GB1587769A

GB1587769A - Process for the stabilisation concentration and purification of insulin

Info

Publication number: GB1587769A
Application number: GB5352777A
Authority: GB
Original assignee: University of Minnesota
Current assignee: University of Minnesota
Priority date: 1976-12-27
Filing date: 1977-12-22
Publication date: 1981-04-08
Also published as: AU3172477A; FR2375193A1; DE2757377A1; JPS53115809A; DK580077A; AR221693A1; NL7713966A

Description

(54) PROCESS FOR THE STABILISATION, CONCENTRATION AND PURIFICATION OF INSULIN (71) We, REGENTS OF THE UNIVERSITY OF MINNESOTA, a corporation of Minnesota, whose address is 1400 University Avenue, Southeast, Minneapolis, Minnesota, United States of America, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: This invention relates to a process for the commercial scale stabilization, concentration and purification of insulin. More specifically, the invention relates to the use of ion ex change materials to recover insulin from acid-alcohol extracts of pancreas tissue from either fresh or frozen pancreas glands. The use of the preferred basket centrifuge permits processing of large quantities of materials.

The recovery and purification process is much simplified and more rapid. Yields are higher. The products are recovered in a pure unaltered stable state. The pancreatic enzymes which are normally discarded can -be recovered.

It is standard practice to remove the pan creatic glands from animals, primarily beef and pork, at the time of their slaughter for meat for human consumption. The glands are frozen for shipment to a processing laboratory and storage until the insulin and enzymes can be removed from the tissue.

After thawing, the pancreatic tissue is mace rated and homogenized and the insulin and enzymes are extracted with acidified aqueous alcohol, after which the extract is concen tral-ed, the insulin is precipitated and re covered for crystallization. The insulin ex tract contains proteolytic enzymes (trypsin, chymotrypsin). These enzymes are detrimen tal to insulin and should be separated from the insulin immediately.

The standard method for eliminating the undesirable contaminating enzymes is to -heat the alcohol extract to 60"C for thirty minutes. However, undesirably part of the insulin is hydrolyzed due to the time required to inactivate the enzymes by heat.

This results in the formation of insulin by-products such as desamido-insulin and arginine-insulin which cause the formation of antibodies to insulin in diabetics taking insulin injections over extended periods.

The standard method of concentrating the insulin from the acid-alcohol extract is to evaporate the extract to reduce the alcohol content and to concentrate the solution and add solid sodium chloride to precipitate the insulin as a salt cake. The evaporation of alcohol and salt cake formation causes additional losses of insulin. After the salt cake formation by the standard procedure, the insulin is further concentrated and purified by several crystallization (iso-electric, zine acetate) steps.

It has been proposed (U. S. Patent No.

3,069,323) to contact an aqueous alcoholic solution of crude insulin with an aminocellulose anion exchanger at a pH of about 5.5 to about 8.0 to cause at least a portion of the insulin to be absorbed on the anion exchanger. Thereafter, the exchanger is separated from the mixture and the insulin eluted from the exchanger with either an acidic or a basic eluant, after which the insulin is crystallized by known procedures. However, there is no disclosure in this patent of the elimination of proteolytic enzymes orthe absence of degradation products in the resulting crystallized insulin.

It is also known (South African Patent No. 69/5280) that crystalllne insulin dissolved in 1 M acetic acid may be fractionated into various components by passage through a molecular sieve, columns of cross linked dextran forming a three-dimensional network of defined pore size (Sephadex G50).

In this manner it is possible to remove from the insulin used as a starting material proteins of pancreatic origin having a molecular weight above about 6,000. However, it is then necessary also to remove the insulinlike substances having almost the same molecular weight as insulin but slightly differing in their composition, for example, desamido-insulin and arginine-insulin. This is done preferably by column chromatography on an ion exchanger. It is also known to purify crystallized insulin directly by column chromatography by subjecting impure crystalline insulin to a column chromatographic anion exchange. Apart from requiring the preparation of crystalline insulin in the ordinary manner, the chromatographic column purification proposed is slow, is not adapted to large scale production and produces low yields.

