FI64508C - FOERFARANDE FOER FRAMSTAELLNING AV RENT INSULIN SAOSOM INSULINAV SVIN UR BUKSPOTTKOERTLAR - Google Patents

Description

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Ma iBj (11) utlAggni NGSSKRIFT 64 50 8 C (45) PaL.t ^ (51) ic ».ik .-] im.ci.3 A 61 K 37/26, C 07 C 103/52 FINLAND — FINLAND ( 21) Pttenttlhakcmut - P * tmtani6knlng 7 7 Oil 2 (22) HakamltplM - Anc5knlftfad «g lU.01.77 (23) Aikupilvt - Glltl | hettdag lU. 01.77 (41) Become Public - Blhrlt offvntllg 17.07 77

Patent registered .... ..........,. .. ...

_ *, (44) Date of first issue. -

Patent · och registerttyrelten 7 Anaekan utlagd och otl. * Krlftan publkerad 31.08.03 (32) (33) (31) Pyydetty «uoikeui - Baglrd prloritet l6.01. J 6

United Kingdom-Storbritannien (GB) 17 ^ + 0/76 (71) Laboratorios Leo S.A., Avda. de Pio XII No. 99, Madrid, Spain-Spain (ES) (72) Willy Henry Jensen, Madrid, Gregorio Ramon Cebrian, Madrid, Jose7Adrian Aguas, Madrid, Spain-Spain (ES) (7 ^) Oy Kolster Ab (5 * 0 Method of pure insulin , such as porcine insulin, from pancreatic glands - Förfarande för framställning av rent Insulin, säsom Insulin av svin, ur bukspottkörtlar

The invention relates to a process for the preparation of insulin, such as porcine insulin, which has been purified to such an extent that only a single component is detectable by polyacrylamide gel electrophoresis using a 15% polyacrylamide gel, which process produces an insulin extract from the pancreas which is further processed into pure insulin. . Insulin prepared by the method of the invention does not generate insulin-destroying antibodies.

It is now generally assumed that the tendency of insulin preparations to form antibodies is largely due to impurities in the preparations. In the past, it was believed that insulin itself caused antibodies specific for itself. These impurities may include pancreatic extraneous proteins, insulin precursor proinsulin, insulin intermediates, dimers, insulin arginine, insulin ethyl ester, desamidoin insulin with varying degrees of deamidation, and other insulin variants.

Thus, the aim is to prepare insulin preparations which contain only pure insulin, the so-called one-component insulin without impurities and excipients, see e.g., English Patent Publications 1,285,023 and 1,285,024.

This can be accomplished by carrying out a thorough further purification of the amorphous or crystalline insulin prepared in the usual way, e.g. by gel filtration and / or ion exchange.

The tendency of the thoroughly purified insulins formed to form antibodies is significantly reduced, but not completely eliminated. The reason is undoubtedly that, despite the purification steps, the purified products still contain impurities.

It has been found that some of the insulin impurities are generated during the insulin isolation step, in which case, for example, the insulin may decompose or aggregates may form. In the pancreas from which insulin is isolated, it is present as a pure monomer (molecular weight about 6000), and from the pancreas the monomer is extracted as usual with an acidic 60-80% aqueous alcoholic solution. In the usual further processing, which includes e.g. separation of the fat from the extract by vacuum distillation at 25 to 30 ° C to evaporate the alcohol to give an aqueous extract with very little alcohol, however, the monomer can be easily degraded by enzymes that are effective in aqueous solution (less than 50% alcohol) but ineffective in alcoholic solution ( alcohol content over 50%); aggregation may also occur, etc.

The process according to the invention is characterized in that the insulin is extracted with an extractant which does not undergo enzymatic degradation of insulin or the formation of aggregates, such as a mixture of water and an organic solvent, preferably alcohol or acetone, containing at least 50% by volume of organic solvent. the water is derived from the pancreas and is kept dissolved in said solvent throughout the treatment process or dissolved in several different solvents of the same type, the extraction treatment process comprising the following main steps: degreasing insulin, preferably by cooling the extract vigorously, e.g. to below -25 ° C, and then separating the fat crystals formed, concentrating the non-fat insulin, preferably by reverse osmosis, further purifying the concentrated solution, preferably by treatment with ionic acid. until pure insulin dissolved in an impurity-free solvent results, after which any insulin is precipitated.

