EP0954533A1 - Derive du facteur de von willebrand ainsi que procede pour l'isolation de proteines - Google Patents

Derive du facteur de von willebrand ainsi que procede pour l'isolation de proteines

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Publication number
EP0954533A1
EP0954533A1 EP97913009A EP97913009A EP0954533A1 EP 0954533 A1 EP0954533 A1 EP 0954533A1 EP 97913009 A EP97913009 A EP 97913009A EP 97913009 A EP97913009 A EP 97913009A EP 0954533 A1 EP0954533 A1 EP 0954533A1
Authority
EP
European Patent Office
Prior art keywords
vwf
derivative
proteins
fraction
factor viii
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97913009A
Other languages
German (de)
English (en)
Inventor
Hans-Peter Schwarz
Peter Turecek
Johann Eibl
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Baxter AG
Original Assignee
Baxter AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Baxter AG filed Critical Baxter AG
Publication of EP0954533A1 publication Critical patent/EP0954533A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/36—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors

Definitions

  • the invention relates to a von Willebrand factor derivative and a method for isolating proteins that bind to vWF.
  • Von Willebrand Factor is a glycoprotein that circulates in plasma in a series of multimers of approximately 500 to 20,000 kilodaltons in size.
  • the multimeric forms of vWF consist of 250 kD polypeptide subunits, which are linked to each other via disulfide bridges.
  • vWF plays a role in binding platelets to the subendothelium of a damaged vascular wall, with only the largest multimers also showing haemostatic activity. It is believed that the endothelial cells secrete large polymeric forms of vWF and that those forms of vWF which have a lower molecular weight (low molecular weight vWF, LMW) have arisen from proteolytic cleavages.
  • vWF is formed in the vascular endothelial cells, which are the main source of this plasma protein, by constitutive or stimulated release, but is also synthesized to a small extent by the megakaryocytes.
  • the biosynthesis of vWF is very complex and therefore results in a large number of different vWF molecules with different structures, tasks and properties.
  • vWF can bind to different receptors in different tissues, whereby the binding to proteins such as glycoprotein Ib, the glycoprotein complex II / IIla, factor VIII: C, to platelets, to the subendothelium is one of the most important physiological activities of the vWF .
  • the factor VIII complex or factor VIII: C / vWF complex is formed, which contains factor VIII: C as a stabilized protein.
  • a vWF deficiency inevitably leads to a reduction in the factor VIII: C concentration in the blood, since the stabilization there is no effect of the vWF.
  • this vWF-Sepharose contained factor VIII in addition to vWF due to the plasmatic starting material, which contains the factor VIII: C / vWF complex. Contamination with the plasmatic factor VIII: C would pose a major problem in the production of pharmaceutical preparations.
  • the object of the present invention is therefore to provide a chromatography material with which proteins which bind to vWF can be isolated, even if vWF is present in the solution from which the proteins are to be isolated.
  • the object of the present invention is to provide a material with an avidity which is higher than that of the vWF in association with its binding partners, in particular than that of the vWF in the factor VIIl / vWF complex.
  • a vWF derivative consisting of vWF, immobilized on a particulate carrier or a carrier gel (carrier), in particular on a chromatographic Phiematerial, which is characterized in that the vWF is a recombinant vWF (r-vWF).
  • the vWF derivative is free of blood coagulation factor VIII and, because of the avoidance of anti-vWF antibodies, free of xenogenic material, such as antibodies.
  • an r-vWF is free of blood coagulation factor VIII, and in particular free of plasmatic proteins, which would be disruptive in an isolation process for vWF-binding proteins.
  • the r-vWF is fixed by chemical binding to the chromatography material.
  • the chemical fixation stabilized the chromatography material, but surprisingly it did not affect the nativity of the vWF.
  • the use of corresponding antibodies which mediate the binding of the vWF to a solid phase can be dispensed with. Antibody binding would have the disadvantage of instability and increased leakage, i.e. removing the immobilized vWF at the same time as acquiring the binding partner for vWF.
  • the avidity of the vWF derivative can be further increased through the targeted selection of the rWF fraction. Surprisingly, it has been shown that just from an r-vWF fraction with a low primary hemostatic activity, in particular from a low molecular weight r-vWF fraction with a molecular weight below about 1.5 million Da, preferably below 1 million Da, a material can be manufactured with high avidity for factor VIII.
  • the availability of recombinant von Willebrand factor in high purity and unlimited quantity enables the vWF derivative according to the invention to be widely used as a ligand in affinity chromatography or related affinity methods in which a molecule to be bound is linked by specific interaction with the ligand.
