Separation of proteins using and ion-exchange resin

The present invention relates to a process for separating proteins · from their solutions, with the aid of ion exchangers. ^{: r} ·. '<

There are various methods of se- 5 known; large-scale protein pairing (being used - extraction with various · solvents or solvent mixtures (Patents, USA, No. 36040374; 3031478):^!¹ Also known is the rare separation of proteins by ion exchange, reacting cellulose or dexthane, over which are either "tertiary amine or quaternary ammonium," or acid functions. However, 'these changers_JSRtu ions have good mechanical properties and, consequently, cannot be used in the column; their volume changes with the ionic strength and pH of the environment of use. On the other hand, they¹ they are biodegradable and cannot be sterilized. 20

Process. according to the invention, it removes the disadvantages mentioned by the fact that the protein solutions are passed through an ion exchanger - consisting of a mineral support based on silica 25 porous alumina salts, with a granulation between 4 ι m and 5 mm, with 0 surface area specifies on the order of 5.:150 m²/ G. a .diamertri / pores of 500 ... 2 500 'A,' uiij volume of the sores^: 0.4 to 30

2Saî / ά which is covered with a pelicu.ujnnifffon, Jn-jz Jk hes; eh ΗπΡυυιυί) -cj ăJiMps-, 'efzo soRAii oJc / ;; enjoying.riiOî ·? ιοίΗ «& ί oiți / loz ij?^: jeg r, ί; ! , 0. onbiothiophene% wool of crosslinked polymers having anion exchange groups of form. <; ih · ..- Η XTb- · or -CH, —N + (R $ XR wherein R is identical "or different and represents alkyl or hydroxyalkyl with 1 ... 4 atoms," carbon, and .X is a mineral or organic anion, or cation exchange groups representing —COOH, —SOsH, or POfOH — groups.

14 examples of carrying out the process according to the invention are given below. <

Example 1. Preparation of ion exchange resin,

An amount of 100 g silica, having a granulometric composition. from 100 up to 200μ m, a specific surface of 24 m '/ g, a diameter, average pore size of 1,400AcSL a volume, of pore size

it._!:ml / g, dried sesame at 150 ° C, under pressure. reduced, 1 for 5 h. Dried silica, obtained_IT It is. . introduced in a solution of 250 ml of methylene chloride, 60 ml of distilled styrene, 20 ml of vinylthiethoxysilane and 0.5 g of azo-base-isobutyronitrile. The methylene chloride is evaporated. at room temperature, then · the impregnated silica is heated. to l20 ° C, for 6 b, under a prehistoric o.iȘp'iotg? a ··: ··,. ι 11; ι.; · ι, · .ob u.,

MCiStei or im _? no pREȚfjCLEf'26 {85

-SOS) O'iofr'of. nr '.'iLOiie O f): li

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3 bar bar, ^ to obtain the retention ^^ ίτϊΠ / -Η -777After that, the syrup is suspended in 300 ml of xylene and boiled for 2 hours. After filtration, the silica is washed cd. acetone, then dried.

The analysis shows a carbon content of 4% by weight, in relation to the coated silica, g of the obtained silica are suspended in 180 g of chloromethyl ether, which contains 6 g of stannous chloride, then the mixture is heated under re. flow for 4 hours in anhydrous medium. After cooling, the silica is dried, washed with 200 ml of 50:50 dioxanapane mixture, which contains 10 ml of hydrochloric acid, then with water, until neutral and, in reaction. Finally, it is dry, the carbon content is now 4.1¼ and that of chlorine, l, 90 »/ o. The obtained product is suspended in: 150 ml. ; aqueous solution of -30% trimethylamine and left in contact for 8 days at '·' / ambient temperature. ~ Du '+ for drying and washing', you get a resin-changing 'resin' comprÎrizBrid working groups

CHt ~

CT-DCi '-', resin and gargoyle beard-characteristics:

- carbon content - content of. chlorine accounting for '/ ol: bâhdÎalba .rie'. '7'polimefr fbfa-t¹ ’’ 7‘^; - i apaeitâteă ',' '· -'lie Exchange' 0.

