Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/16—Extraction; Separation; Purification by chromatography
  • C07K1/18—Ion-exchange chromatography
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• • 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 present invention relates to the field of purification of factor VIII and von Willebrand factor, for their use as drug active ingredient.
• Factor VIII is a plasma protein present in low concentrations in human plasma. Yet it is the focal point of the coagulation cascade. Indeed, this protein acts as factor IX cofactor (or "FIX") in order to activate factor X (or "FX"). Once activated, factor X converts prothrombin to thrombin, which in turn converts fibrinogen to fibrin, resulting in the formation of the hemostatic fibrin clot.
• factor IX cofactor or "FIX”
• Factor VIII concentrates are most often prepared from a fraction of cryoprecipitated human plasma.
• the purity of Factor VIII concentrates generally obtained in industrial centers for the treatment of human plasma is often of the order of 1 IU / mg and generally does not exceed the limits of 10 to 20 IU / mg.
• Conventional production techniques involve precipitation steps that aim to eliminate, often very imperfectly, protein contaminants such as fibrinogen, fibronectin, and immunoglobulins. These techniques can use or combine precipitation at low temperature (10 0 C), or the addition of protein precipitation agents; thus hydrophilic polymers such as PEG (Newman et al., Br. J.
• Factor VIII concentrates were also produced by incorporating into the production protocol a contact with porous silica beads to trap low molecular weight protein contaminants (Margolis et al., Vox Blood 46: 341-348, 1984). .
• the specific activity of the product remains relatively low: 1 IU / mg.
• Factor VIII of very high purity totally devoid of proteins of foreign origin such as antibodies of animal origin, by means of processes applicable on an industrial scale.
• EP 0 343 275 discloses a method for preparing Factor VIII from a cryoprecipitate which is characterized in that, prior to the viral inactivation treatment, the cryoprecipitate is suspended in water containing 1 to 3 U / ml of heparin at pH 6.5-7.5 is reacted with a suspension of aluminum hydroxide and after cooling to 10-18 ° C. and adjusting the pH to 6-7, centrifuging or filtering and then the purification is continued by a subsequent treatment, in particular by chromatography on an ion exchange resin such as Fractogel-DEAE (today DEAE-TOYOPEARL®, marketed by Tosoh Bioscience), of hydrophilic type.
• Fractogel-DEAE today DEAE-TOYOPEARL®, marketed by Tosoh Bioscience
• the document EP 0 359 593 describes a method of purification by anion exchange chromatography, which makes it possible to separate the desired proteins in a single chromatographic column under conditions sufficiently gentle to render the subsequent treatments useless. This process allows separation.
• Factor VIII, fibrinogen, fibronectin and von Willebrand factor proteins of human or animal plasma This method can be summarized thus: the fraction of the cryoprecipitate resolubilized in water is subjected to a single chromatographic separation on an anion exchange resin whose matrix is a macroreticulated vinyl polymer type gel capable, by virtue of its properties of porosity, to retain the Factor VIII-von Willebrand factor complex, and then selectively recover the different proteins by successive increases in the ionic strength of the elution buffer.
• Von Willebrand factor (hereinafter also referred to as "VWF”) plays an essential role in hemostasis by two distinct functions: as adhesion protein, it allows the dispersion, adhesion and aggregation of blood platelets. on the vascular subendothelium, and thus participates in the process of rapid healing of the injured vessels and, on the other hand, it ensures the stabilization and transport of Factor VIII in the bloodstream, which it is associated non-covalently.
• Congenital VWF deficiency or structural abnormality of VWF causes von Willebrand disease, which is manifested by cutaneous haemorrhages and mucous membranes. This disease is very heterogeneous in its clinical expression and poses serious problems in case of surgery.
• vWF-enriched human plasma derivatives eg, the cryoprecipitated fraction of plasma or Factor VIII concentrates containing sufficient VWF associated therewith.
• VWF is a protein difficult to purify.
• the Von Willebrand factor is the largest known protein circulating in the plasma. It consists of a set of multimers linked by disulfide bridges, whose base element has a molecular weight close to 260 kilodaltons (kDa).
• the smallest form of vWF in plasma is a dimer of 440-500 kDa and the largest forms are multimers of this dimer whose molecular weight can reach 20 million daltons.
• This assembly of the subunits into multimers may be specific for the cells in which it is operated, the VWF being synthesized and polymerized in the megakaryocytes and in the endothelial cells.
