FR2842746A1 - Immunoaffinity column, useful for removing antibodies, e.g. for treating familial hypercholesterolemia, contains polymeric microparticles containing antigens associated with phospholipids - Google Patents

Abstract

An immunoaffinity column (A) of adsorbent material (B) for eliminating antibodies (Ab) from a liquid that circulates through the column, where (B) comprises at least one set of polymeric microparticles (MP) that encapsulate phospholipid-associated antigens (Ag) and are produced by removal of at least one organic solvent in a double water-in-oil-in-water emulsion (C), is new. Immunoaffinity column (A) of adsorbent material (B) for eliminating antibodies (Ab) from liquid that circulates through the column. (B) comprises at least one set of polymeric microparticles (MP) that encapsulate phospholipid-associated antigens (Ag) and are produced by removal of at least one organic solvent in a double water-in-oil-in-water emulsion (C). (C) is prepared by combining one liquid phase containing Ag, in aqueous buffer, corresponding to Ag-containing membranes and a second liquid phase comprising at least one water-immiscible solvent (S1) and at least one polymer, soluble in S1 but not in water, to form a primary water-in-oil emulsion (C1); and treating C1 with a third aqueous phase containing at least one surfactant to form (C). The polymer that forms MP allows inwards diffusion of Ab but prevents release of membrane antigen or of Ab/Ag complexes bound to membranes. Optionally a gel is combined with MP in the column. ACTIVITY : Immunosuppressive; Antilipemic. No biological data given. MECHANISM OF ACTION : Specific binding of antibodies.

Description

The present invention relates to an immunoaffinity column for purification.

  of antibodies comprising polymer microparticles encapsulating antigens associated with phospholipids. It has applications in the therapeutic field and more particularly as a device for the extraction or the extracorporeal purification of human or animal of pathological products or not. It uses an adsorbent composition which can be made compatible with the biological substances of the organism and allows operation both in closed circuit with return to the organism and in open circuit, without return. It can thus also allow the preparation of therapeutic products, whether by eliminating undesirable products or by adsorption / recovery of useful products. It can be adapted in specificity and affinity depending on the antigen used. Finally, it can be applied to fields other than therapeutics and, for example, in biological, chemical analysis or other fields in which it is necessary to be able to separate in a specific way from chemical bodies.

  Many genetic or immunological pathologies (familial hypercholesterolaemia, myasthenia, fetomaternal incompatibility, antiphospholipid syndrome, lupus erythematosus, etc.) or immunological reactions (heterografts with incompatibility) arise from the presence in the blood of antibodies or pathogenic antigens that must be eliminated.

  Different therapeutic techniques are currently used:

  - Plasmapheresis or plasma exchange: this is a nonspecific technique which eliminates all the constituents of blood plasma with in particular a significant depletion of immunoglobulins, which can lead to risks of infectious development. Indeed, the loss of 50 to 70% of the immunoglobulins can be responsible for recurrent infections, which is likely to worsen the prognosis.

  - immunoadsorption on protein A: this is a selective but non-specific technique which consists in fixing the Fc fragment

  IgGl, IgG2, IgG4 but not IgG3.

  These two techniques are often associated with

  corticosteroid therapy and / or immunosuppressants.

  - the specific immunoadsorption relating to the formation of an antigen / antibody complex: a ligand (antigen or antibody) is fixed on a chromatogiraphic support (immunoadsorbent) so as to fix the refining immunoaffinity (antibody or corresponding antigen) contained in a solution or a biological medium eluted through a column containing the immunoadsorbent. The specific immunoadsorption technique has already been used in particular for the treatment of familial hypercholesterolemia and lupus erythematosus. However, the immunoadsorbent is prepared by chemical activation of the support so as to fix the ligand by covalent bond. The use of chemical activators such as the most widely used cyanogen bromide can cause toxicity problems. On the other hand, the stability of the covalent bond not being absolute, the ligand, often of animal origin, can be released at the outlet of the column in the purified medium intended to be re-administered to the patients. This contamination can cause immunological disorders in patients whose

  the most serious is anaphylactic shock.