  • r-vWF is preferably found in mammalian cells, e.g. CHO cells. Such a vWF is described in patent application WO96 / 10584.
  • vWF is described in patent application WO96 / 10584.
  • Heparin affinity chromatography results in fractions which contain molecules with a relatively low molecular weight for vWF (up to approx. 1 million Daltons). While these fractions per se have a relatively low primary hemostatic activity, they are still able to bind proteins that interact with vWF, e.g. Factor VIII to bind.
  • a gel is preferably used as the chromatography material which has good affinity chromatographic properties, i.e. low back pressure corresponding to high flow rates, high binding capacity for the ligand to be immobilized, low bleeding behavior and the possibility of disinfection with e.g. Sodium hydroxide solution.
  • the r-vWF is coupled to the solid matrix in such a way that the binding properties for the proteins to be bound in affinity chromatography are not lost.
  • standard immobilization techniques can be used for this purpose, for example in Woodward "Immobilized Cells and Enzymes", IRL. Press, Oxford, Washington (1985).
  • a chromatographic gel has good reusability properties and stability, so that even with repeated After constant use, there is a constant binding capacity for the molecules to be isolated.
  • a preferred vWF derivative therefore comprises an organic polymer, in particular an organic polymer based on carbohydrates, as the chromatography material.
  • Suitable gel matrices or particulate carriers are e.g. Sephacele, Sephadexe, Sepharosen,
  • ® also synthetic polymers, such as Toyopearl gels (Tosohaas)
  • the vWF derivative according to the invention is preferably subjected to virus inactivation treatment, so that it can be ensured that the chromatography material is not a virus contamination factor when isolating proteins which bind to vWF.
  • the treatment for inactivating viruses is preferably carried out before the derivatization by a chemical and / or physical process, for example by treatment with surfactants, polyethylene glycols, chaotropes, by heat treatment or by radiation treatment.
  • the finished derivative preferably in lyophilized form, can also be subjected to a treatment such as that of heat treatment or radiation.
  • the vWF derivative according to the invention is made available in a storage-stable form, in particular as a lyophilisate, which significantly facilitates trading, distribution and storage.
  • the present invention also relates to a device comprising a container and a vWF derivative according to the invention contained therein, the container having an inlet opening and an outlet opening which is suitable for the passage of liquids.
  • the device according to the invention is a column, in particular an affinity acid or chromatography column, trained which, if desired, can be made available as a ready-to-use product in a storage-stable form, so that only swelling of the material has to be carried out by the respective user before protein isolation.
  • the present invention also relates to a method for isolating proteins that bind to the vWF, which is characterized by the following steps:
  • This process can be carried out batchwise, that is to say as a “batch” process, or else as a column chromatography process.
  • Proteins that can be isolated using the method according to the invention are, above all, the physiological binding proteins of vWF, that is to say glycoprotein Ib, the glycoprotein Ilb / IIIa complex, collagen and in particular factor VIII, but of course also recombinant derivatives and analogs of these proteins , vWF antigens, vWF antibodies, vWF multimerases or vWF depolymerases and even enzymes that recognize the vWF as a substrate, and other natural or synthetic peptides and proteins that have an affinity for vWF.
  • vWF-binding saccharides such as e.g. Heparin to be isolated using this procedure.
  • the vWF derivative according to the invention Due to the high avidity of the vWF derivative according to the invention, it is possible to specifically bind and recover the proteins to be isolated in yields of more than 60%, preferably more than 80%, most preferably almost quantitatively, on the chromatography material according to the invention and in purified form Elute form of the vWF derivative, with which the preparation of concentrates of the isolated proteins by simple elution without subsequent the concentration step is possible.
  • the method according to the invention is particularly suitable for the production of biologically active proteins with factor VIII activity, in particular of plasmatic or recombinant factor VIII and their mutants or analogs. This can be isolated with the method according to the invention even if a starting solution which contains the factor VIII / vWF complex is used.
  • the elution of the proteins is preferably carried out with a buffer containing calcium ions.
  • factor VIII from plasma fractions containing factor VIII or from cell culture supernatants of cells which express factor VIII.
  • vWF acts as a carrier protein of factor VIII, i.e. factor VIII is bound to vWF.
