CZ211299A3 - Derivative of von willebrand factor, apparatus in which said derivative is contained and protein isolation process - Google Patents

Abstract

Disclosed is a von Willebrand Factor (vWF) derivative consisting of vWF, immobilized in a carrier, which is characterized in that the vWF is r-vWF. Also disclosed is a method for isolating proteins which bind to the vWF when the vWF derivative is used.

Description

A von Villebrand factor derivative, an apparatus comprising the derivative and a method of protein isolation

Technical field

The present invention relates to a von Villebrand Factor (vVF) derivative, to a device comprising the derivative and to a method for the isolation of proteins which are produced. bind to vVF.

BACKGROUND OF THE INVENTION

Von Villebrand factor (vVF) is a glycoprotein that circulates in plasma in a number of multimers of about 500 to 20000 kilodaltons. Miltimeric forms of vVF consist of 250 kD polypeptide subunits that are linked to each other via disulfide bridges. vVF plays a role in the binding of blood iček platelets to subendothelium of damaged vessel walls, with only the largest multimers also showing hemostatic activity. Entothelial cells were thought to secrete large polymeric forms of vVF and that those forms of vVF that have a low molecular weight (VMF) are due to proteolytic cleavage.

vVF is formed in vascular entothelial cells, which are the major source of this plasma protein, but are also synthesized in a minor proportion by megakaryocytes. The vVF biosynthesis is very complex and therefore results in a large number of different vVF molecules with different structure, tasks and properties.

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This results in vVF being able to bind to different receptors, with the most important physiological activities of vVF binding to proteins such as glycoprotein Ib and glycoprotein complex II / IIIa, factor VIII: C, thrombocytes and subendothelium.

When vVF is bound to blood clotting factor VIII, a Factor VIII complex or a Factor VIII: C / vVF complex is formed which contains Factor VIII: C as a stabilized protein. The lack of vVF also inevitably leads to a reduction in the concentration of factor VIII: C in the blood, as there is no stabilizing effect of vVF.

Recombinant factor VIII is known to be purified through a column containing plasma von Villebrand factor coupled to Sepharose (vVF-Sepharose) (Vood et al., Nature 312 (1984), 330-337). However, the avidity of the immobilized plasma vVF has been shown to be low and, for example, it is not possible to obtain factor VIII with the described vF-sepharose from a solution containing the factor VIII / vVF complex in acceptable yields.

In addition, this vVF-sepharose contains, in addition to vVF, factor VIII based on a plasma starting material containing the factor VIII: C / vVF complex. However, plasma factor VIII: C contamination is a major problem in obtaining pharmaceutical preparations.

Binding studies investigating the inhibition of factor VIII binding to plasma vVF immobilized on microtiter plate dishes by recombinant vVF showed that recombinant vVF could achieve even worse avidity in solution with respect to the factor * h binding partner ♦ »» · · W · • Í 44 '·.!, · * · · ♦ * ♦ · ··· · · # ·

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VIII.-C (Leyteacol., Biochem. J., 274 (1991), 257-261). Thus, this material is not suitable for preparative recovery of factor VIII.

It is therefore an object of the present invention to provide chromatographic material with which vVF-binding proteins can be isolated, even if vVF is present in the solution from which the proteins are to be isolated. In other words, it is an object of the present invention to provide an avidity material that is higher than the avidity of vVF in association with its binding partners, especially the avidity of vVF in the Factor VI / vVF complex.

The essence of the invention

The above object was solved by finding a vVF derivative consisting of vVF immobilized on a particulate carrier or carrier gel (carrier), in particular on a chromatographic material, characterized in that vVF is recombinant vVF (r-vVF). Preferably, the vVF derivative is devoid of blood clotting factor VIII and, by the exclusion of anti-vVF antibodies, devoid of xenogeneic material such as antibodies.

Surprisingly, it has been shown that, unlike plasma vVF, r-vVF has a surprisingly high avidity against vVF-binding proteins, although it is bound to chromatographic material.

