Note: Descriptions are shown in the official language in which they were submitted.
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BACKGROUND
The purifica~ion and properties of bovine thymosin
have been described by Hooper et al, ~nnals New York ~cademy
of Sciences, Vol. 249 p. 125-144 (1975). A process of pre-
paring thymosin has also been described in BritishPatent -
1,195,980.
A crude thymosin which is referred to as "thymosin-
fraction 3" is described by Hooper et al.
Thymosin-fraction 3 is a prime intermediate in the
preparation of pure thymosin from calf ~hymus glands. Thymosin-
fraction 3 contains all the thymosin originally present in the
calf thymus glands but contains only 1% o the original weight
of the glands. The greatly reduced bulk and the 100-fold
purification allows the application of known and usual protein- ~ '
purification procedures for the preparation of pure thymosin.
Purified thymosin is known to consist of several
proteins with molecular weights ranging from -l,200-14,000.
It has been shown to stimulate lymphoid tissue proliferation
and to thereby increase immunity to infectious processes.
Recent clinical trials involving immunodeficiency diseases
have sh~w~ that thymosin increases the number of peripheral
blood T-cells thereby partially restoring immunological
competence.
Previous methods of extracting thymosin from
thymus glands involved homogenizing the glands in saline so-
lution and centrifuging the homogenate. Such processes are
laborious for the extraction of large quantities (500-1,000
pounds) of thymus glands and result in the extraction of
undesirable fatty constituents of the glands which interfere
with subsequent processing. In the published procedures the
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heat-labile proteins in the saline extract are precipitated by
heating the extract -to 80G in a water bath. In previous
procedures, the extract obtained after removing the heat-
labile proteins is mixed directly with five or ten volumes of
cold acetone to precipitate thymosin-fraction 3.
OBJECTS
One of the objects of -the present invention i.s to
provide a new improved process for preparing thymosin in which
thymosin-fraction 3 is prepared in a more simple manner than
in previous processes and witha minimal production of inter-
fering substances such as fats and undesired or inactive pro-
teins.
Another object of the invention is to provide a
process of the type described which is more economical than ,.
previous procedures. Other objects will appear hereinafter.
BRIEF SUMMARY OF THE INVENTION
Thymosin is produced by an improved process in which
thymus glands are coarse grolmd, extracted in saline solution
which is .iltered and the filtrate or extract is injected with .-:
steam for a brief period of time to denature undesired proteins
without denaturing the thymosin, whereby a precipitate is
formed which is separated by filtration. The filtrate is con- ~'
centrated and cooled and crude thymosin is precipated by adding
the concentrate to acetone.
DETAILED DESCRIPTION OF THR INVENTION
In the practice of the invention frozen thymus
, glands such as, for example, calf thym~ls glands, are ground
; in a meat grinder so as to produce relatively coarse ground
particles having diameters of about 1/16 inch to 3/16 inch.
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These coarse paxticles are then extracted by stirring with a
saline solution in a conventional manner. The solids are
filtered preferably by using a filter aid such as diatomaceous
earth or any oth~r suitable fllter aid, and the filter cake
is discarded. The filtrate in the form of a flowing stream
is then brought into contact with steam by injecting steam
under superatmospheric pressure into the flowing stream so as
to bring the filtrate instantaneously to a temperature of around
80~85C. The time during which the filtrate is held at this
temperature should be at least sufficient to denature the :
undesired protein but not long enough to denature thymosin.
Usually instantaneous heating :Eor a period of at least one
minute is required.
Ei.ther low pressure steam, for example, at 15 psig,
or high pressure steam, for e~ample, at 60 psig, can be used
to provide instantaneous heating. Usually not more than
several minutes are required. This produces a suspension of ~ ;~
precipitated protein which is then cooled, preferably by
discharging it on ice, and filtered, preferably with the
assistance of a filter aid such as, for example, diatomaceous
earth. The filtration is preferably accompli~hed by using
a filter press and the filter cake is discarded.
The resultant filtrate is preferably concentrated
to about one-half its volume and cooled to a relatively low
temperature, usually around 4C. The concentrate is then
added to a larger volume of acetone which has been cooled to
a lower temperature than the concentrate, preferably to a
temperature around -10C. This gives a precipitate of crude
thymosin. The supernatant liquid can be decanted and the
thymosin collected by filtration, washed with fresh acetone
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and dried under vacuum drying conditions.
