WO2000074733A1

WO2000074733A1 - Process for removing viruses from biological samples

Info

Publication number: WO2000074733A1
Application number: PCT/EP2000/005246
Authority: WO
Inventors: Djuro Josic; Lothar Biesert; Rolf Willi Karl Klengel; Andrea Buchacher; Horst Schwinn
Original assignee: Octapharma Ag
Priority date: 1999-06-08
Filing date: 2000-06-07
Publication date: 2000-12-14
Also published as: AU5678500A

Abstract

A process for removing or separating viruses from potentially virus-containing samples using gel permeation chromatography on hydrophilic supports.

Description

PROCESS FOR REMOVING VIRUSES FROM BIOLOGICAL SAMPLES

A Process for Removing or Separating Viruses from Potentially Virus-Containing Samples

The present invention relates to a process for removing or separating viruses from potentially virus-containing samples using gel permeation chromatography.

Frequently, valuable substances can be isolated only from samples or sources of biological origin. In particular, these include medicaments which contain, for example, factors of the blood clotting cascade. Although many effective proteins have become available by genetic engineering, in these cases as well, the corresponding active substance is produced by transformed cells. The active substance is then obtained from the transformed organisms by isolation procedures. A risk remains in that infectious particles might be carried over from the biological sources.

However, there is a substantially higher risk in the isolation of active substances directly from biological sources. Thus, for example, a high risk of infection can be seen in the processing of blood products derived from pooled donations. There may be mentioned the infection of hemophilic patients by factor VIII preparations contaminated with HIV.

In the past, there have been many attempts to reduce or even eliminate the risk of infection by such active substances. Currently, a process has proven useful in which the active substance to be isolated is treated with chemicals prior to, during or after separation, which results in the inactivation of any viruses contained in the sample. Such a method is described in EP-A-0 131 740. It is desirable to provide a process which allows the removal or separation of viruses from potentially virus-containing samples wherein the active substance to be isolated is not adversely affected. In addition, it is desirable to provide a method which allows the removal or separation of viruses within a short period of time. Surprisingly, it has been found that this is possible, according to the invention, by a process for removing or separating viruses from potentially virus-containing samples using gel permeation chromatography on hydrophilic supports.

Gel permeation chromatography is a per se known method by means of which mixtures of biopolymers, in particular, can be separated into fractions, wherein molecules contained in the mixture are separated by size.

To date, gel permeation chromatography was considered unsuitable for separating infectious materials, such as viruses, from sources containing biopolymers and being potentially contaminated with infectious materials in such a way that infection by administration or other application of the material obtained from such sources is no longer possible.

Surprisingly, it has been shown according to the invention that gel permeation chromatography on hydrophilic supports can be employed for removing or separating viruses from potentially virus-containing samples.

If plasma fractions are employed for recovering components of the blood clotting cascade according to Josic et al., J. Chromatogr. A 796 (1998), 289-298, there are obtained, for example, factor IX preparations which are free from infectious particles, especially viruses, that can be carried over through blood or blood products (blood-borne virus). However, after leaving the column, the factor IX preparations may still be positive in PCR tests for viral DNA. This is due to nucleic acid contaminations by viral DNA/RNA which is no longer infectious, i.e., viral fragments, as can be shown by a DNA/RNA degradation. In a digestion by DNase or RNase, only said viral fragments are degraded while the intact and potentially infectious viruses can still be detected in a fraction separated from biopolymers.

The process according to the invention enables the separation of infectious particles and viruses from samples potentially containing infectious particles using gel permeation chromatography on hydrophilic supports. As hydrophilic supports, polysaccharides, especially dextranes, cellulose, agarose, modified polysaccharides, hydrophilic synthetic polymers, preferably so-called tentacle chromatographic materials, silica gels modified with hydrophilic groups, or combinations thereof are preferably employed.

Polysaccharides are employed, for example, as commercially available supports, such as Sephadex, Sepharose, agarose etc. For separating substances, dextranes have long been known to those skilled in the art. In addition to cellulose and agarose, modified polysaccharides may also be used. Synthetic polymers may also be employed. Preferred synthetic polymers include those based on polyglycidyl methacrylate, especially those modified with hydrophilic arms (tentacles). For a further description, reference is made to EP-A-0 337 144 and EP-A-0 320 023, which are incorporated herein by reference. As tentacle chromatographic materials, especially for the separation of biopolymers, support materials have been used which are further described in EP-A-0 337 144, which is incorporated herein by reference. In addition to the organic hydrophilic support materials, inorganic materials such as silica gels modified with hydrophilic groups may also be employed. There may be mentioned, for example, TSK gels and so-called SW Series (silica wide pore, Toso Haas, Stuttgart).