In addition, r-vVF, in contrast to plasma vVF, is free of blood clotting factor VIII and particularly free of plasma proteins that would be in the isolation process.

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In a particular embodiment of the vVF derivative of the present invention, the r-vVF is fixed by chemical bonding to the chromatographic material. This ensures that the high avidity of the RF derivative of the present invention is maintained with high stability, which greatly increases the ability to use the material of the present invention. Chemical fixation results in stabilization of the chromatographic material. At the same time, the use of corresponding antibodies that mediate binding of vVF to the solid phase can be excluded. Antibody binding has the disadvantage of instability and an increase in leakage, i.e., elution of immobilized vVF while obtaining a binding partner for vVF.

The avidity of the vVF-derivative can be further increased by the targeted choice of the r-vVF-fraction. Surprisingly, it has been shown that a material with a high avidity for factor VIII can be produced directly from the r-vVF-fraction with low primary hemostatic activity, in particular from the low molecular weight r-vVF-fraction with a molecular weight below about 1500,000 Da, preferably below 10,000 Da.

The acceptability of the recombinant von Villebrand factor in high purity and unlimited quantity allows the broad use of the RF derivative of the present invention as a ligand in affinity chromatography or related affinity methods in which the bound molecule is coupled to the ligand by specific interaction.

r-vVF is preferably expressed in mammalian cells, for example CHO cells. Such vVF is described in the patent application

VO 96/10584. In the purification of r-vVF using, for example, heparin5

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affinity chromatography yields fractions containing molecules having a relatively low molecular weight (up to about 1000000 Dalton) for vVF. While these fractions themselves have relatively low primary hemostatic activity, they are still capable of binding binding proteins that enter specific interactions with vVF, such as factor VIII. Since these side-fractions of r-vVF production are not used for therapeutically utilized high-multimer RF vF-concentrates, they are therefore a by-product that can be used according to the present invention for immobilization and subsequent use of immobilisate to produce affinity matrix.

As the chromatographic material according to the present invention, a gel having good affinity-chromatographic properties, i.e. low back pressure corresponding to high flow rates, high binding capacity for immobilized ligands, low reaction to color transition and the possibility of disinfection with e.g. sodium hydroxide, is preferably used.

Coupling of r-vVF to a solid matrix is accomplished by not forgetting the binding properties of affinity chromatography of the binding proteins. Surprisingly, standard immobilization techniques such as those described by Voodward in Immobilized Cells and Enzymes, IRL, may be used. Press, Oxford, Washington (1985). Furthermore, such a chromatographic gel has good properties for reuse and stability, so that it also has the same binding capacity for isolated molecules also when used repeatedly.

Thus, the preferred RF derivative contains as the chromatographic material an organic polymer, in particular organic poles.

Flfl flfl fl fll fl fll fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fll fl fll fl fll fl fll fll fl fll flll basis basis on carbohydrate. Suitable gel matrices or RR particulate carriers are, for example, sephacels, sephadexes, sepharose®, Fast Flow-Media, High Performance-Media, pp sepharose Big Beads (all from Pharmacia), tris-acryl media, spherodex® or hyper-D®. -media (all by Sepracor), macropep®-gel (BioRad), but also synthetic polymers such as toyopearl®-gel (Tosohaas) and fractogel® (Merck).

In order to prevent contamination, in particular by human pathogenic viruses, the vVF derivative of the present invention is preferably subjected to a virus inactivation treatment, so that it can be guaranteed that the chromatographic material has no virus-contaminant in isolating vVF-binding proteins. The virus inactivation treatment is preferably carried out prior to derivatisation by chemical and / or physical processes, for example treatment with surfactants, polyethylene glycols or chaotropes, heat treatment or irradiation treatment. However, the finished derivative, also preferably in lyophilized form, can also be subjected to a treatment such as heat treatment or irradiation.

In a preferred embodiment, the vF-derivative of the present invention is available in a storage-stable form, in particular as a lyophilisate, thereby achieving a significant ease in sales, operation and storage.

