CA1041090A - Process for purifying insulin - Google Patents

Abstract

ABSTRACT OF DISCLOSURE

The invention relates to a process for purifying alkali metal or ammonium insulin by use of gel filtration at a pH from 2.0 to 3.5.

Description

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~04109(3 The invention relates to a process for purifying alkali metal or ammonium insulin by use of gel filtration at a pH from 2.0 to 3.5.
The invention provides a process for purifying alkali metal or ammonium insulin which comprises swelling a gel having in the dry state a water regain of at least about , ~ .
four percent by weight and particle diameters smaller than about 100 microns; packing a column with the swollen gel;
:1 .
- ~ adding to the packed column an aqueous solution of an alkali metal or ammonium insulin having a purity of at least about :, .
80 percent, wherein the insulin concentration is in the range of from about one to about eight percent, weight per volume, the total amount of insulin is sufficient to provide a column loading in the range of from about 0.8 to about 6.7 grams per liter of bed volume, and the pH of the solution is in the range of from about 2.0 to about 3.5; and eluting the insulin .. . .
from the column, at a temperature in the range of from about 5 to about 30C., with an aqueous eluant having a pH in the .
i same range as that of the insulin solution.
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The drawing is an elution diagram for Example 1.
~- The diagram is a plot of the optical density at 280 m~ of certain fractions of the insulin solution during the course of the gel filtration.

Since its discovery in 1921 as a component of the , pancreas, insulin has assumed world-wide importance in the ' treatment of diabetes mellitus. Because the extraction pro-, cedures employed to remove insulin from pancreas tissue also remove substantial amounts of noninsulin protein, considerable effort has been expended in developing methods for purifying , ~,. .
insulin; i.e., methods for separating insulin from noninsulin " ' ~