  The inventors have shown, surprisingly, that polymer microparticles encapsulating antigens present on a phospholipid membrane prepared according to a particular technique and introduced into a column make it possible to reduce the titer of the antibody specific for the encapsulated antigen when a solution or a biological medium containing the antibody is eluted through the column. Thus, the present invention relates to the biological and medical, or even more generally chemical, field and therefore relates to an immunoaffinity column for purification of antibodies comprising microparticles loaded with membrane antigens distributed in a polymer matrix. The polymer microparticles are preferably non-biodegradable in the case of a medical or biological application. The technique for preparing microparticles uses non-biodegradable and water-insoluble polymers, alone or as a mixture, and a double emulsion, then the columnarization of these particles intended to be used as an immunoaffinity chromatographic support. The microparticles have a diameter between 10 μm and 1 mm, and preferably between 200 μm and 600 μm. Finally, the products used in the invention are preferably biocompatible in medical or biological applications. The invention therefore relates to an immunoaffinity column with an adsorbent composition for purifying antibodies contained in

  a fluid circulating in said column.

  According to the invention, the adsorbent composition comprises at least one set of polymer microparticles encapsulating antigens associated with phospholipids, the microparticles being obtained by elimination of at least one organic solvent in a double water-in-oil-in-water emulsion. , the double emulsion being obtained in two stages: the first stage consisting in preparing a primary water-in-oil emulsion by combining a first liquid phase with a second liquid phase, the first liquid phase comprising at least associated phospholipids to antigens in a buffered aqueous solution, the phospholipids and antigens of the first phase corresponding to membranes carrying antigens, the second liquid phase comprising at least one organic solvent immiscible with water and at least one polymer soluble in said solvent and not soluble in water, the second step being to add the primary emulsion into a e third aqueous phase comprising at least one surfactant in order to form the double emulsion, the polymer forming the microparticles allowing the diffusion of the antibodies of the fluid inside the microparticles while preventing the release of the membrane antigens and antigen / complexes antibodies attached to the membranes, a gel can be associated with microparticles in the column. In various embodiments of the invention, the following means, which can be combined according to all the technically possible possibilities, are used: - in addition to water, the organic solvent is removed from the double emulsion by evaporation and / or drying, drying being obtained in particular by nebulization or lyophilization, or by the use of a supercritical fluid, - the membranes are biological and extracted from biological cells, - the membranes are artificial, - the antigen is an erythrocytic antigen, - the antigen is an antigen of the HLA system, - the antigen is P2-glycoprotein 1, - the polymer is non-biodegradable and chosen from the group 15 of cellulose derivatives including ethylcellulose, cellulose acetate, cellulose triméllitate and the group of polyacrylic derivatives, - the organic solvent is volatile and insoluble in water (<0.5%) and is chosen from solvents or mixtures of solvents such as chloroform, dichloromethane, ethyl acetate, and any other organic solvent capable of dissolving the polymer (s), - the gel is chosen from agarose, dextran, silica or other product compatible with the fluid, - the or the polymers represent 1 to 20% by volume in the primary emulsion and, preferably, 4 to 10%, the organic solvent (s) represent 10 to 90% by volume in the primary emulsion and, preferably, from 50 to 90%, - the primary emulsion representing 0.1 to 10% by volume in the double emulsion and, preferably 1%, - in addition at least one of the two liquid phases intended to form the primary emulsion further comprises at at least one surfactant, said surfactant being chosen from natural, synthetic ionic, synthetic nonionic or amphoteric surfactants, surfactant or mixture of agents

  surfactants representing more than 0 to 10% by weight of the corresponding phase and, preferably, from 0.1 to 2% by weight.

  The column can be applied to treatments by extracorporeal purification of human or animal blood plasma, in particular to hyper-immunized people awaiting a transplant. More particularly, it can be applied to the treatment of patients suffering from familial hypercholesterolaemia in order to remove apolipoproteins B from their plasma. It is also possible to apply the invention to the preparation of blood fractions by excluding anti-A or anti-B antibodies in patients with

  major ABO incompatibility in transplant or transplant operations.