According to a further aspect, the present invention also relates to an apparatus comprising a reservoir and a vF-derivative according to the present invention contained therein, the reservoir having an inlet opening and an outlet opening which are arranged appropriately for fluid flow. In practice, the device according to the present invention is designed as a column, in particular as an affinity frl fr fr fr · · ·

frfr fr · column or chromatographic column, which can be made available as a useful product in a storage-stable form as required, so that only material swelling at the user has to be carried out before protein isolation.

The present invention also provides a method for isolating vVF-binding proteins, characterized by the following steps:

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- preparation of a fraction containing vVF-binding proteins,

- contacting the vVF derivative fraction of the present invention, wherein the proteins bind to the vVF derivative,

separation of unbound components, and

eluting proteins from the vVF-derivative.

This process can be carried out batchwise, i.e. as a batch process or also as a column chromatography process.

Suitable proteins which can be isolated by the method according to the invention are, in particular, the physiological vVF binding proteins, i.e. glycoprotein Ib, glycoprotein IIb / IIIa complex, collagen and in particular factor VIII, but also recombinant derivatives and analogues of these proteins, vVF-antigens , vVF-antibodies, vVF-multimerases or vVF-depolymerases and even enzymes that recognize vVF as a substrate and other natural or synthetic peptides and proteins having affinity for vVF. Furthermore, vF-binding saccharides, such as heparin, can be isolated in this manner.

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Due to the high avidity of the vVF derivative of the present invention, isolated proteins can be specifically bound and recovered in the chromatographic material of the present invention in yields of more than 60%, preferably more than 80%, mostly preferably quantitative, and eluted in purified form from the vVF derivative. it is possible to produce isolated protein concentrates by simple elution without a subsequent concentration step.

The process according to the invention is particularly suitable for obtaining biologically active proteins with activity of factor VIII, in particular plasma or recombinant factor VIII and their mutants or analogs. These can be isolated by the process according to the invention, in particular when a starting solution containing a Factor VI / vFF complex is used.

The elution of the proteins, especially in the recovery of factor VIII proteins, is preferably carried out using buffers containing potassium ions.

Of particular importance here is the isolation of Factor VIII from Factor VIII-containing plasma fractions or from cell culture fluid from cells expressing Factor VIII. When isolating Factor VIII from plasma fractions, care should be taken that vVF functions as a Factor VIII carrier protein, i.e., Factor VIII appears to be vVF-bound. The separation of factor VIII and vVF can otherwise only be accomplished using costly methods, for example by binding factor VIII to the immobilized anti-factor VIII driven monoclonal or polyclonal antibodies to which factor VIII (the vVF complex binds and then vVF is eluted intentionally). that the binding of the antibody to Factor VIII would be impaired.

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In the following, factor VIII is eluted from the chromatographic gel. Indeed, such processes are expensive, but lead to highly pure factor VIII preparations. Using the immobilized r-vVF of the present invention with a high specific binding capacity and high avidity for Factor VIII, the Factor VIII and vVF complex present in plasma fractions can be dissociated, Factor VIII binds to the immobilized vVF with higher avidity. In this way, high purity vFF-free factor VIII can also be isolated from the plasma fraction.

In addition to factor VIII, other proteins with affinity for vVF can also be bound to the immobilized r-vVF and isolated from complex mixtures, for example factor VIII-hybridproteins, in particular factor VIII-heparin-cofactor II-hybridprotein according to US Ser.No. 08 (558,107 or factor VIII-factor 5-hybridprotein according to WO 90/05570 or chimeric human / serving factor VIII according to WO 94/11503, factor VIII mutants, such as in AT A 921/96 (factor VIII dB695-R2307Q ) described factor VIII mutants with Arg -> substitution

Gins that have reduced inhibitor binding for C-procoagulatory activity and vVF-binding activity, von Villebrand factor-degradable enzymes, for example vVF-specific depolymerase or vVF-multimerase or thrombocyte receptors such as GPIIb / HIa or GPIb IX-complex, the provision of which in pure form is of biochemical-analytical, diagnostic or therapeutic interest.

