CA2007236C - Process for preparing virus-free natural substances - Google Patents

Abstract

There is described a viral decontamination process for the preparation of essentially virus free natural substances which involves treating the natural substance with a mixture comprising a halogenated aliphatic hydrocarbon and an lower alcohol and, optionally, water.
The treatment kills off or inactivates coated and uncoated viruses, and may be used for example in the treatment of organ preparations.

Description

200'~~36 The invention relates to a process for preparing virus--free natural substances, from natural substances which may contain coated or uncoated viruses.
As is well known, lipid extraction using halogenated hydrocarbons, such as chloroform or Freon, inactivates coated viruses by destroying the caat (see EP-B-111549). Hitherto, howevE:r, uncoated 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 from tissues is achieved if the tissue is homo-genised with a mixture of chloroform and methanol to produce a singly=-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/wat:er phase contains the non-lipids.
The study performed by Bligh and Dryer utilised substrates that were :not virally contaminated and so makes no mention of the inactivation of viruses, particularly uncoated viruses.
Surprisingly, it has now been found that uncoated viruses which are resistant to halogenated aliphatic hydrocarbons (such as chloroform) and alcohols (such as methanol) on their own 1a 20 0 7 2 3 8 can be killed off or inactivated with a mixture of a halogenated aliphal_ic hydrocarbon, Cl_6 alcohol and water.
Thus viewed from one aspects, the invention provides a use of a mixture of a halogen-containing aliphatic hydrocarbon, an alcoho:L having up to 6 carbon atoms and water, for inactivating non-enveloped viruses in natural substances.
Viewed from another aspect., the invention provides a viral decontamination process for preparing a substantially virus-free natura:L substance, which process comprises treating a natural substance, in particular one contaminated or suspected to be contaminated with coated or more particularly uncoated viruses, with a mixture of a halogenat:ed aliphatic hydrocarbon, a Cl-6 alcohol and water.
Viewed from another aspect:, the invention also provides a substantially virus-free natural substance whenever prepared by treatment of a virus-contaminated natural substance according to the pr~~cess of the invention.
Viewed from yet another aspect, the invention provides a process for obtaining a surfactant that is free from active non-enveloped viruses, which process comprises washing the alveolae of cattle lungs with saline solution, thereafter adding chloroform and methanol to the saline solution to form a homogenous organic-aqueous mixE:d phase and a protein precipitate, separating the protein precipitate and then adding further saline solution and chloroform to cause phase separation and isolating the surfactant from the organic phase.
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Mixtures of chloroform with one or more alcohols selected from methanol, 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 rE~sults are obtained with a methanol/chloroform/water mixture, preferably in the form of ternary phase.
E;~amples of halogenated aliphatic hydrocarbons include in particular mono, di or tri-halo C1-4 alkanes, such as 1,2-dichlo:roethane, 1,1-dichlorobut.ane, 1,1,1-trichloroethane and trichloroethylene, and in particular chloroform, whilst examples of C1-~~ alcohols include ethanol, p.ropanol, isopropanol, n-butano.l, n-hexanol and in particular methanol. The inclusion of water ~~r saline, e.g. physiological saline, is particularly useful if ternary phases can then be obtained.
T:~e viruses are inactivated or killed off generally by homoge:zisation or dissolving in or simply treating the substrate or material in question (e.g. t:he 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 C
a 200"~~36 --20 to 10°C.
It is particularly advantageous to treat or dissolve the substrate or' material in a homogeneous phase consisting of one part by volume of physiological :saline solution, 1.1 parts by volume of chloroform and :?.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.
rdo further virus activity can be detected either in the aqueous or in the organic phase, irrespective of whether coated or uncoated viruses were present.
The process according to the invention for preparing essentially virus-free natural substances 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 =>ubstances which are simply resistant to solvents, since t:he aqueous phase, for example, shows no further viral activity either.
The feasibility of the process according to the invention is demonstrated by means of the following non-7_imiting 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 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. Cattle herds may be affected, for 200'236 example, by bovine respiratory syncytial virus (F~aramyxovirus), infectious bovine rhinotracheitis (I:ferpes virus), parainfluenza {Paramyxovirus) as well as bovine leukaemia (Retrovirus), foot and mouth disease (F~icornavirus) or rabies (Rhabdovirus). 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 coated and uncoated test viruses were added to the test material before processing and this material was then tested again for viral activity after decontamination according to the invention.
The test viruses used were:
a) a coated DNA virus occurring in cattle populations (in this case Bovine Herpes Virus Type .C
(hereinafter referred to as BHV 1 or IBR-IPV)) and b) an uncoated RNA virus (in this case ECBO Virus, strain LCR-4 of the University of Giessen, Institute of Virology of the Veterinary Medical Faculty).
These test viruses, which are representative of coated viruses which occur in cattle populat_Lons and of the uncoated viruses which occur relatively rarely in cattle, were added to the crude surfactant obtained by centrifuging in separate studies.
The DNA virus used, BHV 1 (or IBR-IPV) virus, has a lipoprotein coat and is unstable in the presence of chloroform and ether and is of strictly bovine origin and widespread among calf populations. The diameter of the virion is 150 to 180 nm; it cannot be removed by sterile filtration.
The RNA virus used, the ECBO virus mentioned above, was selected as the official test strain of the 200'236 DE:utsche Veterinarmedizi.nische Gesellschaft (the German Veaerinary Association) within the scope of the testing of chemical disinfectants. Classification of the virus i~~ as follows genus: enterovirus, family:

