Note: Descriptions are shown in the official language in which they were submitted.
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METHODS OF PRESERVING MICROORGANISMS
The present invention relates to methods of preserving
micro-organisms such that they retain their in~ectivity.
In particular the invention relates to methods o~
preserving viral particles.
Storage/viability problems occur in relation to micro-
organism storage. In particular problems occur in
relation to viral storage where the virus particles are
employed ~or uses such as:-
(a) viral vectors ~or use in, e.g. gene
therapy;
(b) storage of viruses ~or general research
progress, e.g. in culture banks;
(c) viruses to be used ~or release into the
environment ~or control o~ agricultural pests;
and
(d) vaccines.
Vaccines comprising viral particles have been in use ~or
a number o~ years. It is, however, essential that such
vaccines can be stored, sometimes ~or long periods,
without the viral component losing its in~ectivity.
Common storage methods include ~reezing or ~reeze-drying,
the latter usually involving reconstitution using water
at a later stage. Un~ortunately, certain viruses display
reduced viability/in~ectivity when subjected to these
processes.
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One virus which is not suitably stored as described above
is polio virus. This virus is readily degraded at room
temperature in aqueous suspension, is stable ~or only two
weeks at 0~C and is destroyed by lyophilisation. For this
particular virus pre~erred methods of storage involve
~reezing at -70~C or re~ridgeration at 4~C. However, such
s~orage conditions are not particularly suitable ~or use
in tropical countries or indeed countries where the
required ~acilities and equipment are scarce.
International Application No PCT/GB94/02495 discloses
compositions comprising a hydrophilic species solubilised
in a hydrophobic phase, as well as methods ~or their
preparation. UK appliçation no. 9424901.8 discloses
compositions as - described in PCT/GB94/02495 which
incorporate additional components which aid retention of
the hydrophilic species in the hydrophobic phase. UK
application no.9424902.6 discloses compositions as
described in PCT/GB94/02495 which incorporate moieties
which aid ~ormation o~ the composition.
In addition, UK patent application no. 9422990.3
discloses immunogenic compositions which comprise an
immunogen solubilised, suspended or otherwise dispersed
in a hydrophobic phase. The immunogen can be a virus and
the compositions are use~ul as vaccines.
It has now been ~ound that micro-organisms, particularly
virus particles, such as polio virus particles, may be
converted to a ~orm suitable ~or long term storage at
ambient temperature, with retention o~ in~ectivity a~ter
reconstitution in aqueous medium. Thus, such compositions
have particular advan~ages ~or use in countries where the
ususal storage methods are less appropriate, and provide
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an e~ective means by which such viruses can be
transported and stored without the need ~or extreme
~ ~reezing or prolonged re~ridgeration.
~ 5 Thus, in a first aspect, the present invention provides
a method of storing micro-organisms such that they
maintain in~ectivity, which method includes the steps
o~: -
(i) bringing the micro-organisms into
association with an amphiphile; and
(ii) solubilising, suspending or otherwise
dispersing the micro-organisms in a hydrophobic
phase.
In one pre~erred embodiment the micro-organisms are virus
particles particularly polio virus particles.
Suitable methods ~or carrying out the above method are
those described in PCT/GB94/02495, UK 9424901.8, UK
9424902.6 and UK 9422990.3.
The hydrophobic solvent could for example be a long chain
~atty acid, a medium chain alcohol, a branched long chain
alcohol, a monoglyceride, a diglyceride, a medium chain
triglyceride, a long chain triglyceride, a halogenated
(e.g. ~luorinated) analogue thereo~, or a
polyoxyethylene-containing lipid.
In particular embodiments the hydrophobic solvent is a
mono-, di- or tri-glyceride, or oleic acid.
In one pre~erred embodiment the method comprises:
-
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(i) co-dispersing the micro-organisms with an
amphiphile in a liquid medium;
(ii) removing the liquid medium to leave an
array o~ amphiphile molecules with their
hydrophilic head groups orientated towards the
micro-organism; and
(iii) providing a non-aqueous solvent around the
micro-organisms/amphiphile array.
The liquid medium can be water, and it can be removed by,
e.g. ~reeze drying, centri~ugal vacuum drying or any
other suitable method.
Suitably, in the above methods the amphiphile will be a
phospholipid, ~or instance one with a phosphatidyl
choline head group, eg phosphatidyl choline (PC),
lysophosphatidyl choline(lyso-PC), sphingomyelin or a
derivative of one o~ these such as hexadecyl
phosphocholine or an amphiphile polymer containing
phosphoryl choline. A bile salt, a glycolipid, a
polyoxyethylene containing sur~actant, a lipophilic
sulphate, betaine, a sarcosine containing surfactant,
Solulan C24, polyoxyethylene 40 stearate, one o~ the
Tween series o~ sur~actants, one o~ the Span series o~
sur~actants or a pegolated castor oil derivative, e.g.