Ul · - '7 07

Trâterefi'una .'s solutions. of

4,8%; &lt; 2,0%; .. 1).9¼ ;

3.3 mg / rii ';

0, b fny-al7'g. albumin

- r 40 sg &lt;from the resin; gearboxes^: Are the ions obtained inserted into a column with a diameter of 1 cm and are mentioned as ctotijrriimrite?^jhp0r'in resin .is * j &amp; JîâÎă 'at pbț ^ .6,5 wi th a' Pb'sîaț'OrOi 'pad todl. ,E, '' 'fittffea £ ă * o s'Oluţie gives' hlbunîihă^;of 4%^:t) freed jn hCelăȘi tăm ^ Î&gt; n, '^:'' with all 'wishes /! /' tffâ 'ml / h, pine to sautriraiAa epToânei, say' 'te' '(cbde'spune. cry * ă'proxțbiătiv ^bml ^flS0 'bibliiţie. ^ Ră'și'niâ e ^ Fe ăpbi. ^ stain pu fdo ml from / âcdlaȘi^!tSm'gtoi. AÎbi5mina^!· Junk. hste ^ / ățtei dlfthtă ^^ p'pâddpl'ârea '' iihei. Iriotarh goalkeepers ib ^ cdiâ ^ '/ Fog / tooth of barrels flowed $ P^zdîiVl ($ ml / hyAS'înr / ăbliitte '. defendant i ^ duțyeitoVa blbuin'i'iiăi' tebiție ile îh gfffeuÎăîfe, '' Se 'jtetoe ron'chi'do da'^):The href = "gfffil de terii" shows a 'ca'pacitateotfe albumin' of .450 mg / g and that they were, therefore, able to concentrate, the albumin solution. "The same opecration is retained ^ Oj ^ p times and no 'swelling is observed, Here an agin g (scale-1. - activity.) Of the exchanger of good.

Example 2. Under the same conditions as in example 1, an albumin solution of 0.2% by weight, instead of 1%, is used. The same results are obtained. that is to say, the same concentration is obtained in the albumin, whatever is concentrated in the cut solution.

3. The conditions of Example 1 are repeated, but the addition of the protein is performed with a 0.05 mol encryption buffer solution at pH = 6.5. 59 ml of 2.5% by weight albumin solution are obtained. This sample proves the influence of the nature of the elution buffer on the concentration of the solution obtained.

Example 4. The solution of albumin rh solution is treated in the same way as Example 1, but using 41 g of the resin. ions, in a column with a diameter of 1 cm and elution flow rate of 80 ml / h, instead of 180 ml / h -.- The concentration of the albumin solution obtained is 7% by weight. This - proves that by increasing the usef ul height of the column and decreasing the elution speed, the concentration of the obtained solution is increased.

. iExemphii 5.. Prepararea-, resin; '; (η1450 units of strong methylene chloride contains 6.5 g of N, N -bis-t-2,3-epoxypropyl) -ethionine^;and / 3 g of&gt; trietrleîltetrhîhi.nă 'sequence: -50 / gedintr a silica given has: fo composition / 40'1100 grit - specific rhco sripraphy of 37 m²/ G ,. diameftrri hours: of porlloî - of • 1 100 Â. and · un -volhm. . &gt; .al ·. .for the pores. 1705 ml / g. Chloride chloride is then evaporated to teiriphraturia-, the surrounding environment, silica. impregnated is: -heated .43-450 ^ times 60: hm to obtain 'cross-linking' / is washed with boiled water. after that cry - acetone. The resin-shifting resin obtained is composed of coated silica: «di a cross-linked polymer, which -includes fused -CH-N-CH-zb- and burns the following-: -. 7 ..- 1 .. '.- ν u,, ::., ι.η, ο. . .Cil -.......

r'ele fea tures: ,, _sj, ..