• VWF concentrates typically involve steps of precipitation of a plasma fraction, intended for the removal of most of the unwanted proteins (fibrinogen, fibronectin etc.), and / or chromatographies (exchange of ions, affinity, immunoaffinity, steric exclusion etc.) which aim to obtain very high purity concentrates with a high specific activity, and which make it possible to preserve the integrity of the multimeric forms, especially those high molecular weight whose biological importance is fundamental in the healing processes.
• Patent EP 0 503 991 discloses a process for the preparation on an industrial scale of a FvW concentrate comprising a step of prepurification of a cryoprecipitated fraction of plasma and three successive stages of chromatography, the third being an affinity chromatography on gelatin column immobilized on agarose.
• the FvW concentrate thus obtained has a specific activity greater than 100 VWF: RCo / mg expressed in units of ristocetin cofactor activity per mg of protein and a level of multimers of high molecular weight comparable to that of the starting plasma.
• Patent Application EP 0 934 748 describes a process for the preparation of VWF comprising the combination of anion exchange chromatography and cation exchange chromatography.
• the VWF fractions obtained have a specific activity greater than 100 IU FvW: Ag / mg expressed in units of VWF antigen per mg of protein, but contain still significant proportions of Factor VIII.
• US Pat. No. 6,579,723 describes a process for preparing a highly purified vWF by immunoaffinity chromatography, the immunoadsorbents of which are anti-FvW antibodies. It can also be provided an additional purification step by affinity chromatography on heparin.
• the disadvantage of immunoaffinity purification is the possible presence of residual antibodies that can lead to immunological reactions.
• EP 0 383 234 teaches the preparation of a FvW concentrate by anion exchange chromatography, carried out with acid solutions (pH 5.5 to 6.5) containing carbohydrates, for fixing the factor VIII on the anion exchanger.
• acid solutions pH 5.5 to 6.5
• EP 1 632 501 discloses a process for the preparation of a very high purity von Willebrand factor concentrate from a von Willebrand factor-containing organic fraction comprising anion exchange chromatography separation using a carrier. of vinyl polymer, weak base type. This method has the advantage of being very simple to implement, resulting in a Von Willebrand factor of high specific activity and containing very little factor VIII.
• FVIII and VWF are very important plasma proteins, and their deficiency in some individuals leads to severe hemostasis disorders. That is why it is of paramount importance to develop processes for the preparation of these proteins, making it possible to obtain products of purity adapted to their repeated use in patients.
• the invention relates to a method for purifying a solution containing a mixture of FVIII and FvW, or a solution containing vWF or a solution resulting from a secretion of an animal, in particular a human, or a plant extract containing FVIII, characterized in that it comprises a chromatography step on an ion exchange chromatography filter membrane for adsorbing at least one protein selected from FVIII and VWF.
• the subject of the invention is a process for purifying FVIII or FvW from a solution chosen from (i) a solution containing a mixture of FVIII and FvW, (ii) a solution containing FvW, (iii) a solution derived from a secretion of a non-human animal and (iv) a solution derived from a plant extract containing FVIII, said method being characterized in that it comprises a step of adsorption of FVIII or VWF on an ion exchange chromatography membrane filter.
• purification process is meant a process for separating FVIII from other molecules in the medium, or a method for separating FvW from other molecules in the medium, or a method for separating FVIII from VWF. , or a method for separating the FVIII / FvW complexes from other molecules contained in the medium.
• These molecules may be different proteins from FVIII and VWF, viruses, bacteria, spores, culture medium, fetal calf serum, this list not being limiting.
• FVIII any form of FVIII, in particular capable of acting as a cofactor in the activation of FIX and having the capacity to form a complex with VWF, in particular mature FVIII, biologically active derivatives of FVIII.
• pro-FVIII which contains the pro-peptide (pro-FVIII)
• protein constructs comprising immature VWF, precursor of FVIII (pre-pro-FVIII), mature FVIII obtained after cleavage of the signal peptide and pro-peptide.
• Other biologically active derivatives of FVIII included in the invention are pro-drugs which undergo post-translational modifications or which are converted into biologically active forms, such as truncated forms, deleterious forms, for example FVIII deleted.
• FVIII one or more amino acids located in the region between Arg-759 and SeM 709 described in EP 218,712, chimeric forms, and forms that have different post-translational modifications of mature natural plasma forms.
• FVIII may for example be manufactured by modifying mature FVIII or any other form naturally present in the blood.
• the nucleotide sequence coding for such an FVIII can come from different sources, preferably mammalian, including human, porcine, ovine, bovine, equine, and goat versions, this list not being limiting.