  Thus the present invention relates to a column based on porous microparticles loaded with antigens conserved in a phospholipid environment and intended to extend the application of immunoadsorption to fields in which it is not currently used. This is the case of the problem of incompatibilities during organ transplants (HLA system) and of the antiphospholipid syndrome due to the presence of pathogenic antiphospholipid and / or antiprotein autoantibodies including the anti-132-glycoprotein 1. In particular, there is a catastrophic form of antiphospholipid syndrome characterized by a number of patients in therapeutic failure and for which the elimination of antibodies

  anti-j32-glycoprotein 1 appears an effective alternative.

  The present invention and its variations will now be exemplified and explained by the non-limiting description which

  follows and in relation to: FIG. 1 which is a first illustration of the adsorption capacity of erythrocyte stromas, FIG. 2 which is a second illustration of the adsorption capacity of erythrocyte stromas,

  Figures 3 and 4 which respectively give the adsorption capacities in multiple passages for three concentrations of membrane proteins and three antibodies (anti-A, anti-B, anti-Kell).

  In the following description, we will first explain the technique for preparing the microparticles and then the column, and we will finally give the results obtained.

  With regard to the microparticles 1) a first liquid phase is prepared, consisting of a

  suspension of biological or artificial membranes ("ghosts") carrying antigens in a buffered aqueous solution and which may contain at least one surfactant.

  2) a second liquid phase consisting of an organic solvent immiscible with water and containing the one or more is prepared

  polymers and optionally a surfactant.

  3) the suspension of biological or artificial membranes carrying antigens is added to the organic solution of polymer (s) with stirring. A primary emulsion 15 of the water in oil (W / O) type is thus obtained. The solvent can constitute from 10 to 90% by volume of the mixture forming the primary emulsion and, preferably, from 50 to 90%.

  4) this primary emulsion is added with stirring to an optionally buffered aqueous phase and containing at least 20 surfactant. The primary emulsion can constitute from 0.1 to 10% of the final mixture, preferably 1% of the final mixture. A double emulsion of the water in oil in water (W / O / W) type is thus obtained. Stirring is continued until total extraction / evaporation of the organic solvent, thus leading to the precipitation of the spherical microparticles encapsulating the membranes carrying antigens. ) The microparticles thus formed are collected after

  filtration or centrifugation or any other suitable process.

  In step 4, a powder of 30 microparticles can also be obtained by using a drying technique (nebulization, lyophilization) after addition of stabilizing substances. In another advantageous process, it is also possible to obtain microparticles by technology in supercritical fluid with or without organic solvent. Thus, other methods can also be envisaged, in particular nebulization, coating, extrusion, lyophilization, use

supercritical fluids.

  The microparticles are formed from a polymer matrix

  comprising at least 1 non-biodegradable polymer.

  Advantageously, the non-biodegradable polymer is chosen from the group of non-biodegradable polymers which are insoluble in water, preferentially cellulose derivatives (ethylcellulose, cellulose acetate, cellulose trimellitate, etc.) and polyacrylic derivatives. The use of polymers considered to be biocompatible even if they are not biodegradable is a

  guarantee of the absence of toxicity of said microparticles.

  The membranes used can be of biological origin or of artificial origin. The suspensions of biological membranes carrying the antigens are produced according to techniques well known in the field and using cell lysis followed by successive washing by centrifugation or tangential filtration and optionally by a sonication step. Suspensions of artificial membranes use a solution of phospholipids mixed by sonication in an aqueous solution. Techniques for eliminating infectious or pathogenic agents, including prions, are preferably used for therapeutic applications. In the case of artificial membranes, the antigen is added extemporaneously in the suspension of phospholipids in order to be fixed on these

last.

  The organic solvent, preferably volatile, is insoluble in water and preferably chosen from solvents or mixtures of solvents such as chloroform, dichloromethane, ethyl acetate, and any other organic solvent capable of

dissolve the polymer (s).

  Advantageously, the concentration of the polymer (s) in the organic solvent or the mixture of organic solvents of the first emulsion is preferably between 1 and 20% by volume and preferably between 4 and 10%.

  The surfactants used can be natural, synthetic ionic and synthetic nonionic or amphoteric surfactants. In each of the phases, the surfactant or the mixture of surfactants may be present in an amount from 0 to 10% by weight, preferably from 0.1 to 2% by weight.