One aspect of our invention provides a method of stabilizing, concentrating and purifying insulin from pancreatic glands, either freshly excised or frozen, which method comprises: (A) preparing an acid-alcohol extract of minced homogenized pancreas gland tissue, (B) first stabilizing, concentrating and puri fying the insulin by contacting said acid alcohol extract with an acidic cation exchanger to adsorb said insulin on the cation exchanger, and washing with acidic aqueous solution to separate alcohol and pancreatic enzymes from the cation exchanger, ç eluting the stabilized, concentrated and purified adsorbed insulin from the cation exchanger with a buffered alcohol solu tion at pH 6.5 to 9, (D) then, further concentrating and purify ing the insulin contained in the eluant from the cation exchanger by contact ing said eluant with a basic anion ex changer to adsorb the insulin on the anion exchanger and separating the alcohol eluant, (E) washing the insulin adsorbed on the anion exchanger with an equeous buffer at pH 6.5 to 9 to remove remaining alcohol, and (F) eluting the further stabilized, concen trated and purified adsorbed insulin from the anion exchanger with dilute add.

In one embodiment the method comprises (A) preparing an extract of minced homo genized pancreas gland tissue with a mixture of about 65 /O by volume ethanol and about 35% water at pH about 2.8, < B) first stabilizing, concentrating and puri fying the insulin bv contacting said ex tract with an acidic cation exchanger characterized by functional sulphopropyl groups attached by ether linkages to the glucose units of cross-linked dextran chains, in a basket centrifuge to absorb the insulin -on the cation exchanger, washing with acidic aqueous solution and spinning off the separated alcohol and pancreatic enzymes, (C) eluting the stabilized, concentrated and purified adsorbed insulin from the cation exchanger with a mixture of about 65% by volume ethanol and about 35% water at pH 6.5 to 9, (D) then, further concentrating and purify ing the insulin contained in the eluant from the cation exchanger by contact ing said eluant with a basic anion exchanger characterized by functional diethyl-(2-hydroxypropyl) aminoethyl groups attached by ether linkages to the glucose units of cross-linked dextran chains, in a basket centrifuge to adsorb the insulin on the anion exchanger, and spinning off the alcohol, (E) washing the insulin adsorbed on the anion exchanger with an aqueous buffer at pH 6.5 to 9 to remove any remaining alcohol, and (F) eluting the stable, concentrated and purified insulin from the anion ex changer with dilute acid.

In a typical embodiment of our invention, an acid-alcohol extract of minced homogenized pancreas gland tissue is first prepared in the conventional manner. The extract is optionally neutralized, and the liquid extract carrying the insulin is separated e.g.

by filtration, decantation, or centrifugation, and the precipitated solids are discarded.

The liquid extract is then reacidified. The insulin undergoes a first stage concentration and purification by contacting the liquid extract with an acidic cationic exchanger preferably in a basket centrifuge or optionally in a chromatographic column or batch process to absorb the insulin on the cationic exchanger and separate the contaminating proteolytic enzymes and other contaminating substances. The concentrated insulin is eluted from the cationic exchanger with a buffered alcohol solution at pH 6.5 to 9.

The insulin is then subjected to further concentration and purification by contacting with a basic anion exchanger to adsorb the product on the exchanger and separate most of the alcohol. The adsorbed insulin is then washed with an aqueous buffer at pH 6.5 to 9 to remove the remaining alcohol. The concentrated purified product is eluted from the anion exchanger with dilute acid, e.g. a low molecular weight organic acid such as acetic acid; the insulin can then be crystallized or further purified by gel filtration chromatography.

-The initial adsorption can easily be carried out at the slaughterhouse where the pancreases. are excised. This eliminates the need for the standard practice of freezing and thawing the pancreas which is a major cause of loss of insulin. The adsorbed insulin can be transported directly to a pharma- ceutical laboratory for the elution of insulin from the cation exchanger followed by the subsequent concentrating and purifying pro- cess steps, followed by recycling and reutilization of the cation exchanger. The enzymes separated from the adsorbed insulin can be separately recovered.

