CA1069843A

CA1069843A - Method of separating proteins by ion exchange

Info

Publication number: CA1069843A
Application number: CA260,064A
Authority: CA
Inventors: Francois Meiller; Bernard Mirabel
Original assignee: Rhone Poulenc Industries SA
Current assignee: Rhone Poulenc Industries SA
Priority date: 1975-08-28
Filing date: 1976-08-27
Publication date: 1980-01-15
Also published as: IE43536B1; DE2638764A1; NO762959L; DK156577B; NL178294C; ES451047A1; AU500134B2; JPS5257200A; AR209193A1; NL7609562A; YU209676A; NZ181884A; RO78229A; MX4333E; IE43536L; BR7605661A; DE2638764C3; AU1721776A; NO145508B; SE426911B

Abstract

Abstract of the Disclosure A method of separating proteins by ion exchange comprising putting a solution of proteins into contact with an ion exchange resin consisting of a porous inorganic carrier coated with a cross-linked polymer containing or carrying anion exchanging groups - tertiary amines or salts of quaternary ammonium - or cation exchanging groups and application of the process to the food, pharmaceutical and veterinary field.

Description

10~3~l~3 S P E C I F I C A T I O N

The invention relates to a method of separating proteins ~y ion exchange.
It is known to separatè pro~ein~ by ion exchange, using cellulose or dexkran, having fixed thexeon either ter-t~ary amines, ~aternary ammonium, or acid ~unctions. How-ever, these ion exchangers have no mechanical properties and consequently cannot be used in a column; their volume under-goes changes with the ionic forces and the pH o the medium of use. Moreover they are biodegradable and cannot be ster~
ilized.

~ ..
It is an object of this invention to provide ion exchange resins which do not have these drawbacks; have good mechanical properties, are not affected by the ionic ~orce or pH of the medium of use, are not biodegradable and can be ~ ~:
15 sterilized. In addition, they make it possible to obtain very pure proteins.
The protein-separating method according to the in .
vention comprises putting a protein solution into contact with an ion exchange resin and is characterized in thak the exchang-er consists o a porous inorganic carrier having a particlesize from 4 ~m to 5 mm, a specific sur~ace area o~-~approxim-ately 5 to 150 m2/g, a pore diameter o 500 to 2500 A and a pore volume of 0.4 to 2 ml/g, that the carrier be coated with less than 15 mg/m2 of a film of cross-linked polymer, contain-25 ing or carrying either anion-exchange groups, repreeented by tertiary amines or quaternary ammonium salts, or cation-exchange groups, represented by acid unctions, and th~t the exchanger has an exchange capacity o~ le~s khan 2 meq/g~
. The porous inorganic carriers used are mekallic : 3 oxides such as titanium oxide, aluminas and more parti~ularly 1~ ~

- ~1698~13 silicas. These carriers have average pore di~met~rs of 500 to 2500 A and pre~erably from600 to 1500 A, a speci~ic sur-face area of 5 to 150 m2/g and preferably ~rom 20 to 50 m2/g, and a particle size of 4 ~m to 5 mm, according to the applica-tion envisaged. Thus the finest particles are used for analy-sis and the coarsest for preparing substances.
The fun~tional groups, tertiary amines or quaternary ammonium salts are repsented by general formulae -CH2-~-CH~-or -CH2-N(+)-(R)3X( ) w~erein R, which may be iden~ical or dif~erent, represents an alkyl or hydroxyalkyl group with l to 4 carbon atoms and x represents an inorganic or organic anion, such as a chloride, sulphate, nitrate, phosphate or citrate.
The acid functional groups are represented by car-boxylic, sulphonic or phosphonic groups having the general formulae -COOH, -so2H~ -PO (OH)2-These functional groups either form part of the chain - o~ the cross-linked polymer or are fixed to the cross-linked polymer which covers the whole surface of the carrier The cross-linked polymers, which cover the sur~ace of the inorganic carrier, are products which are known per se - and obtained by any conventional methods o~ polymerization.; They are prepared from monomers which can be cross-linked either alone or with another mDnomer, often in the presence of a catalyst. The monomers include: epoxy compounds which can be cross-linked with polyamines as catalysts; the formaldehyde, which can be cross-linked by polycondensation without a cata-- lyst with urea~ melamine, polyamines, phenols; and vinyl mono-mers; vinylpyridine, styrene and derivatlves, vinylbenzoic acid or acrylic and/or methacrylic acid3, which can be cross-3o linked with polyfunctional monomers, ~ono- ox polyalkylene-- .