  As surfactants, polyvinyl alcohol (PVA, MW 30,000, 88% hydrolyzed) and a polyoxoethylenated sorbitan derivative of the Tween type will preferably be used. Other nonionic surfactants can be used such as, for example, copolymers of ethylene and propylene oxide (of Pluronic type) or of esters of fatty alcohol and of polyoxyethylene glycol, or of sorbitan derivatives (of of Span type). ).

  The polymers used are polymers which are not biodegradable and are not soluble in water. Without this list being limiting, mention may be made of cellulose polymers of different molecular weights, silicones, acrylic derivatives, vinyl polymers, polyamides, polyureas, polyurethanes, polyethylenes, polycarbonates, polysulfonates. There are thus obtained non-biodegradable and insoluble microparticles serving as

  reservoir of membrane antigens.

  The porosity of the microparticles allows the diffusion of plasma antibodies within the microparticles while it prevents the release of membrane antigens due to their larger size. It can be modulated by incorporating into the internal aqueous phase or the organic polymer phase, biocompatible substances of small molecular mass. Likewise, the antigen / antibody complex fixed on the membranes cannot be released due also to its larger size than the diameter of the pores of the polymer wall and

  the amphiphilic nature of the encapsulated complex.

  As regards the preparation of the immunoaffinity column intended for extracting from the circulating fluid the antibodies and its use, the microparticles which have been prepared according to the preceding description are introduced with or

  frost-free in a cylindrical column of volume and dimensions suitable for use. The gel is chosen so as not to exhibit any interaction with the blood plasma (agarose, dextran, silica). It makes it possible to perfectly homogenize the filling of the adsorbent composition in the column. Each column is equipped with a filter at the inlet and at the outlet of the column, with a filtration diameter smaller than the diameter of the microparticles in order to prevent the microparticles from escaping from the column. The columns are made of glass or treated plastic in order to reduce the adsorption specific to this type of material. They can also be made of steel or any other material compatible with biological substances and, for example, blood plasma. Finally, these various materials can be combined within

of the same column.

  The column is connected at a first of its ends to a

  peristaltic pump allowing circulation and elution of the fluid, for example a patient's blood plasma after separation of the blood cells in the case of extracorporeal circulation in humans. In this type of application, the column is preferably disposable for a patient (it will be seen that it is possible to use it over several cycles) without desorption of the antibody fixed on the encapsulated membrane antigen. In addition, the materials and products used for the gel, the adsorbent substance and the column are sterilized.

  As indicated, the membranes used can be of biological or artificial origin, these two types possibly being combined in the same column. It is also possible to have one or more types of antigens in the same column. These combinations of 30 types of membranes and / or antigens in the column can be homogeneous, differential or segmented. In the first case, the microcapsules are distributed homogeneously along the column, in the second case, the concentration of microcapsules can be variable depending on the position along the column, finally in the third In this case, the microcapsules can be distributed in homogeneous zones or not along the column. For the second case, in certain configurations related to the size of the column and to the flow rate, it is also possible to use a differential concentration perpendicular to the direction of circulation of the fluid to be treated to take account of the difference in speed of the fluid between the center of the flow and the column wall (s). The third case may, for example, be useful for extracting a complexified product, for example a first protein naturally attached to a site of a second protein, the complexification making it difficult to extract the first protein, in this example, the first part of the column contains an antigen with high affinity for the second protein and the second part of the column an antigen for the first protein. It is also possible to arrange columns of different types in series in order to obtain the same result. However, in the example given above, if the second protein must be returned to the fluid, this operation subsequent to adsorption will be easier with independent columns in series. In a typical application, it is also possible to combine the columns in series and / or in parallel as required. It is also possible to use polymers of different type for each type of antigen, the polymers having exclusive specific solvents allowing recovery of the specific antibodies within the same column. It has been shown that, surprisingly and unexpectedly,

  the microparticles thus used in a column gave more satisfactory results than the same types of microparticles in tubes, the mass ratio microparticles / volume of antibody or plasma solution being identical. In a comparative experiment, the antibody titer decreased from 128 to 8 after one hour in the column whereas it only decreased after 3 hours from 128 to 64 in the tube.