Thus a further aspect of our invention is a method of stabilizing and concentrating insulin from freshly excised pancreas glands in a form suitable for transport and storage for later purification and further concentration, which method comprises: (A) preparing an acid-alcohol extract of minced homogenized freshly excised pancreas gland tissue, (B) concentrating the insulin by contacting said extract with an acidic cation ex changer to adsorb said product on the cation exchanger, and separating the alcohol and pancreatic enzymes from the cation exchanger by washing with acidic aqueous solution and (C) removing the cation exchanger with adsorbed stabilized, concentrated and partially purified insulin and packing -for transport and storage.

Alternatively, instead of transporting the adsorbed insulin, it can be eluted into an aqueous solution for subsequent purification.

Thus another aspect of our invention provides a method of stabilizing, concentrating and partially purifying insulin from freshly excised pancreas glands for later further purification and concentration, which method comprises: (A) preparing an acid-alcohol extract of minced homogenized freshly excised pancreas gland tissue, said extract con taining insulin and constituents dele terious to insulin, (B) preparing a compacted bed of acidic cation exchanger, said resin being ad sorptive relative to the insulin and non adsorptive to said deleterious consti tuents, (C) stabilizing and concentrating the insulin by forcing said extract through said compacted bed of cation exchanger to separate crude insulin from said extract by adsorption on the exchanger, (D) washing said bed with acidic aqueous solution to remove the alcohol and deleterious constituents, and thereafter (E) eluting the resulting stabilized, concen trated and partially purified adsorbed insulin from the cation exchanger with an aqueous solution of higher pH in the range 6.5 to 9 for subsequent purifica tion.

The invention is further illustrated by the drawing which is a flow diagram of a pre ferred process showing alcohol removal, stabilization, concentration and purification of insulin from pancreas glands.

The remaining description relates to a preferred embodiment of our invention.

The major source of insulin is from bovine or porcine pancreas glands excised from these animals at the time of slaughter for meat for human consumption. Although the process of this invention is equally suitable to the treatment of insulin extracted from frozen pancreas glands, it is preferred to avoid the loss of insulin due to freezing and thawing by extracting the insulin from freshly excised pancreas glands. However, whether fresh, or frozen and thawed, the glands are minced or otherwise macerated and homogenized in an acidic alcohol solution containing about 65 per cent by volume ethanol and about 35 per cent water at pH of about 2.8. This mixture is optionally neutralized by adjustment of its pH to about 8.2 by addition of e.g. about 20 per cent by weight sodium hydroxide solution to precipitate the solids, including the enzymes, which are removed by e.g. filtration, decantation, or centrifugation. The filtrate, the liquid acid-alcohol extract, contains insulin in the amount of generally 20 to 80 mg per liter, along with the proteolytic enzymes, such as trypsin and chymotrypsin. Because these enzymes are detrimental to insulin, they should be separated immediately. Instead of utilizing the standard method of elimination involving heating and the resulting detrimental hydrolysis of some of the insulin and production of undesirable insulin by-products, e.g. desamido-insulin and arginine-insulin, these contaminants are removed by ion exchange techniques preferably in a basket centrifuge. Although the invention is described with particular reference to the use of a basket centrifuge for ion-exchange, it will be understood that the process may also be carried out using column or batch ion-exchange techniques.

Many types of ion exchange materials may be used in a basket centrifuge to recover biological products. Such exchange materials include cellulose, cross-linked dextrans, agarose, hydroxy-methacrylates, methly-methacrylates and styrene-divinyl benzene ion exchange materials. These ion exchange materials have been used in chromatographic columns and other batch procedures to recover biological compounds. However, by using the ion exchange material in the basket centrifuge, the time required to recover the stabile biologicals is reduced substantially.

Larger volumes of product are recovered in shorter periods of time. Regeneration of the ion exchange material is simplified and speeded up in the basket centrifuge. Basket centrifuges range in size from 48 inch baskets down to 12 inch baskets, having bed capacities from 450 liters down to 5 liters. Processing biological products in a basket centrifuge takes advantage of the simplicity of batch ion exchange, with the separation efficiency of column ion exchange and can be carried out in much shorter periods of time. Maximum yield and efficiency are obtained by combining highly specific ion exchange media with high speed recovery on the basket centrifuge.