'' 104~090 ~, ` protein. Several of the methods developed for purifying insulin have achieved commercial importance.
; Of these commercially-important methods, one of the `
earliest involved the general phenomenon that a protein has -~minimum solubility at the isoelectric point, other factors being kept constant. Banting, et al., disclosed in ~.S. Patent 1,469,994 a purification process which comprises precipitating at their isoelectric point noninsulin proteins contained in an aqueous extract of pancreas. The isoelectric precipitation of insulin from an aqueous pancreatic extract at a pH of from about 4 to about 7 then was disclosed by Walden in U.S. Patent 1,520,673. -~In another early method, precipitation of insulin was effected by the addition of a sufficient amount of an inorganic salt. Murlin, in U.S. Patent 1,547,515, disclosed the first process for salting out insulin from an extraction ; solvent by the addition of sodium chloride. A differential salting out process was described by Lautenschlager, et al., in U.S. Patent 2,449,076, which process comprises salting out insulin from a neutralized extract by the addition of sodium chloride, filtering, redissolving the residue and again salting -; out the insulin, but with a salt concentration lower than that -employed in the first salting-out step.
-- A third commercially-important method for purifying insulin involves the precipitation or crystallization of insulin from solution as a zinc insulin complex. In general, zinc insulin is precipitated from a buffered, aqueous solution ~-~containing zinc ions. Various buffers have been employed;
Petersen, for example, in U.S. Patent 2,626,228, utilized a citric acid-citrate buffer. -~
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; Present commercial processes for the purification of insulin typically involve all three of the above methods.
Because zinc insulin is the most important commercial form of insulin, the last step in insulin purification normally is a zinc crystallization step.
However, noninsulin proteins, of which proinsulin and proinsulin-like protein are the most prevalent, have been recognized in recent years as minor components of commercial zinc insulins. Although such components generally consti-tute less than about eight weight percent of commercial zinc insulins, some of these components are believed to be anti-genic or immunogenic in nature. See, for example, Chance, et al., Science, 161, 165 (1968); Steiner, et al., Diabetes, 18, 725 (1968); Bromer, BioScience, 20, 701 (1970); and ` Rubenstein, et al., Ann. Rev. Med., 22, 1 (1971).
A continuing problem, then, is the removal on a commercial scale of noninsulin proteins from insulinO
The term "insulin" as used herein includes not only , .
insulin per se, but also all insulin-like proteins, such as ; :;
' 20 desamido insulin. Because insulin and insulin-like proteins have similar hypoglycemic activities, it usually is not required to separate insulin from all other pancreatic pro-teins; i.e., a homogenous insulin normally is not required.
Techniques for the resolution of biological sub-stances on an analytical scale, such as electrophoresis and ion-exchange chromatography, are known to separate noninsulin components from the insulin component of commercial (or even crude) insulin preparations. These tPchniques can even resolve the insulin component into insulin ~ se and desamidated X-4018 ~4~
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insulins. However, such procedures have questionable utility on a large scale.
A procedure more readily adaptable to large-scale use is gel filtration ~a term synonymous with gel exclusion chromatography and gel permeation chromatography). In this method, proteins are separated on a column containing a gel which has been crosslinked in such a manner that pores are formed within each gel particle. These pores have a finite, measurable volume which is directly proportional to the degree of swelling of the gel and inversely proportional to the degree of crosslinking. Because smaller molecules have more complete access to these pores than larger molecules, the progress of smaller molecules through the column is impeded relative to larger molecules which have only partial or no access to the , . - .
pores.
Gel filtration in the past has been employed in the ; -- purification of insulin. In the isolation of insulin from a single cat pancreas, crude insulin was obtained by acid- -"~ alcohol extraction followed by an alkaline precipitation to ; 20 remove inaetive material, concentration, an isoelectric ' preeipitation, and finally a sodium chloride salting-out ~ precipitation. The erude insulin then was applied to a cross-- linked dextran gel column and eluted with 1.0 M aeetie acid.
Davoren, Biochim. BioFhys. Acta, 63, 150 (1962).
In the isolation of insulin from fish, glands were homogenized in water and proteins precipitated with trichloro-aeetie aeid solution. The preeipitate was extraeted with acid-ethanol and the extracts were treated with methylene chloride to remove lipids. The remaining solids were isolated and taken up in 5.0 M acetic acid and filtered on a crosslinked ,' .
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. , : ............ . ~ ~ , ,, dextran gel column with 5.0 M acetic acid as the eluting solvent. Humbel, Biochem. Biophys. Res. Commun., 12, 333 (1963). Yields of about 80 percent were reported. It should - be noted that Humbel states that for good separation of insulin from other proteins, elution conditions should favor dissocia-tion of insulin molecules. He further suggests that 1.0 M
acetic acid is not a satisfactory solvent since, according to Yphantis and Waugh [Biochim. siophys. Acta, 26, 218 (1957)], insulin is not completely dissociated therein.
Crude extract of beef pancreas was filtered on a crosslinked dextran gel by Epstein, et al., Biochemistry, 2, 461 (1963). The eluting solvent was 0.2 M ammonium bicarbo-nate, a solvent also condemned by Humbel, supra. Epstein, et al., reported a yield of about 60 percent.
Insulin has even been isolated from human pancreas.
The glands were first homogenized and extracted. An ammonium sulfate fractionation removed some inactive material. Insulin ":
- then was precipitated, first with sodium chloride, then~with ~-an isoelectric precipitation. The resulting insulin was 20 dissolved in 0.5 N acetic acid and filtered on a crosslinked dextran gel. The filtered insulin then was converted to zinc insulin. Jackson, et al., Diabetes, 18, 206 (1969). The yield of insulin after gel filtration was about 60 percent.
Finally, gel filtration and ion-exchange chroma-tography were combined by Schlichtkrull, et al., in South - African Patent 69/5280 (for an equivalent patentl see Belgian Patent 737,257). In the only gel filtration example (Example I), zinc insulin was filtered on a crosslinked dextran gel column with a yield of 80 percent. The eluting solvent was 1.0 M acetic acid. It should be noted that Schlichtkrull, X-401g et al-, found it ~- .

~: ~o~o9o necessary, after filtration, to go through salting out, iso-electric precipitation, and zinc crystallization procedures in order to obtain zinc insulin.
: Each of the prior art applications of gel filtration to the purification of insulin has resulted in substantial insulin loss. Furthermore, from the prior art, there is no reliable indication that only insulin (including insulin-like proteins as discussed hereinabove) is obtained as a result of the use of gel filtration.
In general, any water-swellable gel suitable for use , with protein solutes can be employed in the process of the .;..... .. - .. , present invention. However, such a gel shall have in the dry or nonswollen state a water regain of at least about four percent by weight, based on the weight of dry gel, and particle -diameters smaller than about 100 microns. Preferably, the water regain of the gel will be in the range of from about -.. ,. :
four to about 98 percent, and most preferably from about five to about 20 percent. Preferably, the diameters of the dry gel particles will be smaller than about 80 microns; a particu- -- 20 larly useful range of particle diameters is from about 20 to about 80 microns.
Examples of suitable gels include, among others, ; starch (including maize starch), crosslinked galactomannan, crosslinked dextran, agar or agarose, polyacylamides, copol~-mers of acylamide and methylene bis-acrylamide, copolymers of methylene bis-acrylamide with vinylethyl carbitol and with vinyl-pyrrolidone, and the like. The preferred gels are crosslinked dextrans, such as the "Sephadex"* series manufactured and SOla by Pharmacia Fine Chemicals, Inc., Piscataway, N.J., U.S.A.