Cremaphor EL35.
Without wishing to be bound by the ~ollowing, it is
believed that in the methods described above the micro-
organisms, eg virus particles, ~irst ~orm an array with
the amphiphile molecules. This array is then in turn
coated with the hydrophobic solvent. In this way access
,
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to the micro-organisms by water is restricted, which in
turn accounts ~or the improved storage properties when
the micro-organism preparation is reconstituted ~rom a
~reeze-dried state.
In a second aspect, the present invention provides a
method o~ storing micro-organisms such that they retain
in~ectivity, which method includes the following steps:
(i) bringing the micro-organisms into association
with an amphiphile in an a~ueous phase; and
(ii) removing the water.
Preferably, the water is removed by ~reeze-drying.
The amphiphile can be a phospholipid, ~or instance one
with a phosphatidyl choline head group, eg phosphatidyl
choline (PC), lysophosphatidyl choline(lyso-PC),
sphingomyelin or a derivative o~ one o~ these such as
hexadecyl phosphocholine or an amphiphile polymer
containing phosphoryl choline. A bile salt, a glycolipid,
a polyoxyethylene containing sur~actant, a lipophilic
sulphate, betaine, a sarcosine containing sur~actant,
Solulan C24, polyoxyethylene 40 stearate, one o~ the
Tween series o~ sur~actants, one o~ the Span series o~
sur~actants or a pegolated castor oil derivative, e.g.
Cremaphor EL35.
In a particularly pre~erred embodiment o~ this aspect the
amphiphile is Solulan C24, polyoxyethylene 40 stearate,
one o~ the Tween series o~ sur~actants, one o~ the Span
series of sur~actants or a pegolated castor oil
derivative, e.g Cremaphor EL35. In particularly pre~erred
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embodiments the amphiphile is Solulan C24 or
polyoxyethylene 40 stearate.
It is possible that upon removal of the water the
amphile/micro-organism array will be in an "open" form.
Thus, upon reconstitution water may still have access to
the micro-organisms and this will lead to loss of
infectivity. Therefore, in another preferred embodiment
of this aspect of the invention the method also includes
the step of elevating the temperature of the mixture
after removal of the water. This ensures that the
structure adopted by the amphiphile/micro-organism array
is more condensed, which in turn results in more
restricted access ~or water upon reconstitution.
When the heating step is employed, the amphiphile will be
one which remains solid after the water removal step, eg
it can be chosen from a phospholipid, for instance
lecithin, a glycolipid, a polyoxyethylene containing
surfactant, a lipophilic sulphate, betaine, a sarcosine
containing sur~actant, Solulan C24, polyoxyethylene 40
stearate, one of the Tween series of surfactants, one of
the Span series of surfactants or a pegolated castor oil
derivative, e.g. Cremaphor EL35.
In other aspects the invention provides:
i) a micro-organism composition obtainable by any of
the methods described herein, particularly a micro-
organism composition comprising virus particles, eg poliovirus particlesi and
ii) the use o~ a composition o~ the invention ~or the
storage o~ virus particles.
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Preferred features of each aspect o~ the invention are as
for each other aspect mutatis mutandis.
The invention will now be described with reference to the
following example, which should not be construed as in
any way limiting the invention.
Example 1
A suspension of 109 polio virus particles (Sabin strains,
Types 1, 2, 3) per ml of culture was diluted lOOO-fold in
distilled water. lml of the diluted suspension was mixed
with lml of adispersion of sonicated soya phospholipid
(at a concentration of lOOmg/ml) in distilled water. A
control vial was prepared which contained virus only,
without the addition of phospholipid.
The contents of both vials were shell-frozen in liquid
nitrogen and lyophilised overnight. The following day,
lml of oleic acid was added to the vial containing virus
and phospholipid, and the contents of the vial wre then
mixed on a roller mixer for several hours. A clear
solution was obtained.
A control vial o~ polio virus was prepared as above. To
this control vial, containing virus alone, was added lml
of culture medium.
10~1 of oil/virus preparation was transferred to a fresh
vial, and lml o~ a 2~ solution of ox bile extract
(containing predominantly sodium taurocholate) was added.