'-The crude acid alcohol insulin extract is acidified to pH about 2.8 by the addition of about 8.5 N sulfuric acid. For commercial scale processing, a typical batch is e.g.

45,000 to 450,000 liters. The extract is contacted, preferably immediately after preparation, with a specific cation exchanger in a perforate bowl basket centrifuge. The pro teolytic enzymes are immediately separated from the insulin. The insulin is adsorbed on the cation exchanger and the enzymes are spun off in the centrifuge along with the alcohol carrying other contaminants. The insulin is thus concentrated from typically about 40 mg/liter to between 16,000 and 40,000 mg/liter and 90 to 95 per cent by weight of the contaminating substances are removed from the adsorbed insulin. Every 1000 liters of acid-alcohol extract requires about 1 liter of swelled cation exchanger or 400 grams of dry exchanger.

The preferred ion exchanger for the initial concentrating and purifying step is SP Sephadex C-25, a strongly acidic cation exchanger derived by introduction of functional sulphopropyl [C3H6SO3] groups at tached by ether linkages to the glucose units of cross linked dextran chains forming a three dimensional polysaccharide network.

(Sephadex is a registered Trade Mark of Pharmacia Fine Chemicals Inc., Piscataway, New Jersey). A basket centrifuge with per forate bowl of appropriate bed capacity (450 liters) is prepared by lining the perforate bowl with a 5 or 10 micron filter (nylon bag or vyon sheet). The SP Sephadex C-25 '(150 kilograms) is equilibrated in 0.1 molar acetic acetic acid in 65 per cent by volume ethanol overnight.

The equilibrated cation exchanger is poured into the basket centrifuge and the centrifuge is started. The speed of the centrifuge is increased until a uniform bed has been spun against the wall of the centrifuge.

This requires a centrifuge force of between 500 and 1000 G's. The acid alcohol extract of insulin, which has been prefiltered to remove particulate matter, is applied to the exchanger bed in the basket centrifuge at a speed of between 100 and 500 G. The centrifugate (liquid coming off) is held for further processing to recover the pancreatic enzymes.

The insulin is selectively adsorbed to the exchanger. About 45,000 liters of acid alcohol extract can be applied to a bed containing 450 'liters of SP Sephadex C-' 25.

This represents approximately a 1000 fold cdncentration. The cation exchanger bed -with the adsorbed insulin is washed with 450 liters of 0.1 molar acetic acid in 65 per cent by volume ethanol to -remove any remaining contaminants (enzymes).

The insulin is then eluted from the cation exchanger bed by slowing the centrifuge to 100 G and adding a high pH (pH 6.5 to 9) buffer in about 65 per cent by volume ethanol and about 35% water. For example, a 0.1 M solution of hydrochloric acid buffered with TRIS (hydroxymethyl) aminomethane, pH 8.2, in 65 per cent by volume ethanol is a preferred eluant. The cation exchanger is then washed with the same solution and reequilibrated with 0.1 M acetic acid in 65 per cent by volume methanol.

If the insulin has been adsorbed at the slaughterhouse, then the cation exchanger is sent to the facility (pharmaceutical company) with the insulin adsorbed. The insulin is eluted by the pharmaceutical company for further processing and the exchanger is regenerated and sent back to the slaughterhouse for reuse on subsequent acid alcohol extracts.

The insulin eluted off the cation exchanger, concentrated 400 to 1000 fold, is then applied to an anion exchanger for further concentration and purification, packed in the basket centrifuge. A preferred anion exchanger is QAE Sephadex A-25, a strongly basic anion exchanger derived by introduction of functional diethyl-(2-hydroxypropyl) aminoethyl [C2H4N+(C2H^)2CH2CH(OH)CH] groups attached by ether linkages to the glucose units of cross linked dextran chains forming a three-dimensional polysaccharide network. This anion exchanger is equilibrated overnight in 0.1 M TRIS HC1, pH 8.2, in 65 per cent by volume ethanol. The bed volume of the QAE Sephadex A-25 should be 4 or 25 per cent of the eluted volume of acid alcohol extract from the SP Sephadex C-25 bed (100 to 115 liters if the SP Sephadex C-25 bed volume was 450 liters').