• vWF is meant any form of vWF, especially mature vWF, biologically active derivatives of mature vWF, such as pro-vWF which contains the propeptide, protein constructs comprising immature vWF, including the precursor of FvW (pre-pro-FvW), propeptide of FvW (pro-FvW), mature vWF obtained after cleavage of signal peptide and pro-peptide.
• Other biologically active derivatives of VWF included in the invention are prodrugs that undergo post-translational modifications or that are converted into biologically active forms, such as truncated forms, deleterious forms, for example vWF lacking a domain.
• vWF can for example be manufactured by modifying mature vWF or any other form naturally present in the blood.
• the nucleotide sequence coding for such a VWF can come from different sources, preferably mammalian, including human, porcine, ovine, bovine, equine, and goat versions, this list not being limiting.
• solution containing a mixture of FVIII and FvW denotes any solution containing FVIII and FvW, in complexed or separated form. These solutions may be of recombinant origin, transgenic or plasmatic.
• the solution is of recombinant origin, it is derived from a unicellular system, in which the expression of FVIII and FvW proteins has been induced.
• the animal or human cell lines transfected with a vector containing the gene coding for each of these proteins, preferably the gene coding for the human protein, said lines being able to be selected especially from the CHO-K, CHO-LeClO, CHO Lec-1, CHO Pro-5, CHO dhfr-, Wil-2, Jurkat, Vero, Molt-4, COS-7, 293-HEK, YB2 / 0 lines.
• the solution is of transgenic origin, it is derived from a multicellular system, in particular an animal or a plant obtained by transgenesis, that is to say in which one or more cells have received a molecule of Recombinant DNA.
• a multicellular system in particular an animal or a plant obtained by transgenesis, that is to say in which one or more cells have received a molecule of Recombinant DNA.
• dogs, cats, mice, rats, hamster, cows, goats, sheep, rabbits and pigs, horses, insects and plants, for example the tobacco, soy this list is not limiting.
• animals producing FVIII and VWF this production can take place in various media secreted by the animal, for example urine, blood, saliva or milk, this list not being limiting.
• Such production methods can be carried out using techniques well known to those skilled in the art.
• document EP 0 741 515 and EP 807 170 may be cited, this list not being limiting.
• the latter describes the production of a transgenic animal which has stably integrated into its genome the DNA molecules encoding FVIII and VWF, so as to both express them and secrete them in their milk.
• the solution in the case where the solution is of plasmatic origin, it can be either plasma, animal or human, or cryoprecipitate, or a fraction obtained by conventional fractionation methods (Cohn et al., J. Am. Chem. Soc. , 68, 459, 1946 and Kistler et al., Vox Sang., 7, 1962, 414-424). These fractions may optionally have undergone a prepurification treatment such as by adsorption on aluminum hydroxide.
• solution containing FvW any solution containing vWF, and essentially free, that is to say containing little or very little of FVIII. This solution may be of recombinant origin, transgenic or plasma origin.
• the solution is of recombinant origin, it is derived from a unicellular system, in which the expression of the FvW protein has been induced.
• a vector containing the gene coding for this protein preferably the gene coding for the human protein, said lines being able to be selected in particular from CHO-K lines, CHO-LeCI O, CHO Lec-1, CHO Pro-5, CHO dhfr-, Wil-2, Jurkat, Vero, Molt-4, COS-7, 293-HEK, YB2 / 0, BHK , K61-I6, NSO, SP2 / 0-Ag14 and P3X63Ag8. 653, SK-Hep, HepG2, as well as plant cells, bacterial systems, for example E. CoIi, fungal systems, systems using viruses, in particular baculoviruses, this list not being limiting.
• Such cell systems express the FvW protein and can be obtained using techniques well known to those skilled in the art.
• US 5,198,349 and WO 89/06096 may be cited, this list not being limiting.
• US 5,198,349 thus describes the expression of human VWF in COS cells, by inserting the cDNA encoding human vWF into an expression vector.
• the solution is of transgenic origin, it is derived from a multicellular system, in particular an animal or a plant obtained by transgenesis, that is to say in which one or more cells have received a molecule of Recombinant DNA encoding VWF.
• a multicellular system in particular an animal or a plant obtained by transgenesis, that is to say in which one or more cells have received a molecule of Recombinant DNA encoding VWF.
• transgenic animals producing VWF this production can take place in various media secreted by the animal, for example urine, blood, saliva or milk, this list not being limiting.
• Such production methods can be carried out using techniques well known to those skilled in the art.