-2~

1~69i3~3 glycol diacrylats or dimethacrylate, divinylbenzene, vinyl-trialkoxysilane, vinyltrihalogenosilane, bis methyleneacryl-amide, in the pxesence of an initiator to liberate free radi-cals like organic peroxides and aæonitriles, or UV radiations.
~o obtain the coating of cross~linked polymer on the inorganic carrier, the carrier is impregnated with a solution of the monomer or monomers and possibly the catalyst in a sol~
vent, thus enabling the monomers to be distxibuted evenly over ~ ;
th~ entire sur~ace o~ the inorganic carrier. The solvent is then evaporated and the monomers cross-linked by known methods The solvent used may be any substances which will dissolve the monomers and catalyst, having a boiling point preferably as low as possible in order to encourage subsequent evaporation.
Some examples of such solvents are methylene chloride, ethyl ether, benzene, acetone and ethyl acetate.
A method particularly adapted to the preparation of an anionic exchanger, by coating carriers with epoxy com-poun~s, has been described in the copending application Serial ~o.234,62g filed August2g , 1975, entitled "Modified Mineral Supports".
In cases where the polymer cross-linked to the sur-face of the inorganic carriex does not have any functional groups in the chain, as defined above, it has to be modified;
this applies particularly to cross-linked polymers based on 2~ styrene and derivatives, and polymers of formaldehyde W7 th urea, melamine, polyamines, or phenols.
In the case of styrene or phenol-formaldehyde polymers, such modification comprises ~ixing either carboxylic, sulphonic, or phosphonic group~ on the polymer by any known method or by ~ixing chlorome~hy] groups on the polymer and

-3-~0~

then reacting them with a seconc~ary or ter~iary amine by any known process.
When fixing chloromethyl groups on the polymer, it is advantageous, in the case of styrene polymers, to disperse the inorganic carrier coated with polymer hot in chloromethyl ether in the presence o~ a Lewis acid. In the case of a phenol-~ormaldehyde resin, on the other hand, the inorganic carrier coated with polymer may e.g. be dispersed in epi-chlorohydrin and reacted at elevated temperature.
In the case of polymexs of formaldehyde with urea, melamine, or polyamines, the modification comprises converting the primary amines present in the chain into tertiary amines or salts of quaternary ammonium by any conventional method, e.g. by reaction with an alkyl sulphate or halide.
In the operation o~ coa~ing the inorganic carrier, the quantity of monomer(s) used must be such that the quantity of cross-linked polymer with functional groups, distributed over the surface of the inorganic carrier, is less than 15 and preferably from 1 to 8 mg/m2.
~he resultant inorganic carriers, coated with cross-linked polymers having functional grGups, have an exchange capacity belo~ 2 meq/g and preferably from 0.3 to 1.2 meq/g.
The method of the invention applies to all proteins soluble in an aqueous medium, whatever their isoelectric poLnt.
The following are some examples of such proteins, which include polypeptides and enzymes; al~umin, lactalbumins, egg albumin, serum albumin, haemoglobin, a, ~ and~y globulins, lactoglobulins, ~ibrinogen, urease, tr~psin, lys~xyme, pepsin, proteases, and cytochrome.
The method o~ the invéntion enables proteins to be a,_ ~ 0~ 8 ~ 3 separated very easil~ from ~heir solutions, su-h as milk 5~rum, beer, blood, extract~ from organs and from any industrial effluent; waste water from slaughter houses, the ood industry or potato starch works. It thus provides a means o~ purify-ing such ef~luents and hence a means of avoiding pollution.
Separation is obtained by putting the so~ution to be treated into contact with the ion exchange resin at a tempera-ture, ionic force and pH compatible with the protein or pro-teins, the resin being selected according to the separating conditions. Either the required protein, or the other protein or proteins contained in the solution, or all the proteins in the solution are then ~ixed on the ion exchange resin.
Separation may equally be obtained under the sa~e conditions by putting the solution to be txeated into contact with one or more anion-exchange and/or cation-exchange resin;
in succession. The proteins in the solution are then fixed selectively on each resin.
In eases whexe the required protein is fixed on a resin from a protein solution, it is then separated by elution with a solution that has a pH and/or an ionic force dirferent ~rom those o~ the fixing solution yet compatible with the pr9tein. This leads not only to separation o~ the protein from the solution but also to its purification and concentra~
tion. In this way, albumin, pepsin and proteins from milk ~ e~J-/onlc serum may e.g. be separated and concentrated with ~U~iY~Li~L~-~;. ~n an /onlc resin and lysozyme with ~ ionia resin.
In cases where the required protein remains in theprotein solution treated according to the invention and the other proteins in the mixture are fixed, the required protein is separated from the other proteins and thus purified.