The aforementioned British Patents 1,285,023 and 1,285,024 describe a process for preparing very pure insulin starting from amorphous or crystalline insulin prepared by a conventional process. It has been mentioned that purified insulin is, according to polyacrylamide gel electrophoretic analysis, substantially one-component. However, this insulin always contains a small amount of insulin monodesamido, and if polyacrylamide gel electrophoresis is performed using a gel containing 15% polyacrylamide, two bands, namely a monoinsulin band, and in addition a monodesamido insulin band are visible.

Low levels of insulin monodesamido cannot be detected by polyacrylamide gel electrophoresis using a gel containing 7.5% polyacrylamide. Such a gel does not achieve complete separation of the various components, but the insulin and monodesamide insulin run as a single band.

Desamido insulins are formed in conventional purification and concentration steps, for example by evaporation in vacuo, whereby the insulin at the end of the evaporation is dissolved in water at a relatively high temperature. Once insulin desamido is formed, it is very difficult to separate it from insulin, and it will also be present as an impurity in both amorphous and crystalline insulin, which are derived from the methods of the said English patents. This impurity remains in the final products purified by the methods of said patents.

The method according to Finnish patent application 760058 also starts from crystalline insulin, and even then a final product containing insulin monodesamido is obtained.

Insulin is very readily soluble in an acidic alcoholic extract containing, for example, about 55 to about 80% by volume of alcohol, and when properly treated, the insulin in such an extract does not form aggregates with itself or with the impurities of the alcoholic extract.

Of course, extraction fluids other than an acidic aqueous alcoholic solution can be used, in which the insulin is readily soluble and in which it, 64508 4, does not form aggregates with itself or with the impurities of the extract, e.g. an aqueous acetone solution.

In the present embodiment, it is not a question of purifying the insulin itself, but of the insulin extract obtained being purified by extraction from soluble impurities and fat until the extracts contain only pure insulin.

The method can be implemented, for example, as follows:

The finely divided, frozen pancreas is extracted with 60 to 80% by volume of ethyl alcohol, the pH of which has been adjusted, e.g. with hydrochloric acid, to about 3. After separation of the pancreatic mass, the pH of the extract is adjusted to about 8, precipitating various proteins which are removed. The pH is then adjusted to about 3. Instead of the above precipitation, the proteins can be separated by deriving the extracts (pH

2-3) through an ultrafiltration and hyperfiltration device (described later) using a suitable membrane that retains said proteins, e.g. enzymes.

The resulting clear extract is cooled in a special fat cooling device to a temperature of about -30 ° to -45 ° C, preferably to about -35 ° C, whereby the fat crystallizes as dense, small-area fat crystals. These are separated at the same low temperature, e.g. by centrifugation. At this low temperature of -30 ° to 45 ° C and the alcohol content remaining at 60 to 80% by volume, the insulin is not damaged, which occurs in conventional degreasing by vacuum distillation at 25 ° to 30 ° C to reduce the alcohol content of the extract. decreases.

Low temperature fat dissolution is described in more detail in German application DE 2 531 804.

When the fat is removed in a non-insulin damaging manner, a relatively large amount of extract is obtained, which must also be concentrated in a non-insulin damaging manner.

Gel filtration and / or ion exchange can be used, but according to the invention the following method is recommended: An ultrafiltration and hyperfiltration device is used, which operates according to the membrane method, i.e. according to reverse osmosis (cf. e.g. U.S. Patent 3,623,610).

Using this method, in addition to liquid volume concentration, insulin can be separated from impurities and excipients in the extract, as well as from substances with a larger or smaller molecular size than insulin, using suitable membranes. Not only are the 5 64508 pancreatic, undissolved and unwanted contaminants from the pancreas removed, but also any other non-pancreatic non-insulin molecules from the pancreas that have been involved in the pancreas by mistake or carelessness in slaughterhouses.

In order to prepare a one-component insulin, it is crucial that most of these larger or smaller molecules of insulin can be separated at this stage, because then the impurities are dissolved and have not been able to damage the dissolved insulin due to e.g. from the low temperature of the previous process steps.

