WO2020002512A1 - Method for grafting a fibrous element for eliminating antibodies from blood or a blood component - Google Patents

Method for grafting a fibrous element for eliminating antibodies from blood or a blood component Download PDF

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Publication number
WO2020002512A1
WO2020002512A1 PCT/EP2019/067176 EP2019067176W WO2020002512A1 WO 2020002512 A1 WO2020002512 A1 WO 2020002512A1 EP 2019067176 W EP2019067176 W EP 2019067176W WO 2020002512 A1 WO2020002512 A1 WO 2020002512A1
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WIPO (PCT)
Prior art keywords
fibrous element
grafting
solution
blood
oligosaccharide
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Ceased
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PCT/EP2019/067176
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French (fr)
Inventor
Laurent Ducoroy
Leena PINON
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Maco Pharma SAS
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Maco Pharma SAS
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Publication date
Application filed by Maco Pharma SAS filed Critical Maco Pharma SAS
Priority to EP19742528.3A priority Critical patent/EP3813999A1/en
Publication of WO2020002512A1 publication Critical patent/WO2020002512A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
    • B01J20/3217—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond
    • B01J20/3219—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond involving a particular spacer or linking group, e.g. for attaching an active group
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
    • B01J20/28023—Fibres or filaments
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B01J20/3255—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising a cyclic structure containing at least one of the heteroatoms nitrogen, oxygen or sulfur, e.g. heterocyclic or heteroaromatic structures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/3278—Polymers being grafted on the carrier

Definitions

  • the invention relates to a method of grafting a fibrous element for the removal of antibodies from the blood or of a blood component.
  • the invention applies to the field of filtration of blood or a blood component, and more generally to the field of blood transfusion.
  • Plasma is a constituent of the blood in which blood cells are suspended.
  • Blood plasma consists mainly of water, plasma proteins (including albumin and antibodies) and coagulation factors.
  • Plasma is used in therapy to treat in particular acute hemorrhages, for example after trauma.
  • the patient's blood group (A, B, AB or O) must be compatible with that of the donor in order to prevent the antibodies present in the donor's plasma, namely anti-A and / or anti-B antibodies attack the recipient's red blood cells.
  • contact of these antibodies with the recipient's red blood cells could lead to agglutination and / or hemolysis of the red blood cells, which could lead to the death of the recipient.
  • the document WO99 / 07390 proposes a method for obtaining a universal plasma by neutralization of the antibodies.
  • This universal plasma is obtained by mixing in particular between 6 and 10 units of group A plasma, between 1 and 3 units of group B plasma and between 0 and 1.5 units of group AB plasma.
  • the universal plasma thus obtained has an antibody titer of less than 16 for an anti A / anti B IgM and less than 64 for an anti A / anti B IgG.
  • Document WO2016 / 055647 also proposes a process for preparing a universal plasma by mixing non-universal plasmas, the process further comprising the elimination of anti-A and anti-B antibodies by immunoaffinity chromatography or by depletion in batch, then optionally lyophilization or atomization of the universal plasma.
  • the immunoaffinity chromatography step is carried out in particular on a column comprising crosslinked cellulose beads onto which are grafted, by means of a spacer, trisaccharides corresponding to an epitope of blood group A and / or B.
  • the column notably comprises particles of crosslinked agarose and activated by NHS (N-hydroxysuccinimide), linked via a spacer, to a saccharide such as a blood group determinant A or B.
  • NHS N-hydroxysuccinimide
  • WO2016 / 191691 describes a method for preparing a universal blood product by contacting whole blood or the plasma of a single donor or several donors, with hydroxyapatite, porous carbon particles and a matrix chemically associated with a blood group determinant A or B.
  • the matrix can be a bead, sheet or hollow fiber membrane.
  • Document EP2556848 proposes a separation material for eliminating in particular anti-A and / or anti-B antibodies from whole blood or from plasma, said material being of the saccharide-linker-matrix type.
  • the matrix is in the form of a ball, flat membrane or hollow fiber membrane.
  • the coupling reaction notably involves a matrix carrying amine, azide, carboxy, aldehyde, diimide, acetylene or epoxy functions with a functionalized saccharide with carboxyl function, amine, acetylene, azide, diimide, or hydroxy.
  • the coupling reaction is carried out in a single solvent or a mixture of solvents chosen from water, alcohols, DMSO, DMF, tBuOH, acetone or 1,4-dioxane.
