IE900045L - Viral decontamination of natural substances - Google Patents

Abstract

Method for preparing virus-free natural materials uses a mixture of a halogenated, aliphatic hydrocarbon, a lower alcohol and optionally water for killing or deactivating coated and uncoated viruses, for example during the treatment of organ preparations.

Description

6301 i £f-s/*io .

VIRAL DECONTAMINATION OF NATURAL SUBSTANCES The invention relates to a process for preparing a natural substance free from active non-enveloped viruses.

As is well known, lipid extraction using halogenated hydrocarbons, such as chloroform or Freon, inactivates coated viruses by destroying the coat (see EP-B—111549) . Hitherto, non-enyeloped viruses have been regarded as resistant to numerous organic solvents and may therefore constitute a possible source of contamination of substances of biological origin, e.g. natural substances intended for parenteral use in humans, such as organ extracts and hormone preparations.

E.G. Bligh and W.J. Dyer have described a method of lipid extraction using mixtures of chloroform, methanol and water (see Can. J. Biochem. Physiol. 37: 911-917 (1959)). Optimum lipid extraction . is achieved if the tissue is homogenised with a mixture of chloroform and methanol which produces a single-phase mixture with the water contained in the tissue. The homogenate can then be diluted with water and/or chloroform to form a two-phase system, in which the chloroform phase contains the lipids and the methanol/water phase contains the non-lipids.

.. This study, malces no mention of the inactivation of viruses, particularly non-enveloped viruses.

EP-A-0 112 563 describes a process for deactivating viruses which contain lipids in their structural composition in plasma protein products. The process consists of treating the product with organic solvents in which the structural lipids of the viral contaminants are soluble, unlike the plasma protein. Solvents which may be used include inter alia chloroform, ether, aromatic hydrocarbons and short-chained alcohols. There is no mention of the addition of water..

WO 85/05273 describes a process for deactivating lipid-containing viruses, particularly hepatitis B and non-A, non-B hepatitis viruses, in lyophilised plasma protein products by treating the product with tk 7 n I S n_/ \J ' - solvents which are 75 to 100% saturated with water at the relevant treatment temperature. The preferred solvents mentioned are chloroform, optionally mixed with a short-chained alcohol such as methanol or ethanol, and chlorofluorohydrocarbon.

The two patent specifications mentioned above make no mention of any deactivating effect on lion-enveloped viruses.

Surprisingly, it has now been found that non-enveloped viruses which are resistant to halogenated aliphatic 10 hydrocarbons (such as chloroform) and alcohols (such as methanol) on their own can be killed off or inactivated with a mixture of a halogenated aliphatic hydrocarbon and an alcohol having up to 6 carbon atoms.

Mixtures of chloroform with one or more alcohols selected from ethanol, propanol, and butanol, with or without the addition of water, have proved suitable for this purpose, especially mixtures of chloroform and methanol. Particularly good results are obtained with a methanol/chloroform/water mixture, preferably in the form of the ternary phase.

Examples of halogenated aliphatic hydrocarbons include 1,2-dichloroethane, 1,1-dichlorobutane, 1,1,1-trichloroethane and trichloroethylene, and in particular chloroform, whilst examples of alcohols include ethanol, propanol, isopropanol, n-butanol, n-hexanol but' particularly methanol. The addition of water is particularly useful -if ternary phases can then be obtained.

The viruses are inactivated or killed off generally by homogenisation or dissolving in or simply treating the substrate or material in question (e.g. the natural substances or compositions or substrates, e.g. tissues, containing them) in or with a mixture consisting of the halogenated aliphatic solvent and an alcohol and, optionally, water, e.g. a mixture consisting of water or physiological saline solution, chloroform and methanol, the treatment mixture conveniently being stored for about 1 to 3 hours at low temperatures, for example at 3 -20 to icrc.

It is particularly advantageous to treat or dissolve the substrate or material in a homogeneous phase consisting of one part by volume of physiological 5 saline solution, 1.1 parts by volume of chloroform and 2.2 parts by volume of methanol, with one further part by volume of saline solution and 1.1 parts by volume of chloroform being added after a treatment time of, for example, 2 hours. Separation into two phases occurs. 10 No further virus activity can be detected either in the aqueous or in the organic phase.

The process according to the invention for preparing a natural substance free from active, non-enveloped viruses which has been discovered can be used to treat all natural raw materials which may be used in humans or animals. The process may be used not only for lipid-extractable materials but also for substances which are simply resistant to solvents, since the aqueous phase, for example, shows no further viral 20 activity either.

