Note: Descriptions are shown in the official language in which they were submitted.
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
1 1
- 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
0
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~ '
859
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:
I9
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.
31359
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.
