CA1223859A - Surface-grafted particulate support, process for its preparation and adsorbants for affinity chromatography incorporating such support, and their use, particularly in biology - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A particulate support for the manufacture of adsorbants for affinity chromatography on a column, said support being essentially constituted by particles of a basic polymeric material adapted to be used in column chromatography and on which are grafted, essentially at the surface, molecules of at least one unsaturated monomer capable of forming a copolymer with the basic material and bearing at least one chemical function capable of forming, subsequently, under non-denaturing conditions, a covalent bond with the ligand used in the adsorption envisaged, the amount of grafted monomer possessing a chemical function accessible per unit of surface area (monomer assayed per unit of surface area) being from 10 to 500 µg per cm2 of surface area of the support and the ratio of the amount of grafted monomer possessing an accessible chemical function to the total amount of grafted monomer (assayed monomer/weighed monomer) being from 0.05 to 0.5. The support of the invention may be obtained by radiochemical grafting, using radiations emitted by electron accelerators or cobalt 60 sources. An adsorbant for affinity chromatog-raphy is obtained by covalent fixation on the support of a ligand specific of the substance to be extracted or eliminated, in particular from blood.

Description

122~35~3 Background of the Invention The invention relates to a support for adsorbants useful in affinity chromatograph~, particularly in the field of biology.
More precisely, the invention relates to a particu-late support, obtained from particles of an inert poly-meric material, which has at the surface chemical func-tions adapted to form covalent bonds with ligands useful in affinity chromatography, particularly for the sepa-ration or the elimination of biological substances.
It is also an object of the invention to provide a process for the preparation of this support.
It is in addition an object of the invention to provide adsorbants for affinity chromatography obtained by the covalent bonding of a suitable ligand to such support.
Finally, it is an object of the invention to pro-vide a method of using such adsorbants, particularly for the recovery or elimination of biological substances, particularly from blood.
The use of biofunctional element bearing inert supports fixed by covalent bonds is generalised and extends to a large number of biological and biomedical uses. In particular, it has already been proposed to use, in affinity chromatography, as adsorbants, inert supports bearing suitable ligands for the purification of biological substrates or the extraction of valuable biological substances from biological substrates such as, particularly hlood. The separating power of the adsorbant depends on the specificity of the ligand fixed to the support with respect to the substance to be eliminated or extracted. It is in this regard i2~3859 essential that the physical and chemical properties of the support should not have an unfavorable effect on the interaction between the ligand and the molecule to be fixed.
Taking into account these consid e r a tions, it has already been sought to exploit numerous supports having various properties.
Thus. there exists at present a large number of supports used in affinity chromatography. The majority are in the form of gels which differ according to the size of the meshes and the chemical composition of the macro-molecular chains which constitute them (cellulose, dextran, agarose, polyacrylamide, polystyrene, divinyl benzene...).
In addition, certain solid supports such as glass beads, ceramic, etc, holding a specific ligand, are used for the same purpose by exploiting the absorption phenomenon.
However, none of the supports proposed until now has shown itself satisfactory, in particular to form adsorbants useful in affinity column chromatography.
It has been possible to establish that the various drawbacks presented in affinity column chromatography by the supports of the prior art could be eliminated by resorting to a particulate support (presented in the form of powder or of beads according to the size of the particles), having good dimensional stability whilst possessing good chemical reactivity, practically limited to its surface, with respect to the specific ligand of the subs-tance, espec~ ally biological, to be recovered or removed.
Certainly numerous supports are already knownwhich in particulate form, have the required mechanical properties.
However, until now the problem of fixing the selected ligand on such a support has not been satisfactorily resolved.

~223~359 The invention contributes a satisfactory solution to this problem by providin~ a modified particulate support, which after covalent fixing of a suitable ligand, leads to the obtaining of an effective and stable adsorbant for affinity column chromatography.
This result is obtained by means of the particular structure of this support in which the "branches" are distributed on the surface and have a certain length, so that it is possible to fix molecules, particularly biological ones, by covalency whilst preserving their, if not native, at least, still functional structure, with an accessibility of their reactive chemical func-tions sufficient to react effectively with the compound to be separated by affinity chromatography on a column.
General Description of the Invention More precisely, according to the invention there is provided a particulate support for the manufacture of adsorbants for affinity chromatography on a column, characterized in that it is essentially constituted by particles of a basic polymeric material adapted to be used in column chromatography and on which are grafted, essentially at the surface, molecules of at least one unsaturated monomer capable of forming a copolymer with the basic material and bearing at least one chemical function capable of forming, subsequently, under non-denaturating conditions, a covalent bond with the ligand used in the adsorption envisaged, the amount of grafted monomer possessing an accessible chemical function per unit of surface area (monomer assayed per unit of sur-face area) being from 10 to 500 ~g per cm2 of surface area of the support and the ratio of the amount of grafted monomer possessing an accessible chemical func-tion to the total amount of grafted monomer (assayed monomer/weighed monomer), being from 0.05 to 0.5.

- 4 - i223859 Preferably, the amount of monomer assayed per unit of surface area is 30 to 350 ~g per cm2 and the ratio assayed monomer/weighed monomer is 0.05 to 0.3.
By "particles of a basic polymeric material adapted to be used in column chromatography" is meant here parti-cles of material having particularly and simultaneously, in the state of particles, the following properties:
- dimensional stability under pressure, - crush resistance, - density higher than unity, - sufficient chemical and physico-chemical inertia under the conditions of use, - insolubility in the majority of solvents, par-ticularly in water and aqueous solutions, - low swelling ratio in water and aqueous solutions.
Among the materials which, in the particulate state, respond to this definition may be mentioned particularly:
polyvinyl chloride, polystyrene, poly-chloro-trifluoro-ethylene, polyamides and polyethylene, and their co-polymers having the above-indicated characteristics.
According to the size of these particles, the terms "powders or micro-beads" (size less than lmm) or "beads"
(practically spherical particles having diameters greater than 1 mm), will be used.
The "surface grafting" (bonding) of the unsaturated monomer leads to the more or less considerable "encapsu-lation" of the particle in a copolymeric layer of the basic material and of the one or more grafted unsaturated monomers, called below for simplification, "the monomer".
The grafted monomer must besides an unsaturation, for example a carbon-carbon double bond capable of forming, by grafting, a copolymer with the basic ma-terial, have at least one chemical function capable of subsequently forming a covalent bond with the ligand whose use is envisaged, and this under nondenaturating conditions for the ligand, ______A_-,r~
..