*Trademark .d~
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The type of column employed in the process is not critical. The choice of column height, diameter, or configura-tion will depend upon the opera-ting parameters desired. Of - course, as column height increases the flow rate decreases;
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stated differently, column back-pressure is directly propor-tional to column height. For this reason, a stacked column configuration is preferred, such as the Pharmacia Sectional Column KS-370. For normal production purposes, a stack of six sections serves quite well.
As an approximation, satisfactory insulin purifica--~ tion can be accomplished at column loadings of about 4.5 grams of alkali metal or ammonium insulin per liter of bed volume, . .
which loading is most preferred. However, column loadings generally can range from about 0.8 to about 6.7 grams per liter of bed volume, while the preferred range is from about 3.5 to about 5.0 grams per liter of bed volume. Of course, optimum conditions can be determined readily for any given column.
The gel can be swollen and the column packed with the swollen gel by any of the various methods known to those skilled in the art. In general, the gel will be swollen in the eluting medium. Alternatively, the gel can be swollen in 30 percent aqueous ethanol, the fines decanted, and the swelling medium replaced with eluting medium.
The alkali metal or ammonium insulin solution can be prepared in any convenient manner. For example, the insulin can be dissolved in an appropriate quantity of eluting medium.
Or, the insulin can be dissolved in an aqueous medium which is somewhat more acidic (but still within the eluting medium pH range) than the eluting medium. For example, if the . .

'' ~041090 ~ eluting medium is 0.5 N acetic acid with pH of 2.5, the :~ insulin might be dissolved in an aqueous medium having a pH
of about 2.3; i.e., 1.0 N acetic acid.
he alkali metal or ammonium insulin normally is alkaline or basic in nature. Consequently, upon dissolving the insulin in a neutral or acidic aqueous medium the pH of .. ; ....................................................................... .. ~ ., the resulting solution will rise. Thus, if the insulin is .::
dissolved in 1.0 N acetic acid, the pH of the resulting solu-tion will increase to about 2.7. However, depending upon the -concentration of insulin employed, the insulin solution can have a pH as high as 3.2. This increase in pH is normal and causes no problems provided the pH of the insulin solution does not exceed about 3.5. Alternatively, the insulin can be dissolved in 0.5 N acetic acid and the pH adjusted to less than about 3.2 with dilute hydrochloric acid.
In general, the alkali metal or ammonium insulin can be prepared by any of the known methods. It is preferred, however, that -the alkali metal or ammonium insulin be prepared by the process of U.S. Patent 3,719,655~ As indicated hereinbefore, however, said insulin must have a purity of at least about 80 percent. That is, the content of insulin, including insulin-like proteins having hypoglycemic activity, must be -at least about 80 percent of the total proteins present. ~
The concentration of alkali metal or ammonium insulin ~ -in the insulin solution applied to the column will be, as --~
- stated hereinabove, from about one to about eight percent, . .
weight per volume. The preferred concentration is from about four to about six percent.
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` For satisfactory results, the alkali metal or ammo-: nium insulin should be dissolved in a volume of solvent which is less than the separation volume, discussed hereinbelow.
In chromatography, the distribution coefficient, Kd, is defined as the ratio of the concentration of solute in the , mobile phase to the concentration of solute in the stationary phase. In gel filtration, the mobile phase is the solvent , .
moving in the void space between gel particles and the ~~ stationary phase is the solvent imbibed in the gel particles, , .
- 10 i.e., trapped in the pores within each gel particle. Thus, Kd indicates that fraction of imbibed solvent which is pene-tratable by a solute.
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In terms of Kd, the elution volume, Ve, of a solute can be expressed by the equation, - V = V + K . V.
- e o d ; where VO is the void volume of the column and Vi is the volume of imbibed solvent. Solving for Kd, the equation becomes, Kd = e If the density of water is assumed to be unity, Vi = a.Wr, where a is the weight of dry gel and Wr is the water regain.
Thus, Kd becomes ,: V -- V
. Kd =
. ~' aWr which can be determined experimentally by those skilled in - the art.
Assuming a solution contains two solutes, the elution :
- volume fox each solute is expressed as follows:
Ve ' = VO + Kd Vi Ve " = VO + Kd Vi .. . . . .
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; The separation volume, V , is the difference between the -elution volumes of the two solutes:
.~ V = V " - V ' . s e e VS = (Kd - Kd ) i , From the foregoing, it is apparent that the degree ; of separation of two (or more) solutes is in part dependent upon the column load. As the amount of lower molecular weight i solute approaches the limit of accessible pores, the separa-tion between two solutes necessarily must decrease. Within the load limits specified as a part of the present invention, the degree of separation can vary. In some instances, a higher ; column loading may be selected to balance separation versus productivity.
As stated hereinbefore, the eluting medium can have a pH in the range of from about 2.0 to about 3.5. In general, the eluting medium can be an aqueous solution of any inorganic ~-or organic acid toward which proteins are stable. Examples ;~ of such acids include, among others, hydrochloric acid, phosphoric acid, formic acid, acetic acid, propionic acid, n-butyric acid, isobutyric acid, pentanoic acid, and the like.
Acetic acid is preferred. If hydrochloric acid is employed, a preservative, such as phenol, must be included in the elutlng medium. It may be noted that acetic acid acts as a preserva-tive. The preferred pH is about 2.5. Consequently, the most preferred eluting medium will comprise a 0.5 N solution of - acetic acid.
If desired, the eluting medium can contain up to about 0.02 moles per liter of inorganic salt, such as sodium chloride, potassium chloride, ammonium chloride, and the like.
- 30 The preferred concentration is 0.01 M and the preferred salt - . . ::

