WO2000049047A1 - PROCEDE DE PRODUCTION D'UNE PREPARATION CONTENANT DU FACTEUR DE VON WILLEBRAND (vWF) - Google Patents

PROCEDE DE PRODUCTION D'UNE PREPARATION CONTENANT DU FACTEUR DE VON WILLEBRAND (vWF) Download PDF

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Publication number
WO2000049047A1
WO2000049047A1 PCT/AT2000/000039 AT0000039W WO0049047A1 WO 2000049047 A1 WO2000049047 A1 WO 2000049047A1 AT 0000039 W AT0000039 W AT 0000039W WO 0049047 A1 WO0049047 A1 WO 0049047A1
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WIPO (PCT)
Prior art keywords
vwf
pro
thrombin
preparation
treated
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PCT/AT2000/000039
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German (de)
English (en)
Inventor
Katalin Varadi
Peter Turecek
Hans-Peter Schwarz
Original Assignee
Baxter Aktiengesellschaft
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Application filed by Baxter Aktiengesellschaft filed Critical Baxter Aktiengesellschaft
Priority to AU26459/00A priority Critical patent/AU2645900A/en
Publication of WO2000049047A1 publication Critical patent/WO2000049047A1/fr

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    • 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)

Definitions

  • the invention relates to a method for producing a von Willebrand factor (vWF) preparation.
  • vWF von Willebrand factor
  • Von Willebrand factor is a glycoprotein that circulates in the plasma as a series of multimers in the size range from about 500 to 20,000 kD.
  • Multimeric forms of vWF consist of 250 kD polypeptide subunits that are connected by disulfide bridges.
  • vWF mediates the initial platelet adhesion to the sub-endothelium of the damaged vessel wall, whereby only the larger multimers also have hemostatic activity.
  • Endothelial cells are thought to secrete large polymeric forms of vWF, and those forms of vWF that have a low molecular weight (low molecular weight vWF) are formed by proteolytic cleavage.
  • the multimers with large molar masses are stored in the Weibel-Pallade bodies of the endothelial cells and released when stimulated.
  • vWF is synthesized by endothelial cells and megakaryocytes as pre-pro-vWF, which consists largely of repeated domains.
  • pro-vWF dimerizes through disulfide bridges at its C-terminal region.
  • the dimers serve as protromers for multimerization, which is controlled by free end disulfide bridges between the termini.
  • the arrangement to multimers is followed by the proteolytic removal of the pro-peptide (Leyte et al., Biochem. J. 274 (1991), 257-261).
  • the entire length of vWF c-DNA was cloned.
  • the pro-polypeptide corresponds to amino acid residues 23 to 764 of the pre-pro vWF of the entire length (Eikenboom et al., Haemophilia 1 (1995), 77 to 90).
  • pp-vWF pro-peptide of vWF
  • pp-vWF pro-peptide of vWF
  • vWD the pro-peptide of vWF
  • pp-vWF is identical to that of Willebrand antigen II, the second identified antigen, which is absent in the plasma and in the platelets of patients with severe von Willebrand disease (vWD) is.
  • pp-vWF is specifically synthesized in platelets and endothelial cells.
  • the molecular ratio of pp-vWF to vWF in plasma is 1:10, the plasma concentration of pp-vWF is about 5 nmol / 1.
  • pp-vWF is released from endothelial cells after activation by various agonists.
  • Von Willebrand's disease (vWD; von Willebrand syndrome; Willebrand-Jürgens syndrome) is generally characterized by a decrease or a structural defect in vWF, which can result in a lack of platelet aggregation and adhesion to the sub-endothelium and thus defects in primary hemostasis . These defects cause prolonged bleeding times in vWD patients.
  • vWF For the purification of vWF, a whole series of different, mostly chromatographic processes have been described.
  • the source material for the vWF preparation often contains a mixture of mature vWF and vWF precursors such as pro-vWF, or even contains only (pre-) pro-vWF (e.g. in the case of recombinantly produced preparations) it is necessary in the production of vWF - Preparations to convert the (pre-) ro-vWF into mature vWF.
  • protease-processing proteases are also described in AT-PS 404 554 and AT-PS 404 359.
  • Thrombin (factor Ha of blood coagulation) is an enzyme from prothrombin in the blood plasma with a molecular weight of approximately 35,500. Thrombin converts the fibrinogen molecules into fibrin monomers by splitting off fibrinopeptides A and B, which spontaneously aggregate into fibrin polymers. Furthermore, factor XIII, factor VIII and protein C are activated by thrombin. Thrombin therefore finds a role as a proteolytic enzyme in the production of these activated proteins (cf. EP 0 416 890 A regarding the production of active recombinant human protein C by treatment with immobilized thrombin).
  • the methods for activating pro-vWF described in the prior art are either very cumbersome or were associated with low yields or could not be used on an industrial scale, or only to a limited extent, owing to the insufficient commercial or biological availability of the process components.
  • the object of the present invention is therefore to avoid the disadvantages of the prior art and to provide a method for activating pro-vWF which is highly selective and can nevertheless be used on an industrial scale.
  • a method for producing a vWF preparation from pro-vWF which is characterized in that pro-vWF is treated with thrombin, whereby vWF is generated and a preparation of mature vWF can thus be obtained.
