Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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This invention relates to a method in the recovery
of insulin from an insulin and fat containing pancreas ~t~t
- The conventional method applied in the preparatio-.
of insulin comprises extraction of the minced frozen pancreas
glands by means of aqueous acidified ethanol or any other
suitable organic solvent, precipitation of insoluble proteins
different from insulin by adjustment of the pH of the extract
to approx. 8, filtration and reduction of the pH of the filt~
te to approx. 3. This leaves a clear extract consisting of
60-65~ aqueous ethanol containing a small amount of acid,
lnsulin and other proteins, enzymes and impurities in solution
as well as abt. 5-10% fatty matter. This fat, being dissolved
in the ethanol, is a very disagreeable component which, frorn
a productional point of view, gives great trouble during the
progress of the classic processes. The usual further treatment
of the extract consists in evaporation under vacuum at a
temperature of 25-30C for removal, and recovery, of ethanol
and for separation of fat gradually as the solvent is separated
off from the raw extract; m e fat is removed after the vacuum
distillation e.g. b~ filtration or centrifugation or by
decantation followed by filtration. The remaining aqueous
fluld is further processed to purified insulin.
Apart from the removal of fat not being complete
by this ~ethod, the fat together with other substances exerts
a decomposlng effect on the insulin during the vacuum
destillation, especially in the concluding phase of this
where the alcohol concentration is low. The violent motion
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in the liquid during the vacuum distillation al~o accelerates
the reaction between fat and insulin, resulting in a reduced
yield.
Besides the above mentioned, entirely decisive draw~acks
of the classic method, other, economic, disadvantages have to
be taken into account, since vacuum distilling unlts are
costly to buy and expensive in operation and maintenance.
Accordlng to the invention, an entirely different
method is applied for removing fat from a pancreas extract,
e.g. the extraxt prepared in the manner outlined above or
any other extract containing insulin and fat.
The method according to the invention consists in that
the insulln and fat containing, ~aw extract, which may
for instance be an aqueous alcoholic extract, is cooled to a
temperature at which all the fat freezes and separates out,
whereupon the extract is separated from the fat, e.g. by
filtration, under contlnued cooling. The raw extract may be
cooled e.g. at p~ 2-3 after the pH-8 precipitation, but the~
is nothing to prevent the cooling from being effected a~ p~-8
and in such case preferably after the filtration from the pH-8
precipltate. The yield, however, will be sllghtly smaller by
this latter method. In return, a pH-3 adjustment is saved.
By means of this method, the fat is removed easily and
completely a~ready in one of the initial phases, and harmful
effects on the insulin by the fat in the following process
stages are avoided.
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According to one broad aspect, the invention relates
to a method in the recovery of insulin from an insulin and
fat-containing pancreas extract in an organic solvent and
water, characterized in that the extract is cooled to a
temperature at which all or practically all the fat freezes
and separates out, whereupon the fat is removed from the
: extract under continued cooling, and the extract is worked up
to in~ulin without evaporative removal of the organic solvent.
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The cooling down and the separating out of the fat
can be carried out 9imply by placlng the clear extract ln the
cold store e.g. at -30C to -35C. This wlll cause the fat
to be frozen out and be ~uspended in the extract as solid
crystalline particles.
After having been left, without stirring, for a~out
12-24 hours in the cold store, depending on the ~emperature
and on the amount of extract the extract l~ filtered or
centrifuged. If the extract is agitated during the cooling the
time can be considerably shorter, e.g. 2-3 hours only. The
filtering or centrifuging is carried out in the cold store
proper, where the filtering device or the centrifugal
separator has the 8ame low temperature as the extract. In
thls manner, all the fat is ~eparated from the extract. If the
separation i8 effected outside, or if the filtering device is
taken lnto the cold store from the outs1de and has norma-l room
temperature part of the fat will melt and be carried along
wlth the filtered extract.
When proceeding as described above, however, crystals
Of rather dlfferent slzes, which crystals moreover are often
of rather loose structure and hence include a relatively great
amount of mother liquor are frequently formed.
lt is desirable that the crystallization of the fat
- be carrled out ln such a way that uniform, compact crystals
with a relatlvely great volume/surface ratio are obtained.