The fat-free, clear extract is pumped through an ultrafiltration device where the extracts can be sufficiently cooled, e.g. to a temperature of about + 10 ° to -10 ° C, preferably 0 ° to -10 ° C, at a pressure of 2-20 atmospheres, preferably 6 atmospheres. In this case, a film type, e.g. GRX-6 or GRX-7 (polysulfone films, manufactured by De Danske Sukkerfab-rikker A / S), is chosen in such a way that unwanted molecules larger than insulin can be separated as accurately as possible. Large molecules remain in the concentrate, and insulin and impurities of smaller molecular size than insulin enter the permeate. This solution is then passed through a hyperfiltration device whose membrane type, e.g. GR8-9 or 930 (cellulose acetate, manufactured by De Danske Sukkerfabrikker A / S), has been selected so that most of the undesired molecules smaller than insulin can be separated as accurately as possible. The insulin remains in the concentrate, and the starting extract can be concentrated as desired, e.g., 1/5 to 1/40 of the original volume, and the alcohol-to-water content ratio remains the same as in the starting extract.

The membranes used in the ultrafiltration and hyperfiltration equipment must be able to withstand concentrated, eg 62%, alcoholic solutions. In addition to the polysulfone and cellulose acetate films mentioned above, films made of other polymers, e.g. certain polyamides, can be used.

With regard to ultrafiltration and hyperfiltration devices, reference may be made, for example, to the following patents: U.S. Patent 3,872,015, English Patent 1,390,671 and Swiss Patent 542,639, which describe the structure of the device and / or its parts.

The obtained concentrate, which contains almost all the insulin of the crude extract, is transparent, but due to the concentration, its color is more intense than that of the original extract. Dyes are removed by washing with e.g. 62% alcohol using membranes that retain insulin but pass dyes through. All of the detergent and dyes enter the permeate while the volume of the concentrate remains unchanged.

In addition to the dyes, the salts that have become involved in the extraction of the pancreas are also removed from the extract.

The relatively few impurities still remaining in the insulin-containing extract can preferably be removed by ion exchangers, both cationic and anionic. Separation can also be performed with a molecular sieve, e.g. Sephadex® G-50, manufactured by Pharmacia Fine Chemicals, Uppsala, Sweden, or by other known separation methods, but the use of ion exchangers is recommended.

Ion exchange methods for purifying insulin by both the column method and the batch method are known, and in particular the column method is quite well known.

However, these methods have not been used to purify a concentrated, non-fat alcoholic extract of impure insulin. In particular, in the column process, impurities, dyes and fats of impure insulin from conventional dissolved alcohol-containing alcohol extracts bind to the ion exchangers to such an extent that the ion exchangers after one or two adsorptions and elutions are so impure that they are purified and regenerated.

Ion exchange and molecular sieve processes require that the extract to be treated be purified from fat, in part also from dyes, foreign proteins and other impurities, to the extent that the ion exchangers and molecular sieves can be regenerated and purified for long-term use. Otherwise, their use will be unprofitable.

Such adequate and necessary pre-purification of the crude insulin alcohol extract can be accomplished by the method just described above. In this case, the fat is removed by crystallization at a low temperature, i.e. -40 ° C, and some of the dyes and a considerable part of the molecules, which are larger or smaller than the insulin molecule, are removed as accurately as possible by ultrafiltration in a reverse osmosis device.

V 64508

Most of the available types of ion exchangers can be used, e.g. resin and cellulose based ion exchangers and the known Sephadex ion exchangers SP and QAE, which are cation and anion exchangers, respectively. They are modified, cross-linked dextran chains with a three-dimensional polysaccharide network (manufactured by Pharmacia Fine Chemicals AB, Uppsala, Sweden). Ion exchangers SP-Sephadex® C-25 (cation exchanger) and QAE-Sephadex® A-25 (anion exchanger) are preferred.

Column chromatography or batch separation in an ion exchanger, or possibly both, may be used.

The batch method has been shown to have considerable advantages in larger industrial applications with medium to fine cleaning. Thus, it is located between reverse osmosis separation and concentration and chromatographic purification with an ion exchanger according to the column method. Batch separation is a very fast method and does not involve technical difficulties due to swelling or shrinkage of the ion exchanger. With an ion exchanger, the separation is performed by binding the impurities to the ion exchanger, leaving the insulin free in solution. Once the ion exchanger is in equilibrium in the chosen, suitable buffer at the isoelectric point of insulin pH 5.2, the concentrated, pre-purified crude insulin extract at pH 5.2 is contacted with the ion exchanger and stirred for e.g. two hours, after which the mixture is filtered. All impurities bind to the ion exchanger, and pure insulin remains dissolved in the alcohol extract because insulin at the isoelectric point does not bind to the ion exchanger.