10t~9~3 Elution o the proteins ~ixed leads to their selective or non-selective separation and concentration. This applies inter rr ~ ~ o~ ,~
alia to y-globulin with _n ~ienia resin.
In cases where a plurality of required proteins are fixed simultaneously on a resin, elution at a pH and/or an ionic force different from those of the fixing solution causes the proteins to be separated from the solution and concentrated.
Elution by solu~ions with an increasing pH and/or ionic ~orce leads not only to selective separation buk also to purifica-tion and concentration. This is particularly the case withhuman serum.
In cases where the proteins have to be eliminated, they are fixed on a resin from their solution. This gives solutions whi~h are deproteinized, that is to say purified.
; ~5 Elution of the proteins ~ixed ena~les the resin to be re-used.

This is particularly the case with clarification of beer and a~/0,7/~
treatment of solutions containing haemoglobin with ~h~;L~L-resins.
Separation may be carried out discontinuously or continuously, with identical results. ~
In continuous operations,the resins allow easy fill-ing of the column, a large output and easy elution.
The results obtained are virtually independent of the concentration of the solution treated, but are a function 2~ of the nature of the exchanging group in the resin, the pH, the ionic force and the flow rate of both the solutions to~be treated and that used for elution.
The method of the invention may be applied to the food indu~tries, particularly the dietetic, pharmaceutical and veterinary branches.
-~6-~o~g8~3 The following examples are given by way o illustra-tion, but not by way of limitation of the invention:
EXAMP~E 1:
Preparation of the ion exchanqe resin S 100 g o~ silica with a particle size o~ 100 to 200 ~m, a specific surface area of Z4 m2/g, an average pore diameter of 1400 ~ and a pore volume of l ml/g is dried at 150C at reduced pressure for 5 hours~
The dried silica is placed in a solution of 250 ml of methylene chloride, 60 ml of distilled styrene, 20 ml of vinyltriethoxysilane and 0.5 g of azobis-isobutyronitrileO
The methylene chloride is evaporated at am~ient tem-perature, then the impregnatea silica is heated at 120C and 3 bars for 6 hours to bring about cross-linking.
The silica is then suspenaed in 300 ml of xylene and heated at boiling point for 2 hours. When it has been filtered the silica is washed with acetone, then dried.
Analysis reveals a carbon content of 4% by weig~t relative to the coated silica.
. 20 50 g of the coated silica is suspended in 180 g o~
chloromethyl ether, containing 6 g o stannic chloride, then the mixture is heated under reflux for 4 hours in an anhydrous medium, After cooling, the silica is drained and washed with 200 ml of a 50-50 mixture of dioxane and water containing lO ml of hydrochloric acid. It is then washed with water until nnutral and finally dried.
~he carbon content i~ then 4.1% and the chlorine con-tent l~9~/o~
The product obtained is suspended in 150 ml o~ a 30O/o - _7_ "