1223~359 that is to say conditions which do not largely modifythe aptitude of the ligand once fixed to react selectively with the substance to be adsorbed.
The grafted monomer comprises~ as chemical function corresponding to this definition, for example, and preferably, at least one chemical function selected from among:
- carboxylic acid functions activatable by carboiin~des, - amide functions activatable by glutaraldehyde, - amine functions activatable by glutaraldehyde, - aldehyde functions that it is not necessary to activate, - hydroxyl functions activatable by cyanogen brol~i~e.
As unsaturated monomers corresponding to the definition given above may be mentioned:
- acrylic acid - acrylamide - allylamine - acrolein - ethylene-glycol acrylate and - dimethylaminoethyl methacrylate.
The chemical functions accessible for subsequent covalent reaction of the grafted monomer may be assayed chemically by methods known to the technicians skilled in the art, if necessary after modification (hydrolysis of the amide functions into acid functions for example).
The number of accessible chemical functions thus de~termined by assay enables to calculate easily 30 from the molecular weight of the grafted monomer and knowing the surface area of the basic polymeric material, the weight of grafted monomer possessing an accessible chemical function (assayed monomer)per unit of surface area of the support before grafting.
3~ The total amount of monomer fixed (weighed monomer ) is the difference between the weight P1 of the support 6 ~223~3~9 after grafting and the weight Po of the basic polymeric material.
It is also an object of the invention to provide a process for the preparation of the particulate support according to the invention. This process uses, as the energy source, ionizing radiatior,s, in the absence of any chemical catalyst.
More precisely, the process according to the invention for the preparation of a grafted particulate support, defined previously, is characterised in that it consists essentially of effecting radio-chemical grafting, on particles of a 9 e 1 e c t e d b a s i c polymeric material, of the one or more unsaturated monomers chosen, in solution in a solvent non-aggressive with respect to the reagents or in the absence of solvent.
According to this process, recourse is had essentially to three types of known radiochemical grafting techniques, namely:
- grafting after pre-irradiation in the presence of oxygen, _ grafting after pre-irradiation in the absence of oxygen, - direct grafting under radiations.
The principle of each of these three techniques will here be recalled briefly.
1) Grafting after pre-irradiation in the presence of oxygen (grafting on peroxidized polymcrs):
This method consists of peroxidizing the basic polymer by irradiating it in the presence of oxygen and then carrying out the grafting in a second step in the course of which the peroxides of the macromolecules formed are decomposed, particularly by heat, in the presence of the monomer to be grafted.

122385~

2) Grafting after re~irradiation in the absence P
of oxygen:
This method consists in irradiating, in a first step, a polymer in a vitreous or crystalline state.
5 The macroradicals formed have a very low mobility and om e of them remain "trapped" without recombining.
In a second step, the monomer to be grafted is allowed to diffuse into the thus treated polymer; the macroradicals previously formed initiate the polymerization and a grafted copolymer is obtained.

3) Direct grafting under radiations:
This method consists in using the macroradicals formed directly on the radiolysis of the basic polymer to initiate the polymerization of the polymer to be grafted present in the medium at the moment of irradiation.
In all cases, the ionizing radiations are advan-tageously derived from electron accelerators or cobalt 60 sources.
According to the invention, it is established that particularly satisfactory results are obtained when the grafting is carried out:
- on basic polymer particles whose size is in the range of about 10 to about 5000~1;
- in a solvent such as water, acetone or ethanol, pure or in admixture;
- at a concentration of the monomer in the solvent of 10 to 80% by weight;
- for a period of 1 to 6.5 hours;
- at a temperature i n t h e ra n g e f r o m room temperature to the boiling temperature of the solvent used; and - with a radiation dose comprised in the range of 0.3 to 10 Mrad.
According to a preferred embodiment, the process according to the invention for the preparation of the gra~ted particulate support, defined previously, is hence characterized in that it consists essentially in carrying ~ .

i2~3~359 out the radiochemical grafting, on particles of a selected basic polymeric material w h o s e s i z e i s i n t h e range of about 10 to about 5000 ~, of the selected unsatur-ated monomer, in solution at a concentration of 10 to 80% by weight, in water, acetone or ethanol, pure or in admixture, for a period of 1 to 6.5 hours, at a tempera-ture i n th e r a n g e f r o m room temperature to the boiling temperature of the solvent used, with a radiation dose comprised in the range of 0.3 to 10 Mrad.
The surface grafted particulate support according to the invention, obtained as has just been described, or according to any other method, is stable and can be preserved as such.
This grafted particulate support is advantageously used for preparing adsorbants for chromotography, parti-cularly in the field of biolo~y.
In this field, for a certain number of years already, intensive research has been carried with a view to perfecting devices enabling the extraction or elimina-tion, sa fe ly and efficiently, of different substancesfrom biological materials, in particular from blood.
To do this, one of the most effective means contemplated hitherto consisted in adsorbing the substance to be extracted or to be removed on an adsorbant provided with a substance adapted to react specifically with said substance to be extracted or removed.
Thus~ it is possible, for example, to remove from blood, in vitro or by extracorporal circulation ex vivo, an antibody, an antigen or an enzyme substrate by causing the blood to be purified to pass over an adsorb-ant provided respectively with the corresponding antigen, antibody or enzyme.
` This method can also be used, particularly in vitro, to recover from blood desired biological consti-tuents, particularly in therapeutics, such as anti~ensor antibodies.

12238~i9 The surface grafted particulate support according to the invention is particularly well suited to the prepara-tion of such adsorbants.
In fact, such a support has at its surface a 5 large number of reactive sites with respect particularly to the ligands of biological substances. ~ince one can, by varying particularly the size of the particles, the nature of the grafted monomer and the length of the branches by means of a suitable choice of grafting conditions, 10 regulate on this support the distance, the density and the distribution of the reactive sites, it is possible to fix thereto by covalence, a very large number of ligands, in particular ligands specific of biological substances.
The covalent fixing of the ligand to the particu-15 late support takes place by known methods, such as described, forexample, in Immunochemistry 1, 219-229, (1964), J. of Immunological Methods 11, 129-133 (1976) and Science 195, 302 (1977), after activation of the reactive sites of the support. It is recalled in this respect that 20 the activation of the acid functions can be done by means of carbodiimides and that of the amide and amine functions by means of glutaraldehyde.
It has in addition been established according to the invention that the reactive sites are rendered 25 more accessible for the covalent fixation of the ligand when a hydration of the support is carried out, prior to activation. I~ence, according to the invention, prefera-ably the support is hydrated before proceeding with its activation,and then with the covalent fixation of the li~and.
30 ~ The duration of hydration depends essentially on the structure and the composition of the grafted particu-late support.
This time can be determined easily by the techni-cian skilled in the art, for each type of support.
35 For example, in the case of a particulate support grafted with acrylic acid or with acrylamide after hydrolysis of the amide functions into acid functions, it is possible :;1223~59 to determine the optimum hydration time (at the end of which the maximum of acid functions are rendered accessible) by following the p~l variations of a sodium hydroxide solution in which a sample of the grafted support is suspended.
In any event, it was possible to establish that according to the invention a prior hydration time of 24 hours was sufficient to render all the useful functions accessible. At this stage the swelling ratio, by volume, of the support was at a maximum of 30 % the most often of the order of 10 %, and the support preserved all its desired mechanical characteristics for adsorption chromatographyonacol~.
It is therefore in addition an object of the invention to provide an adsorbant for affinity ch.romo-tography, characterized in that it results from the covalentfixation of the specific ligand of the substance to be adsorbed, to the particulate support according to the invention.
According to another aspect of the invention there is provided a process for the preparation of this adsorbant for affinity chromotography, characterized in that it consists essentially in fixing by covalence the specific ligand of the substance to be adsorbed, to the support after activation of the latter, preceded preferably by hydration.
Finally, according to another aspect of the invention there is provided a method of using such an adsorbant, particularly in the field of biology, and especially to extract or eliminate a biological substance from the blood, in vitro or by extracorporal circulation ex vivo.
By way of examples, it will be mentioned in this respect that:
- for extracting or eliminating an antigen from the blood, there will be used advantageously an adsorbant according to the invention in ~Yhi.ch the ligand is the correspondin~ antibody, for example monoclonal;