  • factor VIII and vWF can only be separated using complex methods, e.g. by binding factor VIII to immobilized mono- or polyclonal antibodies directed against factor VIII, to which the factor VIII / vWF complex binds and then vWF is specifically eluted without the binding of the antibody with factor VIII being disturbed. Then factor VIII is eluted from the chromatographic gel.
  • factor VIII proteins with affinity for vWF can also be bound to immobilized r-vWF and isolated from complex mixtures, namely, for example factor VIII hybrid proteins, in particular factor VIII-heparin cofactor II hybrid protein according to US Ser.No. 08 / 558,107 or factor VIII-factor V hybrid protein according to WO 90/05570 or chimeric human / porcine factor VIII according to WO 94/11503, FVIII mutants, such as in Austria.
  • factor VIII hybrid proteins in particular factor VIII-heparin cofactor II hybrid protein according to US Ser.No. 08 / 558,107 or factor VIII-factor V hybrid protein according to WO 90/05570 or chimeric human / porcine factor VIII according to WO 94/11503, FVIII mutants, such as in Austria.
  • a 921/96 (factor VIII dB695-R2307Q), described factor VIII mutant with an Arg 23 ° 7 ⁇ Gln substitution, which has a reduced inhibitor binding with constant factor VIII: C procoagulatory activity and vWF binding activity, by Willebrand Factor-degrading enzymes, for example the vWF-specific depolymerase or vWF multimerase or platelet receptors, such as GPIIb / IIIa or GPIb / IX complex, the pure representation of which is of biochemical-analytical, diagnostic or therapeutic interest.
  • Willebrand Factor-degrading enzymes for example the vWF-specific depolymerase or vWF multimerase or platelet receptors, such as GPIIb / IIIa or GPIb / IX complex, the pure representation of which is of biochemical-analytical, diagnostic or therapeutic interest.
  • the specific isolation of therapeutic proteins is currently carried out using the method of immunoaffinity chromatography.
  • the molecule to be isolated is bound to an immobilized monoclonal antibody (which is usually obtained from mouse cells) and washed free of impurities and then eluted in high purity.
  • monoclonal antibodies can be used to deliver mouse proteins into the preparation, which, if the molecule to be bound is used therapeutically, lead to side effects such as e.g. lead to antibody formation against mouse protein.
  • vWF physiological binding proteins for vWF according to the inventive method has the further advantage that the vWF exercises a stabilizer or carrier function for its binding partners.
  • the proteins obtained are therefore protected even during isolation and purification from denaturing conditions which may be granted during the elution.
  • the products obtained are thus obtained not only in terms of their antigenicity in the high yield mentioned, but also in terms of their activity or nativity.
  • vWF multimerase Another particularly preferred protein or protein complex which can be purified with the vWF derivative according to the invention is the vWF multimerase, which degrades the high molecular forms of the vWF into low molecular weight variants (see Austrian applications A 769/96 and A 770 / 96).
  • the multimerase can bind to the immobilized vWF, but the derivatization prevents the degradation of the material according to the invention.
  • elution can be carried out particularly efficiently with a buffer which contains a chelating agent for metal ions, in particular EDTA.
  • the vWF derivative according to the invention is also suitable for optionally extracorporeal immunoadsorption of antibodies which are directed against vWF.
  • the formation of antibodies against vWF is a pathological condition that can occur as an autoimmune disease and lead to a blood clotting defect with increased bleeding tendency or can occur as a side effect of the treatment of patients with preparations containing vWF.
  • the formation of a functional inhibitory antibody makes substitution therapy impossible or leads to drastic dose increases in order to maintain the hemostatic effect of the coagulation factor concentrate. In such cases, the circulating functional antibody against the coagulation factor has been removed in the past by plasmapheresis or by extracorporeal immunoadsorption on proteins directed against IgG, for example protein A or protein G.
  • Another application of the method according to the invention for the production of antibodies lies in the preparation of polyclonal or monoclonal anti-vWF antibodies for diagnostic purposes.
  • An elution buffer which is preferably used in the elution of antibodies from the vWF derivative according to the invention, has an acidic pH, in particular a pH in the range from 2 to 5.
  • One of the main advantages of the present invention is that it is possible to purify the proteins from any starting material.
  • Particularly preferred starting fractions form fractions which are derived from a body fluid of a mammal or from a cell culture. Because of the avidity of the chromatography material according to the invention, fractions which contain the vWF and / or the factor VIII / vWF complex are also preferred.
  • a preferred embodiment variant of the method according to the invention is that it is carried out using the device according to the invention with the vWF derivative contained therein.
