FI116569B - Method for Preparation of Virus-Activated Factor VIII-Containing Fraction by Chromatographic Methods - Google Patents

Abstract

A process for producing a virus-inactivated, factor VIII-containing fraction by chromatographic methods in which, starting with cryoprecipitate or blood plasma, possibly followed by a treatment with aluminium hydroxide, at least one separating operation is performed by membrane chromatography after the dissolution of the cryoprecipitate. The fractions are subjected to virus inactivation and preferably an additional pasteurisation step.

Description

116569

Method for Preparation of Virus-Inactivated Factor VIII-Containing Fraction by Chromatographic Methods

The invention relates to a process for the preparation of a viral inactivated fraction containing factor VIII, by chromatographic methods, and to a fraction containing factor VIII which can be obtained by the process of the invention.

Factor VIII is a vital substance that plays an important role in blood coagulation. Thus, coagulation disorders in the blood can be treated by administering factor VIII. There is, therefore, a great need for administrable formulations of factor VIII. There has been no shortage of attempts to isolate Factor VIII in a highly enriched form from natural sources. Thus, chromatographic methods are currently known for purifying Factor VIII from a cryoprecipitate. This is a fraction that can be obtained by cold treatment of plasma. EP 367 840 B1 relates to a chromatographic method for isolating Factor VIII from blood-20 plasma without prior cryoprecipitation. The factor VIII-containing fraction is then separated by chromatographic separation with a hydrophilic chromatography material modified with ion exchange groups. EP 0 238 701 relates to a process for the preparation of a highly pure infectious anti-haemorrhagic factor wherein the pre-treated fractions are a cryoprecipitate which is liberated from ethanol by precipitation of fibrinogen, globulin, albumins and other interfering components. EP-A-88 108 458.6 describes performing chromatographic separation after viral inactivation of the cryoprecipitate fraction with ion exchangers. EP 0 173 242 A describes a process for the chromatographic preparation of factor VIII preparations with anion exchange materials based solely on carbohydrates. The carbohydrate matrix is then modified with DEAE groups. In particular, »116569 2 DEAE Sepharose and DEAE Cellulose are described as suitable. GB-A 1 178 958 describes purification of factor VIII on ECTEOLA cellulose columns. Modified cellulose contains basic substituents incorporated by the epichloro-5 hydrin and triethanolamine reaction. Depending on the state of the art, either chromatographic separation in the form of a batch method or column chromatography is used. However, these methods provide quite good results, but it is still desirable to increase the yields of biologically valuable Factor VIII for both economic and ethical reasons.

Thus, the technical problem underlying the invention is to provide a method which, starting from the prior art in the art, allows for better production of factor VIII in terms of yield and biological activity.

Unexpectedly, the problem is solved by a method starting from a cryoprecipitate or blood plasma, possibly with aluminum hydroxide, and after dissolving the cryoprecipitate, a separation operation is performed by membrane chromatography.

The process of the invention may also be performed with a commercial cryoprecipitate or blood plasma. Preferably. '; *. to pre-concentrate the Factor VIII molten cryoprecipitate., is treated to further pre-purify the sample with aluminum hydroxide.

Prior to the actual chromatographic purification, the materials contained in the membranes or membranes are subjected to viral inactivation. Virus inactivation occurs I | |

• 'J! 30 according to the methods described in EP

131,740 Ai, by treatment with biocompatible organic solvents (detergents), Triton * X-100 / TNBP, preferably TweenR / TNBP (tri-n-butyl phosphate). Well done; results are also obtained with sodium cholate / TNBP. Preferably, the detergent is used in amounts of up to 15% by weight.

116569 3

The chromatographic separation operation to purify Factor VIII from the samples may be performed with ion-exchange-modified base materials, in particular anion exchangers, or immuno-affinity ligands on mo-5 diffused materials. What is decisive is that said materials are in membranes. Preferably, the membranes consist of a base material such as modified cellulose or synthetic fibers. Particularly suitable membranes and sealed sheets of porous polyglycidyl methacrylates and / or other porous hydrophilic polymers having a similar structure as well as hydrophilic polystyrene.