~2238~;i9 - to extract or remove an antibody from the blood, there will be used advantageously an adsorbant according to the invention in which the ligand is the corresponding antigen; and - to remove an enzyme substrate from the blood, there will advantageously be used an adsorbant according to the invention in which the ligand is the corresponding enzyme.
It is self evident that to treat complex media such as blood, the technician skilled in the art will have to select, on the basis of his general knowledge in the field, from among the numerous particulate supports according to the invention, those which are compatible not only with the ligand to be fixed, but also with the complex medium to be treated with which they must not give interfering side reactions, in particular in the case of the purification of the blood by "ex vivo" extra-corporal circulation.
Description of Preferred Embodiments The invention will be better understood by means of the following non-limiting examples, given purely by way of illustration.
I Examples of the Preparation of Supports Example 1 Polyvinyl chloride powder grafted with acryla-mide, after irradiation in the absence of oxygen.
The basic support was a polyvinyl chloride powder (marketed under the name PVC Lycovyl~ GB 1220) f ~
of which the granulometry was comprised between 100 and 30 200 microns. The acrylamide grafting was carried out in the absence of oxygen. The powder was first irradiated in a nitrogen atmosphere under gamma radiation. A dose of 0.57 Mrad was applied at a delivery rate of 0.l9 Mrad/hr.
The powder was then contacted with the monomer 35 in solution in a water-acetone mixture (3:5) containing 15% of acrylamide for lh30 at ambiant temperature, still shielded from oxygen and with shaking.

12 1223~359 The grafted powder was then washed in a water-acetone mixture (3:5) and dried.
The graftin~ by weight which is defined as being equal to x 100 (Pl b ei ng t h e final weight S Po and P0 the initial weight) is 12.7%.
The hydrolysis of the accessible arnide functions carried out in a 3N hydrochloric acid solution for 3 hours under reflux enabled the carboxylic acid functions 1~ obtained to be then estimated and to be determined with sodium hy~roxide. In this way the determination led to an evaluation of 2.35% (by weigllt) of chains derived from acrylamide having an active site.
example 2 Polyvinyl chloride powder grafted with acryla-mlde, after i,rradiation in the absence of oxygen.
The basic support was the same as in Example 1. The acrylamide grafting was carried out according to the same process and under the same irradiation condi-tions as in Exarnple 1. The grafting solution contained 20% of acrylamide and the grafting at ambiant temperature lasted 1 hour. The washed and dried powder was thus grafted to 24% by weight and 5% of active polyacrylamide could be determined.
Example 3 Polyvinyl chloride pos~der grafted with acrylic acid, after irradiation in the absence of oxygen.
The basic support was the same as in Example 1'. The acrylic acid grafting was effected in a nitrogen atmosphere. The irradiation was done in a first stage, a dose of o.6 ~1rad being applied under electrons. Thc grafting was then carried out by shaking the powder, still in a nitrogen at-nosphere, suspended in a solution containing 10% of acrylic acid in water in the presence 35 of 0.15% of inhibitor (~lohr salt) for 1 hour at 85 2C.

13 ~223~59 T~e ~raftir~ was evaluated at 3.5% by determining the carboxylic acid functions with sodium hydroxide.
Example 4 Polyvinyl chloride powder grafted with allylamine~
S after irradiation in the absence of oxygen.
The basic support was the same as in Example 1. The irradiation and the allylamine grafting were carried out in the absence of oxygen, under vacuum. The powder waff pre-irradiated under electrons having an energy of 3 MeV. The applied dosewas 5 Mrad. The grafting was carried Ollt by leavirlg the powder in contact with an allylamine 601ution at a concentration of 50% in ethanol, with stir-rin~, for 1h at 40 - 2C. The washings were carried out in ethanol.
The ~afting measured by weight was6%.
Example 5 Polystyrene beads ~rafted with acrylic acid, after irradiation in the presence of oxygen.
-The support, in the form of beads of diameter 3.2 mm, was constituted by polystyrene. It was peroxidized by irradiation in air, under ~ radiations. A dose of 7.5 Mrad was applied at a flow rate of 0.44 Mrad/hour.
The support was then stirred in a solution containing by weight 79.6% of acrylic acid, 20% of water, 0.4% of 25 Mohr salt, for 6h30, at 87 - 2C, under a nitrogen atmos-phere.
The grafting ratio by weight was 2.8%; it was evaluated, by titration, at 0.29%.
Example 6 r 3 Polystyrene beads Prafted with acrylamide, after irradiation in the presenee of oxygen.
Polystyrene beads of 3.2 mm diameter ~ere grafted with acrylamide under the following conditions;
The peroxidation of the support was carried out by irradiation, in air, under electrons (3 MeV) with a dose of 7.5 ~Irad.