  • a solution containing pro-vWF is preferably treated with thrombin, in principle any solution containing pro-vWF is suitable.
  • Pro-vWF can also be matured as an immobilized product on a solid support.
  • pro-vWF is preferably treated with immobilized thrombin, since this enables the protease reaction to be controlled even more efficiently and the frequent use of the immobilizate and the continuous implementation of the treatment are made possible in a simple manner.
  • Methods for immobilizing thrombin are well known in the art. The fact that the immobilization of thrombin leaves its pro-vWF-cleaving activity essentially unaffected presents another decisive advantage - 5 - part of this embodiment.
  • the starting material containing pro-vWF for the process according to the invention is not critical for the present invention.
  • Possible starting materials are blood serum, blood fractions, plasma, colostrum or milk from transgenic animals or cell culture solutions, in particular cells that were generated using recombinant DNA technology (see, for example, FEBS letters 351 (1994) 345-348 or Blood 88 (8) 1996 2951 -2958).
  • the pro-vWF-containing starting material is preferably subjected to one or more purification steps before the thrombin treatment, so that the pro-vWF-containing solution contains pro-vWF in a purified form.
  • Suitable pre-purification steps include precipitation (for example with ethanol, ammonium sulfate or PEG), adsorbing (for example treatment with aluminum hydroxide) or chromatographic processes (for example affinity, gel or ion exchange chromatography).
  • Such pretreatment methods are particularly favorable if the source material containing pro-vWF contains substances which can interfere with the proteolytic processing of pro-vWF by thrombin, for example inhibitors of thrombin or the like.
  • thrombin inhibitors in the starting material does not in principle have to hinder the successful implementation of the process according to the invention (since thrombin is generally prescribed in excess quantities anyway), such inhibitors can, for example, improve the yield of the process or reduce the lifespan of the thrombin immobilizate so that a simple pre-cleaning step often appears advisable (especially if thrombin inhibitors are present).
  • pro-vWF it is also possible for pro-vWF to be present in the vWF-containing solution which is subjected to the thrombin treatment in a mixture with other, preferably also at least partially purified proteins. This is particularly the case if pro-vWF has been separated from other proteins or components of the starting solution by partial purification, but other proteins, especially those that naturally have a certain affinity for pro-vWF or vWF, in the same way as with thrombin solution to be treated are available.
  • Such proteins preferably include activators and inhibitors of thrombin, such as, for example, heparin, calcium, thrombomodulin, antithrombin III, peptide inhibitor, non-specific thrombin inhibitors, thrombin mutants, prothrombin, alpha 2 -macroglobulin, factor VIII, collagen, surface proteins of platelets.
  • thrombin such as, for example, heparin, calcium, thrombomodulin, antithrombin III, peptide inhibitor, non-specific thrombin inhibitors, thrombin mutants, prothrombin, alpha 2 -macroglobulin, factor VIII, collagen, surface proteins of platelets.
  • pro-vWF or the pro-vWF-containing solution can be bound to collagen and then processed with thrombin. It has been shown here that by binding the pro vWF to the collagen, the pro-peptide cleavage can take place with reduced concentrations of throbine or the processing process can be accelerated.
  • the mature vWF no longer has to be separated from the pp-vWF by chromatographic methods after the vWF has been processed using thrombin.
  • pp-vWF can be separated using a suitable chromatography material, and it has been shown that the purification of pp-vWF using lentil-lectin-Sepharose is particularly advantageous.
  • the pro-vWF-containing solution is produced by a biotechnological logical method, for example by a cell or tissue incubation method. It is particularly preferred that pro-vWF is expressed by a transformed cell. In the case of pro-vWF-containing solutions produced using biotechnology, pro-vWF is preferably expressed by a cell culture, in particular by a mammalian cell culture.
  • a chromatographic purification step for vWF is particularly preferred.
  • cleaning processes are, for example, in EP 0 503 991 A, in WO 89/12 065 A, in EP 0 469 985 A, in EP 0 383 234 A, WO 96/10 584 A, and EP 0 705 846 A and WO 98/38 219 A described.
  • vWF and thrombin together, for example after co-expression or concerted purification and cleavage from a source material which contains both pro-vWF and thrombin, since it has been shown according to the invention that vWF formed is not further proteolytically degraded by the mere presence of thrombin.
  • Obtaining vWF and thrombin in a combination preparation is therefore possible in a simple manner by not separating thrombin from the resulting vWF after proteolytic treatment to generate vWF.
  • the process product - ie the mature vWF - with or without thrombin can optionally also contain a cleavage product of the pro-sequence of the vWF generated by the thrombin treatment.
  • This fission product characterizes the new process product.