In the first case, the separating membrane consists of either interconnected thin cellulose-15 or synthetic fiber films, or in the second case of dense silica gel or polymer carrier plates. The base materials of the membranes or plates are provided with corresponding anion exchange groups or immuno affinity ligands. Particularly suitable ion exchange groups are anion-20 exchange groups such as quaternary amine or diethylaminoethyl groups. Cation exchangers are mainly weak and strongly acidic cation exchangers, such as materials modified with sulfonic acid. with polar or phosphoric acid groups.

Ion exchange groups may be bonded, with or without the so-called t * spacer groups, to the fibers of the base matrix1. Materials with intermediary groups are also called "tactile horns". DE 4 204 694 mentions the corresponding mediator groups i 30 and ligands. The exemplary *, f, glucosamine residue may also serve as a mediator group. Also, anion exchange groups such as DEAE or quaternary amines may have been bound to membranes from porous poly · ···, glycidyl methacrylate or other materials mentioned. The binding of the anion exchange groups ta-35 is then effected either directly on the membrane-forming material or also via a mediator group, e.g., a glucosamine residue.

In another embodiment of the method of the invention, affinity membrane chromatography is used with immobilized agents that show high affinity for factor VIII. In particular, monoclonal and / or polyclonal antibodies or antibody-VIII binding fragments (immunoaffinity membrane chromatography) are suitable. The antibodies are preferably of human or murine origin.

Agents having affinity for factor VIII are immobilized on the carrier by chemically active groups. Preferably, the active moiety does not adhere directly to the carrier material but to the end of the mediator moiety. Immobilization of substances having affinity for factor VIII is accomplished by binding to active groups such as tosyl, tresyl, hydrazide and the like. Similar methods are known in T.M. Phillips's article "Affinity Chromatography" in "Chromatography", E. 20 Heftmann (Ed.), 5th Edition, Elsevier, Amsterdam, 1992.

Antibodies may also be pre-adsorbed to membranes by protein A or protein G ligands. This is followed by **,. abrasive covalent cross-linking can prevent antibody elution (column leakage). Antibodies'. For cross-linking protein A or protein G membranes, a method similar to the ir

* <I

for rally carriers. The advantage of immobilizing protein * 'A' or protein G is that the antibodies become immobilized solely on the persistent segment (Fc) of the molecule. The antigen-binding portion (F ^) is then left free and is not prevented from interacting with factor VIII.

In a further preferred embodiment, materials that may allow hydrophobic interaction are used to separate Factor VIII. Hydrophobic mate-35 materials are acyclic and / or cyclic alkyl chains, for example C 1-18 alkyl chains, and aromatic substances. Suitable materials for hydrophobic interaction are also preferably those with a stepwise hydrophobicity. Hydrophobicity 5 can be graded by incorporation of polar groups such as polar-protic or polar-aprotic, such as hydroxy, amino, cyano. Preferably, this is adapted to the particular separation conditions.

Viral inactivation can also occur by heat concept. Preferably, after the first membrane chromatography of the eluted sample containing the dough, the pasteurization step is performed. A similar method is proposed in P 4 318 435.9. The Factor VIII-enriched fractions are then contacted with di- or trialkyl phosphates and optionally crosslinking agents in the presence of stabilizers and treated simultaneously or sequentially at an elevated temperature of about 55-67 ° C for 5 hours to 30 hours. It may be advantageous to combine both viral inactivation methods, detergent treatment and heat treatment.

Another membrane chromatography may also be performed to remove the chemicals used in the pasteurization step. Advantageously, the added stabilizers are separated by DEAE or quaternary ammonium compounds on the surface of the chromato- graphic support, modified membrane *. It is also possible to place the corresponding ligands without spacer groups on the surface of the carrier material.

Stabilizers do not remain in these anion exchange materials under selected conditions, while factor VIII is adsorbed onto the chromatographic material.

Factor VIII is then eluted with an aqueous solvent system using gradually increasing saline. opportunities.

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The factor VIII-containing fraction thus obtained is concentrated, filled and optionally lyophilized by conventional methods.

Preferably, Factor VIII is transferred from the low ionic strength solution in the first 5 membrane separation steps. Preferably, the ionic strength of the aqueous system corresponds to the ionic strength of 0-150 mM sodium chloride solution. At these ionic strengths, Factor VIII is still adsorbed onto the chromatographic material, while less binding impurities can be washed away with aqueous systems of similar ionic strength.