• the documents WO 2001/022810 and WO1999 / 058699 can be cited, this list not being limiting.
• WO 2001/022810 describes the production of transgenic mice producing human vWF in female milk.
• the solution in the case where the solution is of plasmatic origin, it may be a fraction of animal or human plasma, either cryoprecipitate, or a fraction obtained, obtained by conventional fractionation methods (Cohn et al., J. Am. Chem. Soc., 68, 459, 1946 and Kistler et al., Vox Sang., 7, 1962, 414-424). These fractions may optionally have undergone a prepurification treatment such as by adsorption on aluminum hydroxide.
• solution derived from a secretion of a non-human animal containing FVIII is meant any solution resulting from any secretion, for example plasma, saliva, urine or milk, produced by an animal transgenic manufactured so that it expresses a molecule of FVIII in one of the secretions mentioned above. In this case, VWF is not expressed by the transgenic animal.
• solution from the plasma containing FVIII is also meant any solution from plasma naturally containing FVIII, human or animal, and essentially free of VWF. It can be derived from a fraction of animal or human plasma, either cryoprecipitate, or a fraction obtained by conventional fractionation methods (Cohn et al., J.
• fractions may optionally have undergone a prepurification treatment such as by adsorption on aluminum hydroxide.
• Such an extract can be made by introducing into a plant cell a nucleotide vector containing the gene encoding FVIII.
• Such methods are well known to those skilled in the art, and can be illustrated by many documents of the state of the art, such as for example the document US 2005/0060775.
• ion exchange chromatography filter membrane any semi-permeable physical barrier having the ability to adsorb FVIII and / or VWF by ion exchange when these proteins are entrained through the membrane.
• this membrane has the ability to pass FVIII and VWF when the ion exchange interaction between the membrane and FVIII and / or VWF is no longer sufficient to retain them on the membrane.
• an ion exchange filter membrane consisting of a macroporous support comprising, immobilized on said support, a negatively charged or positively charged coating, said coating imparting to the filter membrane the ion exchange properties.
• the negatively or positively charged coating is immobilized on the macroporous support by chemical grafting.
• the macroporous support has a porosity, that is to say an average pore size, such that the filtering membrane allows FVIII and FvW to pass through.
• a first advantage related to the use of such a membrane is the possibility of using disposable exchange membrane filters, which increases the level of safety of the process.
• a second advantage related to the use of such a membrane is the possibility of carrying out the purification process of the invention, and more precisely the ion exchange chromatography step (s), with a very high flow rate. high of the solution to be purified.
• any type of physical barrier support may be suitable for a filter membrane adapted to the implementation of the invention, for example a polymer film, capillary network, hollow fiber, stabilized cellulose, polyethersulfone, or any three-dimensional structure having the capacity of pass the FVIII and the VWF.
• the ion exchange interaction between the filter membrane and the FVIII and FvW proteins is due to the positively or negatively charged coating which is immobilized on the macroporous support, said coating having basic or acidic functional groups that can be exchanged.
• the ion exchange coating may be of the monofunctional type, that is to say comprising only a variety of functional group, or of polyfunctional type if it comprises different types of functional groups.
• the filter membrane allows simultaneous capture or adsorption of the FVIII and VWF proteins, thanks to the interaction between these proteins and the membrane.
• VWF and FVIII are retained by the membrane through ion exchange interactions.
• This pre-purification step may be advantageous especially in the case of complex solutions, for example from milk or plasma, which is a medium containing many proteins.
• a pre-purification step may in particular comprise a clarification step, for example as described in document WO 2004/076695, or an extraction step as described for example in documents FR 06 04864 or FR 06 1 1536 , this list not being limiting.
• the document FR 06 04864 describes a process for extracting at least one protein present in milk, said protein having an affinity for the complexed or non-complexed calcium ions of said milk, comprising the following steps: ) release the protein by precipitating calcium compounds obtained by contacting the milk with a soluble salt, for example sodium phosphate, whose anion is chosen for its ability to form in such a medium said insoluble calcium compounds to thereby obtain a liquid phase enriched in the protein,
• a soluble salt for example sodium phosphate
• the document FR 06 1 1536 describes a method for extracting a protein present in milk, having at least one hydrophobic bag and a negative charge at the natural pH of the milk, comprising the following steps of: a) Skimming and delipidation of said milk, b) Passage of the delipidated and skimmed fraction containing said protein on a chromatographic support on which is grafted a ligand having both a hydrophobic character and an ionic character, for example 4-Mercapto-Ethyl-Pyridine under pH conditions allowing said protein to be retained on said support, c) eluting the protein, d) purifying the eluted fraction by removing milk proteins from said eluted fraction, and e) recovering said protein.