  For FIGS. 1 to 4, the preparation of the microparticles containing membrane antigens of red blood cells from he donors of blood groups A and B was carried out as follows: Erythrocyte concentrates from healthy blood group B donors are hemolyzed in an isotonic solution at pH 7.4 and then washed by successive centrifugations in a physiological buffered solution until the elimination of hemoglobin is complete. The concentration of membrane antigens is evaluated by an assay of the total membrane proteins with bicinchoninic acid and varies between 6 10 and 10 g / L. The activity of antigenic sites, verified by the method

  of hemagglutination, is kept during preparation.

  The suspension of membrane antigens (1 ml, 2 g / l) is emulsified with magnetic stirring for three minutes in a solution of dichloromethane (10 ml) containing 500 mg of polymer (ethylcellulose, molecular mass 146000). This first emulsion (water / oil) is then poured into a volume of water (1500 ml) containing two surfactants, polyvinyl alcohol (0.4%) and Tween (1%), making it possible to obtain by stirring mechanical using a marine propeller a second water / oil / water emulsion. After two hours, the stirring is stopped and the microparticles are collected by filtration and dried at room temperature. The size of the microparticles is evaluated by laser diffusion with a MasterSizer. The average size is 400 µm.

  Figure 1 illustrates the adsorption capacity of stromas

  red blood cells (2, 4 and 6 mg / ml total protein) carrying group A antigens encapsulated in ethylcellulose microparticles (25 mg) after contacting for 1, 2, 3 and 4 hours with 500 μl of a solution of anti-A polyclonal antibodies.

  The antibody titer was determined by hemagglutination. With regard more particularly to FIG. 1, the adsorption capacity of the encapsulated membrane antigens is tested in a tube by gently stirring 25 mg of microparticles with 500 μl of an anti-A antibody solution for 1 to 4 hours.

  The initial antibody titer then after adsorption on the membrane antigens was tested by hemagglutination.

  FIG. 2 illustrates the adsorption capacity of erythrocyte stromas (2, 4 and 6 mg / ml in total proteins) carrying group A antigens encapsulated in ethylcellulose microparticles (750 mg) introduced into a chromatographic column on which is eluted using a peristaltic pump at a flow rate of 1 ml / min a solution of anti-A polygonal antibodies (15 ml). The antibody titer is determined by hemagglutination at the entry and exit of the column during the various phases of elution on the column. With regard more particularly to FIG. 2, the adsorption capacity of the encapsulated membrane antigens is tested in a column, after having packed a chromatographic column with 750 mg of 15 microparticles and 15 ml of a solution of anti-A antibodies are eluted on the column at a flow rate of 1 ml / min. The antibody titer is evaluated by hemagglutination at the entry and exit of the column. FIG. 3 illustrates the adsorption capacity of erythrocyte stromas (2, 4, 6 and 10 mg / ml in total proteins) carrying group B antigens encapsulated in ethylcellulose microparticles (750 mg) introduced into a column chromatography on which are eluted, using a peristaltic pump at a flow rate of 1 ml / min, 15 ml of human blood plasma supplemented with anti-B antibodies. The antibody titer is determined by hemagglutination at the entry and exit of the column during the various phases of elution on the column. With regard more particularly to FIG. 3, the adsorption capacity of the encapsulated membrane antigens is tested as for FIG. 2 but with 15 ml of blood plasma

  human supplemented with anti-A antibodies.

  Tests have also been carried out with erythrocyte membranes from healthy group A donors as well as with leukocyte or fibroblast membranes to purify anti-HLA antibodies. Likewise with platelet vesicles 35 obtained after activation of platelets and on which the P2-glycoprotein I is fixed to purify the antip2-glycoprotein 1 antibodies. Finally, with artificial membranes obtained by mixing using an ultrasound probe phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine, f32glycoprotein 1 is fixed on these artificial membranes to purify anti-p2-glycoprotein 1 antibodies. In the two examples which follow in relation to FIGS. 3 and 4, a column in glass 1.5 cm in diameter and 10 10 cm in length was used. The column is used for several consecutive chromatographic cycles. It is therefore possible in medical or biological applications to use smaller columns than conventional columns operating only for one cycle and which have a larger volume.

  large in the range of 200 to 300ml.