~1[169~3~3 aqueous so~.uticrl c)f trimethylamine and left in contact for 8 days at ambient temperature.
After draining and washing, an ion exch~nge resin is obtained, carrying OEI3 --~ CH2--~ ( )--CH3 Cl ( ~unctional groups and with the following properties:
- ca~bon content 4~/O
- chlorine content 2 %
- nitrogen content o~/O
quantity or polymer fixed 3.3 mg/m2 - exchange capacity 0O5 me~/g~
Treatment of an aIbumin solution 10 g of the ion exchange resin obtained is placed in 1~ a column 1 cm in diameter and kept compressed, then the resin is put into equilibrium at pE 6.5 with a 0.01 M phospha~e buffer.
A l~/o by weight albumin solution in the same bu~er is percolated at 180 ml per hour until the col~Mn is saturate~;
this represents about 200 ml of solutionO The resin is then washed with 100 ml of ~he same buffer.
The albumin fixed is then eluated by percolating an M solution of NaCl into the same bu~fer, at a flow rate o~
180 ml per hour~ 45 ml of solution enables the albumin to be 2~ recovered in a 3.3% by weight solution.
The ion exchange resin consequently has an albumin capacity of 150 m~ per g and has enabled the albumin solution to be concentrated.
~ he same operation is repeated 30 t:imes, and no swell-ing or aging o~ the ion exchaDge resin is observed.

.; - , ~01i9~3 Example l is repeated with a 0.2% instead of a 1% by weight solution of albumin.
The same results are obtained, that is to say the same concentration of albumin is obtained whatever the concen-tration of the solution treated.
EX~MPLE 3:
Example 1 is repeated except that the protein is eluated with a 0.05 M, pH 6.5 citrate buf~er. 59 ml of 2.5%
by weight albumin solution is obtained.
This test shows the effect that the nature of the elution buffer has on the concentration of the solution ob-tained.
EXLMPLE 4:
~n alb~min solution is treated in the same way as in Example 1 but using 41 g of the ion exchange resin in a column 1 cm in diameter and with an elution flow rate of 80 ml per hour instead of 180 ml per h~ur.
The concentration of the al~umin golution obtained is 7% by weight. This ~hows that by increasing the working height of the column and reducing the elution speed, th~ con-centration of the resultant solution can be increased.

.
Preparation o~ the resin 50 g of a silica, having a particle size of 40-100 ~um, a specific surface area of 37 m2/g, a pore diameter of llO0 A and a pore volume of 1.05 ml/g, i9 placed in 150 ml of met~ylene chloride with 6.5 g o N,N-bis(2,3-epoxy propyl) ethylamine and 3 g of triethylenetetramine dissolved in it.
~he methylene chloride i.8 then evaporated at ambient .
`~ _9_~

,. ~ .

temperature. The impregnated silica is he~ted at 60C for 60 hours to bring about cross-linXiny. It is washed with boiling water and then with acetone.
The ionic exchange resin obtained is made up o~
silica coated with a cross-linXed polymer containing -CH2~-Ç~2-functional groups and has the following properties: C2H5 carbon content 8 . 8%
- nitxogen content 2.4%
- guantity of polymer fixed 3-3 mg/~2 - exchange capacity 1 meq/g Separation of y-globulin 10 g of the ion exchange resin ~btained is placed in a column ' cm in diameter and kept compressed. The resin is put into equilibrium in 0.1 ~ hydrochloric acid, then in 0.02 N, pH 6.5 phosphate buffer.
20 ml of a 1% by weight solution of delipidized and .lyophilized human sexum in the same phosphate buffer is per-colated at 100 ml per hour thxough the column. The resin is then washed with 50 ml of the same p~osphate buffer.
~he solution emerging from the col~mn contains the r -globulin present in the initial solution, in an electro-phoretically pure state.
The other proteins- ~-globulins, ~-globulins and albumin, also present in the initial solution, remain fixed on the resin. They are recovered by elution with a 3 ~ solu-tion of NaCl in the same phosphate buffer.
` If elu~ion is effected by buffexs of increasing ionic force, with an increase in the concentration of NaCl, solutions enriched with a globulin~, p-globulins and albumin 3 are o~tained.

