CA1041902A - Method in the recovery of insulin - Google Patents

Abstract

Abstract of the disclosure: A method in the recovery of insulin, particularly a method for the removal of fatty matter from an insulin and fatty matter containing pancreas extract. The extract, which may be the raw extract as separated from the residue of the comminuted pancreas glands after extraction, or an extract from which certain proteins different from insulin have been removed by precipitation by adjustment of the pH of the extract to about 8 and filtration, or any other extract containing insulin and fat, is cooled to a temperature at which all the fat freezes and separates out, whereupon the fat is separated from the extract, e.g. by filtration, under continued cooling. The extract can e.g. be cooled down to -30°C to -35°C. The crystallization of the fat can advantageously be carried out at a pH-value of about 3 under cautious agitation, the cooling time, until the final temperature of e.g. -30°C to -35°C is reached, being at least 2-3 hours.

Description

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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.

1041~
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, 1041!~0~

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.

Claims (6)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. 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 insulin without evaporative removal of the organic solvent.

2. A method according to claim 1, characterized in that it is applied to an aqueous alcoholic raw extract which has been subjected to precipitation of proteins at a pH of approximately 8.

3. A method according to claim 1, characterized in that it is applied to an aqueous alcoholic raw extract which has been subjected to precipitation of proteins at a pH of approximately 8 and has been afterwards adjusted to a pH of approximately 3.

4. A method according to claim 1 characterized in that the extract is cooled down to -30° to -35°C.

5. A method according to claims 1, 2 or 3 characterized in that the cooling and crystallization conditions are controlled such that uniform, compact fat-crystals having a relatively great volume/surface ratio are obtained.

6. A method according to claim 4, characterized in that the cooling time, until the final temperature of -30°C to -35°C is reached, is at least 2-3 hours.