While the insulin is adsorbed on the anion exchanger, the alcohol is removed by washing the exchanger with an aqueous buffer at pH 6.5 to 9.0. A preferred buffer is 0.1 M TRIS HC1, pH 8.2, without ethanol. (100 to 200 liters of TRIS buffer should be sufficient). Insulin is normally unstable at high pH. However, when adsorbed' on an anion exchanger according to the present invention, insulin is stable at high pH within the indicated range.

The insulin is eluted from the anion exchanger with dilute acid, preferably acetic acid, e.g. 1.0 M acetic acid of ptI 3.2-3.6 (100 to 130 liters). The final insulin concentration is between 16,000 and 64,000 mg/li,ter with 95 to 99 per cent of the contaminants removed. The resulting stable concentrated and purified insulin can be crystallized from the acetic acid or may be further purified to e.g. 99.0 to 99.9per cent pure insulin by gel filtration chromatography. The anion exchanger is reequilibrated and is ready for the next charge from the cation exchanger.

The ion exchangers can be regenerated in the basket centrifuge enabling large volumes of regenerating buffers to be passed through the ion exchange beds in short periods of time making the use of "low flow" ion exchangers feasible on a commercial scale.

The process of the present invention has a number of advantages over standard in sulin recovery procedures. The following steps have been eliminated: (1) heat activa tion of proteolytic enzymes, (2) evaporation of alcohol, (3) salt cake precipitation, (4) iso electric crystallization, and (5) zinc acetate crystallization. By eliminating these standard procedures and replacing them with ion ex change processes in a basket centrifuge, a much greater yield of insulin is obtained.

By eliminating procedures that denature insulin, the yield may be 85 to 95 per cent compared to 40 to 60 per cent. The pan creatic enzymes are not only removed at an early stage without hydrolyzing of the insulin and formation of undesirable insulin bypro ducts, but the enzymes may be recovered as well. Since the enzymes have been removed during the cation exchange adsorption, the insulin is stable and not subject to hydro lysis. This makes possble carrying out the initial cation exchanger adsorption at the slaughterhouse, elimination of freezing and thawing - of the pancreatic glands with re sulting insulin loss, and subsequent process ing at the site where pharmaceutical pre parations of insulin are prepared. The cation exchanger can be regenerated by the phar maceutical company and recycled to the pancreas supplier for further insulin re covery.

WHAT WE CLAIM IS:- 1. A method of stabilizing, concentrating and purifying insulin, which method com prises: (A) preparing an acid-alcohol extract of minced homogenized pancreas gland tissue, (B) first stabilizing, concentrating and ,puri fying the insulin by'contacting said acid alcohol extract with an acidic cation exchanger to adsorb said insulin on the cation exchanger, and washing with acidic aqueous solution to separate alcohol and pancreatic enzymes from the cation exchanger, (C) eluting the stabilized, concentrated and purified adsorbed insulin from the cation exchanger with a buffered- alcohol solu tion at pH 6.5 to 9, (D) then, further concentrating','and -purify-' ing the insulin contained in the' eluant from the cation exchang r by cntact- ing said eluant with a basic anion ex changer to adsorb the insulin on the anion exchanger and separating the alcohol eluant, (E) washing the insulin adsorbed on the anion exchanger with an equeous buffer at pH 6.5 to 9 to remove remaining alcohol, and (F) eluting the further stabilized, concen trated and purified adsorbed insulin from the anion exchanger with dilute acid.

2. A method according to claim 1 wherein said acid-alcohol extract of pancreas gland tissue is prepared in a mixture of about 65% by volume ethanol and about 35% water at pH about 2.8.

3. A method according to claim 1 or 2 wherein said acid-alcohol extract is neutralized, separated from precipitated solids and re-acidified before step (B).

4. A method according to claim 3 where in said acid-alcohol extract is neutralized to about pH 8.2 with sodium hydroxide solution.

5. A method according to any of the preceding claims wherein said cation exchanger is characterised by functional sulphopropyl groups attached by ether linkages to the glucose units of cross-linked dextran chains.

