubilization on sulfated compounds in the presence of glycine (Berntorp et al., Vox Sang. 1989, 56: 212), sulfated compounds (Winkelman et al., Vox Sang. 1989, 57: 97) and by using different methods of chromatographic separation, such as molecular size exclusion (Perret et al., Haemostasis 1984, 14: 289) and ion exchange (Austen et al., Thromb Haemostas. 1982, 48: 295). Hovvever, these techniques give either low yields of vWF or have a low gel capacity, or do not make the simultaneous isolation of FVIII and vWF possible, which make them less convenient for an industrial application.

In addition, Berntorp et al. (Vox Sang. 1989. 56: 212) obtain a v\VF of low purity : 45 U Ag/mg protein (p. 213) vvhereas the Applicants obtain 205 U Ag/mg protein. Similarly, Winkelman et al. (Vox Sang. 1989, 57: 97) obtain 10 U Ag/ml protein (p. 101).

Perret et al. (Haemostasis 1984, 14: 289), perform a defibrination step (to eliminate fibrinogen as fibrin molecules) with the use of calcium as well - 4 - as enzymes frora snake venon. This renders the preparation obviously unsuitable for therapeutic purposes. Moreover, gel filtration systems as the one they used are hardly compatible with industrial scaling up since allowing a flow rāte of only 10 cm/h or less and shotving a high risk of plugging, especially in the presence of fibrinogen and fibronectin. Also the purification factor is known to be usualiy low due to the poor resolution of proteins in this chromatographic system.

Austen et al. (Throm. Haemostas. 1982, 48: 46) also obtain a low purity concentrate (8 U Ag/mg protein) and relatively low yield probably due to their drastic chromatographic conditions (pH 5.5).

Harrisson et al. (Thromb. Res. 1988, 50: 295) use dextran sulfate-sepharose as a chromatographic matrix ; this material has a lovv retention capacity for the vWF. As a result, they obtain a vWF preparation of low specific activity : 2-4 U/mg protein (p. 301).

Finally, most of these products contain a rather large proportion of denatured or inactive forms of vWF as evidenced by the ristocetin cofactor activity (RCo)/antigen ratio ranging from 0.08 to 0.8 (Lawrie et al., Br. J. Haematol. 1989, 73: 100). This makes them less efficient for therapeutic use in von Willebrand disease. On the contrary, the Applicant's procedure allows the recovery of vWF with a RCo/antigen ratio higher than unity which is comparable to that of native v\VF from normai pool plasma. - 5 - LV 10193

Summarv of the Invcntion

The present invcntion relates to an industrial process for preparing a· vWF concentrate for therapeutic use as a by-product of a high-purity FVIII production process, enabling standardized batches characterized by a high content in high molecular weight multimers, to be produced from very large volumes of plasma (4000 liters or more), and allowing to make an optimal use of cryoprecipitate.

More particularly, the present invention relates to a process for preparing a vWF concentrate that comprises the combination of three successive chromatographic steps aIIowing an enrichment in high molecular weight multimers vvhich are related to the vWF biological activity. The starting material is the cryoprecipitated fraction of human plasma subjected to a conventional prepurification step involving adsorption on aluminium hydroxide. This material then undergoes virai inactivation, for example using a solvent-detergent treatment, bcfore it is purified.

DETATLED DESCRTPTTON OF THE PREFERRED F.MBODTMENTS

The purification process according to the present invention comprises a combination of three successive chromatographic steps from a by-product fraction of a FVIII production process, the first two involving ion exchange chromatography, and the third, affinity chromatography.

The two ion exchange chromatography steps are carried out on the same vinyl polymer resin onto which are fixed diethylamino ethyl (DEAE) groups, more particularly on columns of DEAE-Fractogel® TSK 650 (Merck), equilibrated with a buffer solution containing 0.01 M trisodium citrate. - 6 - 0.11 M sodium chloride, 0.001 M calcium chloride, 0.12 M glycine and 0.016 M lysine, pH 7. DEAE-Fractogel TSK® 650 is a synthetic hydrophiIic gel medium. The support is a copolymer of oligoethyleneglycol, glycidinemethacrylate and pentaerythritol-dimethacrylate to which diethylaminoethyl groups, i.e., -0-CH2-CH2N+(C2H5)2HCI, are attached, resulting in a weakly alkaline anion exchanger. DEAE-Fractogel® TSK 650 is available in two particle size ranges (when moistened with water) : Type S (0.025 - 0.050 mm) and Type M (0.045 - 0.090 mm). Both types are useful in carrying out the present invention.