Currently, specific isolation of therapeutic proteins is performed using immunoaffinity chromatography. In this case, the immobilized monoclonal antibody (which is usually obtained from mouse cells) binds isolated

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4 • 4 in 4 molecules, washed to the absence of impurities and then eluting in high purity. The disadvantage of using monoclonal antibodies is that murine proteins can be introduced into the preparation, which, when bound therapeutically, result in side effects, such as the generation of antibodies against the murine protein. By using immobilized specific ligands of human origin or a molecule equivalent to a human protein and eliminating xenogeneic antibodies, the immanent problem of contamination by affinity column contamination is so reduced that possible contamination is also of human origin in the formulation and the reported side effects, namely heterologous antibody formation. Another advantage is that the use of a highly specific non-antibody ligand eliminates the non-specificities that occasionally occur with antibodies.

Obtaining the physiological vVF binding proteins of the present invention further has the advantage that vVF acts as a stabilizer or carrier for its binding partner. Thus, the proteins obtained are even protected during the isolation and purification from denaturing conditions that may occur during elution. The products obtained are thus obtained not only with regard to their antigenicity, but also with regard to their activity or nativeness, in the said high yields.

Another particularly preferred protein or protein complex that can be purified by the vVF derivative of the present invention is vVF-multimerase, which degrades high molecular weight vVF forms to low molecular weight variants (see AT A 769/96 and A 770/96). In doing so, the multimerase can bind to immobilized vVF, which suppresses derivatisation

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However, it promotes the degradation of the material of the present invention.

Particularly in the isolation of vF-multimerase, it can be eluted particularly efficiently with a buffer containing chelating agents for metal ions, in particular EDTA.

The vVF derivative of the present invention is also suitable for optionally extracorporeal immunadsorption of antibodies that are directed against vVF. it may occur as an autoimmune disease and leads to blood clotting defects with an increased tendency to bleed, or may be a side effect of treating patients with vVF-containing preparations. The production of a functionally inhibitory antibody renders replacement therapy impossible or leads to drastic dose increases to maintain the hemostatic effect of the clotting factor concentrate. In such cases, circulating functional anti-clotting factor antibodies have been removed in the past by plasmaferesis or extracorporeal immunadsorption to anti-IgG-directed proteins, such as protein A or protein G. A similar method is known to remove antibodies directed against factor VIII from Nilsson and Berntrop. In doing so, the patient's plasma is pumped through an immobilized, recombinant Factor VIII column to remove Factor VIII-inhibitory antibodies from the bloodstream (Nilsson et al., Thromb. Haemostas. 70 (1993), 56-59). However, the immobilized ligand is free of von Villebrand factor. Correspondingly, it is not possible to carry out extracorporeal immunoadsorption of antibodies directed against vVF.

Further use of the method of obtaining the antibodies of the prior art 4 fcfcfc fcfcfc fcfcfcfc fcfcfc fcfc fcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfcfc The invention is based on the preparative acquisition of polyclonal or monoclonal anti-vVF antibodies for diagnostic purposes.

The elution buffer, which is preferably used to elute antibodies from the vVF-derivative of the present invention, has an acidic pH, in particular a pH in the range of 2 to 5.

One of the essential advantages of the present invention is that it is possible to purify proteins from each starting material. Particularly preferred starting fractions are those derived from mammalian body fluids or cell cultures. On the basis of the avidity of the chromatographic material of the present invention, fractions containing vVF and / or Factor VIIΙ / vVF complex are also preferred.

A preferred variant of the process according to the invention is that it is carried out using the device according to the invention with the RF derivative contained therein.

The present invention is illustrated by the following examples and figures, but is not limited thereto.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1 and 2di show the purification of recombinant factor VIII with the vVF derivative of the present invention.