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~ 1041V90 is sodium chloride. The use of such a salt is preferred to improve resolution and to prevent basic substances from being absorbed by the gel.
Elution of the column can be carried out at tempera-tures in the range of from about 5 to about 30C. Preferably, the process temperature will be at ambient temperature or ; lower.
The course of elution is followed by any convenient means. A particularly useful method, however, consists of measuring the ultraviolet absorption at 280 m~ of each frac-tion. Those fractions representing the desired purified - insulin component are combined and, usually, converted to zinc insulin.
As indicated hereinbefore, zinc insulin is the most important form of insulin. Consequently, the high-purity - alkali metal or ammonium insulin obtained by the process usually will be converted to zinc insulin. Conversion to zinc insulin preferably is made at pH 6.0 in the presence of ammonium acetate buffer. It is not necessary to isolate the ; 20 alkali metal or ammonium insulin from the eluting medium, or to concentrate the solution of purified insulin before crystallizing with zinc. An advantage of the present process . . .
is that the insulin in solution as eluted is sufficiently con-centrated that salt or isoelectric precipitations are not re-quired, although such procedures can be employed, if desired.
The zinc insulin thus obtained has an improved purity as a result of the process. This improved purity can be demonstrated by several methods. The zinc insulin can be analyzed directly for noninsulin protein components, such as proinsulin and glucagon. The presence of high molecular weight - - . - -, . , : - ," ~ .

~.04~090 ~- proteins, which typically include protaminase, a proteolytic - enzyme, can be determined by measuring the stability of pro-tamine insulin suspension at elevated temperatures; if pro-taminase is present, the protamine insulin complex will dis-sociate because of protamine degradation. Physical properties, such as solubility of the zinc insulin at neutral pH and color of the resulting solution, can be measured. Finally, the ' .~
specific activity of the insulin can be measured by biological and immunological assays.
Because of increased purity, the zinc insulin, obtained after carrying out the processj is a desirable material for formulation into the various pharmaceutical forms ; of insulin, e.g., regular insulin, isophane insulin, protamine zinc insulin, zinc insulin suspension, globin insulin, and the like. Further, the purer zinc insulin permits the prep-aration of a neutral insulin which is more stable than acidic .~
; insulin; impurities that heretofore were present in zinc in-sulin often partly precipitated at a neutral pH. Such impuri-ties often would include insulin-degrading enzymes, such as trypsin and chymotrypsin, which decreased the potency of the insulin preparation. The purer zinc insulin also permits the formulation of extended action pork and beef insulin prepara-; tions without additional purification as required in the past.
Finally, the preparation of isophane insulin from the purer zinc insulin requires less protamine since the absence of acidic protein impurities permits the use of only that amount - of protamine required to precipitate the insulin.
The process can be combined as desired with other - purification procedures. For example, the alkali metal or ammonium insulin solution can be subjected to one or more : - . . . . .

