Note: Descriptions are shown in the official language in which they were submitted.
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- 1 - 27169-170
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
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3
--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
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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
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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
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6
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
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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).
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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.