  • 1 and 2 show the purification of recombinant factor VIII with the vWF derivative according to the invention.
  • a CHO cell clone that produces recombinant von Willebrand factor is described in FEBS.Lett. 351 (1994), 345-348.
  • the cell line was brought to co-expression of human furin by transfection with a vector coding for the cDNA human furin (van den Ouweland et al., Nucleic Acids Res. 18 (1990), 664).
  • Such stable cell clones were fermented on a large scale in perfusion reactors on microcarriers (Blüml et al., In: Spier RE, Griffith JB, Berthold W, eds. Animal cell technology. Oxford, London: Butterworth-Heinemann, (1994), 267-269) .
  • the purification was carried out by a 2-stage chromatographic method according to Thromb.Haemost. 73: 1160 (1995).
  • the fraction desorbed by elution with sodium chloride was obtained and buffered by gel filtration through Sephadex G25 (Pharmacia) in a buffer containing 20 mM Tris-HCl, 150 mM NaCl, pH 7.5.
  • the preparation was then concentrated by ultraconcentration over an Amicon YM30 membrane (cut-off: 30,000 D) to a protein concentration of 3 mg / ml.
  • the vWF concentration in this preparation was 60 U vWF antigen / mg protein. This preparation did not contain factor VIII due to the avoidance of serum or plasma components during the production in cell culture and during the cleaning.
  • the preparation of the recombinant vWF from Example 1 was diluted to 1.5 mg / ml with a buffer containing 20 mM Tris-HCl, 150 mM NaCl, pH 7.5.
  • a preactivated gel suitable for affinity chromatography (Actigel, ALD-Superflow, from Sterogene) was excessively prewashed with a buffer containing 20 mM Tris-HCl, 150 mM NaCl, pH 7.5.
  • One volume part of the pre-washed gel was mixed with 1.1 parts by volume of protein solution to be immobilized and then 0.15 parts by volume of a solution of 0.1 M cyanoborohydride (NaCNBH 3) in 0.1 M phosphate buffer, pH 7, 0, is added.
  • NaCNBH 3 0.1 M cyanoborohydride
  • the gel was shaken suspended in this buffer and incubated for 16 hours at room temperature with further shaking.
  • the gel was then placed on a sintered funnel with 10 times the volume of a buffer containing 20 mM Tris-HCl, 150 mM NaCl, pH 7.5 and with 5 times the volume of a buffer containing 20 mM Tris-HCl, 2 M NaCl, pH 7.5.
  • the mixture was then equilibrated again with 5 parts by volume of the buffer, 20 mM Tris-HCl, 150 mM NaCl, pH 7.5 and the gel was transferred to a chromatographic column with a dimension of diameter to a gel bed height of 1: 4.
  • a recombinant factor VHI preparation (Recombine, Baxter) was mixed with 10 ml A.dest. reconstituted.
  • This solution contained 50 IU FVIII / ml, 12 mg human albumin / ml, 1.5 mg polyethylene glycol 3350 / ml and traces of vWF in a histidine saline buffer at physiological pH.
  • the low molecular weight components were separated from this solution by gel filtration on Sephadex G25 (Pharmacia) and the factor VIII / vWF / albumin mixture was brought into a buffer containing 20 mM Tris-HCl, pH 7.5.
  • a mouse monoclonal antibody directed against vWF (MAb 03768/3, from Chemicon International, Inc.) with an IgG concentration of 7 mg / ml was gel-filtered via Sephadex G25 (from Pharmacia) in a buffer, 20 mM Tris-HCl, 150 mM NaCl, pH 7.5, buffered. The protein concentration was adjusted to 0.5 mg / ml by dilution with the same buffer. 4 ml of this solution were pumped at a flow rate of 0.5 ml / min over the column containing immobilized recombinant vWF from Example 3 and the optical density at 280 nm was determined in a run.
  • the UV absorption was measured at 280 nm and the activity of the vWF-degrading enzyme was determined as follows.
  • a preparation of the recombinant vWF from Example 1 was repuffed in a buffer, 5 mM Tris-HCl, 1.5 M urea, containing 0.2% (w / v) bovine serum albumin. finished and brought to 0.4 vWF E / ml.
  • Aliquots of the fractions to be examined of 100 ⁇ l were each mixed with 5 ⁇ l of a 50 mM aqueous PPACK solution and 12.5 ⁇ l of a 200 mM BaCl 2 solution and incubated for 5 min at 37 ° C.
  • the sample 1 + 1 prepared in this way was mixed with the substrate (vWF) and 15 h at 37 ° C. on a floating dialysis membrane (Millipore VSWP) according to the method of Marusky and Sergeant (Anal. Biochem. 105 (1980), 403) dialyzed against a buffer containing 5 mM Tris-HCl, 1.5 M urea, pH 8.0. The dialysate was then analyzed for its residual vWF activity in an ELISA, in which the collagen binding activity of the vWF and its antigenicity are determined.
  • a floating dialysis membrane Millipore VSWP
  • Buffer 1 (for collagen coating and antibody dilution):
  • Bovine serum albumin pH 7.3
  • the reciprocal of the collagen binding activity is a direct measure of the enzyme activity of the vWF-degrading enzyme.
  • a plasmatic factor VHI / von Willebrand factor concentrate (IMMUNATE; Fa. Immuno) was reconstituted with 5 ml of distilled water. This solution contained 25 IU factor VHI / ml and 10 U vWF / ml (measured with the ristocetin cofactor method) in a citrate-glycine-lysine buffer, pH 7.4. The factor VHI / von Willebrand factor complex was buffered against a buffer containing 20 mM Tris / HCl, pH 7.5, by gel filtration on Sephadex G25. 3 ml of this solution were then applied directly to the column from Example 2. The flow rate was 0.1 ml / min.