- the colleague Jn:, carbon 7 ',' 7_; . ,..0,8¼ ;

. "Cheek-mole azol.,. 2,4¼; ... "- p the quantity of.,, .., 7, +. polymer -,. fixai, .3, -3. mg / m²;

disability. 7 and exchange .. ... .1 mval / g.

Gamma-globulin separation. .

10. p from the resin .seTuimbatbare.xthe ions. obtained, spt ₍ introduction Jiitr-p Column with a diameter of 1 cm and are compressed. The resin is adjusted with._chydrochloric acid 0.1 n, then with 0.02 mol phosphate buffer solution, at pH = 6.5. 20 ml of a 1% by weight solution of human serum (blood serum) delipidated and lyophilized, in the same phosphate buffer solution, with a flow rate of 100 ml / h, is percolated. The resin is then washed with 50 ml of the same phosphate buffer solution. The solution coming out of the column contains in pure electrophoretic state the gamma-gulin in the starting solution. .

The other proteins: al-u-globulin, befa-globulin and albumin, also present in the initial solution, are fixed on the resin. They are recovered by elution, using a 3 mol NaCl solution, in the same phosphate buffer solution. In case the elution is performed with buffers with increasing ionic strength, increasing the concentration of NaCl, enriched solutions are obtained in affa-globulins, befa-globulins and albumin.

Example 6. Preparation of the resin It is worked as in Example 5, but with a silica having a granulometric composition. from 100 to 200 - m and with 6.5 g of N, N-bis- (2,3-epoxy-propyl) -butylamine, instead of 6.5 g of Ν, Ν-bîs (2.3-epoxy- propyl) - ethylamine. The obtained ion exchange resin consists of silica coated with a cross-linked polymer, which comprises functional groups - CH? - N - CHz - and has urI

CAL · the following characteristics:

- carbon content - nitrogen content - amount of polymer fixed - exchange capacity

9,2% ;

. 2,5%:

3.2 mg / m⁵ ; 1.1 mval / g.

Extraction of protein g from the obtained ion exchange resin is introduced into a column with a diameter of 1 cm and tablets are maintained. The resin is treated successively with 0.1 n hydrochloric acid, then with 0.01 mol phosphate buffer solution at ρΉ = 7.5. At a flow rate of 80 ml / h, 30 ml of a solution of 1% by weight, in the same solution of ammonium phosphate, whey powder (I'actozeir) ultrafiltered, containing 75% is percolated. by weight protein. The resin is then washed with 100 ml of the same phosphate buffer solution. The solutions coming out of the column include fat and lactose, existing in the solution from which it started. Proteins, lactalbumin, lactoglobulin, serum albumin and a small_:parts of, immunoglobulins, existing in. şoliiţia. dc, at which it was started, is fixed on the resin, By elution, using a 0.05 mol buffer solution, at pH = 4, the separated and purified proteins are recovered.

₅ Example 7. Extraction of g proteins from an ion exchange resin similar to that of example 1, but whose particle size is 200 ... 500 · m, are introduced into a column, with a diameter of 2.5 cm and ¹⁰ they are kept, tablets. The resin is treated successively with 0.1 n hydrochloric acid, apo in the 0.01 mol hydrochloric trisacid buffer solution, at pH = 7. It is percolated, with a flow rate of 300 ml / h,

600 ml of a solution consisting of 300 mol of the same solution iris-hydrochloric acid and 300 ml of whey delipidated with 0.5% by weight soluble proteins. Then the resin is washed with 100

2Θ ml of the same buffer solution. The solutions coming out of the column contain lactose existing in the solution from which it was started. Proteins: lactalbumin, lactoglobulin, serum albumin and a small portion of the immunoglobulin present in the solution from which they are originated are fixed on, - the resin. They are eluted by passing through a column solution of 0.1 mol sodium citrate buffer at pH = 7. The solution obtained con30 contains all the proteins.fixed, with a concentration of 4% by weight.

The operation · allowed to obtain a mixture of pure proteins, free of lactose, in a much more concentrated solution since those days that started. After 30 successive operations, there is no aging of the resin.

