The method of obtaining, using chromatographic methods of virus-inactivated fraction containing factor viii

 

(57) Abstract:

The invention relates to medicine, in particular to Hematology. The essence of the invention lies in the fact that the described method of obtaining virus-inactivated fraction containing factor VIII using the methods of chromatography. As raw materials use cryostat or blood plasma, if necessary with subsequent processing of aluminium hydroxide, after dissolution krivoshapka is at least one operation of the division. Another aspect of the invention is a fraction containing factor VIII, subject to viral inactivation and preferably additional pasteurization. The invention expands the Arsenal of tools for the treatment of disorders of the blood coagulation system. 2 C. and 12 C.p. f-crystals.

The invention concerns a method of obtaining using chromatographic methods of virus-inactivated fraction containing factor VIII, as well as containing factor VIII fraction, which can be obtained by the method according to the invention.

Factor VIII is essential for life substance that plays an important role in blood coagulation. So, for example, blood clotting disorders can be treated by administering factor VIII. Therefore, itoc to isolate factor VIII in heavily saturated form from natural sources. So, already known chromatographic methods for purification of factor VIII from cryostat, which is a fraction that can be obtained by using the plasma processing at low temperatures. European patent EP 367840 B1 - concerns chromatographic method of separation of factor VIII from blood plasma without the prior deposition. When this fraction containing factor VIII is separated by chromatographic separation of hydrophilic chromatographic material, which modified ion-exchange groups. European patent EP 0238701 - concerns a method for obtaining ultra-pure infectious antihemophilic factor, and pre-treated fractions are krishika, which are released from fibrinogen, globulin, albumin and other interfering components in the deposition process ethanol. In the European application for patent N 88108458.6 describes the division of fractions of krisada using ion-exchange chromatography after inactivation of the virus. European patent EP 0173242 AND describes how to obtain preparations of factor VIII chromatographic method for anyoneeven materials that are based solely on carbohydrates. When this carbohydrate Matritsa purification of factor VIII using columns ECTEOLA cellulose. Modified cellulose contains basic alternatives introduced by the reaction of epichlorohydrin and triethanolamine. These prior art uses or chromatographic separation periodic manner, or column chromatography, using these methods get pretty good results, but there is a need to increase the yield of biologically valuable factor VIII, based on both economic and ethical reasons.

The technical problem underlying the invention is thus to provide a method which will create opportunities based on prior art, to obtain the factor VIII with a large output and biological activity.

The problem is solved by a method in which, based on cryostat or blood plasma, if necessary, by treatment with aluminum hydroxide, after dissolution krivoshapka is separation using membrane chromatography.

In the method of the invention may use commercially available krivoshapka or blood plasma. The preferred way for pre-concentration factor VIII thawed cryostat processing is graficheskiy cleaning materials, located in the membranes or membranes, is a viral inactivation. Virus inactivation is carried out in accordance with the method described in European patent EP 131740 A1, by treating the biocompatible organic solvents (detergents), Triton X - 100/TNBP, preferably Tween/TNBP (tri-n-butylphosphate). Good results are achieved using cholate sodium/TNBP. A preferred use amount of detergent to 15 wt.%.

Chromatographic separation for cleaning of factor VIII in the sample can be made, first, on the basic materials modified ion-exchange groups, in particular anion-exchange substances, or materials modified with immune affinity ligands. Decisive is the fact that these materials are placed in the membranes. The preferred way membrane is composed of a base material, such as a modified cellulose or synthetic fiber. In particular, suitable membranes, as well as compact disks of porous polyglycerylmethacrylate and/or other porous hydrophilic polymers with the same structure as, for example, from hydrophilizing policy is PS or artificial fibers or in the second case of compact discs silica gel or carrier polymer. Main material of diaphragms or disks provided with a corresponding anion exchange groups or with immune affinity ligands. As the ion exchange groups are taken into account, in particular, anion-exchange groups such as Quaternary amines or diethylaminoethylamine group. As the cation-exchange substances are taken into account, mainly weakly and strongly acidic cation-exchange materials, such as materials that are modified by sulfonic acid groups or phosphoric acid.