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DETAILED DESCRIPTION OF THE INVENTION

Example 1

Production of recombinant von Villebrand factor from CHO cells

A CHO-cell clone that produces the recombinant von Villebrand factor is made as described in FEBS, Lett. 351 (1994), 345-34. A cell line for co-expression of human furin was prepared by transfection with the cDNA of a human furin encoded vector (van den Ouweland et al., Nucleic Acids Res. 18 (1990), 644). Such stable cell clones are extensively fermented in fusion reactors on microcarriers (Bluml et al., In: Spier RE, Griffith JB, Berthold V, eds. Animal Cell Technology, OxfordLondon: Butterworth-Heinemann, (1994), 267-269 ).

Purification is carried out by the two-step Thromb chromatographic method. Haemost. 73 (1995), 1160. The fraction desorbed by elution with sodium chloride is obtained and transferred by gel filtration through sephadex G25 (Pharmacia) to a buffer containing 20 mM Tris-HCl, 150 mM NaCl, pH 7.5. Thereafter, the preparation is concentrated by ultra-concentration through an Amicon YM30 membrane (cut-off: 20,000 D) to a protein concentration of 3 mg / ml. vVF-Concentration in this preparation is 60 E vVF-antigen / mg protein. This preparation contains no factor VIII due to the excretion of serum or plasma components in production in cell culture and purification.

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Example 2

Immobilization of recombinant Villebrand factor

The recombinant vVF preparation of Example 1 is diluted with a buffer containing. 20 mM Tris-HCl, 150 mM NaCl, pH 7.5, to 1.5 mg / ml. Preactivated gel (Actigel, ALD-Superflow, Sterogene company) suitable for affinity chromatography, ^it předpromyje excessive buffer containing 20 mM

Tris-HCl, 150 mM NaCl, pH 7.5. One volume of pre-washed gel is mixed with 1.1 volume of immobilized protein solution and then 0.15 volume of 0.1 M cyanoborohydride (NaCNBH 4) solution in 0.1 M phosphate buffer, pH 7.0 is added. The gel is suspended by shaking in this buffer and incubated for 16 hours at room temperature with further shaking. Then, the gel is washed on a sintered well with a 10-fold volume of buffer containing 20 mM Tris-HCl, 150 mM NaCl, pH 7.5, and a 5-fold volume buffer containing 20 mM Tris-HCl, 2 M NaCl, pH 7.5.

It is then equilibrated once more with five volumes of a buffer containing 20 mM Tris-HCl, 150 mM NaCl, pH 7.5, and the gel is transferred to a diameter dimension chromatography column to the bed height of gel 1: 4. By determining the concentration of the protein in the solutions of the incubation mixture, the vF solution and the affinity gel, as well as the wash solutions separated on the sintered resin, a coupling value of over 90% of the protein used can be determined.

Example 3

Purification of recombinant factor VIII

Preparation of recombinant factor VIII (Recombinate, *, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 999, 999, 9, 9, 9, 9, 9 Baxter) is reconstituted with 10 ml of distilled water. This solution contains 50 IU Factor VIII / ml, 12 mg human albumin / ml, 1.5 mg polyethylene glycols 3350 / ml, as well as traces of vF in the histidine-sodium chloride buffer at physiological pH. Gel filtration on Sephadex G25 (Pharmacia) separates the low molecular weight components from this solution to give a Factor VIII / vVF / albumin mixture in a buffer containing 20 mM Tris-HCl, pH 7.5. 7 ml of this solution is then applied to the column of Example 2 with a flow rate of 0.5 ml / min. Then, at the same flow rate, 10 ml of a buffer containing 20 mM Tris-HCl, pH 7.5 are washed at the same flow and further eluted on vVF bound a factor VIII fraction using 250 mM calcium chloride in the same buffer. During total chromatography at room temperature, the fractions were collected to a volume of 500 μΐ and the optical density at 280 nm was determined after passing through the column. The fractions are then determined by a chromogenic test for. factor VIII (Immunochrom FVIII: C (Immuno)) factor VIII content. The result is shown in Figure 1.