Example 8. Treatment of a solution of pepsin g from an ion exchange resin, similar to that of Example 1, is introduced into a. column with the diameter of the cm Resin is washed with

1 00 ml of distilled water, then 150 ml of a crude pepsin solution with 20 units of pepsin / ml, with a flow rate of 100 ml / h, is percolated. The resin is washed that dune with 20 ml of distilled water.

The solution coming out of the column and the washing water has no activity; all pepsin is fixed to the resin. · Impurities remained in solution, as evidenced by the research carried out on the dry extract. The fixed pepsin is then eluted by percolating a NaCl solution with a flow rate of 100 ml / h.

ml solution allows the recovery of pepsin in solution with 170 units of pepsin / ml. We note the important concentration of the solution obtained. The resin is reused after washing with 50 ml of distilled water. After 10 operations. successively, there is no aging, a

65- resins.

t

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Example 9. Preparation of ion exchange resin

100 g of silica, with a particle size of 100 to 200 &lt;nț ,. a specific surface of 25 m '/ g ·, an average pore diameter of 1,400 Â and a volume, of the pore of 1.1 ml 7g are impregnated with a solution of 200 ml of methylene chloride, 24, cp of acid acrylic, 6 g, diethylene glycol dimethacrylate and 0.4 g benzoyl peroxide,

The methylene chloride is evaporated at ambient temperature and at • atmospheric pressure, until the weight is constant afterwards, the impregnated silica is heated to 80 ° C for 6 h to obtain the polymerization. The silica is then suspended in 30Θ ml of water and heated to boiling for 1 h. After filtration, the silica is washed with acetone, then dried under vacuum at 80 ° C. S «obtain- an ion exchange resin-, as-comprising functional groups - CQQH and which have the following characteristics:

• - carbon content. 10.55%;

- the amount of polymer fixed, 7.2 mg / m²;

- exchange capacity. . . l, 05 mval / g.

The treatment of a solution. Of lysgzma dO g φη the resin-exchange resin obtained is inserted into a column i the diameter of 1 cm and the resin is kept compressed, then it is brought into shape? of? NHH by percolation of 2 1 of an aqueous solution of 0.5 mol ammonium asphaltene, buffered to pH = 8.2. The resin is then adjusted to pH = 6.5 with- 100 ml of buffer solution / s-maleic acid &amp;, G2 mol. It is percolated - a lysozyme solution t% by weight in the same buffer, with the flow rate of 180 mf / h, until the column saturation, which corresponds to approximately 200 ml of the resin solution and is then washed with 100 ml of the buffer solution / zis-malic acid 0.02 mol, pH = 8.2.

The fixed lip is then eluted by quenching with a molar solution of NaCl · in. the same buffer solution, with a flow rate of 180 ml / h, 50 ml of solution allows to recover lysozyme and a solution of 2.5% by weight. Ppe ppAîe choir, chidje that. the ion exchanger has a lysozyme capacity. 125 mg / g and ca pi; allowed the concentration of lysozyme solution.

Example 10, Preparation of the ionic resin resin) · 'FOO cj .dg alloys with a' granutotairical position '^anddte '100 /./ 200 /' ni, '6 specific surface of 37 m²/ g, an average pore diameter of 200 Â and n volume, of 0.95 ml / g pores are impregnated with a solution of 150 · ml of methylene chloride, 60 ml of distilled styrene, 20 m of vinylthytriloxysi lane and 0.5 g of azo-bis-isobutyronitrile, methylene chloride, is evaporated at ambient temperature and atmospheric pressure, up to constant weight; In this case, the impregnated silica is heated at 2 ° C for 6 hours to obtain the reticulation. The silica is then suspended in 30.0 ml of xylm and heated to a boil for 6 hours. After drying, the silica is washed with acetone, then dried at 80 ° C.

g of the modified silica obtained is suspended in 500 ml of chloroform and 50 g of HSO; C1, in a solution of 50 ml of chloroform, is added dropwise. Hydrochloric acid release occurs. After the addition is complete, the stirred mixture is heated at 50 ° C for 4 hours. After drying, I wash water until neutral, then with acetone and dry under vacuum at 80 ° C. se · oEțțțîto o - ion exchange resin ·. cane i'oiiinct /, 'functional groups - SO.iH and seems to have the following characteristics: - ^rcarbon nut. , 4%;

- sulfur content, , J., 4% „- quantity of polymer fixed. . 2.8 mg / m²;

- exchange capacity. . . 0.43 mval / g.