5 Picornaviridae. ECBO is a single-strand RNA virus, with an. uncoated, cubic capsid, stable in the presence of chloroform, 24-30 nm in diameter.
The test viruses were coated, as culture supernatants, with 105 TCIDSO/ml (BHV-1) or 106-8 TCIDSO/ml. The filters were autoclaved before use.
(TCIDSO 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 with both test viruses. 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 wa;~-mixed 1:1 by volume with 9 g/1 of saline solution to pr~~duce the material of :3ample 1. Filtering through a 0.22 ~,in filter yielded the material from which Sample la wa;~ taken. Sample 1a serves as a control in place of the contaminated starting material since native surfactant cannot be sterile-filtered as its particles arE~ larger than bacteria and consequently block the fi=Lter.
The crude surfactant is obtained from the washings of cattle lungs by centrifuging. The moist pellet obtained contains cell debris and bacteria in addition to the surfactant. At this point, 23 ml of moist surfactant pellet was tal~:en from the production process and suspended in 23 ml of: the virus-containing culture 200~~36 supernatant.
This contaminated ~.urfactant 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 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 50.6 ml of chloroform, phase separation was carried out.
The aqueous phase was separated off and sterile-filtered through a 0.22 ~m membrane filter (Sample 2). The organic phase was also sterile-filtered through a 0.22 ~m filter. The solvent was distilled off in vacuo 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 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
virus) were mixed with 4 ml of 9 g/1 saline solution and sterile-filtered (Sample 1).
5 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).
5 ml of virus suspension (ECBO virus) were mixed 200'~~36 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-f: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 chloroform, phase separation took place. The aqueous phase was separated off and sterile-filtered (Sample 5).
Example 3: Viral activity investigation All the samples of Examples 1 and 2 were investigated on MDBK cell cultures (Nadin and Darby, Esovine Kidney: ATCC CCL 22) by the adsorption method and by inoculation into the culture medium. A1.1 the samples apart from 1 (la) were tested for BHV 1 or ECBO virus-specific changes only after several sub-passages owing t:o their cell-toxic properties.
By mixing the crude surfactant materia:L in the volume ratio 1:1 with culture supernatant (:105 and 1.06'8 TCIDSO/ml) a high contamination of the atarting material was simulated. The limits of proof for the test viruses used is about 10 TCIDSO/ml. The coated BHV-1 virus or the uncoated ECBO virus could only be detected in Samples 1 and 1a according to Example 1.
The test viruses could not be detected in Samples 3 and 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 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).

200'7236 s This finding was expected in the case of the coated and chloroform-sensitive BHV-1 virus but it 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 ~;olution and with chloroform/methanol/saline solution-~~F-RI 1, in Example 2. The cytopathogenic effects of t:he ECBO virus were detectable in the positive control and in the samples with chloroform or methanol (samples 1. to 3 of Example ?.). Samples 4 and 5 of Example 2, on t:he other hand, showed no virus-specific effects on MDBK
c:el l cultures .
This result shows that the organic solvents chloroform or methanol on their own will not inactivate E;CBO viruses, nor will they affect the test results.
C>nly the combined action of the halogenated solvent and t:he alcohol (in this case in a homogeneous organic-aqueous mixed phase) inactivated the uncoatc~d test virus.
As demonstrated by this test model, thc~ process according to the invention is generally suitable for killing or inactivating viruses, but particularly L~coated viruses, in organic preparations.

Claims (17)

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

2. Use of a mixture of chloroform, one or more alcohols selected from the group consisting of methanol, ethanol, propanol and 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 any one of claims 1 to 3, in which the water is in the form of a physiological saline solution.

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

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

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

8. A process for obtaining a surfactant that is free from active non-enveloped viruses, which process comprises washing the alveolae of cattle lungs with saline solution, thereafter adding chloroform and methanol to the saline solution to form a homogenous organic-aqueous mixed phase and a protein precipitate, separating the protein precipitate and then adding further saline solution and chloroform to cause phase separation and isolating the surfactant from the organic phase.

9. A process according to claim 8, wherein the surfactant obtained from the alveolae comprises phospholipids, cholesterol, glycerides, fatty acids or surfactant-associated proteins of type B and C.

10. A process according to claim 8 or 9, wherein the homogeneous organic-aqueous mixed phase is cooled to facilitate precipitation of the protein.

11. A process according to claim 8, 9 or 10, wherein the saline solution used to wash the alveolae is physiological saline solution.

12. A process for inactivation non-enveloped viruses in a natural substance which process comprises treating the natural substance with a mixture of a halogenated aliphatic hydrocarbon, a C1-6 alcohol and water.

13. A process as claimed in claim 12, wherein as said mixture is used a mixture of chloroform, at least one alcohol selected from the group consisting of methanol, ethanol, propanol and butanol, and water.

14. A process as claimed in claim 12, wherein as said mixture is used a ternary phase comprising chloroform, methanol and waiver.

15. A process as claimed in claim 14, wherein the water is in the form of a physiological saline solution.

16. A process as claimed in any one of claims 12 to 15, wherein the virus that is inactivated is a virus of the Picorna viridae family.

17. A process as claimed in any one of claims 12 to 15, wherein the virus that is inactivated is an ECBO virus.