The invention will be further illustrated but is
not limited by -the following example in which the quantities
are given in parts by weight unless otherwise indicated.
EXAMPLE
500 pounds of frozen calf thymus glands were ground
in a meat grinder and the ground glands were mixed with 185
gallons of 0.15M sodium chloride at 4C. for 5-30 minutes.
250 pounds of filter aid were stirred into the
mixture and the mixture was filtered on a filter press. The
filter cake was discarded. The ~iltrate was brought to 80-
85C. by injecting steam into a flowing stream of the extract ~ `
and allowing the steam and extract to remain in contact with
each other for ].-2 minutes. The resultant suspensi.on of pre-
cipitated protein was discharged onto 200 pounds of ice, 80
pounds of filter aid were added and the resulting slurry was
~iltered on a filter press. The filter cake was discarded.
The 200 gallons of ~ltrate were concentrated to 100
gallons and cooled to 4C. The concentrate was added to 400
gallons of acetone cooled to -10C. ThiS gave a precipitate
of crude thymosin. The supernatant was dec~lnted and the -
thymosin was collected by filtration, washed with fresh acetone
and dried in vacuo. The dried powder weighed 2,500 g.
As contrasted with previous processes in which
thymus glands have been homogenized in saline solution and
centrifuged, the present process is much simpler in that the
frozen thymus glands are ground with a meat grinder and the
ground glands are extraced by stirring with saline solution.
By this method 500:pounds of calf thymus glands can be ground
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and extracted in one hour. Furthermore, by coarse grinding
rather than homogenizing, -the release of interfering sub-
stances, especially fat, is kept at a minimum, and the
extrac.t is substantially free of fatty material and can be
readily filtered on a filter press.
Thymosin is soluble in dilute saline solution such ::
as for example, 0.15 molar sodium chloride in water. Any
dilute aqueous saline solution capable of extracting the
thymosin can be used in the initial extraction.
Whereas in the published procedures the heat-labile
proteins in the saline extract are precipitated by heating the .
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extrack to 80C.in awater bath, in the present invention the :~
heat-labile proteins are precipitated by injecting steam into ~ ;
a ~lowing stream of the extract, thereby raising the temperature
of the stream to 80C. instantaneously and holding the tempera-
ture of the extract at 80C. for 1-2 minutes. The stream is : :~
then pumped onto ice to reduce the temperature, mixed with
filter aid and filtered on a filter press. This process provides
for the precipitation o the unwanted heat-labile proteins but
the rapid heating (as contrasted to heati.ng on a water bath)
minimizes the time period during which the extract is held at a
high temperature, thus lowering the risk of denaturing the
thymosin and preserving the integrity of the thymosin.
While in previous procedures the extract obtained
after removing the hea~-labile proteins is mixed directly with
5 or 10 volumes of cold acetone to precipitate thymosin-
fraction 3, in the present invention it is concentrated in vacuo
at 25-30C., preferable to one half of its original volume. The
final concentrated extract is then added to four to ten times
its volume of cold acetone to precipitate thymosin-fraction 3.
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This process yields the same quantity of fraction 3 as the
published procedures but reduces the quantity of acetone
required by 50% and greatly simplifies the precipitation :~
and collection procedures.
It is believed to be apparent, therefore, that the
present invention greatly facilitates the preparation of
thymosin. While crude thymosin (thymosin~fraction 3) usually
contains less than 1% by weight thymosin, it will be under-
stood that this can be further purified in any suitable ma~ner,
for example, by the procedures described in the previously
mentioned references.
It will also be understood that the invention is
susceptible to some variation and modification in its practical
application. Thus, while instantaneous heating of the saline
solution extract is preferably accomplished by injection of
steam into a stream of said solution, the heating might also -
be accomplished by high intensity rays or by means o~ conven-
tional heat exchangers or in any other manner which does not
inactivate or denature the thymosin. -
The concentration of the filtrate prior to addition
to acetone is preferably accomplished by evaporation in vacuo .
usually at a temperature around 30C.