The advantage of this method is that no insulin needs to be eluted from the ion exchanger and that the insulin extract can be passed to the next step immediately after filtration.

The ion exchangers are regenerated in a known manner.

On the other hand, insulin and other proteins can be bound to an ion exchange and then eluted from the fractions by resuspending the mixture in a buffer of higher ionic strength or second pH. In the latter method, the insulin loss is lower than in the previous method and is therefore recommended.

The method can be broadly performed, for example, as follows: β 64508

300 g of dry SP-Sephadex® C-25 ion exchange pulp in 1000 ml of 0.1 M acetic acid buffer containing 62% ethanol and having a pH of 3 were swollen for two days. The used buffer solution was replaced several times with fresh buffer. The swollen SP-Sephadex weighed 720 g.

The pH was adjusted to 3.0 with 1000 ml of an extract concentrated ten times after osmosis and containing 18,000 units of insulin and stirred in a rotating tank (12 rpm) for one and a half hours. Half of the swollen ion exchanger required for adsorption was present. After vacuum filtration, the adsorption of the filtrate was continued for one and a half hours using a 360 g swollen ion exchanger. The insulin content of the surface fluid was 288 units, corresponding to an adsorption loss of 1.6%. Such double adsorption has been found to reduce the loss by one third compared to single adsorption.

1000 ml of a solution containing 0.1 m HAc and 0.075 m NaCl in 62% ethanol, pH 3.0, was changed to buffer. Impurities were eluted with this buffer from the ion exchanger by stirring for two hours at which time no insulin eluted.

After filtration, 1000 ml of 0.1 M tris (hydroxymethyl) aminomethane (tris) in 62% ethanol, pH 8.0, was exchanged for buffer and insulin was eluted from the ion exchanger SP. The insulin concentration was 17,000 units and the elution loss was 4%. The total loss was 5.6%.

The ion exchanger was washed twice to remove trapped insulin residues.

In polyacrylamide gel electrophoresis (15% polyacrylamide), there were four partially weaker bands above the insulin zone, but no zone at all below. This indicates that the partially purified insulin is not deamidated in the purification step. It has no desamid components, nor does it contain insulin mono-desamido, which is completely new in the manufacture of insulin.

After elution and washing, the ion exchanger was treated with slow stirring for 2-3 hours with 62% ethanol supplemented with 0.2-0.3 mol / l NaCl. The ion exchanger was then regenerated with an equilibrium buffer of pH 3 to give the ion exchanger completely pure chalk white and ready for the next charge.

The batching process can advantageously be carried out in a specially designed Y-shaped device with adjustable speed.

9 64508 This form allows a very efficient and careful handling of the ion exchanger during adsorption and elution. At the same time, the loss in both adsorption and elution is reduced to a minimum. The entire method can be carried out without disconnecting the ion exchanger from a device equipped with a filtration system and a vacuum tank for rapidly filtering the liquid from the ion exchanger.

The method comprises the following steps: 1) Adsorption of insulin and possibly other proteins in an ion exchanger, pH 3, from a concentrated impure insulin extract in two steps with vacuum filtration in between.

2) The protein-free extract is vacuum filtered and removed.

3) The ion exchanger is washed twice with 62% ethanol buffer, pH 3, and the washings are removed by filtration.

4) Impurities are eluted twice with pH 3 buffer, ionic strength 0.075 m NaCl, stirring speed 12 rpm.

5) The eluate and its impurities are filtered.

6) The ion exchanger is washed twice with 62% ethanol buffer, pH 3, and filtered.

7) Insulin is eluted with a buffer containing 0.1 M tris- (hydroxymethyl) aminomethane in 62% ethanol, pH 8. The pH of the whole mixture of elution liquid and ion exchanger is adjusted to 8 with tris buffer. The pH of the ion exchanger was 3.

8) The insulin-containing eluate is filtered.

9) The ion exchanger is washed twice to remove trapped insulin residues.

10) The ion exchanger is cleaned and regenerated.

The entire production phase 1-9 takes 12 hours and the cleaning and regeneration of the ion exchanger takes 4 hours, ie a total of 16 hours.

After adjusting the pH and ionic strength, the elution extract obtained from step 7 can be passed directly to a cation or anion exchanger according to methods known in the art of column technology. This method is best suited for programmed columns with automated fractionation to isolate the middle portion of the insulin curve containing pure insulin and in which 15% polyacrylamide has only one zone in polyacrylamide gel electrophoresis.