The cryoprecipitated plasma fraction, which has been prepurified and has undergone virai inactivation treatment according to conventional procedures, is applied to the first chromatographic column which rētains a large proportion of the vWF. vWF is then eluted by inereasing the sodium chloride concentration of the buffer solution to 0.14-0.15 M.

The fraction thus eluted, enriched in vVVF, is applied to the second chromatographic column under the same conditions as the first. Since many of the proteīns (especially FVIII and fibronectin) which competed for the adsorption sites have already been eliminated from this fraction during the first chromatographic step, the capacity for adsorbing the vWF on the second column is advantageously far greater. After the filtrate has been removed and the column has been rinsed with the equilibration buffer solution, the adsorbed vWF is eluted by inereasing the sodium chloride concentration of the buffer solution to 0.15-0.17 M. Due to the excellent capacity and efficiency of the DEAE Fractogel® resin for vWF, vWF can be - 7 - LV 10193 eluted from the column at a very high potency (> 150 U RCo/ml). Thus, the mechanical stress of ultrafiltation that would be needed to concentrate the product· can be avoided.

The fraction thus eluted is subjected to affinity chromatography on a gelatin-derived gel in the same equilibration buffer solution thus avoiding any dialysis or ultrafiltration step to modify the salt coraposition ; this column is essentiel to retain the molecules of residual fibronectin that stili contaminate the vWF. The choice of gelatin-derived gel is not critical, however : Gelatin-Sepharose, Gelatin-Ultrogel®, Gelatin-Spherodex® and Gelatin-Fractogel® are ali suitable for this purpose. Gelatin-Sepharose may be the best choice since it fixes 5 to 10 mg fibronectin/ml gel under the conditions used for the present process.

In the conditions used the highly purified vVVF does not bind on the gel and is thus eluted in the filtrate ; as the gelatin affinity step does not inducē extensive dilution of the vWF fraction, the product can be directly dispensed without any need for a concentration step by e.g. ultrafiltration. The absence of proteolytic enzymes in the final product makes it very stable during the sterile filtration and freeze-drying steps, without any need for stabilizing aģents.

The von Willebrand factor concentrate obtained using the process according to the present invention has an exceptionally high purification factor of > 10,000 fold in relation to the initial plasma, and its specific activity is 345 U CBA/mg protein (units of measurement for the collagen binding activity), and > 100 U RCo/mg protein (units of ristocetin cofactor - 8 - activity). The contribution of each chromatographic step to purifying v\VF is illustrated in Figurē 1.

Quite iraportantly, improvement in the quality of the product during the successive purification steps was monitored as a function of the proportion of high molecular weight rnultimers (the molecular forms of vWF having high biological activity) as detected by electrophoretic analysis.

Interestingly, this analysis reveals a Progressive enrichment in rnultimers > 4 (Figurē 2), which represent 79 % of the vWF polymers eventhough cryoprecipitation eliminates half of them. Unexpectedly, it is the chromatography on DEAE-Fractogel TSK 650 that favors this selective retention of the very large rnultimers and eliminates vvith the filtrate the forms of small size, abnormal structure (having undergone partial proteolysis) and low activity.

The standardized vWF concentrate of high purity, high specific activity and high content in high molecular v/eight rnultimers, obtained by the process according to the prcsent invention is thus particularly well suited to therapeutic use in the different forms of von Willebrand disease, as confirmed by preliminary clinical studies.

Preliminary clinical tests have shown that this concentrate led to an efficient shortening of the bleeding time during hemorrhages. LV 10193 - 9 -

In vitro tests have confirmed that its biochemical and physiological properties are identical to those of the native molecule, in particular its ability to fix blood platelets in a perfusion device, and its ability to bind in-vivo endogeneous Factor VIII.

Due to its high purity, the v\VF obtained during the process according to the present invention could also be considered for various laboratory applications (fine s.tructural analysis, functionality studies, diagnoses, etc.) and for the production of specific antibodies.

The concentrate according to the present invention can also be used as a stabilizer during the production of Factor VIII by celis transformed by genetic engineering as well as during the purification of the Factor VIII thus produced.