~04109~
filtration operations prior to carrying out the gel filtration.
Also, the eluted insulin can be subjected to~one or more fil-tration operations prior to conversion to zinc insulin.
Example 1 A crosslinked dextran gel having a water regain of 5 percent and particle sizes of from 20 to 80 ~ was equili- -brated in 0.5 N acetic acid. A K100/100 Laboratory Column (Pharmacia Fine Chemicals, Inc.) having a cross-sectional diameter of 10 cm. and a length of 100 cm. was packed with 'he swollen gel to a bed volume of 7 liters (bed depth, 89 cm.). Sodium insulin (from beef pancreas), 17.5 g., prepared by the process of U.S. Patenh 3,719,655 and having a potency of 23.8 Units/mg. and a proinsulin content of 4.21 percent by weight, was dissolved in 400 ml. of 0.5 N acetic acid. The insulin solution was applied to the column and eluted with 0.5 N acetic acid under a pressure of 5 psig., resulting in a flow rate of 900 ml./hr. (linear flow rate, 0.19 ml.~cm.2/min.).
Following elution of the void volume (1725 ml.), fractions of about 23.5 ml. each were collected; protein content was estimated by measuring ultraviolet absorption at 280 m~.
Solids concentrations in mg./ml. also were determined. A
total of 240 fractions was collected. The fractions were combined into four groups, as summarized below: -~
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`: 10~0~0 Vol., Percent ;~ Group ml. Fractions Solidsa Identification : A 140510- 69 1.4 High molecular weight proteins and enzymes B-l 119370-119 2.4 Primarily Proinsulin B-2 978120-160 9.7 Proinsulin and Insulin inter-mediates C 1520161-225 86.4 Insulln a Percentage of total solids eluted To Group C was added 3.0 ml. of a 20% zinc chloride `; solution and the resulting solution was diluted to a volume of 3025 ml. with 0.5 N ammonium hydroxide. The pH of the final ` solution was adjusted to pH 6.0 with dilute ammonium hydroxide.
The zinc insulin which precipitated was isolated by centrifu-gation and washed successively with water, absolute alcohol, and ether, then dried ln vacuo. The yield of zinc insulin was , .
13.9 g.; the potency of the insulin was 25.6 Units/mg. and the proinsulin content was 0.44 percent.
Since the solids content of Group C was 86.4 percent of the total solids eluted, the maximum yield of solids from Group C would be 86.4 percent of 17.5 g., or 15.1 g.; on a - Unit basis, the maximum yield should be about 360,000 Units.
; Thus, the yield of zinc insulin from Group C corresponds : to 91.9 percent recovery on a weight basis and 98.9 percent recovery on a Unit basis. Based on starting material, the weight recovery and Unit recovery are 79.4 percent and 85.4 percent, respectively.
Group B-2 was precipitated with excess zinc ions as described above for Group C, redissolved in 0.5 N acetic acid, ~o~io~, and refiltered on a smaller column. The insulin peak was recrystallized as described above for Group C, giving an additional 1.0 g. of zinc insulin having a potency of 26.7 Units/mg. Thus, the overall yield of zinc insulin, based on starting material, was 85.1 percent on a weight basis and 91.8 percent on a Unit basis.
A better understanding of the process described in Example 1 will be had by referring to the drawing. The drawing is an elution diagram for Example l; i.e., a plot of the optical density at 280 m~ of various fractions v~rsus frac~ion number. Thus, the diagram indicates protein content of the fractions collected during the course of elution. The collec-tion of various fractions into the several groups described . .
; in Example 1 is shown by the dashed vertical lines. From the diagram, it is clear that the starting material consisted substantially of insulin and that the insulin component is ;~ effectively separated from proinsulin and similar proteins.
Example 2 s ~ The procedure of Example 1 was repeated, except that ,- 20 the beef sodium insulin was replaced with 30 g. of pork sodium ~:, insulin and the amount of 0.5 N acetic acid in which the insulin was dissolved was increased to 600 ml. The pork sodium insulin had a potency of 23.3 Units/mg. and a proinsuli~
content of 2.42 percent by weight. The following results were obtained:

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Group Vol., ml. Fractions % Solidsa A 1410 10- 69 1.3 i~ B-l 1200 70-119 2.7 B-2 820 120-154 6.0 C 1920 155-235 90.0 a Pereentage of total solids eluted ~; Group C yielded 26.5 g. of zinc insulin having a ; potency of 25.3 Units/mg. and a proinsulin content of 0.57%.
-~ Thus, the yield of zine insulin corresponds to a recovery of ', 10 98.1% on a weight basis or 95.9% on a Unit basis.
Zinc insulin prepared from the same batch of panereas, ,~ in the proeessing of whieh alkaline erystallization and gel ' filtration process were replaced with conventional isoeleetrie '- preeipitations from aqueous and aqueous aleohol solvents, was obtained in the same yield per pound of panereas. However, the potency was 24.3 Units/mg. and proinsulin eontent was 3.18%.

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Claims (18)

The embodiments of the invention for which an ex-clusive property or privilege is claimed are defined as follows:

1. A process for purifying alkali metal or ammonium insulin which comprises swelling a gel having in the dry state a water regain of at least about four percent by weight and particle diameters smaller than about 100 microns;
packing a column with the swollen gel; adding to the packed column an aqueous solution of an alkali metal or ammonium in-sulin having a purity of at least about 80 percent, wherein the insulin concentration is in the range of from about one to about eight percent, weight per volume, the total amount of in-sulin is sufficient to provide a column loading in the range of from about 0.8 to about 6.7 grams per liter of bed volume, and the pH of the solution is in the range of from about 2.0 to about 3.5; and eluting the insulin from the column at a temp-erature in the range of from about 5 to about 30°C., with an aqueous eluant having a pH in the same range as that of the in-sulin solution.

2. The process of claim 1, wherein said gel has a water regain in the range of from about four to about 98 per-cent.

3. The process of claim 2, wherein said gel has a water regain in the range of from about five to about 20 per-cent.

4. The process of claim 3, wherein said gel is a crosslinked dextran.

5. The process of claim 4, wherein said gel has a water regain of about five percent.

6. The process of claim 5, wherein the particle diameters of said gel are in the range of from about 20 to about 80 microns.

7. The process of claim 1, wherein the column loading is in the range of from about 3.5 to about 5.0 grams per liter of bed volume.

8. The process of claim 7, wherein the column loading is about 4.5 grams per liter of bed volume.

9. The process of claim 1, wherein the insulin is dissolved in 1.0 N acetic acid.

10. The process of claim 1, wherein the insulin is dissolved in 0.5 N acetic acid and the pH is adjusted with dilute hydrochloric acid.

11. The process of claim 1, wherein the eluting agent comprises 0.5 N acetic acid.

12. The process of claim 1, wherein the eluting agent contains an inorganic salt at a concentration of up to about 0.02 M.

13. The process of claim 12, wherein the inorganic salt is present at a concentration of about 0.01 M.

14. The process of claim 13, wherein said inorganic salt is sodium chloride.

15. The process of claim 1, wherein elution is carried out at ambient temperature.

16. The process of claim 1, wherein elution is carried out at 5°C.

17. A process for purifying beef sodium insulin which comprises swelling a crosslinked dextran gel having a water regain of 5% and particle diameters in the range of 20 to 80 µ; packing a 10 cm. x 100 cm. column having a bed volume of 7 liters with the swollen gel; adding to the packed column a solution of beef sodium insulin in 0.5 N acetic acid and having an insulin concentration of 4.4%; and eluting the in-sulin from the column at ambient temperature with 0.5 N acetic acid solution.

18. A process for purifying pork sodium insulin which comprises swelling a crosslinked dextran gel having a water regain of 5% and particle diameters in the range of 20 to 80 µ; packing a 10 cm. x 100 cm. column having a bed volume of 7 liters with the swollen gel; adding to the packed column a solution of pork sodium insulin in 0.5 N acetic acid and having an insulin concentration of 5%; and eluting the insulin from the column at ambient temperature with 0.5 N acetic acid solution.