• the prepurification step may be performed immediately after the cell culture step itself.
• the composition of the cell culture medium can be controlled so that the proteins produced by the cell are excreted in the extracellular medium.
• the choice of cells can be made in such a way that the protein produced is excreted in the medium.
• a depth filtration step or a tangential micro-filtration may be suitable to obtain a pre-purification adapted to the implementation of the steps of the purification process of the invention.
• the filter membrane is a cation exchange membrane.
• the positive counter-ions of the functional groups carried by the membrane are exchanged by charges of the same sign on VWF and FVIII.
• CM carboxymethyl
• SP phosphoryl and sulfopropyl
• S sulfate
• the buffers that may be used are Tris-hydroxymethyl-aminomethane, carbonate, ethylene diamine, imidazole or triethanolamine, this list not being limiting.
• the pH of the buffer will be chosen according to the isoelectric point of the protein, so that the protein is at this overall positive charge pH, according to routine techniques well known to those skilled in the art.
• the filtering membrane used is an anion exchange membrane. The negative counter-ions of the functional groups carried by the membrane are exchanged by charges of the same sign on the VWF and the FVIII.
• the buffers that may be used are acetate, citrate, phosphate, glycine or barbiturate, this list not being limiting.
• the pH of the buffer will be selected according to the isoelectric point of the protein, so that the protein is at this overall negative charge pH, according to routine techniques well known to those skilled in the art. .
• the filter membrane comprises an ion exchange coating consisting of a strong anion exchanger.
• strong anion exchanger any anion exchange membrane capable of adsorbing weakly ionized proteins.
• Such filter membranes are commercially available.
• membranes carrying QAE, Q groups in particular the Mustang Q® membrane (PaII) or the Sartobind Q membrane (Sartorius), this list not being limiting.
• the Mustang Q membrane (PaII) is particularly advantageous because it has a high adsorption capacity, high volume chromatography flow rates, a single-use or multi-cycle capability. Moreover, it makes it possible to get rid of the steps of validations of cleaning of the support, such as the regeneration and the sanitization (study viral security, prions, this list not being limiting).
• the surface of the filter membrane in contact with the solution to be purified comprises an anion exchange coating comprising quaternary ammonium groups which are immobilized on the macroporous support by chemical grafting.
• the filtering membrane is of macroporous type.
• macroporous is meant a pore system included in the membrane, whose size is between 0.3 microns and 1.0 microns.
• the pore size is between 0.5 ⁇ m and 0.9 ⁇ m.
• the pore size is 0.8 ⁇ m.
• the support of the membrane is polyethersulfone.
• such a polyethersulfone membrane may be a Mustang Q® membrane, distributed by PaII. This membrane has pores of size 0.8 ⁇ m, and is grafted with quaternary amino groups.
• the solution to be purified contains FVIII or VWF, and is of plasmatic origin.
• the solution to be purified contains a mixture of FVIII and FvW, and is preferably of plasmatic origin.
• the solution is derived from a natural human or animal plasma, that is to say containing in the natural state of FVIII and FvW, human or animal respectively.
• a natural human or animal plasma can be taken from pigs, rabbits and goats, this list not being limiting.
• the filter membranes and in particular the Mustang Q® membrane, have a higher capacity for adsorption of FVIII and / or VWF than that of a resin or a gel.
• adsorption capacity is meant the amount of the target proteins fixed on the gel; it is generally expressed by the supplier of gels or resins by the amount of BSA (bovine serum albumin) fixed on the gel for the anionic resins.
• BSA bovine serum albumin
• it is between 25 and 35 mg / ml for the EAE-TOYOP EARL® gel D, usually used for the purification of FVIII and / or VWF, while it is greater than or equal to 30 mg / ml for the Mustang Q® membrane.
• This greater adsorption capacity of the filter membrane results in the possibility of using a smaller amount of gel equivalent to adsorb the same amount of FVIII and / or VWF as on a gel or a resin.
• the greater adsorption capacity of the membrane membrane compared to a gel or a resin could be explained by a better accessibility of ionized sites for FVIII and / or VWF, especially when they form FVIII / VWF complexes. .
• these proteins have a high size, especially when they form FVIII / FvW complexes, and it can be hypothesized that the ionized sites are more easily accessible when they are grafted onto the filter membrane. macroporous, because of pore size, only on gels or resins, whose ionized sites are encased in canals, making them less readily accessible to larger proteins.