The mixture was shaken well to disperse the oil in water,
with the intention of releasing particles into the
aqueous phase. Ten-~old serial dilutions were made in
culture medium, and 0.5ml o~ each dilution was added to
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con~luent monolayers o~ Viro cells, and incubated ~or
~our days, to test ~or the presence o~ intact virus. An
identical procedure was ~ollowed ~or the contents of the
control vial. Growth was assessed by visual observation
o~ virus-induced cell lysis in each monolayer. Growth was
recorded in the two series o~ dilutions as ~ollows:
Dilution of lyophilisatelo2103 104 105 lo6
Virus particles present104103 1o2 10
(per ml)
Oil-based lyophilisate + + + +
Oil-~ree lyophilisate + +
These results indicate that the method o~ the present
invention clearly improves the viability o~ stored viral
preparations, when compared to lyophilisation alone.
Example 2
A virus suspension (Sabin strains, Types 1, 2, 3)
containing 5X108 particles/ml (spun to remove
contaminating protein) was diluted 50-~old by addition o~
200~1 o~ the suspension to 9.9ml o~ distilled water,
yielding a concentration of 107 particles/ml. The
suspension was divided into ~our equal aliquots o~ 2.5ml,
and dispensed into 7ml screw-capped glass vials. One
aliquot was employed in the experiment described herein,
while two were used in the experiment described in
example 3.
2.5ml o~ sonicated phospholipid dispersion (lOOmg/ml) was
added to the aliquot o~ diluted virus particles with
gentle mixing. 200~1 o~ this mixture was dispensed into
~reeze-drying vials, and the remainder was
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transferred, In l00~l aliquots, into other tubes as "pre-
drying" controls. The controls were stored overnight at
+4~C. The freeze-drying vials were placed in the
centrifugal rotor of the freeze-dryer and lyophilised
overnight.
On the following day l00~l of culture medium was added to
the contents of ten o~ the vials freeze-dried overnight,
while l00~l of oleic acid (B.P.) was added to the other
ten. The groups were labelled "M" and "O" respectively.
l0~l of samples from two "M" labelled tubes were
transferred to ~resh lml vials, and lml of 0.lM
bicarbonate solution containing 25mg/ml sodium
taurocholate was added and mixed well. Under these
conditions the oil was dispersed well to give a clear
solution.
4 x 20~1 aliquots of sample were transferred ~rom the
pre-drying control group stored overnight at +4~C to
fresh lml vials. To two of these vials was added lml of
medium, while to the other two was added lml of 0.lM
bicarbonate solution containing 25mg/ml sodium
taurocholate. The contents o~ each of the vials was mixed
well.
The suspensions prepared above were used to per~orm l0-
fold dilutions in Vero cell monolayer cultures, in order
to measure the viability o~ the polio virus present. the
results were expressed as the highest dilution at which
50~ cytopathic e~fects were observed.
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Nature o~ sample Highest dilution at which
50% CPE obser~ed
Non-dried control in medium 10-4/10-5
Non-dried control in taurocholate 10-3/10-3
oil-free lyophilate in medium 10-1/10~
Oil-free lyophilate in taurocholate 10-1/lO-
Oil-based lyophilate in taurocholate 10-6/10-6
ExamPle 3
2.5ml of distilled water was added to one aliquot of
virus particles prepared as described in example 2, and
this group was labelled "W". 2.5ml of Solulan C24
(lOOmg/ml) was added to ~nother aliquot and mixed gently.
Thls group was labelled "S".
200~1 of each preparation was dispensed into lO freeze-
drying vials, and the remainder in 100~1 aliquots into
other tubes as "pre-drying" controls. The controls were
stored overnight at +4~C. The ~reeze-drying vials were
placed in the centri~ugal rotor of the freeze-dryer and
lyophilised overnight.
On the following day 100~1 of culture medium was added to
each vial in group "W~' and mixed gently. The vials in
group "S" were sealed and heated to 60~C in a hot water
bath ~or 5 seconds to melt the Solulan C24, which
resulted in a claer solution. Upon cooling to room
temperature this material solidi~ied. 90~1 of medium was
added to the vials of the "S" group to make the total
volume up to 100~1. 10~1 o~ sample was then trans~erred
~rom each of groups "S" and "W" to ~resh lml vials and
lml o~ medium was added to each and mixed well.
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To ~resh lml vials was added 4 x 20~1 o~ samples ~rom
each o~ the pre-drying groups and lml o~ medium was added
to each. The contents of each vial were mixed well.
The suspensions prepared as described herein were used to
per~orm 10-~old dilutions in Vero cell cultures,to
measure the viability o~ the polio virus present. The
results were expressed as the highest dilution at which
50~ cytopathic ef~ects were observed.
Nature o~ Sample Highest Dilution at
which 50% CPE observed
Non-dried control + water 1 0 -4 / 1 0 -6
Non-dried control + Solulan C24 10-5/lO-~
Freeze-dried control + water 1O-2/1O-2
Freeze-dried control + Solulan C24 1O-6/10-8