Fraction analysis shows that more than 90% of the protein is contained in the column and wash phase, while only a small fraction of Factor VIII activity (below 5%) elutes with this protein fraction. The major amount of factor VIII can be desorbed from the high purity column eluting with calcium chloride. The recovery of factor VIII is carried out with a yield of more than 90%.

Example 4

Reusability of affinity gel

After the first purification of the recombinant factor has been completed. 4 444 4 444 4 4 * * 4 4 4 44 4 4 4 4 4 4 4 4

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VIII by affinity chromatography, the affinity gel in the column was washed extensively with buffer, 20 mM Tris, pH 7.5. The vF-antigen was determined in the wash solution by ELISA (Boehringer, Mannheim). No vVF-antigen can be detected, which is an indicator of the slight possibility of affinity column leakage. Purification of recombinant factor VIII by affinity chromatography on immobilized vF was repeated as in Example 3 under identical conditions.

The elution diagram is shown in Figure 2. The elution profile of proteins and activity is comparable except for measurement deviations —........................

k with the course of the first affinity chromatography. Thus, good reusability of the affinity gel can be assumed.

Example 5

Binding of anti-von Villebrand factor-antibodies to immobilized recombinant von Villebrand factor

An anti-vVF-driven mouse monoclonal antibody (MAb 03768/3, Chemicon International, Inc) with an IgG concentration of 7 mg / ml was transferred by gel filtration through Sephadex G25 (Pharmacia) into a buffer, 20 mM Tris-HCl, 150 mM NaCl, pH 7.5. The protein concentration is adjusted to 0.5 mg / ml with the same buffer. 4 ml of this solution is pumped at a flow rate of 0.5 ml / min through the immobilized recombinant vVF column of Example 3, and the optical density at 280 nm is determined in the passed liquid. It is then washed with 20 ml of Tris-HCl buffer followed by 10 ml of Trls-HCl buffer containing 500 mM sodium chloride. No significant amount of protein was eluted in any of the wash steps. It is then eluted with buffer, 100 mM glycine hydrochloride, pH 2.2. By changing the pH value, the protein can be desorbed from the column. The determination of the IgG content shows that <* * 9 99 9 »*« φ «« «« φ · · · ... ... ... 9 * ** · ΦΦ ΦΦ ΦΦ ΦΦ of the 2 mg IgG used again detects 1.75 mg IgG in the glycine eluate. This corresponds to a yield of 88%. The affinity column is rinsed with 20 mM Tris-HCl buffer, pH 7.5 after the acid buffer for further use.

Example 6

The purification of von Villebrand-degrading factor &quot; in the VF-cleavage &quot; human plasma protease described by Furlan et al. (Blood 87 (1996), 4223-4234) is purified according to the method described therein using copper chelate affinity chromatography followed by hydrophobic interaction chromatography on butylsepharose (Pharmacia). The butylsepharose eluate is lyophilized and concentrated into distilled water. It is then buffered over Sephadex G25 (Pharmacia) against buffer, 20 mM Tris-HCl, 50 mM sodium chloride, 10 mM barium chloride, 5 mM PEFA-Block (Pentapharm), pH 7.5. 3 ml of this solution is applied to a column containing the immobilized recombinant vVF of Example 2 at a flow rate of 0.5 ml / min. It is then washed with 10 ml of the same buffer against which the protease fraction has been buffered, and the column is then washed with another 10 ml of buffer containing 20 mM Tris-HCl, 50 mM NaCl, 20 mM EDTA, 5 mM PEFA-Block (Pentapharm). pH 7.5. Fractions of 450 μΐ are collected throughout the chromatography, with 50 μΐ of a 1% bovine serum albumin aqueous solution in each fraction collector tube to stabilize the vF-degrading enzyme known as labile. During chromatography, UV-absorption at 280 nm is measured and the activity of the RF-degrading enzyme is determined as follows. Preparation of recombinant vVF of Example 1 is buffered in buffer, 5 mM Tris-HCl, 1.5, fefe, fefefe, fefe fefe