The feasibility of the process according to the invention is demonstrated by means of the following non-limiting Examples which relate to the viral decontamination of the surfactant SF-RI 1.

SF-RI 1 is prepared from the product of washing out healthy cattle lungs which have been removed by a veterinary surgeon. The washing out of the alveoli is carried out using physiological saline in combination with organic solvents, e.g. chloroform. SF-RI 1 is a 30 mixture consisting of phospholipids, (about 90% by weight), cholesterol (about 0.3% by weight), glycerides (about 4% by weight), fatty acids (about 0.3% by weight) and surfactant-associated proteins of type B and C (about 1% .by weight).

Generally, the lungs are not contaminated with cattle-specific viruses, but contamination cannot be ruled out altogether. However,in order to be able to test the effectiveness of the process according to the invention, a non-enveloped test virus (in this case ECBO Virus, strain LCR-4 of the University of Giessen, Institute of Virology of the Veterinary Medical Faculty) was added to the crude surfactant obtained from the wash-out by centrifuging. This test virus is representative of the non-enveloped viruses which occur relatively rarely in cattle. Cattle herds may be affected, for example, by foot and mouth disease (Picornavirus). In order to guarantee a surfactant totally contamination free it would have to be tested for every possible virus. As this is not possible, in order to be able to test the effectiveness of the process according to the invention, selected exemplary test viruses were added to the test material before processing and this material was then tested again for viral activity after inactivation according to the invention.

The test viruses used .was a non-enveloped RNA virus.

The RNA virus used, the ECBO virus mentioned above, was selected as the official test strain of the Deutsche Veterinarmedizinische Gesellschaft (the German Veterinary Association) within the scope of the testing of chemical disinfectants. Classification of the virus is as follows genus: enterovirus, family: 5 Picornaviridae. ECBO is a single-strand RNA virus, with non-enveloped, cubic capsid, stable in the presence of chloroform, 24-30 nm in diameter.

The test viruses were coated, as culture supernatants, with 10s TCIDS0/ml (BHV-l) or 10 106"8 TCID50/ml. The filters were autoclaved before use. (TCID50 is the Tissue Culture Infectious Dose, whereby 50% of the cells are virus infected) .

A validation study (Example 2) was carried out separately and in parallel to the preparation process 15 with the test virus. The procedure for the preparation process, the addition of the test virus and the taking of samples are set forth in Example 1 with reference to the working up of the crude surfactant from the washings of cattle lungs: Example 1: Test Sample Production As a positive control, a virus culture supernatant was mixed 1:1 by volume with 9 g/1 of saline solution to 25 produce the material of Sample 1. Filtering through a 0.22 /xm filter yielded the material from which Sample la was taken. Sample la serves as a control in place of the contaminated starting material since native surfactant cannot be sterile-filtered as its particles 30 are larger than bacteria and consequently block the filter.

The crude surfactant is obtained from the washings of cattle lungs by centrifuging. The moist pellet obtained contains cell debris and bacteria in addition 35 to the surfactant. At this point, 23 ml of moist surfactant pellet was taken from the production process and suspended in 23 ml of the virus-containing culture 6 supernatant.

This contaminated surfactant suspension was extracted by the method of Bligh and Dyer (supra) . In order to do this, 50.6 ml of chloroform and 101.2 ml of 5 methanol were added and the homogeneous organic-aqueous mixed phase produced was stored in a cold place for 2 hours, expediently at -10 to -20"C. The protein precipitate formed was removed by centrifuging. After the addition of 46 ml of 9 g/1 saline solution and 10 50.6 ml of chloroform, phase separation was carried out. The aqueous phase was separated off and sterile-filtered through a 0.22 /xm membrane filter (Sample 2). The organic phase was also sterile-filtered through a 0.22 /xm filter. The solvent was distilled off in vacuo 15 at 10°C and the lipid was dried at ambient temperature. 0.95 ml of distilled water were added for every 50 mg of dry lipid. By shaking for about 10 minutes, liposomes were obtained with a vesicle size of about 1000 nm (Sample 3) . Some of the liposomes were bombarded with 20 ultrasound at an output of 20 Watts for 4 minutes (Branson Sonifier, Microtip). Vesicles were formed measuring 200 nm (Sample 4).