Ion-exchange groups can be associated with the fiber base material without the so-called spacer or with his help. Materials provided with the spacer, denoted as materials with sensitive fibers. In the Federal Republic of Germany patent DE 4204694 - called appropriate spacers and ligands. As the spacer can be, for example, the glucosamine residue. Also with the membranes of porous polyglycerylmethacrylate or other of the above mentioned materials can be linked anion-exchange groups, such as DEAE or Quaternary amines. Join anion-exchange groups is carried out either directly to the material forming the membrane, or through a spacer, such as the Naya membrane chromatography with immobilized substances, detecting a high degree of affinity for factor VIII. In particular, the calculation monoclonal and/or polyclonal antibodies or binding factor VIII fragments of antibodies (immune-affinity membrane chromatography). Antibodies are the preferred way of human or mouse origin.

Substances with affinity in terms of factor VIII, are immobilized on the carrier by means of chemically active groups. The preferred way active group will impact not directly on the carrier material, and at the end of the spacer. Immobilization of substances for factor VIII is carried out through communication with the active groups, such as tosyl, trail, hydrazide, and others. Corresponding methods are known from T. M. Phillips "Affinity chromatography" in "Chromatography" (E. Heftmann, ed.), 5th ed. Elsevier, Amsterdam 1992.

Antibodies can also pre-adsorbed on the membrane ligands protein a or protein ligands G. By subsequent covalent "stitching" can prevent the elution of antibodies (column leaching). For "stapling" of the antibodies on the membrane protein a or protein G can be used a method similar to that used in unrelated media. The advantage of immobel segment of the molecule (Fc). Thus, part (Fab) that binds the antigen remains free and in their interaction with factor VIII is not limited.

In its preferred form of execution used materials for the separation of factor VIII, which can provide a hydrophobic interaction. As hydrophobic materials are acyclic and/or cyclic alkyl chain, such as alkyl chain C1-C18, and aromatic substances. As materials that provide hydrophobic interactions, are taken into account, the preferred way is also such materials, which are fractionated hydrophobicity. Hydrophobicity can be fractionated by introducing polar, such as polar-proton or polar aprotic groups, such as hydroxyl group, amino group, ceanography. The preferred way, it is subject to the relevant terms of the separation.

Viral inactivation can be achieved by heat treatment. While the preferred way after the first membrane chromatography allerona sample containing factor VIII is subjected to the operation of pasteurization. The appropriate method is proposed in P 4318435.9. When this fraction, the cat is ri the necessity of wetting agents and processed simultaneously or sequentially at an elevated temperature in the range from 55oC to 67oC for from 5 hours to 30 hours. Preferred way you can combine both methods of viral inactivation treatment, detergents and heat.

Removal used in the process of technological operations pasteurization chemicals can also be the second membrane chromatography. Preferable, the Department added stabilizers is carried out using a membrane modified DEAE or Quaternary ammonium compounds, through which the spacer is placed on the surface of the chromatographic material of the carrier. It is also possible to place the corresponding ligands on the surface of material carrier without the spacer. The stabilizers of this aminobenzyl material at the chosen conditions is not delayed, while factor VIII is adsorbed on the material used in chromatography.

After that, factor VIII eluted with solvent system of water when increasing superiorcasino concentration of salt.

Thus obtained fraction containing factor VIII, when using conventional methods in a concentrated form is packaged and, if necessary, liophilized.

Preferably autographical separation. The preferred way water system has an ionic strength that corresponds to 0 to 150 mM of sodium chloride solution. When these ion power factor VIII is still adsorbed on the chromatographic material, whereas poorly communicating impurities can be washed out by the water systems of the same ionic strength.

This purging of adsorbed material in another form of execution of the method according to the invention can be performed using the water system, having an ionic strength corresponding to 200 - 400 mM solution of sodium chloride. Desorption of factor VIII and elution of this fraction is then using the water system, having an ionic strength corresponding to 500 - 1500 mM solution of sodium chloride. The pH value is kept in the range of 4 to 9. If you are cation-exchange chromatography, it is preferred, when the pH < 6, meanwhile chromatography using anion-exchange substances is more likely at higher pH values greater than 6.