• the method comprises the following steps: a) Obtaining cryoprecipitate from the plasma, b) Capture, that is to say, adsorption, factor VIII and von Willebrand factor on said ion exchange chromatography membrane, and more particularly on the anion exchange chromatographic membrane, and c) Selective recovery of von Willebrand factor and factor VIII by successive increases in the ionic strength of the elution buffer.
• a prepurification step is carried out by adsorption of factor VIII and von Willebrand factor on an alumina gel, followed by a cold precipitation.
• step c) of the above process the ionic strength value of the elution buffer is easily adapted by those skilled in the art, in view of his general knowledge of the physicochemical properties of each of the FvW and FVIII proteins. and ion exchange properties of the filter membrane, which is generally a commercially available filter membrane, for which there are recommendations for use by the manufacturer.
• those skilled in the art know, from their general knowledge, that FVIII is eluted from an anion exchange chromatography support with a buffer having an ionic strength greater than the ionic strength necessary for the elution of the FvW of the same support.
• step c) of the above method uses an ionic strength buffer suitable for eluting (desorbing from the support) (i) VWF, (ii) VWF and FVIII, or (iii) successively first the VWF, then the FVIII.
• an ionic strength buffer suitable for eluting (desorbing from the support) i) VWF, (ii) VWF and FVIII, or (iii) successively first the VWF, then the FVIII.
• a first embodiment of the alternative (iii) of successive elution of FvW, then of FVIII one skilled in the art can successively use two elution buffers, each elution buffer having an ionic strength adapted to the desorption of VWF or FVIII, respectively.
• the skilled person elutes with a buffer for generating a gradient of increasing ionic strength, with which are successively desorbed FvW, then FVIII.
• a linear increase in the ionic strength of the elution buffer is included, which can be obtained by adding a salt, for example sodium chloride, calcium chloride, this list not being limiting.
• the elution of VWF is first obtained, followed by elution of FVIII by increasing the ionic strength of the buffer. It is therefore possible to either recover the VWF and stop the elution after obtaining the VWF, or to obtain the FVIII while continuing the elution.
• cryoprecipitate is meant a precipitate obtained from a human or animal plasma by a low temperature precipitation technique.
• Cryoprecipitate can be obtained by methods well known to those skilled in the art.
• the frozen plasma is brought to a temperature of approximately -5 ° C. to -15 ° C. and then slowly heated with stirring to a temperature which does not exceed 1 ° C. or optionally 4 ° C. Under these conditions, the frozen plasma melts to give a liquid phase and a solid phase, the solid phase, the cryoprecipitate, being then recovered by centrifugation.
• Cryoprecipitate is composed mainly of fibrinogen, fibronectin, factor VIII and factor Von
• vWF Willebrand
• selective recovery is meant the ability to recover either FVIII or VWF, or a mixture of FVIII and VWF, depending on the elution method, depending on the protein or proteins that are desired.
• the solution to be purified contains a mixture of
• This embodiment comprises the following steps: a) Obtaining a cellular supernatant or a pre-purified solution comprising FVIII and VWF. B) Capturing factor VIII and von Willebrand factor on said membrane exchange chromatography membrane. ions, and more particularly on the anion exchange chromatographic membrane, and c) selective recovery of von Willebrand factor and factor VIII by successive increases in the ionic strength of the elution buffer.
• the solution to be purified contains either
• This embodiment comprises the following steps: a) Obtaining a Cellular Supernatant or a Purified Solution Comprising FVIII or VWF, b) Capturing Factor VIII or Von Willebrand Factor on the Ion Chromatography Membrane , and more particularly on the chromatographic anion exchange membrane, and c) Recovery of von Willebrand factor or factor VIII.
• the method of the invention allows a reduction in the amount of vitamin K dependent factors, fibrinogen and fibronectin.
• the selective recovery of FvW and FVIII is carried out according to the following steps: d) elution of VWF by increasing the ionic strength of the equilibration buffer; said chromatography membrane.
• the ionic strength may be increased by the addition of 0.25 M sodium chloride to achieve an osmolality of about 600 to 660 mOsm / kg.
• the ionic strength can be increased by addition of 0.7 M sodium chloride, or 0.35 M calcium chloride, to achieve osmolality of between about 1400 and 1700 mOsm / kg.
• Step d) is a step of selective recovery of VWF, in which a buffer solution having an ionic strength suitable for desorbing the VWF of the ion exchange membrane is used.
• Step c2) is a step of selective recovery of FVIII, during which a buffer solution having an appropriate ionic strength is used to desorb the FVIII from the ion exchange membrane filter.