Example 2 : Comparative sample production In order to make sure of the virucidal effect of the.process of the invention, the influence of individual process steps was investigated more fully. 4 ml of culture supernatant containing virus (ECBO 30 virus) were mixed with 4 ml of 9 g/1 saline solution and sterile-filtered (Sample 1). ml of virus suspension (ECBO virus) were shaken ; with 1 ml of chloroform. The aqueous phase was separated off and sterile-filtered (Sample 2) . 5 ml of virus suspension (ECBO virus) were mixed with 0.5 ml of methanol and sterile-filtered (Sample 3). ml of virus suspension (ECBO virus) were mixed 7 with 11 ml of methanol and 5.5 ml of chloroform to produce a homogeneous phase. Phase separation was effected by adding 5 ml of 9 g/1 saline solution and 5.5 ml of chloroform. The aqueous phase was sterile-5 filtered (Sample 4) . 1 g of SF-RI 1 lipid were dissolved in a mixture of 5 ml of virus suspension (ECBO virus) with 11 ml of methanol and 5.5 ml of chloroform. After the addition of 5 ml of 9 g/1 saline solution and 5.5 ml of 10 chloroform, phase separation took place. The aqueous phase was separated off and sterile-filtered (Sample 5).

All the samples of Examples 1 and 2 were investigated on MDBK cell cultures (Nadin and Darby, Bovine Kidney: ATCC CCL 22) by the adsorption method and by inoculation into the culture medium. All the samples apart from 1 (la) were tested for ECBO virus- specific changes only after several sub-passages owing to their cell-toxic properties.

By mixing the crude surfactant material in the volume ratio 1:1 with culture supernatant (10s and 106'8 TCIDS0/ml) a high contamination of the starting 25 material was simulated. The limits of proof for the test viruses used is about 10 TCIDS0/ml. The non-enveloped ECBO virus could only be detected in Samples 1 and la according to Example 1. The test viruses could not be detected in Samples 3 and 30 4 according to Example 1, both of which simulate removal from the production process at different times. The studies according to Example 1 show that even in the extraction process according to Bligh and Dyer the test viruses added were inactivated or removed. The test 35 viruses could no longer be detected either in the aqueous phase (Sample 2) or in the dry mass from the organic phase (Samples 3 and 4). 8 This finding was not foreseeable for the uncoated ECBO virus.

In order to pinpoint and make sure of the step which inactivates the virus, ECBO viruses were mixed with chloroform, methanol, methanol/chloroform/saline solution and with chloroform/methanol/saline solution-SF-RI 1, in Example 2. The cytopathogenic effects of the ECBO virus were detectable in the positive control and in the samples with chloroform or methanol (samples 1 to 3 of Example 2) . Samples 4 and 5 of Example 2, on the other hand, showed no virus-specific effects on MDBK cell cultures.

This result shows that the organic solvents chloroform or methanol on their own will not inactivate ECBO viruses, nor will they affect the test results.

Only the combined action of the halogenated solvent and the alcohol (in this case in a homogeneous organic-aqueous mixed phase) inactivated the uncoated test virus.

As demonstrated by this test model, the process according to the invention is generally suitable for killing or inactivating viruses, but particularly non-enveloped viruses, in organic preparations.

Claims (9)

1. Use of a mixture of halogen-containing aliphatic hydrocarbons, alcohols having up to 6 carbon atoms and water, for inactivating non-enveloped viruses in natural substances.

2. Use of a mixture of chloroform and methanol and/or ethanol and/or propanol and/or butanol and water according to claim 1.

3. Use of a ternary phase of chloroform, methanol and water according to claim 1.

4. Use of a mixture according to claims 1 to 3, in which a physiological saline solution is added instead of water.

5. Use of a mixture according to claims 1 to 4 for inactivating viruses of the Picorna viridae family in natural substances.

6. Use of a mixture according to claims 1 to 4 for inactivating ECBO viruses in natural substances.

7. Use according to claims 1 to 4, characterised in that the natural substance is a surfactant obtained from cattle lungs by washing out.

8. Process of preparing a surfactant free from active non-enveloped viruses, consisting of phospholipids, cholesterol, glycerides, fatty acids and surfactant-associated proteins of type B and C, characterised in that a lung surfactant is extracted, which is obtained by washing out cattle lungs by washing the alveolae with physiological saline solution, wherein a homogeneous, organic-aqueous mixed phase is produced by adding chloroform and methanol, a protein precipitate formed at the same time is separated off, then phase separation is effected by the addition of saline solution and chloroform and the surfactant is isolated from the organic phase.

9. Surfactant, characterised in that it is prepared by a process according to claim 8. TOMKINS & CO.