6. A method according to any of the preceding claims wherein said concentrated and purified insulin is eluted from said cation exchanger with a mixture of about 65% by volume ethanol and about 35% water at pH 6.5 to 9.

7. A method according to any of the preceding claims wherein said anion ex changes is characterised by functional diethyl-(2-hydroxypropyl) aminoethyl groups attached by ether linkages to the glucose units of cross-linked dextran chains.

8. A method according to any of the preceding claims wherein said insulins eluted from the anion exchanger with dilute acetic acid. 9. A method according to claim 8 wherein said dilute acetic ,acid is about 1 Molar.

10. A method according to any of the pre- ceding claims wh.erein said pancreas gland tissue, is, from freshly excised - pancreas glands.

11. A method according to any of the preceding claims wherein the ion exchange steps .are carried out in a basket centrifuge.

12. A method according to cT im l of stabilizing, concentr ting and purifying, ,iu- sulin, which inethdd'comprises: (A) preparing an extract of minded homo- genized pancreas gland tissue with' 'a mixture of about 65 % by volume ethanol and about 35% water.at pH.about 2.8, (B) - first stabilizing, concentrating and puri frying the insulin by contacting said ex tract with an acidic cation exchanger

**WARNING** end of DESC field may overlap start of CLMS **.

Claims

**WARNING** start of CLMS field may overlap end of DESC **. gel filtration chromatography. The anion exchanger is reequilibrated and is ready for the next charge from the cation exchanger. The ion exchangers can be regenerated in the basket centrifuge enabling large volumes of regenerating buffers to be passed through the ion exchange beds in short periods of time making the use of "low flow" ion exchangers feasible on a commercial scale. The process of the present invention has a number of advantages over standard in sulin recovery procedures. The following steps have been eliminated: (1) heat activa tion of proteolytic enzymes, (2) evaporation of alcohol, (3) salt cake precipitation, (4) iso electric crystallization, and (5) zinc acetate crystallization. By eliminating these standard procedures and replacing them with ion ex change processes in a basket centrifuge, a much greater yield of insulin is obtained. By eliminating procedures that denature insulin, the yield may be 85 to 95 per cent compared to 40 to 60 per cent. The pan creatic enzymes are not only removed at an early stage without hydrolyzing of the insulin and formation of undesirable insulin bypro ducts, but the enzymes may be recovered as well. Since the enzymes have been removed during the cation exchange adsorption, the insulin is stable and not subject to hydro lysis. This makes possble carrying out the initial cation exchanger adsorption at the slaughterhouse, elimination of freezing and thawing - of the pancreatic glands with re sulting insulin loss, and subsequent process ing at the site where pharmaceutical pre parations of insulin are prepared. The cation exchanger can be regenerated by the phar maceutical company and recycled to the pancreas supplier for further insulin re covery. WHAT WE CLAIM IS:-

1. A method of stabilizing, concentrating and purifying insulin, which method com prises: (A) preparing an acid-alcohol extract of minced homogenized pancreas gland tissue, (B) first stabilizing, concentrating and ,puri fying the insulin by'contacting said acid alcohol extract with an acidic cation exchanger to adsorb said insulin on the cation exchanger, and washing with acidic aqueous solution to separate alcohol and pancreatic enzymes from the cation exchanger, (C) eluting the stabilized, concentrated and purified adsorbed insulin from the cation exchanger with a buffered- alcohol solu tion at pH 6.5 to 9, (D) then, further concentrating','and -purify-' ing the insulin contained in the' eluant from the cation exchangér by cóntact- ing said eluant with a basic anion ex changer to adsorb the insulin on the anion exchanger and separating the alcohol eluant, (E) washing the insulin adsorbed on the anion exchanger with an equeous buffer at pH 6.5 to 9 to remove remaining alcohol, and (F) eluting the further stabilized, concen trated and purified adsorbed insulin from the anion exchanger with dilute acid.

8. A method according to any of the preceding claims wherein said insulins eluted from the anion exchanger with dilute acetic acid.