Purification of the adsorbed material in a further embodiment of the process of the invention may be carried out with an aqueous system having an ionic strength equivalent to 15,200 to 400 mM sodium chloride solution. Desorption of Factor VIII and elution of this fraction is then effected by an aqueous system having an ionic strength of 500-1500 mM sodium chloride solution. The pH is then maintained at 4-9. If cation exchange chromatography is performed, this preferably takes place at pH <6, while anion exchange chromatography is preferably performed at higher pH values greater than 6.

If purification of factor VIII is carried out by immunoaffinity membrane chromatography, a differential treatment is performed; In addition to the above methods, elution with anion exchange materials by chaotropic reagents or ;;; well-concentrated saline solutions. The elution is preferably carried out with concentrations of chaotropic reagents or salts sufficient to release: binding between the high affinity factor VIII and the factor VIII itself. The concentration of these substances in each of the elution systems depends on the potency of the factor VIII and the like binding agents. Preferably, the antibodies used are the immunofibrillar ligands. affinity is not too high, then elution can be carried out with aqueous solutions having lower denaturation capacity Preferably, aqueous solutions containing 1 to 6 M urea, particularly 2 to 4 M urea, or correspondingly concentrated salts are used to elute Factor VIII from the immune affinity membrane. .

In hydrophobic interaction chromatography, the samples are transferred to an aqueous solution of very high ionic strength, such as a highly concentrated solution of ammonium sulphate (concentration up to 4 M) or a highly concentrated solution of sodium chloride (concentration up to 5 M). In particular, the elution is carried out stepwise or continuously with lower saline solutions. Lower aqueous ionic solutions for elution of samples for hydrophobic interaction membrane chromatography may also employ an aqueous solution containing organic solvents, especially dilute alcoholic solution.

The procedure of the invention allows, in a surprising manner, the rapid and uncomplicated purification of factor VIII, which simultaneously results in high purity and high yield. In addition, the specific activity of factor VIII thus obtained is quite high, which can be explained by the slight denaturation of the active factor in the process of the invention. Thus, the invention also relates to a factor VIII fraction which may be obtained by the process of the invention.

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Claims (11)

1. A process for the preparation of a virus-inactivated Factor VIII-containing fraction by chromatographic methods, starting from a cryoprecipitate after being dissolved or from plasma, possibly followed by aluminum hydroxide treatment, and performing a viral inactivation by treating the fraction with di- or trialkyl phosphate. ionic surfactant followed by at least one separation operation by membrane chromatography, membranes or dense plates of porous hydrophilic polymers of poly (glycidyl methacrylates) or hydrophilized polystyrene. 1,16969 The process according to claim 1, wherein the separation operation is performed on an ion exchange material in the membrane or on the membrane, in particular anion exchange material. The method of claim 1 or 2, wherein the viral inactivation occurs in a pasteurization step, optionally followed by another separation operation by membrane chromatography. The method according to claim 1 or 3, wherein the membrane chromatography is carried out on a material that exhibits a high affinity for factor VIII. The method of claim 1 or 3, wherein the high affinity for factor VIII is shown by h! The material has been modified with high and / or low molecular weight ligands. The method of claim 4 or 5, wherein: the modified material exhibiting high affinity for factor VIII has immobilized ligands with a high affinity for factor VIII. 116569 The method of any one of claims 1 or 3, wherein the chromatographic material permits hydrophobic interaction with resolvable factor VIII or comprises corresponding ligands that mediate hydrophobic interaction. The method of any one of claims 1 to 3, wherein the sample to be purified (transferred to a membrane containing ion-exchange material) in a water system having a low ionic strength corresponding to 0 to 150 mM sodium chloride is optionally washed with an aqueous system having a higher ionic strength corresponding to 200 to 400 mM sodium and then eluted with a high ionic strength aqueous system corresponding to 500-1500 mM sodium chloride, maintaining the pH at 4-9. The method of any one of claims 1, 3 or 7, wherein the sample to be purified is transferred from a solution of very high ionic strength to a membrane comprising hydrophobic ligands on its surface, and eluted with lower solvent systems of ionic strength. The process according to any one of claims 1 to 9, wherein the fraction containing the eluted factor VIII is concentrated, filled and / or lyophilized. The me- * * * JJ according to any one of claims 1-10. from viruses in which viral inactivation occurs by treating 25 up to 15% by weight of detergent. »· • *> * i * *» »» · '»* · * *» * · »» * I I * I i 116569