The follovving example illustrates one form of an embodiment of the present invention v/ithout, hovvever, limiiing the scope thereof. - 10 -

EXAMPLE

Starting Material

The cryoprecipitate is preparcd from frcsh plasma collected in the presence of sodium citrate (4 %) or CPD (citrate, phosphate, dextrose) anticoagulant solution and frozen at the most 6 hours after being obtained. The plasma is deep frozen to -60° C, then preserved at -35° C. The plasma batches contain 1800 to 2000 liters which are pooled into 4000-liters batches for each application of the process. For the purpose of thav/ing, the plasma is placed in a temperature-regulated chamber at for 12 hours to ensure slow, regular warming to - 7° C, then thawed in a thermostatically controlled enclosure at 0° to 2° C with constant stirring. The cryoprecipitate (which represents about 9 g/I plasma) is recovered by cold centrifugation.

After centrifuging, the cryoprecipitate recovered is resolubilized and adsorbed on aluminum hydroxide to remove some contaminants, i.e. the components of the prothrombin complex (particul arly Factor VII) and Factor XII. The supernatant is then cooled to 15° C (which partially removes the fibrinogen and the fibronectin).

This treatment permits the recovery of 80 to 86 % of the Factor VIII/yWF mixture from the cryoprecipitate ; the specific activity of the Factor VIII represents 0.7 IU/mg, and that of the vWF 0.6 U RCo/mg (ristocetin cofactor activity) and 1.2 U CBA/mg (collagen bindi-ng activity).

Virai Inactivation Treatment

The solution containing the Factor VIII/v\VF mixture is subjected to a solvent-detergent treatment known for its efficiency in destroying lipid * - 11 - LV 10193 enveloped viruses (Horowitz et al., Transfusion, 1985, 25: 516) and which includes incubation for 8 hours at 25° C in the presence of 0.3 % of tri-n-butyl phosphate (TnBP) and 1 % of Tween 80.

After this treatment, 95 % of the activity of Factor VIII and vWF measured in the preceding step is recovered. Electrophoresis can be used to confirm that the v\VF is stili in multimeric form.

Chromatographic Separation Process

The purification of the vWF is derived from the Factor VIII purification process disclosed by the Applicant in European patent application No. EP 0,359,593.

The first chromatography is carried out on a column of DEAE-Fractogel® TSK 650 (Type S or M) (Merck). The equilibration buffer solution contains trisodium citrate (0.01 M), calcium chloride (0.001 M), glycine (0.12 M), L-Lysine (0.016 M) and 0.11 M sodium chloride). The v\VF, Factor VIII and fibronectin are retained by the column ; the contaminating proteīns (chiefly fibrinogen and some IgG) IooseIy fixed or not fixed by the column and the virus sterilizing aģents are eliminated by several successive washings with the same buffer solution.

The column is used at a linear flow rāte of 100 cm/h. Under these vvorking conditions, the column used has a vWF retention capacity of approximately 75 % of the amount injcctcd (measured as the antigen, Ag) the remainder being lost in the filtrate. This binding capacity corresponds to 45 U of vWF Ag/ml gel. - 12 -

The vWF is desorbed from the column by increasing the NaCl concentration of the buffer solution to 0.15 M, The fraction of vWF harvested contains 30 to 35 % of the initial vWF while 40 % of it remains co-adsorbed vvith the Factor VIII which will be co-eluted by a second increase in the NaCl concentration of the buffer solution to 0.25 M and then co-purified.

The fraction containing the vWF eluted from this first column is reinjected onto a second identical column, after a slight dilution with the equiIibration buffer, in order to adjust the ionic strength of the vWF fraction down to an equivalent of 0.11 M sodium chloride.

Since the contaminants and the Factor VIII vvhich competed with the vWF for the adsorption sites of the first column were almost eliminated during the first chromatographic step, binding capacity of the second column is much greater : 320 U of vWF Ag/ml gel.

The vWF is desorbed by increasing the NaCl concentration of the buffer solution to 0.17 M. %

This second chromatography permits a concentration rāte 8 to 10 times that of the previous one, vvhich eliminates the need for anv additional concentration steps by ultrafiltration. for example. Using standardized techniques, the eluate is found to contain the following vWF quantities or activities : - Antigen (Ag) 88 ±9 IU/ml - Ristocetin cofactor (RCo) 97 ± 19 IU/ml - Collagen binding activity (CBA) 149 + 13 IU/ml - High molecular wcight multimers 79 % (> 4 multimers) - 13 - LV 10193

The CBA units (collagen binding activity) are quantified by ELISA as described by Brown and Bosak, (Thromb. Res. 1986, 43: 303). A Standard plasma,· calibrated against the 2nd British Standard (86/717), was used as a reference to express the values in terms of international units.