9. A method according to claim 8 wherein said dilute acetic ,acid is about 1 Molar.

12. A method according to cTåim l of stabilizing, concentràting and purifying, ,iu- sulin, which inethdd'comprises: (A) preparing an extract of minded homo- genized pancreas gland tissue with' 'a mixture of about 65 % by volume ethanol and about 35% water.at pH.about 2.8, (B) - first stabilizing, concentrating and puri frying the insulin by contacting said ex tract with an acidic cation exchanger

charaeterized by functional sulphopropyl groups attached by ether linkages to the glucose units of cross-linked dextran chains, in -a basket centrifuge to absorb the insulin on the cation exchanger, washing with acidic aqueous solution and spinning off the separated alcohol and pancreatic enzymes, (C) eluting the stabilized, concentrated and purified adsorbed insulin from the cation exchanger with a mixture of about 65% by volume ethanol and about 35% water at pH 6.5 to 9, (D) then, further concentrating and purify ing the insulin contained in the eluant from the cation exchanger by contact ing said eluant with a basic anion exchanger characterized by functional diethyl-(2-hydroxypropyl) aminoethyl groups attached by ether linkages to the glucose units of cross-linked dextran chains, in a basket centrifuge to adsorb the insulin on the anion exchanger, and spinning off the alcohol, (E) washing the insulin adsorbed on the anion exchanger with an aqueous buffer at pH 6.5 to 9 to remove any remaining alcohol, and Q eluting the stable, concentrated and purified insulin from the anion ex changer with dilute acid.

13. A method according to claim 12 wherein said pancreas gland tissue is from freshly excised pancreas glands.

14. Amethod according to claim 1, substantially as hereinbefore described.

15. A method of stabilizing and concentrating insulin from freshly excised pancreas glands in a form suitable for transport and storage for later purification and further concentration, which method comprises: (A) preparing an acid-alcohol extract of minced homogenized freshly excised pancreas gland tissue, (B) concentrating the insulin by contacting said extract with an acidic cation ex changer to adsorb said product on the cation exchanger, and separating the alcohol and pancreatic enzymes from the cation exchanger by washing with acidic aqueous solution and (C) removing the cation exchanger with adsorbed stabilized, concentrated and partially purified insulin and packing for transport and storage.

16. A method according to claim 15 wherein said extract of freshly excised pancreas gland tissue is prepared with a mixture of about 65% by volume ethanol and about 35 /O water at pH about 2.8.

17. A method according to claim 15 or 16 wherein said cation exchanger is characterised by functional sulphopropyl groups attached by ether linkages to the glucose units of cross-linked dextran chains.

18. A method according to any of claims 15 to 17 wherein pancreatic enzymes separated from the initially concentrated insulin are recovered.

19. A method according to any of claims 15 to 18 wherein the ion exchange is carried out in a basket centrifuge.

20. A method of stabilizing, concentrating and partially purifying insulin from freshly excised pancreas glands for later further purification and concentration, which method comprises: (A) preparing an acid-alcohol extract of minced homogenized freshly excised pancreas gland tissue, said extract con taining insulin and constituents dele terious to insulin, (B) preparing a compacted bed of acidic cation exchanger, said resin being ad sorptive relative to the insulin and non adsorptive to said deleterious consti tuents, (C) stabilizing and concentrating the insulin by forcing said extract through said compacted bed of cation exchanger to separate crude insulin from said extract by adsorption on the exchanger, (D) washing said bed with acidic aqueous solution to remove the alcohol and deleterious constituents, and thereafter (E) eluting the resulting stabilized, concen trated and partially purified adsorbed insulin from the cation exchanger with an aqueous solution of higher pH in the range 6.5 to 9 for subsequent purifica tion.

21. A method according to claim 20 wherein step (E) is performed with the eluant defined in claim 6.

22. A method according to claim 15, substantially as hereinbefore described.

23. A method according to claim 20, substantially as hereinbefore described.

24. Insulin when stabilised, concentrated and purified by the method of any of claims 1 to 14.

25. A cation exchanger having insulin adsorbed therein, obtained by the method of any of claims 15 to 19 or 21.

26. A solution of insulin obtained by the method of claim 20, 21 or 23.