  • oligosaccharides solubilized in an aqueous solution comprising a phosphate buffer or an MES buffer are coupled to balls or membranes of hollow fibers, in the presence of a EDC or diisopropylcarbodiimide (DIC) coupling agent
  • EDC ((1-ethyl-3- (3-dimethylaminopropyl) carbodiimide)
  • NHS N-hydroxysuccinimide
  • Document WO2016 / 177967 provides a purification support for the capture of anti-A and / or anti-B antibodies comprising a solid phase, which may be a particle or a fibrous matrix, onto which are grafted, by covalent bond, oligosaccharide molecules capable of binding to anti-A and / or anti-B antibodies.
  • the solid phase carries free aldehyde functions which can interact with the antibodies and stabilize their affinity binding to the purification support. For example, micronized cellulose is activated by the creation of aldehyde functions. A covalent bond is then produced between part of these aldehyde functions and amine functions of oligosaccharide molecules activated by the addition of an amine spacer molecule.
  • the oligosaccharide molecules are dissolved in water. It is further indicated that this grafting from aldehydes is easier to control than an epoxy amine grafting and simpler than that to a carboxylic acid involving EDC and / or NHS molecules.
  • the coupling reaction involving a carboxyl group and an amine function with EDC and / or NHS is well known, EDC and / or NHS being used to facilitate the formation of the amide bond.
  • This reaction is generally carried out in two stages in aqueous media.
  • the first step is to activate the carboxylic acid with a carbodiimide (such as EDC) to form an O-acylurea.
  • the second step is to react the O-acylurea with the amine to form the amide and urea as a secondary product.
  • additives such as NHS are conventionally used which will react with the O-acylurea to form a more stable active ester.
  • the first activation reaction of the carboxylic acid with EDC / NHS is more effective under acidic conditions (pH 4.6) and is generally carried out in the presence of a buffer solution such as the MES solution ( (2- [morpholino] ethanesulfonic acid)
  • MES solution (2- [morpholino] ethanesulfonic acid
  • the second amine coupling reaction is optimal at physiological pH (Thermo Scientific. "Crosslinking technical handbook.” Thermo Fisher Scientific Inc., Waltham (2012)).
  • the invention thus provides an industrial process for grafting oligosaccharides of blood group A determinant and / or blood group B determinant onto a fibrous element.
  • the invention provides a method of grafting a fibrous element for the elimination of antibodies from the blood or of a blood component, said fibrous element carrying carboxylic acid functions, said method providing impregnating a fibrous element carrying carboxylic acid functions with a grafting solution comprising at least one oligosaccharide derivative capable of binding to one or more antibodies, said oligosaccharide derivative carrying at least one amine function, so as to ensure covalent bridging by forming an amide bond between the oligosaccharide derivative and said fibrous element, the grafting solution comprising said oligosaccharide derivative solubilized in an organic solvent.
  • the invention provides a method of grafting a fibrous element for the removal of antibodies from the blood or a blood component.
  • the fibrous element is capable of removing antibodies from the blood or from a blood component, in particular anti-A and / or anti-B antibodies from the blood plasma in order to obtain a universal plasma.
  • the elimination of the antibodies is carried out in particular by filtration, that is to say by passage of the blood plasma through the fibrous element.
  • Universal plasma means plasma that can be transfused to a patient, regardless of blood type, without the risk of agglutination or hemolysis of the recipient's red blood cells.
  • an AB blood donor does not have anti-A or anti-B antibodies and therefore their plasma is already universal.
  • the fibrous element is capable of retaining the anti-B antibodies.
  • the fibrous element is capable of retaining anti-A antibodies.
  • the fibrous element is able to retain anti-A and anti-B antibodies.
  • the fibrous element is capable of retaining the anti-A and anti-B antibodies in order to obtain a universal plasma from any plasma or from a plasma of which the donor's blood group is not known.
  • Obtaining universal plasma has several advantages, including the elimination of the risk of errors related to incompatibility of blood groups between donor and recipient and the logistical simplification of hospitals.
  • the method allows the grafting of the fibrous element with at least one oligosaccharide derivative capable of binding to one or more antibodies, in particular capable of binding to anti-A and / or anti antibodies -B.
  • the grafting of the oligosaccharide derivative and of the fibrous element is ensured by covalent bridging by the formation of an amide bond between the oligosaccharide derivative and the fibrous element.
  • the fibrous element carries carboxylic acid functions and the oligosaccharide derivative capable of binding to one or more antibodies carries at least one primary amine function.
  • the fibrous element is in particular a porous element used in filters for leukoreducing blood or blood components, such as those described in document EP 1 336 417.