14 ~223~59 After 24 hours, the beads were plunged into a solution containing 59.4~ acrylamide, 40% of water, and o.6% of Mohr salt, by weight, into which nitrogen was bubbled. The temperature of the solution was 92 + 2OC, -the graftin~time was 6h30.
The ratio of grafting by weight was 0.45%.
Example 7 Polystyrene beads ~rafte~ with butyl acrylate and acrylic acid, after irradiation in the presence of lo oxygen.
Polystyrene beads of 3.2 mm diamcter were grafted simultaneously, under the same irradiation conditions as those d~scribed in Exarnple 6, with acrylic acid and butyl acrylate to increase the hydropllile/hydrophobe balance.
The grafting was carried out in a solution contain-ing, by weight, 5% of butyl acrylate, 74.6% of acrylic acid, 20% of water and 0.4% of ~lohr salt, at 87 + 2C
for lh45.
The ratio of ~rafting by weight was 2.4%.
~xasnple 8 Polystyrene beads grafted with acrylic acid, after irradiation in the ~resence of oxy~en.
. , Polystyrene beads of 3.2 mm diameter were grafted with acrylic acid. The method used was peroxidation, irradiation of the support being carried out under a beam of electrons (3 ~leV), in air, wi-th a dose of 7.5 Mrad.
After 24 hours, the beads, plunged into a solution containing by weight 79.6% of acrylic acid, 20% of water and 0.4% of ~lohr salt, were stirred for 4 hours at 87 + 2C,while maintaining in the solution a stream of nitrogen bubbles.
The grafting by weight was 2.6% and the grafting measured by titration of the acid functions was 0.12%.

1 5 ~2231~159 Example 9 Polyvinyl chloride-vinyl acetate copolymer powder grafted with acrylic acid, after irracliation in the presence of oxygen.
The basic support was a copolymer, in the form of microbeads, of polyvinyl chloride containing 10% of ~ vinyl acetate (marketed under the name LucovylW SA 6001), 5-$~ of which the granulometry was comprised between 100 and 200 microns.
The ~aftir1F of the acrylic acid was carried out after irradiation, in air, of the powder, under~
r a d i ations,at a dose of 0.3 ~1rad dispensed in 1 hour.
Thegrafting solution, from which the oxygen was removed by the bubbling of nitrogen, contained, by weight, 9.95%
of acrylic acid and 90% of water, in the presence of 0.05% of ~1ohr salt. It was heated to 80 - 2C for the graftirlg time of 1 hour.
The ratio of ~rafting by weight, measured by weight diffcrence, after washing and dryin~ was 3.6%.
Titration of tl-e carboxylic acid functions corresponded to 1.5% of grafting.
~xample 10 Powder of chlorotrif]uoroethylene and vinylidcne fluoride cor)olymer grafted with acrylic acic1, after irradia-tion in the absence of oxygen.
A basic support constituted by a copolymer ofchlorotrifluoroethyle~e and vinylidene fluoride (marketed under the name Kel-F of granulometry comprised between RO and 160 microns, was grafted with acrylic acid under the following conditions: pre-irradiatio11 of the support was done in the a~sence- of oxygen, under vacuum, under electrons (3 ~1eV) with a dose of o.87 ~1rad. The grafting was then carried out, under vacuum, a-t 90 ~ 2C for lhlO
in contact with a solution of 20% acrylic acid in water in the presence of 0.2% of ~1ohr salt.
The acid functions titrated correspon~d to titrata~le grafting ratio of 8.9%.

1~ ' Examl)le 11 Po~der of polyvinyl chloride and ~inyl acetate copolymer ~raftecl under vacuulTl with acrolein, after irradia-tion Wit}l an clectron bcam.
The basicsupport was a powder of a copolymer of polyvinyl chloride-vinyl acetate containing 10% of vinyl acetate, wllose granulollletry was comprised between 100 and 200~ .
The acrolein grafting was carried out under vacuum, after double dc-gassing. The pow(!er was irradiated by passage under an electron beam (energS~: 3 ~leV; intensity: 40n ~A) at a ~s)se of 2.05 ~Irad. It was then contacted with an acrolein solution composed of:
50% acrolein 25% ethanol 25% water for 17 hours at ambiant temperature. The ~rafted powder was washed with ethanol.
The grafting was evaluated by weight at 5.3%
and by titration of the aldehyde functions at 1.5~.
Example 12 Polyamide powder grafted with ethylene-61ycol acrylate, after irracliation under vacuum by an electron bcam.
The ~asic support was a polyamide 11 of granulo-25 metry comprised between 10 and 100 ~.
The grafted monomer ~as ethylene-glycol acrylate.
The g~afting was carried out on the pre-irradiated powder under a beam of electrons (E=3 ~leV, I=~00 ~) in the absence of oxygen, under vacuum, at a dose of 9 ~Irad, by placing 10 g of powder in suspension for 15 minutes at 75C in 100 ml of a solution containing 50% of ethylene-glycol acrylate in water, in the presence of 0.5% of ~lohr salt. The powder was wa~hed with water and dried.
The ratio of graftin~- by weight was 186%.

~2~59 E~alllple 13 Polyethylene powcier ~rafte~ with ekhylene-glycol acrylate, after irradiatiol- under vacuum by a beam of electrons.
Tlle basic support was a polyethylene (very high molecular weight) of granuloMetry comprised between 10 and 100~ .
The ~rafted monomer was ethylene-~lycol acrylate.
The grafting was carried out by a pre-irradiation method in tl-le abserlce of oxygen, un~cr vacuum. The polyethylene powdcr was irradiated under an electron beam (E=3 MeV, I=400 ~A) at a dose of 9 1~1rad. It was t}len contacted, still undcr vacuum, with a solution of 49.5% of ethylene-~~lycol acrylatc and 0.5% of ~1ohr salt in water, at 75C
for 90 minutes, in a proportion of 10 ml of solution per gram of pow(ler.
The grrafted powder was then washed with water and dried. The ra ti o of grafting by weight was 110%.
Example 14 Powder of polyvinyl chlorideand polyvinYl acetate copolymer ~rafted with dimetllylamino-etllyl methacrylate, aftcr pre-irradiation in the absence of oxy~en, with rays.
The basic support was a powder of polyvinyl cllloride arld ~olyvinyl acetate copolymer with 10% of polyvinyl acetate (marketed ~Indcr the name: Lucovyl SA
6001) whose ~ranulometry was comprised bctween 100 and 200 ~ .
The graftingof the monomer, namely dimet}lyla-30 mino-ethyl methacrylate (~IADA~I) of formula ~C113 C~l2 C~ /C113 C - O - (C~2)2 - ~ , o C~3 ~L2~3~35 ~as carried out by a pre--irra-liation metllod in the absence of oxygen. The powder was irradiated in a nitrogen atmos-phere under ~ radiation at a dose of 1.5 Mrad with a flow rate of 0.3 ~Irad/h, it, was then contacted with the S monomer in solution contain:ing 40% of MADA~IE, 59 . 6% of water and 0.4~ of hydroquinone playing the role of inhibi-tor, in the proportion of 5 ml of solution per gram of support. The reaction was carried out at 30C for 1 hour, in a nitro~en atmospllcre.
1() The powder was tllcn was}1ed in solutions of watcr-ct}1ar1ol and dried a-t 50C. The ratio of grafting by weight ol~taincd was 8.6%.
II ~xaml)1es of ~dsorbant Prel~aration Exanlple 15 I'i,xing of amino acids on a support.
Tl-le support obtained in Example 1 was used.
After hy~ration of the support, the amide func-tions were activatecl by incubation of the grafted support for 17 h at 37C in a solution containing 6% of gluturaldehyde i,n a ~IES buffe~r (0.1 M) at pfl 7.4, in thc proportion of 25 ml of solution for 1 6ram of grafted support.
The support was suspended in the proportion of 1 g/10 ml and shaken in an amino acid solution (50 ~/l) in ~I~Q buffer (0.1 ~1) kept at a temperature of 5C for 5 hours.
The manipulations ;ere carried out at different pll values:6 5;7.5;8.5, with 2 d;,fferent amino acids: lysine and ~lutamic acid.
The coupling ratios were evaluated by titration of the carboxylic acid functions with 0.1 N sodium hydrox-ide.
The validity of thi~s test was proven by confirmin~
on a control the absence o~ any hydrolysis of the amide functions on the same sup1--ort which had not under~one activation and coupling.
The results obtained were tl1e following:

1~23~3S9 _ Number of Moles ¦ Yield with respect ¦
_ Fixe(1 per yram to the G~ftintr 6.5 2.55.10-4 77 %
Lysine 7.5 1.34.10 4 40.5 %
8.5 2.2 .10-4 66.7 ~

Glutamic ~-5 0.88.10 4 26.5 %
ACid 7.5 1.08.10 4 33 %
8.5 1.03.10-4 31.1 %

Exam~)Ie 16 I'ixin~ of an enzyme(chymotry~sin~ on a sup~ort.
The support obtained in Example 1 was used.
After hydration of the support, activation was carrlecl Ollt under the i(]entical conclitions with those ~escri~ed in Examp]e 15~ The couplingS of the chymotrypsin I~as carried out at an optimum p~l of 7 under conditions equivalent to tlIose described for the coupling of the amino acids. The concentration of the chymotrypsin solution ~as only 2 ~/liter.
TlIe support then underwent 4 washings:
1st washing : 17h ill MES bu~fer (0.1 M) 2nd washing : 7h is MES buffer (0.1 ~I) NaCl(lM) at pll 7 3rd washing : 41h in a solution of IICl(10 3M) NaCl(l~l) at pll 3 4th washing : 7h in a TRIS buffer (O.Ol~I)CaCl2 ( O. 05!~1) at pII 8.
The results were evaluated in:
- weight of chymotrypsine per ~ram of suppor~, deduced from ol)tical density measurements on tl-e reaction medium; and - enzymatic activ;ties measured on the different solutions ancl on the powder.

. .

~o ~223~59 The enzymatic activity was expressed in Interna-tional Units (IU) (i-t i$ recalled that an International Unit represents the numl)er of` micromoles of sodium hydroxide added, necessary to neutralize the carboxylic acid functions S liberate~ by hydrolysis of the ethyl ester of acetyl-tyrosine (ATEE) under the action of chymotrypsin~.
In the couplin~r solution the concentration of cllymotrypsin clropl)ed from 1.93 g/l to 1.815 g/l:
an amount o~ 1.15 mlg was henee fixed per gram of support.
I() In para]lel, the enzymatic ae-tivity of the soLution dropped from259 IU to 7.68 IIJ.
Tlle final enzymatic activity mec1sure~ per gram of sul~port was 25.8 IU.

Example 17 lixing of ~-globulins on a support.
The support obtainecl in Example 2 was used.
After hydration of the support, the amide fune-tions were aetivated by stirring the powder in suspension in a solution with 2.5% of glutaraldellyde in a phosphate bllffer at pll 7.2 for 1 hour at 4C. After rinsing the ~owder, hllman G ~ -globulins labelled with iodine 125 were eoupled, by shaking the powdet in suspension in a solution of 1 mg/ml of ~ -globulins ( ~-g) ;n a phosphate buffer at 4C, overnigllt.
Tllorougrh washin~s w:ith phospllate buffer were repeated in order to remove adsorbed proteins.
Under these eondit;ons, the fixin~ of ~ g was evaluated at o.6 mg per gram of support.
~xample 18 Fixingof~ -~lobulins on a support.
The support obtaine(l in Example 3 was used.
Tlle aetivation of tlle earboxylic acid funetions, after hydra-tion~ was earried out in the presenee of 1-eyelohexyl-3-(2-morpholino-etllyl) carbodiimi~e in a ~IES 2-N(morpholino)ethane-sulfonie aeid solution (0.1 ~
at pl-l 4.75, for 1 hour at 20C. The rinsed support was 'I 12~313S9 suspencled in a solution of lluman G ~ -globulins (lmg/ml) and shaken overni~rht at ~lC. The ~ashings were carried Ollt in an ilES buffer. 'l'he fixincr reached 7 m~r of human G ~-olobulills per rram of sul)port.
E,xample 19 Eixirlc~; of ~ -~lobu~ins on a support.
The support obtainec1 in Example 4 was use~
The ~ixin,n,r of humall G ~ -globulins labelled with iod;rle 125, after hydration'arld activation wi~h glutaralde-hy(le reslllt((l, under the usllal conditions, in the fixing O~' 0.7 m~r of ~t''; pel' ,r,ram of slJl)pOrt.
L~allll)le 20 F;Xin~?~ of ~ -n]obu]ins on a support.
T~le support obtain((l in Example 5 was used.
Tlle hulllan G ~ -g'lobulins fixed on that support urlcler conditiolls equivalent ~,o those described in Example 18 ~ere 25 to 35 ~ r~r per cm of surface area of the support.
Example 21 Fixin~r of ~ -~Iobulins on a su~ ort.
The support obtaillerl in Example 6 was usecl.
The fixing of hutnan G ~ -g'lobulins under condi-tions equivalent to those des(ribcd in Exalnple 17 achieved 20 to 30 ~r~,r per cm surface area of the support.
Example 22 Fixin,~ of ~-t~rlobulins on a ~upport.
'rhe support obtai--led in Example 7 was used.
The fixin,~,r of human G ~ -~lobulins under condi-tions customarily employed i'or carboxylic acid functions (Example 18) was ~5 ~/cm s~lrface area of the support.
Example 23 Fixin~r of ~ -~lobulins on a support.
The support obtained in ~xample 8 was used.
'rhe support enable(l under tlle usual conditions (Example 18) the fixin~ of ~0 to 130 ~ ~ of G human ~ rlobulins per cm2 of its surface area.