Hemoglobin extraction of 00 g of the obtained ion exchange resin is introduced into a column with a diameter of 1 cm, after which the resin is adjusted to H&gt; f, 5 with a phosphate buffer solution Q "02 mol. Percolate.SCLQ ml of a hemoglobin solution of 0.2% by weight in. same buffer solution with a flow rate. 100 ml / h. Hemoglobin is absorbed on the tune exchanger. The 'colorless' solution does not contain hemoglobin and it does. purified. Axial adsorbent hemoglobin eluted by percussion of a 0.5 mol solution of ammonium carbonate; that dip, the column, is stripped, successively, with. 30, (1 mj ds. Sodium hydroxide solution. P, -in and gu. 50 · ml of

a.cid cyprlhidric 1 n, it is reached by the meetings' • &lt;has. , -_! ,/ / ·/ ...

The example. whey whey (iacfosernm) '' g from a tohi, / resin-like resin.

1;.^: shit ip.trqrfuse 'into it^! · Column; cp. ' di'a.m^!eițțuÎ / _db 2/5 cnț (c0ițțâiitia''jax.^bll) '.' '' iO '' 'g of an ion-exchange resin, similar to Example 9, was etched in a column with a diameter of 2.5 cm- {column no. 2), The two columns are serialized and the resins are washed with 5Θ ral of water.

500 'ml of whey, adjusted to pH = 7.5 by the addition of 0.1 n sodium hydroxide solution, is filtered to remove insoluble substances, then pierced in the ar column. 1 and in, column no, 2, with a flow rate of 300 ml / h. The protein precipitate sample using trichloroacetic acid proves that whey comes out of the no. 2 no longer contains protein. The resins of the two columns are washed by: passing 100 ml of water.

Proteins: lactalbumin, laetoglobulin, albumin. din zer (serum albumin). and a very small part of the immunoglobu.lin.e, existing in the solution of the one that started, are fixed on the resin of column no. 1, They are eluted as described in Example 7.

The proteins fixed on the resin-column no. 2 are tn. especially the immunoglobulins, which have not been fixed to the resin of the column '' no./1 / They ·, are eluted-by the percolation 'of a solution we offer · -of' which is banned by ammonium. The solution-obtained contains an oglum immunity to a bulletin in a concentration of about 3¾ by weight. Immuneglobulins make up about 1-6% by weight of all the proteins in the die solution on which you started. After the avenue, the columns are washed by passing 500 ml of water, before being reused. After 10 operations' successively ', no aging of the resins is observed.' . Bxena ^ '·' ^) 3_:,_: ExtMition · proieinelex from beer, &lt;·. ,, g; from G: ion-resin resin; like the mist from example 9t they are inserted into a column with a diameter of one meter! 2.5 cm, then it is washed with 250 ml of water. 3 1 unlimited beer steit perks cp. a flow rate of 100 m, l / h. The thirst that comes out; down the column nju. more precipitate; by addition of piuric acid. .and presents, - the characteristics of a cleared drink. ρ,

The products · fixed - on the resin are holy. are you special in protein; They are pissed through the broom; 0, 400, ml de- acijdl, ejostoioxic n / 1.0 ·,: -S- ;; ··; , - .- ·_r. .