In particular, the hydrophilic support material to be used in the process according to the invention has a large-pore structure. Particulate materials have a grain size of from 0.5 to 350 μm, in particular. So-called compact block materials as described in EP-A-0 320 023, which is incorporated herein by reference, may also be employed. The compact block material has an advantage in being pressure-stable due to its more or less monolithic structure and can be operated in radial flow, which results in an advantageous reduction in dead volume.

The pore size of the hydrophilic support, expressed as the maximum of the pore size distribution of the support, can be chosen as a function of the separation problem, within certain limits. In some way, the pore size has an upper limit in that the viruses to be separated cannot or can only partly enter the pores of the hydrophilic support material and thus are only subject to a negligible delay, at best. In particular, this depends on the type of substance to be isolated from the sample potentially containing infectious particles. If it is a substance which is characterized by a low molecular weight, the difference in size between the infectious particles and the substance to be separated is in turn large enough so that larger-pore chromatographic materials may also be used. Although the infectious material may also be subject to some delay then, this delay is short enough, as compared to that of the molecule to be separated, to achieve a sufficiently safe separation of the infectious materiai and the substance to be separated.

There is also a lower limit in the choice of the pore size of the hydrophilic support material. It essentially depends on the size or the molecular weight of the substance to be separated. Typically, the pore size of the hydrophilic support material is larger than 5 nm, especially larger than 50 nm.

The process according to the invention enables the separation of viruses from potentially virus-containing samples which contain biopolymers, such as proteins, polypeptides, oligopeptides, nucleic acids, polynucleotides, oligonucleotides and/or carbohydrates. There may be used, in particular, samples such as blood plasma or plasma fractions which contain substances such as factor IX, antithrombin III, immunoglobulins G and A, factor VIII (devoid of von Willebrand factor), factor VII, oci-antitrypsin, prothrombin, thrombin, factor X, protein C, protein S and other factors of the blood clotting cascade. These substances may also be contained in a sample which is obtained in the genetic engineering of these substances.

The viruses which can be carried over by blood or blood products and can be separated off by the process according to the invention include, in particular, blood-borne viruses, such as parvo viruses, hepatitis A, B, C viruses, HIV, and the like.

The hydrophilic supports to be used in the process according to the invention are employed, in particular, in conventional chromatographic devices or methods. These include, for example, column chromatography in all its aspects as a discontinuous or continuous method. Chromatography on compact block materials may also be employed as a discontinuous or continuous method. Annular chromatography, simulating moving bed (SMB) chromatography, and/or truly moving bed chromatography and combinations of these chromatographic methods may be used as continuous procedures. Annular chromatography or chromatography on compact block materials are further illustrated, in particular, in the publications by K. Reissner et al., J. of Chromatography A, 763 (1997), 49 to 56, and in WO-A-96/06158. These documents are incorporated herein by reference.

Another advantage of the process according to the invention is the simultaneous depletion of accompanying proteins for the preparation of purified protein concentrates. For example, by means of gel permeation chromatography, which is also called molecular size-exclusion chromatography, used for the separation of a previously added test virus from a plasma fraction containing coagulation factor IX derived from pooled human plasma, further purification of coagulation factor IX could also be achieved. Surprisingly, vitronectin could be separated quantitatively and identified as a contaminant. Namely, vitronectin is enriched with factor IX by the usual method of ion-exchange chromatography or affinity chromatography on heparin. By the process according to the invention, a factor IX recovered from plasma characterized not only by the absence of infectious particles, but also by the absence of viral fragments and at the same time the absence of vitronectin was obtained for the first time. Thus, this human material is free from xenogenic components such as murine antibodies and alien nucleic acids. Although depletion of vitronectin from a solution containing factor IX could be optionally achieved in the prior art by the use of murine monoclonal antibodies, murine contaminations of the preparation obtained, i.e., a content of xenogenic biopolymers, had to be put up with.

According to the invention, the separation of vitronectin from the solution containing factor IX results in a surprising quality of a pharmaceutical preparation containing factor IX. Namely, it has been found that vitronectin is contained in factor IX and PPSB preparations, i.e., in prothrombin complex preparations based on coagulation factors II, VII, IX and X, in its active form. These preparations may additionally contain the anticoagulant or fibrinolytically active factors protein C and protein S, which are mostly referred to as coagulation inhibitors.