  • pp-vWF or its cleavage product which arises from the treatment of pro-vWF with thrombin, from the proteolytically treated solution.
  • pp-vWF can be obtained by treatment with immobilized heparin.
  • pp-vWF can be used in accordance with Austrian application A 917/97.
  • Pp-vWF preparations as well as preparations that additionally contain about vWF, factor VIII, other activated blood coagulation factors, blood factors with FEIB activity and FEIBA can also be used for the treatment of patients at risk for blood coagulation disorders or for patients with vWD, phenotypic Hemophilia, Hemophilia A and factor VIII inhibitors can be used.
  • the present invention relates to a vWF preparation which, in addition to (mature) vWF, also contains thrombin.
  • a vWF preparation which, in addition to (mature) vWF, also contains thrombin.
  • Such a preparation can be used, for example, for direct local application to wounds or bleeding tissue to improve or support primary hemostasis and wound healing.
  • Such a combination preparation of vWF and thrombin preferably also contains calcium ions, since the calcium ions present in the wound may not be sufficient for the thrombin or wound enzymes to function optimally.
  • vWF preparation produced according to the invention which can optionally also contain thrombin and pp-vWF, has preferably been made virus-safe by treatment for virus inactivation or removal.
  • the virus inactivation or removal treatment can be carried out by any treatment accepted as efficient.
  • the pharmaceutical composition is treated with surfactants and / or heat, for example by means of heat treatment in solid State, in particular by means of a steam treatment according to EP 0 159 311, EP 0 519 901 or EP 0 637 451, by means of a hydrolysis treatment according to EP 0 247 998, by means of a radiation treatment or by means of a treatment with chemical or chemical / physical processes , for example with chaotropic agents according to WO 94/13 329, by means of a treatment with organic solvents and / or surfactants according to EP 0 131 740 or by means of photo inactivation.
  • Filtration techniques such as depth filtration or nanofiltration, are also a preferred method for virus depletion in the context of the present invention.
  • the vWF (/ thrombin combination) preparation according to the invention is preferably provided as a pharmaceutical preparation and furthermore contains suitable pharmaceutically acceptable carriers and / or suitable buffer, auxiliary, preservation and / or stabilizing substances such as carbohydrates, salts or protease inhibitors - or co-factors.
  • the pharmaceutical preparation is preferably made available by packaging for storage in the lyophilized or frozen state. In the manufacture of a pharmaceutical vWF preparation, further purification and virus inactivation steps are particularly preferred before the final formulation and packaging.
  • the present invention relates to a set for producing the preparation according to the invention or a vWF preparation, which contains component pro-vWF and component B thrombin.
  • components A and B are combined, this combination being possible both by in vitro mixing of the two components, but also in situ, for example on a wound.
  • component A of the set according to the invention can further contain fibrinogen or (fibrin) adhesive protein.
  • pro-vWF Ag ( ⁇ ), pp-vWF-Ag ( ⁇ ), total vWF: Ag (•) and collagen binding activity (CBA, O)
  • rp-vWF human recombinant vWF preparation
  • pro-vWF pro-vWF was made by expression in CHO cells.
  • a buffer containing 25 mM HEPES, 175 mM NaCl and 1 mg / ml BSA, pH 7.35 was used for the dilutions.
  • the thrombin reaction was stopped by adding 450 ⁇ m PPACK and the samples were frozen at -80 ° C. for later analysis.
  • FIG. 3A shows the time curve of the thrombin-mediated disappearance of the pro-vWF antigen in an rp-vWF preparation in a pro-vWF ELISA.
  • the changes in the band pattern of the pro-vWF and mature vWF are shown in Fig. 3B.
  • the upper band represents the pro-vWF monomer
  • the lower band the mature vWF monomer.
  • the time of 120 minutes of the processing mixture (rprovWF and thrombin) showed the SLS_RPPMV sequence of the mature vWF and the AEGT_G_SS sequence of the pro-peptide in approximately equimolar amounts in addition to the heavy and light chain sequences of thrombin, which were also present. Possible other degradation products, if present, were below the detection limit of the system, which confirms that thrombin specifically cleaves the pro-peptide from vWF.
  • vWF is an adhesive protein that can bind to different components of the extracellular matrix. It was tested whether the binding of vWF to a surface ligand leads to a change in the tertiary structure of the vWF multimers, and therefore by. the proteolytic cleavage sites are made more accessible to thrombin.
  • rp-vWF which is bound to a carrier under comparable conditions by means of a monoclonal vWF antibody, is processed only very slowly and an at least 10-fold higher thrombin concentration must be used in order to obtain the same processing effect as with collagen-bound vWF.
  • the solution containing pp-VWF was purified by applying the solution to a Lentil-lectin-Sepharose column. Pp-vWF binds to the column material. After washing the column and eluting the non-binding proteins, pp-vWF was eluted. The fractions obtained were concentrated, dialyzed against physiological buffer and frozen at -80 ° C.