The resin is washed PX in the passage of 250 ml of water, "reusable bits"

EXAMPLE-13, Preparation of the resin shakerib ^ .ap ^ p.ippi, ^ .. ... /. . ,.,.3,,,-,

, 1.00 g, de'aluminum, with a particle size superpâfâi ^ specîÂ'câ '^0r of d i \ 5f ¥ ^ n® ^ 'W⁵ HSA-mejW¹¹ forPP * by ώ

500 And a porous volume of 0, 9 ml / g, peak · dried at t50 ° C under reduced pressure, for 5 hours. The obtained dry alumina · - is introduced into a solution of - 250 ml dc chloride, of upethylene, 60- rqj, d ^. distilled styrene 20. ml of Y.intltri.ethoxysilane and Q, 5 g azo-bisizQ, butyr.onitrile. Methylene chloride is evaporated at room temperature. ambient, and the diurnal toads pa-, impregnated is- Heated Ia, ÎȘ-OÎ'C tămș.p. from -Q h, sub. a 3i bar bullshit, to get it, is the reticulation '. In eontihua, i; e, alumina / fish brought in, suspension in 300 mt of xylene and heated, boil time. After 2 hours, After filtration, the alumina is washed with acetone, then dried, 50 g of the obtained alumina are suspended in suspension. 180 g of elorethyl ether, containing 6. g of tin chloride, then the mixture is heated under reflux for 4 hours in anhydrous medium. After the cold. alumina is dried, washed with 200 ml of one. mixture ch.oxapapa 50/50, concipiid, 10 ml of hydrochloric acid, then with water, up to. pH neutral and · ,,, finally. dry. The obtained product is brought to a suspension in 150 ml of a 30% aqueous solution. of trimethylamine. and I would leave it in contact for 8 days at room temperature. ambience ,. After drying and washing; yes, get an ion exchange r'așiha with functional groups:

- - - in cm which 'possesses the following' characteristics:

- carbon ..... 8.2¾ .;

- chlorine. 3; 7%;

- nitrogen. . Ί, 5%;

- quantity of flxed polymer 1. 1105 mg / m²;

- for a change of mind. . . 1.1 'mval / g.

Treatment of a solution of albumin --10.- g of resin · scbw-hăIore of obtained ions - scabbard placed © in it, column I diameter of -1 pm sr. mention and sub- pressure, 'then the resin is. pH-balanced = 6.5 with a 0.01 M phosphate buffer solution. A 1% albumin solution is brewed in the same droplets as the buffer solution I · 60 ml / h, until saturation · eftlosarae · 'eed eoresp.un.dfe; Take (•• iipcu 140. ml of: the solution Apoii resin is * ·. Your wash with^;lWml from azeea and buffer solution. Fixed albumin 'is ·' then taken; for the surrender.¹ a solution of 'acid · ctorliidrin 0; t · N; with a flow rate of 60hml- / h. 257iil''soluțî &amp; was about to recover the albumin in dn'i4 © / Ch in / 8229 solution and 5 weight. It is concluded that the exchanger has: a. -Capacity - in albumin of) 100 mg / g and. Which allowed the concentration of the albumin solution.

Example 14. The same conditions are repeated with 100 g of albumin having a particle size of 5 to 50 m, a specific surface of 110 m²/ g, an average porous diameter of 750 Â and a porous volume of 1.1 m / g. 30 ml of 4,5¼ weight albumin solution are obtained. The exchanger has an albumin capacity of 135 mg / g and allowed the albumin solution to be concentrated.

As is apparent from the examples, the process of protein separation according to the invention consists in contacting a protein solution with an ion exchange resin, and is characterized in that the ion exchanger is constituted by a porous mineral support, having a gnanulometric composition between 4 - m and 5 mm. a specific surface of the order of 5 ,, 150 m²/ g, a pore diameter of 500 jp. 2 500 Â, a pore volume of 0.4 up to 2 m] / q, covered with a smaller amount. than 15 mg / m² dinir-ο (the film Feticulated polymer He, which contains or 'pointer, either groups of Jde fines' tions represented first tertiary amines or quaternary ammonium salts,. be cation exchange groups, represented by acid functions, and possess a exchange capacity less than 2 mval / g. '.

As a porous mineral support, metal oxides, such as titanium oxide, alumina and, in particular, silica, are used. These supports have average pore diameters of 500 to 2,500 A and; preferably from 600 up to 1 500 Â, a specific area of ​​5 .. 150 m²/ G. and preferably up to 20. Almost 50 m^s/ g and a granulometric composition of 4 ··. m ... 5 mm, depending on the application.,. Thus, the smallest particles are used in analytical applications and the largest in preparation applications.