'2 1223~59 ~xam le 24 Fixing of lluman red blood cells on a support.
The support ob~ained in Example 9 was used.
The irreversible fixin~ of human red bloocl corpuscles to this support w~s c~rried out after hydration and intermediate treatment of the support with ethylene-imine fixed with carbodiimide.
The operationalcon(litions were the following:
Tllc ~rafted microbeacls were plunged into a solution lV o~ ethy]ene-imine of molecular wei~ht 30,000 to 40,000 in an M~ buffer,with rotary stirring,inthe proportion of 50 mg~ of ethylene-iminc pel ~ram of microbeads. This treatnlent las~c~ 90 minut;es at aml~iant tempera-ture.
Tl~c Inicrobeads were then transferred into a solution containing, in final concentrations, the following elements, for 0.25 ~ram of microbeads/mL of solution:
- polyethylene-imil1~ : 12.5 m~/ml - 1-cycl ohexyl-3(2-morpholinoethyl)carbodiimide:
50 mg/ml, Tl1c w}~ole was kept 48h at ambiant temperature under rotary stirring.
The microbeads were rinsed with water and treated a~ain for 4h at ambiant temperature in a solution containing carbodiimide (50 mg/ml) and ammonium chloride (1~1).
After numerous rinsin,Ts, thcy were stored in a TRIS buffer (10 ml~1) at pll 7.4 in the prcsence of ~aN (0.2%).Befc~re pe rm it ti ng the adsorption of the red blood cells on the microbeads, the 2 elements were wasllec1 separately in a sucrose-acetate buffer (7/3) at 310 mOSM at pll 5 (it is recalled that 1 OSM i.c the osmotic pressure exerted by an aqueous solution conta;ning 1 mole of solute (molecule or ion) per liter, separated from pure water by a membrane impermeable to this solute).
i~ 50/50 by volume suspension of microbeads 3~ ~as added drop by drop to a 50/50 suspension by volume of red blood cells, in the same proportions. Very gTentle stirring was applied for lQ minutes. I~lnsin~ was then :~22~ !359 carrie(1 out Wit}l the same bllffer at pE1 7.4. ~ solution of glutaral~ellyde (310 mOS~I - 1%) in a PBS buffer (Phosphate 13uffer Saline : buffered isotonic solution) ~as prepared.
Tle microi~eads ~ere suspen~ecl in the proportion of 10%
of microbea~s in a solution containin~ 0.5% of glutaralde-hyde. Tlc reaction lasteci 1 hour at room temperature.
~arious rinsings were carricd ou-t at 4C:
- glycine (0.1 ~1) - P13S buffer tO followcd l>y incubation for 1 hour in a glycine solution.
Tllc final rinsin~ was carrie~ out with PBS buffer.
Tl-le rcd ~lood cc11s of the reaction medium were coullte(l on a ~lallss(7 cell (blade ~hich enables the cc~lls ~o be countcd) an(l eva1uated in ad(lition after llcmolysis in an amnlonium mc{lium. It was thus possible to cstimlte th<lt there was 8 to 9 x 10 red blood cclls fixed per gran~ of support.
Example 25 Tixin,r of human red blood cells on a sup~ort.
The support obtaj nccl in Example 4 was used on whicll the hurnan red b~ood cells ~ere adsorbed and then fixe(l-~itll~lutara]delly(lc. The opcratiorlal conditions describecl in Ixample 24 were repeated, avoidin-r tlle ethyl-ene-imine treatment.
The fixin~ of the red blood cclls was estimated to be of thc same order of magni-tudc, namely 8 to 9 x 10 red blood cells pcr ~ram of support.
Example 26 Fixin~g of ~lutamic .cid on a support.
The support obtaine(~ in Example 10 was uscl.
The activation of the carboxylic acid functions and thc fixing of rlutamic acid werc carried out in a single step . The pol~der was suspcnde(l for l711 at 4C
in a solution containin~ ~;lutamic acid (50 ~/l) and 1-cyclohexyl-3-(2-morpholinoetllyl)carbodiimi~e (10~) in ~1ES
buffer (O.l ~l) at pll 4.75. For 1 gram of support 10 ml of solution were necessary.

The titration of tl1e carboxylic acid functions on the coupled support enabled with respect to the uncoupled control, theevaluationof a ratio of 14% by weight of glutamic acid fixed by irreversible bonding.
~xample 27 Fixin~ of chymo-try~sin on a support.
The support obtairl(d in Example ll was used.
The coupling of the chymotrypsin was carried out by shalcing I gram of p~afted support for 20h at 20C
l() in lO ml of a potassium phosphate buffer solution (0.05 M
at pll 7) containing 20 m~ of chymotrypsin.
Ihe powder was ther1 washed abundantly in a phosphate buffer medium (pll 7), then in pho~phate-NaCl-buffer medium (l M) in a so1ution of IICl(lO 3 ~1)-NaCl(lM) at pll 3, and finally in TRI~ buffer (O.Ol M) -CaCl (~.05 ~l) at ptl 8.
The en~ymatic activity of the powder so obtained was 22 IU/gram of support (il-litial activity of the chymo-trypsin in solution was l3 IU/mg).
Exam~le 28 Fixln~ of ~ -galactosidase on a support.
The support obtain--d in Example l2 was used.
The hydroxyl functions were activated by cyanogen bromide under tlle following conditions: l g of support was suspended at ambiatlt temperature for lS minutes in a sodium carbonate solution (').02 ~l) whose pll was adjusted to ll witl1 sodium hydroxide and containin~r 10% of cyanogen bromide, in the proportion of 30 millim~les of cyanogen bromide per l g of support.
After rinsing in a sodium bicarbonate solution (O.l ~l), the powder was suspended in lO ml of a ~ -galacto-sidase solution with2 g/l in a bicarbonate buffer pH 9.3, for 24 hours at 4C. The powder was then washed abundantly and successively in 4 solutions:
- NallC03 (O()1 1`1) - IlCl ( 10-3 M~
- NaCl (0.5 ~l) - distillecl water.