The CBA/Ag ratio of 1.69 shows that the activity of the vWF is well preserved. This is in agreeraent with the high percentage in high molecular weight multimers (79 %) and comparcs favorably with that of native vWF (70 %) from plasma.

Electrophoretic analysis of this vWF eluate reveals a slight contamination by fibronectin and inter-alpha trypsin inhibitor, a serine-protease inhibitor.

The second v\VF eluate is then subjected to a third step of purification on a column of gelatin-Sepharose CL4B (Pharmacia) equilibrated with the elution buffer solution of the preceding column, in order to eliminate fibronectin.

This affinity chromatographic gel has a fibronectin retention capacity of > 5 mg/ml, which enables this contaminant to be reduced to undetectable quantities (< 4 mg/1) in the v\VF fraction.

The purified v\VF of the present invention is found in the filtrate of this last step and can be directly dispensed and freeze-dried. - 14 -

Electrophorctic analysis of the final product can no longer detect any contaminants. The vWF content is 205 U Ag/ml protein and its specific activity is 345 U CBA/mg protein and 186-220 U RCO/mg protein.

The total degree of purification in relation to the initial plasma is > 10,000 fold.

Electrophoretic analysis (SDS-agarose and scanning of the bands) demonstrates that the v\VF obtained from this purification procedure is composed of 65 to 80 % of high molecular vveight multimers, i.e. a proportion comparable with that of the initial plasma, which was 70 %.

The stability of the concentrate was studied in a liquid State at room temperature for 24 hours : no sign of proteo!ytic activity or any change in specific activity could be detected.

Absence of thrombogenic activity in the concentrate was verified using the conventional tests like the non-activated partial thromboplastin time (NAPTT). Thrombin, PKA and Kallikrein wers undetectable.

Therefore, no stabilizing aģent needs to be added to the final vWF concentrate.

The possibility of designing a purification process specifically intended for the recovery of vWF as a by-product of a FVIII production process makes thus, for the first time, possible to producē a high-purity, highly effective therapeutic concentrate standardized for the treatment of von Willebrand disease.

Claims (10)