M urea containing 0.2% (G / V) bovine serum albumin and converted to 0.4 vVF E / ml. An aliquot of the test fraction of 100 gl is mixed with 5 μΐ of a 50 mM aqueous PPACK solution and 12.5 gl of a 200 mM barium chloride solution in each case and incubated for 5 minutes at 37 ° C. Thereafter, the sample 1 + 1 thus prepared is mixed with substrate (vVF) and dialysed for 15 hours at 37 ° C on a floating dialysis membrane (Millipore VSVP) according to the method of Marusky and Sergeant (Anal. Biochem. 105 (1980), 403). ) ^J

against a buffer containing 5 mM Tris-HCl, 1.5 M urea, pH -8.0.

Then the dialysate vWF analyzed for their residual activity by ELISA, in which is found a collagen binding vWF AK- ₄ dent, and its antigenicity. To this end, it is added to a polystyrene microtiter plate (96 well, Pierce Reactibind ⁱⁿ / activated with maleic anhydride). for a plate of 100 g of pepsin-digested Type-III-Collagen (Southern Biotechnology) suspension from human placenta at a concentration of 2 gg / ml and incubated for one hour at room temperature. 150 g of blocking buffer (SuperBlock ®, Pierce) are then treated for 30 minutes. 100 g of different dilutions of vVF-containing samples (25 to 250 ng / ml) are then added and incubated with 100 g of peroxidase-conjugated anti-vVF-antibody solution (Dakopatts P226, 1: 1000 dilution). Subsequently, 100 g of substrate (Single Component TMB Peroxidase EIA Substrate, Bio-Rad) was added and the color reaction was terminated after 1 minute by diluting 1 + 1 with 0.18 M sulfuric acid. The extinction is then measured at 450 nm with an ELISA counter and the sample concentration is determined against the standard dilution series. After each incubation step, wash three times with 150 g of buffer (buffer corresponding to the next step).

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Buffer system used: Buffer 1 (for collagen coating and antibody dilution); 8 mM sodium phosphate, 135 mM sodium chloride, 2.6 mM potassium chloride, pH 7.3, 1 ’· * Year Buffer 2 (for sample dilution): corresponds to buffer 1 + 0.05% Tween 20 and 1% albumin

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Reciprocal value of collagen binding activity is a direct measure of the enzyme activity in the RF-degrading enzyme.

Of the amount of enzyme applied to the von Villebrand factor column, about 50% is found in the flowing liquid and wash solutions, while about 50% of the activity can be desorbed by elution with EDTA. However, since more than 95% of the protein was contained in the flow and wash fractions of the chromatographic purification, the isolated enzyme activity could be enriched and purified by a factor of 5 to 10 relative to the starting material.

Example7

Purification of plasma factor VIII

Plasma factor VIII / von Villebrand factor-concentrate (IMMUNATE, immuno) is reconstituted with 5 ml of distilled water. This solution contains 25 IU factor

VIII / ml and 10 E vVF / ml (measured with Ristocetin CofactorMethode) in citrate-glycine-lysine buffer, pH 7.4. Gelovou

7, '· »», · · · · ·,,,, »» »» »» »» »» Filtration on Sephadex G25 buffered Factor VIII / von Villebrand-Factor Complex against a buffer containing 20 mM Tris / HCl, pH 7.5. 3 ml of this solution are then applied directly to the column of Example 2. The flow rate is 0.1 ml / min. It is then washed with 30 ml of Tris / HCl buffer, pH 7.5 and further eluted with a buffer containing 20 mM Tris / HCl and 250 mM CaCl2. Elution collects the fractions and determines the optical density. In the fractions immediately after application of the elution buffer, an increase in UV-absorption of 280 nm can be measured.