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  • Health & Medical Sciences (AREA)
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  • Organic Chemistry (AREA)
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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract

L'invention concerne un procédé de production d'une préparation contenant du facteur de von Willebrand (vWF), à partir d'un pro-facteur de von Willebrand (pro-vWF), qui se caractérise en ce que ledit pro-vWF est traité avec de la thrombine, ce qui provoque la production de vWF et permet d'obtenir une préparation contenant du vWF.
PCT/AT2000/000039 1999-02-19 2000-02-15 PROCEDE DE PRODUCTION D'UNE PREPARATION CONTENANT DU FACTEUR DE VON WILLEBRAND (vWF) WO2000049047A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU26459/00A AU2645900A (en) 1999-02-19 2000-02-15 Method for producing a vwf preparation

Applications Claiming Priority (2)

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AT28399A AT407750B (de) 1999-02-19 1999-02-19 Verfahren zur herstellung einer vwf-präparation
ATA283/99 1999-02-19

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006071801A2 (fr) 2004-12-27 2006-07-06 Baxter International Inc Conjugues entre polymeres et facteurs von willbrand
WO2010075132A1 (fr) * 2008-12-23 2010-07-01 Baxter International Inc. Facteur de von willebrand recombinant utilisé comme marqueur de poids moléculaire dans des analyses par spectrométrie de masse
US8053561B2 (en) 2006-03-31 2011-11-08 Baxter International Inc. Pegylated factor VIII
US8058411B2 (en) 2007-05-18 2011-11-15 Baxter International Inc. Method for producing mature VWF from VWF pro-peptide
US9127264B2 (en) 2007-12-31 2015-09-08 Baxalta Incorporated Substantially animal protein-free recombinant furin and methods for producing the same
WO2019183290A1 (fr) 2018-03-21 2019-09-26 Baxalta Incorporated Séparation de vwf et de propeptide de vwf par des procédés chromatographiques
US11020458B2 (en) 2006-03-31 2021-06-01 Takeda Pharmaceutical Company Limited Factor VIII polymer conjugates