LV 10193 Patentformulas punkti 1. Standartizētā cilvēka plazmas Villebranda faktora koncentrāta, kas bagātināts ar augstmolekulāriem multimēriem iegūšanasjnetode no krioprecipitētās plazmas frakcijas, kas atšķiras ar to, ka ietver triju secīgu hromatogrāfiskās sadales stādīju kombināciju; pirmās divas stādījās ir jonu apmaiņas hromatogrāfija uz makroporu vinila polimēra tipa sveķiem ar DEAE grupām, bet trešā stādīja ir afinitātes hromatogrāfija uz želatīn-sefarozes.EN 10193 Patent Claims 1. A standardized human plasma Villebrand Factor concentrate enriched with high molecular weight multi-dimensional multi-dimensional multi-dimensional multi-dimensional multi-dimensional crystal diffraction, characterized in that it comprises a combination of three successive chromatographic distribution plants; the first two were ion exchange chromatography on macroporous vinyl polymer type resin with DEAE groups, and the third plant was affinity chromatography on gelatin-sepharose. 2. saskaņā ar 1. p. , kas atšķiras ar to, ka izejmateriāls ir plazmas krioprecipitētā frakcija, kas iepriekš attīrīta uz alumīnija hidroksīda.2. in accordance with paragraph 1; characterized in that the starting material is a plasma cryoprecipitated fraction previously purified on aluminum hydroxide. 3. Metode saskaņā ar 1. p., kas atšķiras ar to, ka divas jonu apmaiņas hromatogrāfijas stādījās tiek izpildītas uz kolonnām ar DEAE-Fractogel ^ ΤΞΚ 650 sveķiem, kurus līdzsvaro bufer-šķīdums, kas satur 0,01 M trinātrija citrāta, 0,11 M nātrija hlorīda, 0,001 M kalcija hlorīda, 0,12 M glicīna un 0,016 M L-lizīna.Method according to claim 1, characterized in that two ion exchange chromatographic plants are performed on columns with DEAE-Fractogel ^ ΤΞΚ 650 resin balanced by a buffer solution containing 0.01 M trisodium citrate, 0 , 11 M sodium chloride, 0.001 M calcium chloride, 0.12 M glycine and 0.016 M L-lysine. 4. Metode saskaņā ar 1. p., kas atšķiras ar to, ka iepriekš attīrītā krioprecipitētā plazmas frakcija tiek adsorbēta uz pirmās hromatogrāfijas kolonnas un pirmā Villebranda faktoru saturošā frakcija tiek eluēta nātrija.hlorīda koncentrācijai pieaugot bufer-šķīdumā līdz 0,14 - 0,15 M.Method according to claim 1, characterized in that the pre-purified cryoprecipitated plasma fraction is adsorbed on the first chromatography column and the first fraction containing the Villebrand factor is eluted with sodium chloride concentration in the buffer solution to 0.14-0. 15 M. 5. Metode saskaņā ar 1. p., kas atšķiras ar to, ka Villebranda faktoru saturošais eluāts no pirmās hromatogrāfijas stadijas tiek adsorbēts uz otrās jonapmaiņas hromatogrāfijas kolonnas un Villebranda faktors tiek eluēts pieaugot nātrija hlorīda koncentrācijai buferšķīdumā līdz 0,15 - 0,17 M.Method according to claim 1, characterized in that the Villebrand factor-containing eluate is adsorbed from the first chromatography step on the second ion exchange chromatography column and the Villebrand factor is eluted with increasing sodium chloride concentration in the buffer to 0.15-0.17 M. . 6. Metode saskaņā ar 1. p., kas atšķiras ar to, ka eluātu no otrās hromatogrāfijas stadijas adsorbē uz želatīn-sefarozes hromatogrāfijas kolonnas, kas līdzsvarota ar iepriekšējās hromatogrā-fijas stādījās eluāta buferšķīdumu, kā rezultātā kolonnā selektīvi adsorbējas atlikušais fibronektīns.Method according to claim 1, characterized in that the eluate from the second chromatography step is adsorbed on a gelatin-sepharose chromatography column equilibrated with a pre-chromatographed eluate buffer resulting in selective adsorption of residual fibronectin in the column. 7. Metode saskaņā ar 1. p., kas atšķiras ar to, ka no želatīn-sefarozes kolonnas izvadītajā filtrātā filtrātā esošais Villebranda faktors tiek savākts, sadalīts un liofilizēts.A method according to claim 1, characterized in that the Villebrand factor present in the filtrate from the gelatine-sepharose column is collected, divided and lyophilized. 8. Villebranda faktora koncentrāts ar ļoti augstu tīrības pakāpi, augstu specifisko aktivitāti (RCO un CBA vienībās) un augstu augstmolekulāro multimēru saturu, kas iegūts saskaņā ar jebkuru no punktiem 1-7. LV 101938. Willebrand factor concentrate of very high purity, high specific activity (in RCO and CBA units) and high high molecular weight multimers obtained according to any of claims 1-7. LV 10193 9. Villebranda faktora koncentrāts saskaņā ar 8. p., kas atšķiras ar to, ka tā aktivitātes attiecība (CBA vienībās) pret klātesošo antigēnu ir vismaz 1,5. «The Willebrand Factor concentrate according to claim 8, characterized in that its activity ratio (in CBA units) to the antigen present is at least 1.5. « 10. Villebranda faktora koncentrāts saskaņā ar 8. vai 9. p., kas atšķiras ar to, ka augstmolekulāro multimēru saturs tajā ir vismaz 65 - 80 %. LV 10193 PROCESS FOR AN INDUSTRIAL-SCALE PREPARATION OF A STANDARDIZED HUMAN VON VVILLEBRAND FACTOR CONCENTRATE OF VERY HIGH PURITY AND SUITABLE FOR THERAPEUTIC USE CENTRE REGIONAL DE TRANSFUSION SANGUINE DE LILLEThe Wildman concentrate according to claim 8 or 9, wherein the high molecular weight multimers are at least 65-80%. EN 10193 PROCESS FOR AN INDUSTRIAL-SCALE PREPARATION OF A STANDARDIZED HUMAN VON VVILLEBRAND FACTOR CONCENTRATE OF VERY HIGH PURITY AND SUITABLE FOR THERAPEUTIC USE CENTER REGIONAL DE TRANSFUSION SANGUINE DE LILLE