  • the fibrous element has an average pore size of between 5 and 15 ⁇ m, in particular between 8 and 10 ⁇ m.
  • the fibrous element comprises a basis weight between 20 and 80 g / m2, in particular between 40 and 60 g / m2.
  • the fibrous element is in the form of at least one layer of nonwoven, in particular a nonwoven of fibers blown in the molten state.
  • the fibrous element is made of a material biocompatible with blood and blood components.
  • the fibrous element is made of polyester such as polybutylene terephthalate or polyethylene terephthalate.
  • the fibrous element does not carry, or not enough, carboxylic acid functions, it is functionalized before grafting.
  • the fibrous element is, before the grafting step, treated with a gaseous plasma, in particular with oxygen, argon or a nitrogen / hydrogen mixture, so as to increase the number of carboxylic acid functions .
  • a gaseous plasma in particular with oxygen, argon or a nitrogen / hydrogen mixture, so as to increase the number of carboxylic acid functions .
  • the oligosaccharide derivative is capable of binding to one or more anti-A and / or anti-B antibodies, that is to say that it has an affinity for said antibodies.
  • the oligosaccharide derivative notably comprises an oligosaccharide and an amine function, separated by a link arm, also called a spacer.
  • the oligosaccharide is an antigenic oligosaccharide of a blood group determinant.
  • the oligosaccharide is advantageously a trisaccharide, tetrasaccharide, pentasaccharide or hexasaccharide.
  • antigenic oligosaccharides from a blood group determinant are:
  • the link arm between the oligosaccharide and the amine function especially comprises a triazole function and / or a thiol bond and / or a polyethylene glycol chain and / or one or more alkylene chains.
  • Such oligosaccharide derivatives are in particular commercially available.
  • the method of the invention is intended to be implemented on an industrial scale, that is to say it must meet the constraints of industrial production, in particular in terms of processing time, cost and efficiency.
  • the grafting process developed by the applicant reduces, and even eliminates, the presence of water in the process.
  • water has drawbacks for industrial use since water creates the risk of mold and bacterial contamination. The water must therefore be purified, which complicates the process.
  • water involves long drying times, often longer than 24 hours, which should be minimized.
  • the grafting process comprises impregnating the fibrous element carrying carboxylic acid functions with a grafting solution comprising said oligosaccharide derivative capable of binding to one or more antibodies and carrying at least one amine function, the grafting solution comprising said oligosaccharide derivative solubilized in an organic solvent.
  • organic solvent is meant a carbon-based substance capable of dissolving another substance.
  • Water is the solvent usually used to dissolve oligosaccharides. Surprisingly and unexpectedly, the Applicant has found that the oligosaccharide derivative is soluble in an organic solvent.
  • the organic solvent of the grafting solution is for example an alcoholic solvent such as methanol, ethanol or isopropanol, or a ketone such as acetone or methyl ethyl ketone, and in particular ethanol.
  • an alcoholic solvent such as methanol, ethanol or isopropanol
  • a ketone such as acetone or methyl ethyl ketone, and in particular ethanol.
  • the grafting solution does not contain any added water.
  • the grafting solution consists only of the oligosaccharide derivative and of the organic solvent, which is free of water or contains a minimal amount of water, i.e. less than 5% water .
  • the solvent is ethanol
  • the ethanol is pure ethanol or 96% ethanol.
  • the grafting solution is free of water and consists only of the oligosaccharide derivative and of an organic solvent free of water.
  • the grafting solution consists only of the oligosaccharide and ethanol derivative.
  • ethanol is advantageous in that the drying time of the fibrous element is reduced.
  • ethanol has a greater affinity for the fibrous element and makes the fibers swell more than water, potentially making the carboxylic acid functions of the fibrous element more accessible to a reagent.
  • the concentration of the oligosaccharide derivative in the grafting solution depends on the amount of carboxylic acid functions present on the fibrous element.
  • the molar ratio between the carboxylic acid functions and the amine functions of the oligosaccharide derivative is between 1: 1 and 1: 5, particularly of the order of 1: 1.
  • the carboxylic functions of the fibrous element are activated, that is to say made more reactive vis -with respect to the amine.
  • the activation of the carboxylic functions is carried out by transformation of the carboxylic acids into O-acylurea or active esters by a carbodiimide compound.
  • the O-acylurea is advantageously stabilized with a succinimide compound to form a stable active ester.
  • the fibrous element is impregnated with an activation solution comprising a carbodiimide compound or a mixture of a carbodiimide compound with a succinimide compound, so as to activate at least part of the carboxylic acid functions.