Insulin can be isolated from the concentrated, insulin-containing eluate of the last column in the usual manner, i. by diluting ίο 64508 solution and precipitating e.g. by adding zinc ions.

However, according to the invention, it has been found that it is not necessary to dilute the highly concentrated alcoholic water extract before precipitation. The extract can be directly precipitated, e.g. with zinc ions, when their addition takes place at a low temperature, e.g. -30 ° C to 45 ° C.

Thus, a zinc acetate solution can be added to the filtrate, after which the solution is allowed to stand at -35 ° C for 24 hours. In this case, insulin precipitates quantitatively, e.g. in a 62% alcohol solution. The precipitated insulin is isolated by centrifugation, washed with e.g. acetone and dried. If desired, it can be crystallized later.

As mentioned, the insulin prepared according to the invention is a pure one-component insulin having only one component when analyzed by polyacrylamide gel electrophoresis (DISC PAGE) using 15% polyacrylamide (for the general principle of this method, see Ann. NY Acad. Sci., 121, pp. 321-349). and 404-427 (1964)).

The insulin prepared according to the invention has proven to be very stable compared to known highly purified insulins, in which, for example, additional small amounts of desamido insulin are often formed during storage. The reason is unknown, but any insulin produced, despite careful purification, still contains small amounts of substances that break down insulin.

The insulin according to the invention is not degraded in this way, which may be due to the fact that substances with a different molecular size from the insulin have been more completely removed.

Insulin preparations can be prepared from the described pure insulin in a known manner, e.g. by dissolving and / or suspending amorphous and / or crystalline insulin in an intramuscular or intravenous injection or aqueous solution suitable for implantation techniques.

Example

A crude insulin extract was prepared by treating comminuted porcine pancreas with 85% ethanol adjusted to pH 3 with sulfuric acid. A pancreatic extract containing 65% ethanol was obtained.

A 15 liter aliquot of this extract (840 mg insulin + insulin derivatives and impurities) was treated as pure insulin as follows: n 64508

The pH of the extract was adjusted to 8, at which point the precipitated substance, which contained most of the proteins insoluble in the basic solution, was separated while the insulin remained dissolved.

The pH of the solution was adjusted to 3, after which the extract was cooled to -35 ° C and kept at this temperature for three hours with stirring. The separated fat crystals were then separated by centrifugation at the same temperature in a centrifuge made of a material containing a particularly high amount of alloy steel, in which case it is not expected that cracks would form in the rotating ball due to the low temperature.

The degreased liquid was treated at 6 bar in a reverse osmosis device with GRX-6 type membranes (polysulfone membrane manufactured by De Danske Sukkerfabrikker A / S), after which the insulin-containing permeate was treated at 40 bar in a 4 HM membrane hyperfilter with a membrane. (polymer membrane manufactured by De Danske Sukkerfabrikker A / S). The concentrate contained insulin.

Thus, the insulin-containing starting extract was partially purified and partially concentrated by reverse osmosis to about one-fifth of its original volume, i.e., three liters.

Dyes and salts were partially removed from the concentrate in a hyperfilter with 65% ethanol using the same membrane as in the concentration. This type of membrane retains insulin, but dyes and salts pass through the membrane.

The pH (3000 ml) of the thus-purified concentrate containing 660 mg of insulin and insulin derivatives was adjusted to 3, and the concentrate was treated in an ion exchange apparatus consisting of a Y-shaped stirrer equipped with a filtration apparatus and a separate part which provided a vacuum to accelerate ion filtration. The ion exchanger used was the cation exchanger SP-Sephadex®C-25, which had been allowed to swell before use in a 65% ethanol buffer solution containing 0.1 mol of acetic acid per liter and having a pH of 3.

The ion exchange process (batch method) consisted of the following steps: 1) The dry ion exchange mass (180 g) was allowed to swell in the buffer solution, after which the mass was filtered, 90 g of ion exchange mass was poured into an ion exchange device, 12 64508 of purified, concentrated and concentrated were added. The solution was then vacuum filtered and the process was repeated with 90 g of ion exchange mass. Time 2.5 hours.

2) The product was vacuum filtered, and the protein-depleted extract was passed from an ion exchange apparatus to a distillation column to regenerate the alcohol. Time 2.5 hours.