Functional groups, tertiary amines or quaternary ammonium salts are represented by the formulas: general: -CH? -N-CH? - or -CIî? -N (⁺) - (R), X (~) in ξ ¹ 'Λ.: ;; Î_:; · R. .

which R / -identical '.or different,; represents an alkyl group giving hydroxyalkyl, having 1 to 4 carbon atoms, and X represents &lt;ηη: mineral or organic anion, such as, for example, chloride, sulfate ,. nitrogen, phosphate, citrate._r ,.

: Carboxylic acid and functional groups? Sulfonic or zephosphonic. Correspond to the general formulas: '—COOH,. -riSChH, ^ PQ (&lt;3H) aț5 'mirio? ,, · ni ic-pr'U'fO. ^ np-,

These functional groups are part of the cross-linked polymer chain or are attached to the cross-linked polymer that covers the entire surface of the support. Cross-linked polymers that cover the surface of the mineral support are known products_;, itself, obtained according to any conventional polymerization process. These polymers are prepared starting from monomers capable of cross-linking - either alone, or with another monomer, possibly the presence of a catalyst. Among the same monomers can be mentioned: epoxy compounds which cross-link with polyamines, as catalysts, monomers that cross-link polycondensation without formaldehyde catalyst, such as urea, melamine, polyamines, phenols, vinyl monomers, for example. vinylpyridine, styrene and its derivatives, acrylic, methacrylic, vinylbenzoic acids, which cross-link with polyfunctional monomers, such as diac, Hat or dimethylacrylate of mono- or polyalkylene glycol, divinylbenzene, vinyltrialcoxysilane, tri-vinyl amine, genyl o-triyl , in the presence of an initiator, who puts in: freedom of the radicals, free¹!, such as organic peroxides and azonitriles, or in the presence of ultraviolet rays. .To get the coverage_; mineral support with cross-linked polymer. the support is impregnated with a solution .a, 'monomer .or monomers and possibly the catalyst in a single-solver. which allows a good distribution of the monomers, on the entire surface of the support, mineral, the solvent is then evaporated and the monomers are cross-linked. according to the known processes. As a solvent, any of the products that dissolve the monomers and the catalyst, whose boiling point is preferably as small as possible, is used to favor its subsequent evaporation. These are, for example, methylene chloride, ethyl ether, benzene, acetone, ethyl acetate. '' '' '' if the polymer · 'cross-linked to the surface of the mineral support does not have in its chain groups¹ functional, as defined above, it needs to be modified; it is, in particular, the case of polymers cross-linked with styrene and derivatives, of the formaldehyde polymers with urea, melamine, polyamines, phenols. This modification, in the case of polymers of styrene or phenolformaldehyde, consists in fixing to the polymer, either carboxylic, sulfuric, phsphonic groups, according to any known process, or groups of 'chlormeityl', which are then left to react with -o · '7 ^ 29 animates and they fail. tertiary,. r ^ ety executed according to any known method ... '' ',

To re-attach the clormetft groups to the polymer, it is ai / ante] os, in the case of polyiriylcircrylate Î,% ă It is completed) i&gt;^fer'seiie Șftportul'mineral acripdrțt;^!'cu · t ^ oiiAer in eieV Vlorm ^ Hlfc,' lâ hâM, in prezehțg 'unlif' âcîâ LÎewife '. DiinpotrÎvă / ·· fell ihhhi razșihî'-feTÎhiforhihlțîelÎidÎce one can, for example, disperse sujidrtu1 jhr'rihrăl crushed with po! initiate in the epicTdrhl · · cirin and execute the reaction to warm. This modification, in the case of the polymers of the forhydedehyde of the polyamines, as the urea with melamine consisted in 'transforming the amines; exidlent priiri'are in the chain, animates tertiary salts tertiary salts He ammonium cationic acid, honferih of any classical method, of exehiph, reaction -fcu uhslilfat or with an alkyl halide.