Villebrand factor, measured in von Villebrand-Factor-ELISA (Boehringer Mannheim), while a factor VIII content of about 0.2 E / ml could be detected in the first ten elution fractions by chromogenic factor VIII assay,

Immunochrom FVIII: C (Immuno).

Claims (22)

PATENT CLAIMS A von Villebrand factor derivative consisting of recombinant von Villebrand factor immobilized on a particulate carrier or carrier gel. 2. A compound of von Willebrand Factor according to claim 1 wherein "ic ^- Í ΐ d m that the particulate carrier or the carrier is a gel chromatographic material. The von Villebrand factor derivative according to claim 1 or 2, characterized in that the Villebrand factor derivative is free of a blood clotting factor VIII. The von Villebrand factor derivative according to any one of claims 1 to 3, characterized in that the recombinant von Villebrand factor is fixed to the particulate carrier or carrier gel by chemical bonding. The von Villebrand factor derivative according to any one of claims 1 to 4, characterized in that the recombinant von Villebrand factor is derived from a recombinant von Villebrand factor fraction with low hemostatic activity, in particular from a low molecular weight recombinant von Villebrand factor fraction. The von Villebrand factor derivative according to any one of claims 1 to 5, characterized in that the particulate carrier or carrier gel is an organic polymer, in particular a carbohydrate-based organic polymer. σ * * <# «fl fl fl fl fl fl fl * * * * fl f f f fl f fl f fl f fl fl fl f f fl f f fl f A von Villebrand factor derivative according to any one of claims 1 to 6, characterized in that the recombinant von Villebrand factor or derivative is virally inactivated. A von Villebrand factor derivative according to any one of claims 1 to 7, characterized in that it is present in a storage-stable form, in particular as a lyophilisate. * ¹ - ¹ Apparatus comprising a reservoir and a von Villebrand factor derivative contained therein according to any one of claims 1 to 8, wherein the reservoir has an inlet and an outlet aperture which are configured to be suitable for flowing liquids. Device according to claim 9, characterized in that the reservoir is designed as an affinity column. 11. A method for isolating von Villebrand factor binding proteins comprising: - prepare a fraction containing von Villebrand factor binding proteins, - contacting the fraction with a von Villebrand factor derivative according to any one of claims 1 to 8, wherein the proteins are? bind to this derivative, - unbound components are separated and • proteins from the von Villebrand factor derivative are eluted. The method of claim 11, 4 4 4 i 44 »* 44 fr 44 * 4 • 44a 4 4 4 »44 4 «44 4 * 4 4 4 4 4 4 444 444 Wherein the proteins are eluted from the von Villebrand factor derivative in a concentrated form. A method according to claim 11 or 12, characterized in that a fraction is prepared which contains a biologically active protein with factor VIII activity and is isolated. 14. A process according to any one of claims 11 to 13, wherein the elution is carried out using a buffer containing calcium ions. Method according to claim 11 or 12, characterized in that a fraction is prepared which contains von Villebrand factor multimerase and the protein is isolated. Method according to one of claims 11, 12 or 15, characterized in that the elution is carried out with a buffer comprising a chelating agent for metal ions, in particular ethylenediaminetetraacetic acid. A method according to claim 11 or 12, characterized in that a fraction is prepared which contains antibodies against von Villebrand factor and the protein is isolated. Method according to one of claims 11, 12 or 17, characterized in that the elution is carried out with an acid pH buffer, in particular in the pH range 2 to 5. Process according to one of claims 11 to 18, characterized in that a fraction is prepared; b b * b b b *>> b B bb b which is derived from mammalian body fluid or cell culture. Method according to any one of claims 11 to 19, characterized in that a fraction is prepared which contains von Villebrand factor and / or a complex of factor VIII and von Villebrand factor. to 21. A method according to any one of claims 11 to 20, ff * in ^L 'y is u ic ^ j s * t I'M the proteins obtained 4 ·> in at least 80% yield. Method according to one of Claims 11 to 21, characterized in that it is carried out using a device according to Claim 9 or 10,