~ 10-~06~ 3 l.E G:
Preparation of_the resin The procedure is the same as in Example 5, but the silica used has a particle size of 100 to 200 ~m, and 6.5 g of ~,N-bis (2,3-epoxy propyl~ butylamine is used instead of 6.5 g of N,N-bis I2,3-epoxy propyl) ethylamine.
The ion exchange resin obtained consists of silica coated with a cross-linked polymer containing -CH2-~-CH2-functional groups and has the following properties:C4H9 - carbon content 9.2%
- nitrogen content 2.5%
- quantity of polymer ~ixed 3.2 mghn2 - exchange capacity 1.1 meq/g Extraction of proteins 15 . 10 g of the ion exchange resin obtained is placed in a column 1 cm in diameter and kept compressed. The resin is successively put into equilibxium in 0.1 ~ hydrochloric acid, then in 0~01 N, pH 7.5 phosphate buffer.
30 ml of a 1% by ~eight solution o~ ultrafine-filtered milk serum powder, in the same phosphate buffer con-taining 75% by weight of proteins is percolated through ~he column at 80 ml per hour. The resin is then washed with 100 ml of the same phosphate bu~fer.
The solutions emerging ~rom the column contain the fatty materials and lactose present in the initial solution.
The proteins, lactalbumins, lactoglobulins, serum - albumin and a small part of the immunoglobulins, present in the initial solution, are fixed on the resin. The separated and purified proteins are recover~d by elution with a 0.05 ~I, p~ 4 Mac Ilvaine buffex.

~, ~O~ 3 EXAMPLE 7:
Extraction of proteins 20 g of an ion exchange resin, similar to that in Example 1 but with a particle size from 200 to 500J~m, is placed in a column 2.5 cm in diameker and kept compressea.
The resin is successively put into equilibrium in 0.1 N hydrochloric acid, then in 0.01 M, pH 7 tris HCl buffer.
600 ml of a solution consistiny of 300 ml of the same tris-HCl buffer and 300 ml of delipidized milk serum, containing 0.5% by weight of soluble proteins, is percolated through the column at 300 ml per hour. The resin is then washed with 100 ml of the same buffer.
The solutions emerging from the column contain the lactose present in the initial solution.
The proteins: lactalbumins, lactoglobulins, serum albumin and a small part of the immunoglobulins, present in the initial solution, are fixed on the resin. They are elu-ated by passing a 0.1 ~, pH 7 citrate-caustic soda buffer into the column. The solution o~tained contains all the pro-teins fixed, at a concentration of 4% by weight.
; The operation enables a mixture of pure proteins to be obtained, free from lactose and in a far more concen trated solution than the initial one.
~o aging of the resin is observed after 30 success-ive operations.
EX~MPLE 8:
Treatment of a pepsin solution 3 g of an ion exchange resin similar to that in Example 1 is p~aced in a column 1 cm in diameter.
The resin is washed with 100 ml of distilled water, 10698~3 the~ 0 ml o~ a solution of crude pepsin containing ~0 pepsin units per ml is percolated through the column at 100 ml per hour. The resin is then ~ashed with 20 ml of distilled water.
The solution emerging from the column and the wash-ing water do not sho~ any activity; the pepsin is fixed onthe resin. The impurities have remained in the solution, as shown by dry extract determinations.
The pepsin fixed is then eluated by percolating an M solution of NaCl through it at 100 ml per hour.
16 ml of solution enable the pepsin to be recovered in a solution containing 170 pepsin units per ml.
The high concentration of the solution obtained is noted.
The resin is re-used after being washed with 50 ml of distilled ~ater.
No aging of the resin is observed after 10 success~
~ive operations.
EXAMPLE 9:
Preparation of the ion exchanqe resin a~o g of silica, with a particle size of 100 to 200 ~m, a specific surface area of 25 m2/g, an average pore dia-meter of 1~00 ~ and a pore volume of 1.1 ml/g, is impregnated - - with a solution comprising 200 ml of methylene chloride, 24 g of acrylic acid, 6 g of diethylene glycol dimethacrylate and 0.~ g o~ benzoyl peroxide.
~he methylene chloride is evaporated at ambient - temperature and atmospheric pressure to constant weight; then the impreynated silica is heated at 80C ~or 6 hours to bring about polymerization The silica is ~hen su~pended in 300 ml of water and :. :