~223~35~
The enzymatic activity of the fixed ~ -galactosi-t]ase was measurec1 after incubation of the pow~er at 37C
for 20 minutes in tlle p1csence of orthollitrophenol-D-~alactopyranoside (ONPG), of which su~strate the orthonitro-phellol (ONP) was liberated l~y l~ydrolysis~ proportionallyto the amount of enzyme prese1lt.
T}le activity thus measured was 0.34 micromole of ONP released per ~ram of sl1pport.
Ixample 29 I`ixin~ of ~ -ga]actosit1ase on a sup~ort.
T}lc support obtaine(l in ~xample l3 was used.
Thc activatjon o~ tlle }lydroxyl functions by cyanog~en bromi(le as well as tlle coupling of the p -galacto-sid~se were carried out un(ler the conditions described in ~xaml)Le 28.
In the same way, the enzymatic activity of tlle en~yme fixed was measuIed by hydrolytic action of the enzyme on tl1e substrate: ONPG.
Tht- activity measured was 0.45 microlnole of ONP released per gram of supl)ort.
~xaln~le 30 ~;xin~ of human rcd blood cells on a support.
1he suppor-t obtained in Exanlple 14 was used.
rllc adsorption ancl fastening of the red blood cells was carried out directly on the support, after having waslle(1 the beads and tlle red blood cells separately in sucrose-acetate buffer (7/3) at pll 5.
A 50/50 by volulne suspension of microbeads l~as added dro~ by drop to a 50/50 suspension of red blood cells, in the same proportions.~ery light rotary stirring was applied for 10 minutes. Rinsing was then carried out with the same buffer at pl~ 7.4. A glutaraldehyde solution was prepared: 1% in PBS buffer (buffered isotonic solution). The micro~eads wet-e suspended in the proportion of lO~o of microbeads in a solution contaillirlg 0.5~ of glutaraldellyde. T}~e rcactit-n lasted 1 hol1r at ambiant :>6 ~L2Z3~-29 temperature. Various rinsin~s were carried out at 4C:
- g].yci.ne (0.1`~1) - P~S l>uffer followed by incub~tion for I llour in glycine solution.
Ihe final rinsi.n~r ~as carrie(l out in the P13S buffer.
The number of red hlood eells fixed was evaluated at 8 to 9 x 1 o8 per ~ram of suppor-t (1.8 to 2 x 10 red l~l.oo~ eells/cm 2 ) III Use of the Adsorbants Accordin to the Invention:
- ~
A))licltiorl to Immurlo:lo~ical T'urification.
r Immllr)olo6ie;ll put~.ifieation is a particular appl. i C.ltiOn O~ the prineipl( of affinity ehromoto~raphy.
The removal frol1l the plasmatic medium Ofunclesirable elements (ex. antibocJies~ anti~ens, :immune complexes) is earried 1~ out by contactin~ witll a sllpport wllich possesses at its surface active elements reacting specifically witll tlle eleme-nt to be removed.
The plasmatie pl.lrification techniques most eurrently employed are at present plasmaphereses. In this case the removals are norl-specific with a eonsiderable loss of pl.~sma, expensive ancl a certain number of aceidents have occurred.
T]le manufacture of adsorballts obtlined from ~rrafted po]ymer supports, possessin~ varied mecllanical characteristics and havin r fixed irreversibly various biomolecules, opens a wide route of applieation in tl~is fie].d.
Some of the adsorbants describecl in tlle second part have been use(1 to estal~lish their purification eapa-~ity.
A. "In vitro" pur;.~ieation Example 31 The adsorbant obtained irl Example 18 was used.
Rabbits (~ouscat whi.te) were immunized by intra-dermic injeetion of human (; ~-globulir~s (of tlle Si~ma Company: Co~m fraction II).At reg~lar intervals~samples were taken out to det.ermine antiboclies all(l the constitution of "anti-27 ~22~35~

body pools" used in the "in vitro purifications".
The quantitative assay of the antibodies was carried out by a radioimmullol~gical method of the sandwich type using radioiodized (I125 ) h -~lobulins ( ~GII).
~ y t~lis method, ~GIIs fixed to cellulose particles were contacted simultaneously with the antiserum and the ~GII I125 tracer in excess. The ~GHs fixed to the cellnlose ~ere in excess ~;o trap all the antibodies.
Thc excessive tracer was renloved by washin~. The amount o~ tracer fixe~l to the supl~ort by antibodies and yG11s was proportional to the alll(>unt of antibodies present in thc antis~rurn. The reslllts compared to a standardwere expressed in ~/ml.
The pool of antibodies taken from the rabbit ~as diluted with normal rabbit serum. By mearls of a peristaltic pump (of the ilastuflex type) the solution was perfused in the purifier; the system function~d in closed circuit; successive samples were taken for the determination of the anti ~GII antibodies and total pro-teins.
The operational conditions of the two tests and the "in vitro" purification results are indicated in the following table.

Test ~umber 1 2 - Total Surface Area2 of the Support ( cm ) 4,200 ~,200 - Concentration of Anti-bodies ( ~g/ml) 19.7 ~3 - Plasma Volume (ml) 30 5o - ~ntibody Amount (mg)591 2,150 - ~low ~ate (ml/min) 5 5 - Time (min) 60 60 - % Purification 97 75 -"Purified" Antibodies573 l,612 2s 12~3l!359 Example 32 ~ `he adsorbant used was that obtained in Example 20 to carry out purification te~ts of serum containin~r l~unlan G anti- ~-rlobulin antibodies, under conditions similar to tllose of Exalllple 3l.
Tlle operational conclitions of thetwo "in vi-tro"
purification tests and the results obtained are assembled in the follc~win/r table.
, .