3) The ion exchanger was washed twice in each case with 200 ml of a 65% ethanol buffer solution having a pH of 3, and the washing solution was filtered off. Time 2 hours.

4) The impurities were eluted with 400 ml of buffer with a pH of 6 and an ionic strength of 0.025 mol / l at a stirring speed of 12 rpm. Time 1 hour.

5) The elution solution and the impurities dissolved therein were filtered off. Time 1.5 hours.

6) The ion exchanger was washed twice in each case with 200 ml of a 65% ethanol buffer solution having a pH of 6, and the washing solution was filtered off. Time 1.5 hours.

7) Insulin was eluted with 200 ml of a 65% ethanol buffer solution containing 0.033 moles of phosphate per liter and having a pH of 8. The pH of the whole mixture, the elution solution and the ion exchanger mass was adjusted to 8 with NaOH. The pH of the ion exchange mass was 3. Elution was carried out for 2 hours with slow stirring.

8) The elution solution containing insulin and simple insulin derivatives was filtered off separately. 200 ml of elution buffer solution contained 580 mg of insulin + derivatives. Time 30 min.

9) The ion exchanger was washed twice in each case with 100 ml of the buffer solution mentioned in 7) to remove residual insulin residues in the ion exchanger. The washings were combined in the following batches. Time 2 hours.

10) The ion exchanger was purified and regenerated.

The whole process (steps 1-9) takes 12 hours and the cleaning and regeneration of the ion exchanger takes 4 hours, for a total of 16 hours.

The 200 ml elution extract from step 7 contains high purity insulin after the purification steps described above. The insulin extract purified by the present method does not contain any insulin monodesamido, the zone of which in the electrophoretic analysis is 13 64508 below the zone of insulin, whereas above the zone of insulin there are two additional zones due to insulin arginine and insulin ethyl ester.

The pH of the elution extract from Step 7 (580 mg of insulin and insulin derivatives in 200 ml) was adjusted to 7.5 and the ionic strength to 0.05, after which the extract was further purified in an anion exchanger DEAE Sephadex® C-25 using a programmed column with automatic fractionation. The ion exchange mass was allowed to swell in a 0.033 m phosphate buffer solution containing 65% ethanol and having a pH of 7.5. The diameter of the column was 2.5 cm, it was packed with ion exchange mass to a height of 18 cm.

200 ml of the elution extract from step 7 was poured onto the column. 900 ml of the above-mentioned phosphate buffer (temperature 20 ° C) was used for elution, the elution rate was 100 ml / h, and the eluate was collected in 18 ml fractions. The elution progress was recorded. The fractions containing the most insulin were combined to give 125 ml of an eluate containing 310 mg of monomeric insulin. In electrophoretic analysis on a polyacrylamide gel (15% polyacrylamide), only one band, i.e. the insulin band, was detected.

12 ml of a 1 M zinc acetate solution was added to the eluate, and it was kept at -35 ° C for 24 hours, during which time pure amorphous insulin precipitated quantitatively.

Claims (5)

A process for producing insulin, such as porcine insulin, which has been purified to such an extent that it exhibits only a single component when assayed by polyacrylamide gel electrophoresis using a gel containing 15% polyacrylaxnide, in which process is prepared an extract of insulin from pancreas, which extract is processed into pure insulin, characterized in that the insulin is extracted with such an extractant, in which no enzymatic degradation of the insulin or formation of aggregates occurs, as with a mixture of water and an organic solvent, preferably an alcohol or acetone, which extractant contains at least 50% by volume of organic solvent, a portion of the water contained in the extractant originating from the pancreas, and being poured during the entire processing process of insulin until the separation of the final product dissolved solvents, or successively in several different solvents of the same type, wherein the following process of reprocessing on the extraction comprises the following main steps: removing fat from the insulin, preferably by vigorously cooling the extract, e.g. to a temperature below -25 ° C, and then separating the formed fat crystals, concentration of the fat-liberated insulin solution, preferably by reverse osmosis, further purification of the concentrated solution, preferably by treatment in ion exchanger, until the pure insulin is dissolved in a solvent-free contaminant, whereupon the insulin is possibly precipitated. 2. A process according to claim 1, characterized in that, after removal of the fat, the extract is concentrated and purified by reverse osmosis, whereby membranes can be used which can remove any impurities which have larger molecules than the monoinsulin and impurities respectively. smaller molecules than the monoinsulin. Method according to claim 2, characterized in that the insulin-containing concentrate from the reverse