9~43 heated ~t boiliTIg point ~or 6 hours. Wh~n it has hcen ~iltered, the silica is washed with acetone, then drie~ under vacuum at 80C.
The ion exchange resin obtained carries - COOH
functional groups and has the following properties:
- car~on content 10.55 %
- quantity of polymer fixed 7.2 mg/1~2 - exchange capacity l~O5 meq/g.
Treatment of a lysozvme solution 10 g of the ion exchange resin obtained is placed in a column 1 cm in diameter and kept compressed~ then the resin is put into ~H4~ form by percolating through the column 2 liters of a O.5 M aqueous solution of ammonium acetate buffexed at pH 8.2~ -The resin is then put into equilibrium at p~ 6~5 with 100 ml of 0.02 ~ tris-maleic acid buffer.
A 1% by weight solution of lysozyme in the same buffer is percolated through the column at 180 ml per hour until the column is saturated; this represents about 200~ml of solution. The resin is then washed with 100 ml of 0.02 N, pH 8.2 tris-maleic acid buffer.
The lysozyme fixed is then eluated by percolating an M solution of ~aCl in the same buffer, at a flow rate of lBO ml per hour. 50 ml of solution enables the lysozyme to be recovered in a 2.5% by weight solution.
It follows that the ion exchange resin has a lyso-: . zyme capacity of 12~ mg/g and that it enables the lysozyme solution to be concentrated.
EXAMPLE 10:
Preparation o~ the e~chan~e resin .. ~ ... .
,~ , .

~1~)65~8~3 100 g of silica, Wit]l a particle size o 100 to 200 ~m, a specific surface area of 37 m2/g, an average pore dia-meter of 1200 ~ and a pore volume o~ 0~95 ml/g, is impregnated with a solution comprising 150 ml of methylene chloride, 60 ml of distilled styrene, 20 ml of vinyltriethoxysilane and 0.5 g of azobis-isobutyronitrile.
The methylene chloride is evaporated at ambient temperature and atmospheric pressure to constant weight, thën the impregnated silica is heated at 120C for 6 hours to bring abou~ cross-linking.
The silica is then suspended in 300 ml of x~lene and heated at boiling point for 6 hours. ~hen it has been drained the silica is washed with acetone and then dried at 8 0 C~ C o - 15 50 g of the modified silica obtained is suspended in 500 ml of chloroform, and 50 g of HS03Cl dissolved in 50 . ml of chlorororm is added dropwise. Hydrochloric acid is re-leased. After the addition, the mixture is heated with agi-~ tation at 50C for 4 hours.
- 20 When the product has been drained, washed with wat~r until neutral, then washed with acetone and dried under vacuum at 80C, an ion exchange resin is obtained, carrying -S03H functional groups and having the ollowing properties~
-~ - carbon content -4 %
;~ ~5 - sulphur content 1.4%
-~uantity of polymer fixed 2.8 mg~m2 , . . .
- exchange cap~city 0.43 ~q/g~
xtraction o~ haemoqlobi~
10 ~ o the ion exchan~e resin obtained is placed in a column 1 cm in diamcter, then the resin is put into ~'- ' .

~o~ 43 equilibrium a~ ~H 6.5 with a 0.02 M phosphate buffer.
500 ml of a 002% by weight solution of haemoglobin in the same buffer is percolated throuyh the column at 100 ml/h. The haemoglobin is adsorbed on the ion exchange resin.
The colorless effluent solution no longer contains any haemo-globin and is thus purified The adsorbed haemoglobin is eluated by percolating a 0.5 M solution of ammonium carbonate, then the column is washed successively with 300 ml of 0.1 ~ caustic soda and 50 ml of 1~ ~Cl, beore being used again.
EX~MPLE 11:
Treatment of milk_serum 2~ g of an ion exchang2 resin similar to that in Example 1 is placed in a first column 2~5 cm in diameter.
10 g of an ion exchange resi~ similar to that in Exampl~ ~ is placed in a second column 2.5 cm in diameter.
T~e two columns are axranged in series and the resins are washed with 500 ml of water.
500 ml of milk serum, adjusted to pH 7.5 by the addition of 0.1 N caustic soda, is filtered to eliminate in-soluble materials, then percolated into column ~o. 1 and column ~o. 2 at 300 ml per hour.
The test in which proteins are precipitated by tri-chloroacetic acid shows that the milk serum emerging from colu~ ~o. 2 no longer contains any protein.
The resins in both columns are washed b~ passing 100 ml of water through them.
The protein~: lactalbumins, lactoglobulins, serum albumin and a very small part o ~he immnnoglobulins, present in the initial solution, are fixed on the resin in column ~6-.