Test 'iuml)er 1 2 , - Total ~urface Area2 of tllc Support (CM ) 210 420 - Conccntr~tiorl of Anti-boclies ( ~g/ml) 4.7 4-7 - I']a~ma VO~.llnlC (ml ) 30 3o - Antibody Amoullt (~r) 141 141 ~ F]ow Rate (ml/mill) 15 15 - Time (min) 60 60 - % Purification 10 4o - "Purit`iecl" Antibodies ~rr) 14 56.4 Ixample 33 Tlle aclsorbant obtaine(l in ~xample 22 ~Yas used to carry out a purificati()r- tcst of serum containing l~uman G anti- ~ -glo~u]in antibodies, uncler conditions similar to tllose of ~xample 31.
Tota1 Surface Area of the Support (cm )......... 630 Concentration of Antibodies ( ~g/llt~
Plasma Vo1ume (ml).............................. 30 Amount of t~ntibodies. ~ 330 Flo~ Rate (ml/min)................... ....... 20 Time (min)....................................... 60 Purification ~................................... 33 "Purifieci" Arltiboclies ( ~r) ~ 109 ~ ~ ~22;~S9 E.Yampl e 34 The adsorbant,obta-ined in Example 23 was used to carry out a purificatiorl test of serum containing human anti- ~ -globu]in antiboclies, under conclitions 5 simi]ar to t}lose of E~ample 31.
Total Sur~ace Area of the Support ~cm ).......... 210 Concentration of Antibodies (~/ml)............. 16 Plasma Volume (ml)....~........................................ 30 Amollnt oE Antibodics ( ~g)...................... 480 ilo~ Rltc (ml/min)............................... 5 Tilne (mill)...................................... 60 ,~, Purification.................................. 9 "Puri~ied" Antibodies ( ~)...................... 48 i,xam~)lc 35 rl~e adsorbant obtaine~l in Example 24 was used.
'Ihc-~ specific adsorption capacity of antigen ~ bornei)y the red blood ceJls fixed to the support was checkc,~d with re.spcct to the rlon-specific activity relative to an antibocly n. ior this, the ratios of anti A and anti 13 antibodies contained in the serum were measured before and aftcr incubation for 1 hour at 37C of the support in t}lis serum. The anti A antiboclies present in lIuman fresil bloocl were relnoved.
Ti~e activities wel-e measured i~y the method 25 describc(l by C. I'OPAI~S, A. ~IUii,l,I~, C. IIUREL and J. LE~LANC
in .~1. Transf. arl(l Imlll., Volume XXIV, ~`o. l (19Sl).
The startin~ serum ~as ti-trated at 256. Tlle conclitions of tllc two tests carried out ancl their results are indicated in the followin~ -table:
.
Test '.~umber 2 , - I~'ei~rht of Dry Support (mg) 3~o 630 - Dilution of tl~e Serum l/4 l/~
- Amount of Serum (ml) , - Purification : anti A 93 ~ 37.6 Y~
- Purificatiorl : allti 13 ~ 0 ~ 0 ~o :~23l~i9 The tota1 protein ratio remained practically constant .
ExamT)Ie 36 The adsorbant obtainec1 in Example 30 was used.
The in vitro purification carried out on ml of serum witll 320 mr oi` clry support, according to the technique clcscribed in Example 35, led to an anti ~ specific purification of 85,% and an anti B non-specific puri fication of S ~.
1,0 13. "E,x vivo" r)urification l,xaml)le 37 The a(1sorb~nt obtaine(~ in Example 18 ~as used.
An arterio-venous shunt (ju6rular-carotid ) ~-IS prcviously place-1 on a rllbbit immunized by the intra-derlllic injection of human G ~ -~lobulins and anestheti~ed .~ith .~'eml)utal.
The blood was perfllsed directly onto a column containin~ 25 to 30 ~ of ac3sorbant. The flow rate was 10 to 15 ml/min. Tl1e animal was heparinized previously and by perfusion in tile conrse of thc operation. The extracorporal blood circulation lasted 1h30. l'lle antibody ratio was 22.2 ~r,/ml at tlle start and 2.21 ~/ml after puriEication. Total proteirls droppecl from 56 m~/ml to ~4.8 m~/nll.
The invcntion is in no ~Yay limited to those of its cmbodimel1ts anc1 uses envisa~ed or described in the examples. In particular, satisfactory results are obtainecl by cffectin~ tlle direct graftill~, o~ tl1e monomer on thc basic particulate snpl)ort, espccially under the preferred conclitions wl1icl1 have been previously define(1.

Claims (14)

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

1. Particulate support for the manufacture of adsorbants for affinity chromatography on a column, said support being essentially constituted by particles of a basic polymer having dimensional stability under pres-sure, crush resistance, a density higher than unity, sufficient chemical and physico-chemical inertia under the conditions of use, insolubility in the majority of solvents including water and aqueous solutions and low swelling rates in water and aqueous solutions, said par-ticles being adapted to be used in column chromatography and on which are grafted essentially at the surface, molecules of at least one unsaturated monomer capable of forming a copolymer with the basic material and bearing at least one chemical function capable of forming, sub-sequently, under non-denaturing conditions, a covalent bond with the ligand used in the adsorption envisaged, the amount of grafted monomer possessing a chemical function accessible per unit of surface area (monomer assayed per unit of surface area) being from 10 to 500 µg per cm2 of surface area of the support and the ratio of the amount of grafted monomer possessing an accessi-ble chemical function to the total amount of grafted monomer (assayed monomer/weighed monomer) being from 0.05 to 0.5.

2. Particulate support according to Claim 1, wherein the amount of monomer assayed per unit of surface area is 30 to 350 µg per cm2 and the ratio assayed monomer/weighed monomer is 0.05 to 0.3.

3. Particulate support according to Claim 1 or 2, wherein the basic material is selected from among polyvinyl chloride, polystyrene, polychlorotrifluoro-ethylene, polyamides and polyethylene, and their co-polymers adapted to be used, in the state of particles, in column chromatography.

4. Particulate support according to Claim 1, wherein the monomer bears at least one carboxylic acid function, an amide function, an amine function, an aldehyde function or a hydroxyl function.

5. Particulate support according to Claim 4, wherein the monomer is selected from among acrylic acid, acrylamide, allylamine, acrolein, ethylene-glycol acrylate and dimethylamino-ethyl methacrylate.

6. Process for the preparation of the grafted particulate support according to Claim 1, consisting essentially in effecting radiochemical grafting, on particles of a selected basic polymeric material, of the selected unsaturated monomer, in solution in a solvent non-aggressive with respect to the reagents or in the absence of solvent.

7. Process for the preparation of the grafted particulate support according to Claim 1, consisting essentially in carrying out the radiochemical grafting, on particles of a selected basic polymeric material whose size is in the range from about 10 to about 5000 µ, of the selected unsaturated monomer, in solution at a concentration of 10 to 80% by weight, in water, acetone or ethanol, pure or in admixture, for a period of 1 to 6.5 hours, at a temperature in the range from room temperature to the boiling temperature of the solvent used, with a radiation dose comprised in the range of 0.03 to 10 Mrad.

8. Process according to Claim 6, wherein the grafting takes place after pre-irradiation by ionizing radiations coming from electron accelerators or cobalt 60 sources, in the presence of oxygen.

9. Process according to Claim 6, wherein the grafting takes place after pre-irradiation by ionizing radiations coming from electron accelerators or from cobalt 60 sources, in the absence of oxygen.

10. Adsorbant for affinity chromatography, resulting from the covalent fixation of the specific ligand of the substance to be adsorbed, to the particu-late support according to Claim 1.

11. Process for the preparation of the ad-sorbant according to Claim 10, consisting in fixing by covalence the specific ligand of the substance to be adsorbed, on the support after activation of the latter.

12. Process according to Claim 10, wherein the covalent fixation of the specific ligand of the sub-stance to be adsorbed is preceded by hydration of said ligand.

13. Process for the extraction or elimination of a substance contained in a mixture of other sub-stances characterized in that it comprises the step of submitting said mixture to affinity chromatography on a column comprising an adsorbant according to Claim 10, in which the covalently fixed ligand is specific to said substance to be extracted or eliminated.

14. Process for the extraction or elimination of a substance contained in blood, characterized in that it comprises the step of submitting the blood containing said substance to affinity chromatography on a column comprising an adsorbant according to Claim 10, in which the covalently fixed ligand is specific to said sub-stance to be extracted or eliminated.