This active form of vitronectin binds the plasminogen activator inhibitor-1 (PAI- 1) from the donor plasma. PAI-1 is known to be an inhibitor of fibrinolysis and thus acts as a factor for generating thromboses.

Since it has surprisingly been found that PAI-1 together with active vitronectin is recovered from plasma or plasma fractions simultaneously with factor IX, it is all the more important that vitronectin is depleted as a carrier for PAI-1. By the process according to the invention, the vitronectin can be separated from a factor IX or PPSB preparation, and separation of any PAI-1 present can be detected electrophoretically by immunoblot analysis.

Thus, safe administration of a factor IX or PPSB to patients having an increased risk of thrombosis, such as bed-ridden patients, smokers and patients subjected to hormone treatments, for example, with contraceptives, is possible for the first time with the preparation obtainable according to the invention.

The invention will be further illustrated by the following example.

Separation of viruses from a preparation of factor IX.

Samples containing factor IX were dissolved in 5 ml of a mobile buffer, 200 mM NaCI, 20 mM sodium citrate dihydrate, pH 7.4, and charged onto a Pharmacia XK-26 column (r = 13 mm, h = 680 mm). At a flow rate of 1 ml/min, fractions were collected every 4 min. Separation was effected on Fractogel EMD Bio SEC 650 (S) (Merck Darmstadt). A typical chromatogram is represented in the Figure. The dotted line corresponds to fractions in which DNA could be detected. The detectable DNA eluted about in the range of fractions 16 to 18 can be assigned to intact viral particles whereas the apparent second peak in the range of fractions 26 to 44 is due to viral fragments which are non-infectious. Thus, the viral particles themselves are completely eluted within fractions 16 to 28. For the parameters selected, the factor IX fraction appears in the range of fractions 32 to 40 so that sufficient separation from the virus particles can be observed.

The viral fragments were completely removed by digestion with DNase and were below the detection limit even after amplification with PCR. At the same time, the accompanying protein vitronectin could be separated off to obtain a homogeneous factor IX preparation.

Claims

C L A I M S

1. A process for removing or separating viruses from potentially virus- containing samples using gel permeation chromatography on hydrophilic supports.

2. The process according to claim 1, characterized in that said hydrophilic supports are polysaccharides, especially dextranes, cellulose, agarose, modified polysaccharides, so-called tentacle chromatographic materials, silica gels modified with hydrophilic groups, or combinations thereof.

3. The process according to claim 1 or 2, characterized in that said hydrophilic supports have a large-pore structure, preferably in the form of particulate materials have a grain size of from 0.5 to 350 μm, or consist of a compact block material.

4. The process according to at least one of claims 1 to 3, characterized in that the pore size of said hydrophilic support is larger than 5 nm, especially larger than 50 nm.

5. The process according to at least one of claims 1 to 4, characterized in that the sample contains biopolymers, such as proteins, polypeptides, oligopeptides, nucleic acids, polynucleotides, oligonucleotides and/or carbohydrates.

6. The process according to claim 5, characterized in that the sample contains substances from blood plasma, such as factor IX, antithrombin III, immunoglobulins G and A, factor VIII, factor VII, αi-antitrypsin, prothrombin, thrombin, factor X, protein C, protein S, or the mentioned substances also from a fraction for their production by genetic engineering.

7. The process according to at least one of claims 1 to 6, characterized in that said viruses are blood-borne viruses.

8. The process according to at least one of claims 1 to 7, characterized in that discontinuous or continuous methods or combinations thereof are used.

9. The process according to claim 8, characterized in that annular chromatography, simulating moving bed (SMB) chromatography, and/or truly moving bed chromatography is used as said continuous method.

10. The process according to claim 8, characterized in that chromatography on compact block materials and/or column chromatography is used as a continuous or discontinuous chromatographical method.

11. A pharmaceutical preparation containing factor IX obtainable from blood plasma by a process according to claim 6, said preparation being free from viruses, viral fragments and vitronectin.

12. The preparation according to claim 11 being free from plasminogen activator inhibitor.

13. The preparation according to claim 11 or 12 which additionally contains factors II, VII and X, and optionally the coagulation inhibitors protein C and protein S.

14. Use of the preparation according to any of claims 10 to 13 for the preparation of a pharmaceutical formulation for treating blood clotting disorders in patients subject to an increased risk of thrombosis.