No. 1. They are eluated as described in E~ample 7.
The proteins ~ixed on the resin in colur~ No. 2 are essentially the immunoglobulins which were nct fixed on the resin in column No. 1. They are eluated by percolating 5 an M solution of ammonium carbonate through them. The solu-tion obtained conkains the immunoglobulins in a concentration of about 3% by weight. Immunoglobulins represent about 16%
by weight of all the proteins in the initial solution.
The columns are then washed, by passing 500 ml of water through them, before being re-used.
~ o aging of the resins is observed after 10 success-ive operations.
EX~MPLE 12-_ .
Extraction of proteins from beer 60 g of an ion exchange resin similar to that in Example ~ is placed in a column 2.5 cm in diameter, then washed with 250 ml of water.
5 liters of non-clarified beer i~ percolated through at lOO ml per hour. The beer emerging from the column is no longer precipitated by the addition of picric acid and has the properties of a clarified beer.
The products fixed cn resin are essentially pro-~eins. They are eluated by percolating 400 ml of ~10 hydro-chloric acid.
2~ The resin is washea, by passing 250 ml of water ~ -through it, before being re-used.

. - ' .

Claims

The embodiments of the invention in which an exclu-sive property or privilege is claimed are defined as follows:

1. A method of separating proteins, comprising putting a solution of proteins into contact with an ion ex-change resin, comprising a porous inorganic carrier with a particle size from 4 µm to 5 mm, a specific surface area of approximately 5 to 150 m2/g, a pore diameter of 500 to 2500 .ANG.
and a pore volume of 0.4 to 2 ml/g, coated with less than 15 mg/m2 of a film of cross-linked polymer, containing or carrying either anion-exchanging groups, represented by ter-tiary amines or quaternary ammonium salts, or cation-exchang-ing groups, represented by acid functions, said exchanger having an exchange capacity of less than 2 meq./g.

2. The method of Claim 1, in which the inorganic carrier is selected from the group consisting of metallic oxide: titanium oxide, aluminas, and silicas.

3. The method of Claim 1, in which the anion-exchanging groups are represented by formulae selected from the group consisting of -CH2-?-CH2- and -CH2-N(+)-(R)3X(-) in which R, which may be identical or different, represents a group selected from the group consisting of an alkyl and hydroxyalkyl group having 1 to 4 carbon atoms and X represents an inorganic or organic ion.

4. The method of Claim 1, in which the cation-exchanging groups are selected from the group consisting of carboxylic, sulphonic and phosphonic acid functions.

5. The method of Claim 1, in which the cross-linked polymer results from polymerization selected from the group consisting of epoxy compounds in the presence of polyamines, mixtures of formaldehyde with urea, melamine, polyamines, phenols: mixtures of vinylmonomers: vinylpyridine, styrene and derivatives, vinylbenzoic acid or acrylic and/or meth-acrylic acids with polyfunctional monomers: mono- or poly-alkyleneglycol diacrylate or dimethacrylate, divinylbenzene, vinyltrialkoxysilane, vinyltrihalogenosilane, bis methylene acrylamide.

6. The method of claim 1, in which the proteins, including polypeptides and enzymes, are represented by albumin, lactalbumins, egg albumin, serum albumin, haemoglobin, .alpha., .beta.
and .gamma.-globulins, lactoglobulins, fibrinogen, urease, trypsin, lysozyme, pepsin, proteases and cytochrome.

7. The method of claim 1, in which the protein solutions to be treated are selected from the group consist-ing of milk serum, beer, blood, extracts from organs and industrial effluents.

8. The method of Claim 1, in which one or more re-quired proteins are fixed on the ion exchange resin, then eluated.

9. The method of Claim 1, in which the required protein remains in the solution, and the other proteins in the solution are fixed on the ion exchange resin.

10. The method of Claim 1, in which all the proteins are fixed and the solution purified.