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0416890A1 (fr) * 1989-09-05 1991-03-13 Eli Lilly And Company Procédé d'activation de la protéine C
EP0775750A2 (fr) * 1995-11-24 1997-05-28 IMMUNO Aktiengesellschaft Préparation de protéines à partir de pro-protéines avec des protéines de fusion dérivés de furin ou d'analogues de furin
WO1997041206A2 (fr) * 1996-04-29 1997-11-06 Immuno Aktiengesellschaft Multimerase purifiee

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0416890A1 (fr) * 1989-09-05 1991-03-13 Eli Lilly And Company Procédé d'activation de la protéine C
EP0775750A2 (fr) * 1995-11-24 1997-05-28 IMMUNO Aktiengesellschaft Préparation de protéines à partir de pro-protéines avec des protéines de fusion dérivés de furin ou d'analogues de furin
WO1997041206A2 (fr) * 1996-04-29 1997-11-06 Immuno Aktiengesellschaft Multimerase purifiee

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
PHILIP J.FAY ET AL: "Propolypeptide of von Willebrand Factor Circulates in Blood and Is Identical to von Willebrand Antigen II", SCIENCE, vol. 232, 1986, XP000885936 *
ROB J.HAMMER ET AL.: "The effect of thrombin on the complex between factor VIII and von Willebrand factor", EUR.J.BIOCHEM., vol. 167, no. 2, 1987, pages 253 - 9, XP000886716 *
TANA N.MAYADAS ET AL.: "In Vitro Multimerization of von Willebrand Factor is Triggered by Low pH", J.BIOL.CHEM, vol. 264, 1989, pages 13497 - 503, XP000885940 *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8076463B2 (en) 2004-12-27 2011-12-13 Baxter International, Inc. Polymer-von Willebrand factor-conjugates
US8835388B2 (en) 2004-12-27 2014-09-16 Baxter International Inc. Polymer von Willebrand factor-conjugates
US7884075B2 (en) 2004-12-27 2011-02-08 Baxter International Inc. Polymer-factor VIII-von Willebrand factor-conjugates
WO2006071801A2 (fr) 2004-12-27 2006-07-06 Baxter International Inc Conjugues entre polymeres et facteurs von willbrand
US8357779B2 (en) 2004-12-27 2013-01-22 Baxter International Inc. Polymer-von Willebrand factor-conjugates
US8053561B2 (en) 2006-03-31 2011-11-08 Baxter International Inc. Pegylated factor VIII
US11020458B2 (en) 2006-03-31 2021-06-01 Takeda Pharmaceutical Company Limited Factor VIII polymer conjugates
US8058411B2 (en) 2007-05-18 2011-11-15 Baxter International Inc. Method for producing mature VWF from VWF pro-peptide
US9127264B2 (en) 2007-12-31 2015-09-08 Baxalta Incorporated Substantially animal protein-free recombinant furin and methods for producing the same
EP3521422A1 (fr) 2007-12-31 2019-08-07 Baxalta GmbH Furine recombinante essentiellement exempte de protéines animales et ses procédés de fabrication
WO2010075132A1 (fr) * 2008-12-23 2010-07-01 Baxter International Inc. Facteur de von willebrand recombinant utilisé comme marqueur de poids moléculaire dans des analyses par spectrométrie de masse
WO2019183290A1 (fr) 2018-03-21 2019-09-26 Baxalta Incorporated Séparation de vwf et de propeptide de vwf par des procédés chromatographiques
US10934340B2 (en) 2018-03-21 2021-03-02 Baxalta Incorporated Separation of VWF and VWF propeptide by chromatographic methods
CN112533940A (zh) * 2018-03-21 2021-03-19 百深公司 通过色谱方法分离vwf和vwf前肽

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ATA28399A (de) 2000-10-15
AU2645900A (en) 2000-09-04

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