Crystals having the same or approximately the same size are
easier to 6eparate from the mother liquor, e.g. by filtration,
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than are crystals of different sizes, and the compact form
implies that only relatively little mother liquor is included
1~ the crystals and is lost in this way.
~ he ~tystal form can, as is ~ell-known in the art,
be influenced by regulating the cooli~g and crystallizatloh
conditions.
In a preferred embodiment of the present invention,
therefore, the cooling and crystallization conditions, e.g.
with respect to cooling time,pH-value of the extract and
gpeed of agitation, are controlled with a view to obtaining
uniform, compact fatcrystals having a relatively great
volume/surface ratio.
When an aqueous alcoholic extract is treated i~ has
proved that a pH-value of approximately 3 during cooling
gives the best crystallization and the best yield.
It has generally proved that the cooling time, until
the final temperature of e.g. -30& to -35& is reached,
should be at least 2-3 hours, agitating the liquid.
Stirring of or other agitation of the cooled liquid
is desirable. The stirring rate must not be very high, however,
e.g. at most 50 r.p.m. If a too violent agitation is used the
crystals formed may be shattered. The cooling down o~ the
extract and separating out of the fat and the separation of the
extract from the fat can with advantage be carried out
continuously.
Any suitable crystallization apparatuses can be used
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for carrying out the method. As an example mention shall
be made of vertical or horizontal cooling cylinders, with
cooling jacket and scrapers or with a cooler in the form
of a body of revolution, for producing the supersaturation
and causing the formatlon of crystal nuclei, connected to
one or more crystallization rooms, wherein the formed crystal
nuclei grow to the desired size and form. From the crystalli-
zation room, or the last crystallization room, the crystal
containing liquid passes to the filtering apparatus or the
centrifuge.
The insulin can be recovered from the fat-free extract
in any convenient way.
Below a non-limiting example illustrating the method
accordlng to the invention, carried out in laboratory scale,
is glven.
Example
100 g pancreas extract obtained in the conventional
- manner by extracting minced pig's pancreas glands by means
of aqueous ethanol acidified with hydrochloric acid,
precipitating lnsoluble proteins different from insulin
by adjustment of the pH of the extract to 8, filtering and
reducing the pH to 2,8, were placed in a 600 ml beaker provided
with a stirrer. The beaker was placed in a freezer, wherein
; its contents were in the course of 3 hours cooled down to
-35&, whilst being stirred with a velocity of 50 r.p.m.
Hereby compact crystal aggregates having a great volume/surface
l~i902
ratio were formed, which were easily filtered from the mother
liquor. The filtration was carried out in a coo}ed filter
funnel (-37&, 11 cm filter - Munktell No. oo) using vacuum
generated by a water-jet injector.
The enormous advantaqe obtained by proceeding
according to the invention will be immediately obvious: for
one thing the fat is removed at a very early stage and for
another it is re~oved at a very low temperature of e.g. -30 &
to -35& . At this low temperature the harmful components in
the fat will have no decomposing effect in the insulin or, at
most, a very small decomposing effect in comparison to the
decomposing effect that takes place during the vacuum
distillatlon at a temperature of approx. 25& (i.e. at a
difference in temperature of 55-60& between the known method
and the method according to this specification). It ls
- undoubtedly also of importance that by the present method
a removal of fat by freezing i~ effected with no or with only
a llttle motion of the extract, whereas at the vacuum distil-
lation, violent motion is caused by the boiling which accelera-
tes the decomposition of tha insulin.
It i9 a special advantage of the present method that
it makes it possible at a very early stage of the process of
preparing insulin to choose among the different and recent
processes for purification of insulin such a~ for instance
gel filtration and ion exchange without the risk of the
expensive prcducts used herefore being contaminated by the
fat. Such purification processes cannot or can, at best, only
with great difficulty be used at an early stage of the
conventional processes of preparing insulin.