  In FIG. 3, the adsorption capacity of a column comprising ethylcellulose microparticles (750 mg) including membranes of group B erythrocytes is expressed as a decrease in the antibody titer. For each passage of the antibody solution, the results were given concerning, from left to right, respectively, microparticles corresponding to 2, 6, and 10 mg / ml of total encapsulated proteins. Eight consecutive passages of 15 ml of an anti-B antibody solution with a flow rate of lml / min are shown. The greater the amount of total protein, the faster the decrease. After a number of passages depending on the quantity of total proteins, the antibody binding sites are practically all occupied and the level of antibodies remains in plateau.

  In FIG. 4, the adsorption capacity of a column comprising microparticles of ethylcellulose (750 mg) including membranes of erythrocytes carrying antigens B and Kell is expressed as a decrease in the titer of antibody. For each passage of the antibody solution, the results were given relating respectively to an anti35 B antibody solution and to the right an anti-Kell antibody solution. Eight consecutive 15 ml passages of these solutions with a flow rate of 1 ml / min

are represented.

Claims (10)

  1. Immunoaffinity column with adsorbent composition for purifying antibodies contained in a fluid circulating in said column, characterized in that the adsorbent composition comprises at least one set of polymer microparticles encapsulating antigens associated with phospholipids, the microparticles being obtained by elimination of at least one organic solvent in a double water-in-oil emulsion in water, the double emulsion being obtained in two stages: the first stage consisting in preparing a primary water-in-oil emulsion by combination of a first liquid phase with a second liquid phase, the first liquid phase comprising at least phospholipids associated with antigens in a buffered aqueous solution, the phospholipids and antigens of the first phase corresponding to membranes carrying antigens, the second liquid phase comprising at least one organic solvent immiscible with water and at least one polymer soluble in said solvent and not soluble in water, the second step consisting in adding to the primary emulsion a third aqueous phase comprising at least one surfactant in order to form the double emulsion, the polymer forming the microparticles allowing the diffusion of the antibodies of the fluid inside the microparticles while preventing the release of the membrane antigens and of the antigen / antibody complexes fixed on the membranes, a gel which can be associated with the microparticles in the column. 2. Column according to claim 1 characterized in that, in addition to water, the organic solvent is removed from the double emulsion by evaporation and / or drying, the drying being obtained   in particular by nebulization or lyophilization.   3. Column according to claim 1 or 2 characterized in that the membranes are biological and extracted from cells organic.   4. Column according to claim 1 or 2 characterized in that   that the membranes are artificial.   5. Column according to any one of the preceding claims, characterized in that the antigen is an antigen   Group A or Group B erythrocyte   6. Column according to any one of the preceding claims, characterized in that the antigen is an antigen of the HLA system.   7. Column according to any one of the preceding claims, characterized in that the antigen is the P2-glycoprotein 1.   8. Column according to any one of the preceding claims, characterized in that the polymer is non-biodegradable and chosen from the group of cellulose derivatives including ethylcellulose, cellulose acetate, trimellitate   cellulose and the group of polyacrylic derivatives.   9. Column according to any one of the preceding claims, characterized in that the organic solvent is volatile and is chosen from solvents or mixtures of solvents such as chloroform, dichloromethane, ethyl acetate, or any other organic solvent capable of dissolving the polymer (s).   10. Column according to any one of the preceding claims, characterized in that the gel is chosen from agarose, dextran, silica or other product compatible with the fluid. He. Column according to any one of the preceding claims, characterized in that the polymer (s) represent 1 to 20% by volume in the primary emulsion and, preferably, 4 to 10%, the organic solvent (s) represent 10 30 to 90% by volume in the primary emulsion and preferably 50 90%,   the primary emulsion representing 0.1 to 10% by volume in   the double emulsion and preferably 1%.   12. Column according to any one of the preceding claims, characterized in that at least one of the phases   liquids intended to form the primary emulsion additionally comprises at least one surfactant, said surfactant being chosen from natural surfactants, ionic synthetics, nonionic synthetics or amphoterics, the surfactant or the mixture of surfactants representing more from 0 to 10% in   weight of the corresponding phase and preferably from 0.1 to 2% by weight.