• step c2) a buffer solution having an ionic strength greater than the ionic strength of the buffer solution used for step d) is used.
• step d) The fvW eluted in step d) is containing very little FVIII. It is recovered and a purified FvW solution is obtained.
• the FVIII obtained in step c2) contains less VWF than the starting solution. It is recovered and a solution of purified FVIII is obtained.
• the method of the invention makes it possible to obtain a solution of purified vWF, by implementation, after the step of simultaneous adsorption of FVIII and vWF on said membrane filtering membrane.
• ions of the following steps: d) Elution of VWF by increasing the ionic strength of the equilibration buffer of said chromatography membrane, e) capture of von Willebrand factor on ion exchange chromatography membrane, preferably of the same type as the first, and f) eluting the von Willebrand factor by increasing the ionic strength of the equilibration buffer of said chromatography membrane.
• step d) of eluting FvW by increasing the ionic strength of the equilibration buffer of said chromatography membrane it is possible, after step d) of eluting FvW by increasing the ionic strength of the equilibration buffer of said chromatography membrane, to elute FVIII by an even higher increase in the ionic strength of the equilibration buffer of said chromatography membrane only for the recovery of von Willebrand factor.
• a solution containing a purified VWF and another solution containing a purified FVIII is obtained successively.
• the method of the invention therefore has the advantage of allowing sequential or simultaneous purification of FVIII and VWF from a solution containing FVIII and VWF. If the solution is of plasma origin, the method of the invention is particularly advantageous because it makes the best use of the use of plasma, human or animal.
• this particular embodiment additionally comprises the following steps: g) chromatography of the fraction eluted in step c) enriched in factor Von
• the fibronectin assay can be performed for example by immunonephelometry, according to a technique well known to those skilled in the art.
• an embodiment of the method of the invention makes it possible to obtain a solution of purified FVIII and a purified vWF solution, by implementing, after the step of simultaneous capture of FVIII and FvW on the filtering membrane of ion exchange chromatography, the following steps: a) Elution of the VWF by increasing the ionic strength of the equilibration buffer of said chromatography membrane, b) elution of FVIII by an even greater increase in the ionic strength of the equilibration buffer of said chromatography membrane than for the recovery of von Willebrand factor c) Von Willebrand factor capture on ion exchange chromatography membrane, d) eluting the Von Willebrand factor by increasing the ionic strength of the equilibration buffer of said chromatography membrane. e) chromatography of the fraction eluted in step d) enriched in von Willebrand factor on a gelatin gel affinity gel column, and f) recovery of the non-retained fraction on the affinity gel and
• the method of the invention in its various embodiments, thus makes it possible to separate FVIII and FvW in a simplified manner.
• the purification steps of the invention are the only ones which make it possible to purify factor VIII and von Willebrand factor separately or simultaneously from the plasma by simultaneous capture of the two proteins on anionic exchange chromatography membrane.
• Another object of the invention is a process for obtaining a purified FVIII comprising the implementation of the purification process of the invention.
• Another object of the invention is a process for obtaining a purified FvW comprising the implementation of the purification process of the invention.
• FIG 1 Diagram of the von Willebrand Factor Purification Process
• Cryoprecipitate is prepared by thawing frozen fresh plasma at a temperature of between 10 ° C. and 60 ° C.
• cryoprecipitate containing fibrinogen, fibronectin, von Willebrand factor and factor VIII is recovered and resuspended in an aqueous solution of heparin sodium at 3 IU / ml.
• the pH of the solution is then adjusted to 7, 0 ⁇ 0.1.
• the resuspended cryoprecipitate is pre-purified by alumina gel adsorption to remove vitamin K dependent factors and cold precipitation of fibrinogen and fibronectin.
• alumina hydroxide is added to the stirred suspension for 5 minutes.
• the pH is adjusted to 6.5 + 0.2 with 0.1 M acetic acid and the solution is cooled with stirring until the temperature is between 14 and 18 ° C.
• the solution is then centrifuged at a temperature of 14-18 ° C.
• the supernatant is recovered and clarified by filtration on a 0.22 ⁇ m filter.
• This pre-purified solution is then subjected to a viral inactivation step by solvent-detergent treatment in the presence of effective Polysorbate 80 (1%, w / v) and Tri-n-Butyl Phosphate (0.3%, v / v). on enveloped viruses.
• the solvent-detergent treatment is carried out for a period of at least 6 hours at pH 7.1.