In the cover operation,; a. .for mineral support, the amount of monoraer (s) used must be such that the quantity of crosslinked polymer comprising functional groups distributed over the surface of the mineral carrier is less than 15 and preferably between 1 and 8 mg / ml Mineral substrates coated with crosslinked polymers comprising functional groups thus obtained have an exchange capacity of less than 2 mva / g and, preferably, between 0.3 and 1.2 mval.

The process according to the invention is applied to all proteins which are soluble in aqueous medium, whatever their isoelectric point. Among these proteins, polypeptides and enzymes are included. they can be cited among others: albumin, lactialbumin, ovalbumin. albumin from the set⁴ (serum albumin), hemoglobin, α, β and γ -globulins, lactoglobulins, fibrinogen, urea, trypsychrome.

The process, according to the invention, allows the proteins to be easily separated from their solutions, such as whey, beer, blood, organic extracts and from all industrial effluents: wastewater from slaughterhouses, food industries, starch factories of potatoes, thus constituting a means of treatment of the so-called effluents, therefore, a means of combating pollution. The separation is obtained by contacting, at a temperature, ionic strength and pH comparable to the protein or proteins, of the solution to be treated with the ion exchange resin, chosen according to the separation conditions. Then, fixing on

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1¾ 'profeifee' bdftțiriute '' in solftți'e, 'f'i'e -â of all the proteins in the solution. 'Sgpârârha ή'όΐίίε fi, by^;'' Asbmeneăpoobțiîuta hh. .achTeHși ceindi ^ iiiîh; by 'the lust of the devotion' - of thâtâitY in^: indd successively, with one or more nhllte sthîmbătdW resins; - of aniohi and / saii of kaiihhî; SW / product of the initial selective fixation of the solution; on each river. In 'case the protein sought is lixatrl pp a râ.şhlă, receiving from it' 0- Solution, pro'Îhbșă or nh, it is apbi '' split RrinAfeluaite, - with the help of a solution that has a pH and / or or. an ionic strength different from those of the fixation solution, but still co-compatible with the protein. In this way it is obtained, not only the separation of the protein from. solution, but at the same time purifying and concentrating it 20: it is thus possible, for example, to separate and concentrate the albumin, cucumber, yogurt whey proteins, with the help of an anionic resin, and the lysozyrraa'ciî ajtiidfhl uhei resins. cation.

If the protein followed remains in the protein solution treated according to the invention and the other proteins of the mixture feel fixed, the separation of the protein followed by the other proteins is obtained, so its purification. Elution of fixed proteins leads to selective or not separation of proteins and their concentration. This is the case, inter alia, of the gamma-globulin with an anionic resin.

If more of the targeted proteins are attached at the same time to a resin, elution at pH and / or ionic strength different from those of the fixation solution produces a separation of the proteins from the solution and their concentration. Elution using solutions with increasing pH and / or ionic strength, along with selective separation, purification and concentration₄- iteration. This is the case, especially in the human serum. In case the proteins have to be eliminated, they are fixed on a resin, starting from the other solution. In this way, solutions are obtained₅₀^r'photo: here ·, in other words purified. Elution of the fixed proteins allows the reuse of the resin. This is especially the case with the clarification of beer and the treatment of solutions that. contain hemoglobin, with the help of cationic resins.

The separation can be performed with identical results, in a continuous or continuous process, in continuous process operations, the resins allow easy loading of the column, a high flow rate and an easy elution. The results obtained are practically independent of the concentration of the treated solution, depending on 'nature

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Ιό lys.; Earq.se treats.and of, the solution, elution. . , - /. CJ-ir. ·

The process, [... according to the invention, may be; applied - in the food industries, in particular; in the diet,. 5 and veterinary. ,_; _ ,

The process according to the invention presents as advantages the use of exchange resins with good mechanical properties that are not. force-sensitive¹⁰nothing at pH. the environment, for use, are not biodegradable and can be sterilized and allow the production of very pure proteins.