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Abstract

L'invention concerne un dérivé du facteur de Von Willebrand (vWF), constitué de vWF, immobilisé sur un support, qui est caractérisé en ce que le vWF est du r-vWF, ainsi qu'un procédé pour l'isolation de protéines qui se lient au vWF à l'aide de ce dérivé de vWF.
EP97913009A 1996-12-13 1997-11-19 Derive du facteur de von willebrand ainsi que procede pour l'isolation de proteines Withdrawn EP0954533A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
AT0217896A AT405740B (de) 1996-12-13 1996-12-13 Von willebrand-faktor-derivat sowie ein verfahren zur isolierung von proteinen
AT217896 1996-12-13
PCT/AT1997/000253 WO1998025969A1 (fr) 1996-12-13 1997-11-19 Derive du facteur de von willebrand ainsi que procede pour l'isolation de proteines

Publications (1)

Publication Number Publication Date
EP0954533A1 true EP0954533A1 (fr) 1999-11-10

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Application Number Title Priority Date Filing Date
EP97913009A Withdrawn EP0954533A1 (fr) 1996-12-13 1997-11-19 Derive du facteur de von willebrand ainsi que procede pour l'isolation de proteines

Country Status (6)

Country Link
EP (1) EP0954533A1 (fr)
JP (1) JP2001506987A (fr)
AT (1) AT405740B (fr)
CZ (1) CZ211299A3 (fr)
HU (1) HUP9903789A3 (fr)
WO (1) WO1998025969A1 (fr)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5994310A (en) * 1998-09-03 1999-11-30 Bayer Corporation Peptide ligands for affinity purification of human Factor VIII
EP1835938B1 (fr) 2004-12-27 2013-08-07 Baxter International Inc. Conjugues entre polymeres et facteurs von willebrand
US7645860B2 (en) 2006-03-31 2010-01-12 Baxter Healthcare S.A. Factor VIII polymer conjugates
KR20080108147A (ko) 2006-03-31 2008-12-11 백스터 인터내셔널 인코포레이티드 페질화된 인자 ｖｉｉｉ
AU2009307648C1 (en) * 2008-10-21 2016-12-08 Takeda Pharmaceutical Company Limited Lyophilized recombinant VWF formulations
US20130230901A1 (en) * 2012-02-14 2013-09-05 Portola Pharmaceuticals, Inc. Process for making recombinant antidote to factor xa inhibitor

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL74909A (en) * 1984-04-20 1992-01-15 Genentech Inc Preparation of functional human factor viii and dna sequences,expression vectors,transformed microorganisms and cell lines used therein
DE19521313A1 (de) * 1995-06-12 1996-12-19 Max Planck Inst Fuer Physiolog Verfahren zur affinitätschromatographischen Aufreinigung von Faktor VIII
SE9503380D0 (sv) * 1995-09-29 1995-09-29 Pharmacia Ab Protein derivatives

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9825969A1 *

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Publication number Publication date
HUP9903789A2 (hu) 2000-03-28
CZ211299A3 (cs) 1999-09-15
WO1998025969A1 (fr) 1998-06-18
AT405740B (de) 1999-11-25
JP2001506987A (ja) 2001-05-29
HUP9903789A3 (en) 2002-01-28
ATA217896A (de) 1999-03-15

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