If cleaned factor VIII using immunoaffinity membrane chromatography, in contrast to the above-mentioned method is carried out using anion-exchange materials, elution is carried out using chaotropic reagents or is entrace chaotropic chemicals or salts, which are sufficient to break the link between substance with a high degree of affinity for factor VIII and factor VIII. The concentration of these compounds in the respective lucianic systems will depend on the strength of the affinity of the factor VIII and the corresponding connecting component. The preferred way as immunogenic ligands are antibodies with not too high affinity. In may result in elution of aqueous solutions with minor denaturing ability. For elution of factor VIII with immunoaffinity the preferred membrane is used as aqueous solutions with a concentration of from 1 to 6 M urea, in particular from 2 to 4 M urea, or correspondingly highly concentrated salt solutions.

In the case of hydrophobic interactive chromatography, the sample is introduced into an aqueous solution of very high ionic strength, such as highly concentrated ammonium sulfate (having a concentration of up to 4 M) or sodium chloride (having a concentration of up to 5 M). Elution is carried out, in particular, by stages or continuously using salt solutions having low ionic strength. In solutions with low ionic strength for luteaster with organic solvents, in particular diluted alcohol solution.

The method according to the invention provides a fast and simple purification of factor VIII, which precipitates, having simultaneously a high degree of purity and high yield. In addition, the specific activity of the thus obtained factor VIII is quite high, because of the minor denaturization active factor by means of the method according to the invention. Thus, the subject invention is also obtained by the method according to the invention the fraction of factor VIII.

1. The method of obtaining virus-inactivated fraction containing factor VIII, in which the blood plasma or cryostat after its dissolution by processing di - or trialkylphosphates, if necessary, treated with aluminum hydroxide, is subjected to the influence of non-ionic surfactants for viral inactivation, followed by at least one operation of the separation using membrane chromatography, excluding affinity membrane system, consisting of a hollow fiber.

2. The method according to p. 1, in which the above operation division is placed on the membrane or ion-exchange material, in particular ncert technological operation of pasteurization, if necessary, followed by an additional separation using membrane chromatography.

4. The method according to any of paragraphs.1 to 3, in which membrane chromatography is carried out on the material, which has a high degree of affinity for factor VIII.

5. The method according to any of paragraphs.1 to 4, in which the material having a high degree of affinity for factor VIII modified ligands with high and/or low molecular weights.

6. The method according to p. 5, in which this material is modified antibodies targeted against factor VIII.

7. The method according to any of paragraphs.4 to 6, which is called the modified material having a high degree of affinity for factor VIII, is immobilized ligands with high affinity for factor VIII.

8. The method according to any of paragraphs.1 to 7, in which the chromatographic material allows hydrophobic interaction with detachable factor VIII or has the appropriate ligands, which transmit hydrophobic interaction.

9. The method according to PP.1 to 5 and/or 7, in which the purified sample in the aqueous system to be applied on the ion-exchange material and conduct elution in a gradient of increasing ionic strength using the solution of the RA of sodium chloride from 200 to 400 mm, then in the aquatic environment even higher ionic strength with a solution of sodium chloride in a concentration of from 500 to 1500 mm, at pH 4 - 9.

10. The method according to any of paragraphs.1, 3 and 8, in which the purified sample is applied from a solution that allows you to bind factor VIII due to antibodies to factor VIII, affinity membrane with adsorbed on her factor VIII, then washed with genotype reagents in the appropriate concentration, for example urea, at a concentration of 1 to 6 M or elute salt solution of high concentration.

11. The method according to any of paragraphs.1, 3 and 8, in which the purified sample is applied from a solution of very high ionic strength on the membrane, which has on its surface hydrophobic ligands and eluted with solvent system low ionic strength.

12. The method according to any of paragraphs.1 to 11, in which allerona fraction containing factor VIII concentrates, packaged and/or liophilized.

13. The method according to any of paragraphs.1 to 12, in which viral inactivation is carried out by treatment with detergent in an amount up to 15 wt.%.

14. The fraction containing factor VIII obtained by applying the method according to any one of paragraphs.1 - 13.

 

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