• the protein solution treated solvent-detergent is then passed on a grafted exchange membrane of strong anions, type Mustang capsule Q, previously equilibrated with a buffer solution of base, pH 6,9-7,1 added sodium chloride to reach an osmolality of 370-390 mOsm / kg.
• the capsule After passage of the protein solution, the capsule is rinsed with the same osmolality buffer solution 370-390 mOsm / Kg until the optical density of the column effluent returns to the baseline.
• the protein fraction not adsorbed on the membrane is rich in fibrinogen and contains the added chemical agents for the viral inactivation treatment by solvent-detergent treatment.
• the von Willebrand factor adsorbed on the membrane is then eluted by passage of the basic buffer solution, pH 6.9-7.1 of ionic strength increased by addition of sodium chloride to reach an osmolality of 600-660 mOsm / kg.
• the eluted fraction is then diluted with the basic buffer solution, pH 6.9-7.1 free of sodium chloride, to an osmolality of 370-390 mOsm / kg.
• the diluted fraction is then passed on a strong anion exchange graft membrane, Mustang Q capsule type, previously equilibrated with a basic buffer solution, pH 6.9-7.1 added with sodium chloride to reach an osmolality of 370-
• the capsule is rinsed with the same osmolality buffer solution 370-390 mOsm / Kg until the optical density the column effluent returns to the baseline.
• the von Willebrand factor adsorbed on the membrane is then eluted by passing the basic buffer solution, pH 6.9-7.1 ionic strength increased by addition of sodium chloride to reach an osmolality of 600-660 mOsm / kg.
• the eluted fraction is then chromatographed on gelatine ligand affinity gel previously equilibrated with a basic buffer solution, pH 6.9-7.1 of ionic strength increased by addition of sodium chloride to reach an osmolality of 600-660 mOsm. / Kg.
• the affinity gel is rinsed with the same osmolality buffer solution 600-660 mOsm / Kg until the optical density of the column effluent returns to the baseline.
• the non-adsorbed fraction including gel wash constitutes the high purity von Willebrand factor enriched fraction.
• Cryoprecipitate is prepared by thawing frozen fresh plasma at a temperature of between 10 ° C. and 60 ° C.
• cryoprecipitate containing fibrinogen, fibronectin, von Willebrand factor and factor VIII is recovered and resuspended in an aqueous solution of heparin sodium at 3 IU / ml.
• the pH of the solution is then adjusted to 7.0 ⁇ 0.1.
• the resuspended cryoprecipitate is pre-purified by alumina gel adsorption to remove vitamin K dependent factors and cold precipitation of fibrinogen and fibronectin.
• alumina hydroxide is added to the stirred suspension for 5 minutes.
• the pH is adjusted to 6.5 + 0.2 with 0.1 M acetic acid and the solution is cooled with stirring until the temperature is between 14 and 18 ° C.
• the solution is then centrifuged at a temperature of 14-18 ° C.
• the supernatant is recovered and clarified by filtration on a 0.22 ⁇ m filter.
• This pre-purified solution is then subjected to a viral inactivation step by solvent-detergent treatment in the presence of effective Polysorbate 80 (1%, w / v) and Tri-n-Butyl Phosphate (0.3%, v / v). on enveloped viruses.
• the solvent-detergent treatment is carried out for a period of at least 6 hours at pH 7.1.
• the protein solution treated solvent-detergent is then passed on a grafted exchange membrane of strong anions, type Mustang capsule Q, previously equilibrated with a buffer solution of base, pH 6,9-7,1 added sodium chloride to reach an osmolality of 370-390 mOsm / kg.
• the capsule After passage of the protein solution, the capsule is rinsed with the same osmolality buffer solution 370-390 mOsm / Kg until the optical density of the column effluent returns to the baseline.
• the protein fraction not adsorbed on the membrane is rich in fibrinogen and contains the added chemical agents for the viral inactivation treatment by solvent-detergent treatment.
• the von Willebrand factor adsorbed on the membrane is then eluted by passage of the basic buffer solution, pH 6.9-7.1 of ionic strength increased by addition of sodium chloride to reach an osmolality of 600-660 mOsm / kg.
• Purification of von Willebrand factor can be continued as described in Example 1.
• Factor VIII adsorbed on the membrane is then eluted by passage of the base buffer solution, pH 6.9-7.1 ionic strength increased by addition. of sodium chloride to reach an osmolality of 1400-1700 mOsm / kg.
• the factor VIII is eluted by a buffer solution, pH 6.0, of high ionic strength obtained by addition of calcium chloride.

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