  • the carbodiiimide compound is chosen from dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC).
  • DCC dicyclohexylcarbodiimide
  • DIC diisopropylcarbodiimide
  • EDC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide
  • the succinimide compound is N-hydroxysuccinimide (NHS) or N-hydroxysulfosuccinimide (Sulfo-NHS).
  • the activation solution comprises a mixture of EDC and NHS.
  • the molar ratio between the carbodiimide compound and the succinimide compound is between 5: 1 and 1: 5, particularly of the order of 1: 1. More specifically, the molar ratio between EDC and NHS is about 1: 1.
  • the amount of the carbodiimide compound and the succinimide compound in the activation solution depends on the amount of carboxylic acid functions on the fibrous element.
  • the molar ratio between the carboxylic acid functions and the carbodiimide compound is between 1: 1 and 5: 1, particularly of the order of 1: 1.2. More particularly, the molar ratio between the carboxylic acid functions of the fibrous element and the EDC is of the order of 1: 1.2.
  • the molar ratio between the carboxylic acid functions, the EDC and the NHS is of the order of 1: 1.2: 1.2.
  • the activation solution comprises the carbodiimide compound or the mixture of the carbodiimide compound with the succinimide compound, dissolved in an organic solvent.
  • the organic solvent of the activation solution is for example an alcoholic solvent such as methanol, ethanol or isopropanol, or a ketone such as acetone or methyl ethyl ketone, and in particular ethanol.
  • an alcoholic solvent such as methanol, ethanol or isopropanol
  • a ketone such as acetone or methyl ethyl ketone, and in particular ethanol.
  • the organic solvent of the activation solution is the same as the organic solvent of the grafting solution, in particular ethanol.
  • the activation solution does not contain any added water.
  • the activation solution consists only of a carbodiimide compound or of a mixture of a carbodiimide compound with a succinimide compound solubilized in an organic solvent, which is free of water or contains a minimal amount of water, i.e. less than 5% water.
  • the organic solvent is ethanol
  • the ethanol is pure ethanol or 96% ethanol.
  • the activation solution is free of water.
  • the activation solution consists only of a carbodiimide compound or a mixture of a carbodiimide compound with a succinimide compound, dissolved in an organic solvent free of water.
  • the activation solution consists only of EDC, NHS and ethanol.
  • the activation solution comprises a carbodiimide compound or a mixture of a carbodiimide compound with a succinimide compound, dissolved in an organic solvent / water mixture, such as an ethanol / water mixture.
  • the volume ratio in percentage of the organic solvent / water is between 95/5 and 50/50, in particular 70/30.
  • the activation solution advantageously contains a buffer solution with an acidic pH (from 4 to 6), such as the MES solution, in order to promote the activation of the carboxylic functions.
  • the impregnation of the fibrous element carrying carboxylic acid functions with the activation solution is carried out prior to the impregnation of said fibrous element with said grafting solution.
  • the grafting reaction is monotopic, that is to say that the activation of the carboxylic functions and the amidation reaction are carried out in a single reaction mixture.
  • the activation solution and the grafting solution together form an impregnation solution which comprises a carbodiimide compound or a mixture of a carbodiimide compound with a succinimide compound and an oligosaccharide derivative, dissolved in an organic solvent .
  • the impregnation solution does not contain any added water.
  • the impregnation solution consists only of a carbodiimide compound or of a mixture of a carbodiimide compound with a succinimide compound; an oligosaccharide derivative; and an organic solvent which is free of water or contains a minimal amount of water, i.e. less than 5% water.
  • the organic solvent is ethanol
  • the ethanol is pure ethanol or 96% ethanol.
  • the impregnation solution is free of water and comprises only a carbodiimide compound or a mixture of a carbodiimide compound with a succinimide compound; an oligosaccharide derivative; and a water-free organic solvent.
  • the impregnation solution consists only of an oligosaccharide derivative, EDC, NHS and ethanol.
  • the impregnation of the fibrous element with the activation and / or grafting solution is carried out by padding, thus permitting the impregnation of the fibrous element in depth.
  • the fibrous element is soaked in continuous scrolling in the activation and / or grafting solution, as the case may be.
  • the surplus of the activation and / or grafting solution on the fibrous element is then expressed or wrung out by passing between two rollers whose pressure is between 1 and 5 bars.
  • the fibrous element is conveyed into an oven equipped with mechanical ventilation in order to dry it by evaporation of the solvent.
  • the speed, between 1 and 10 m / min, is regulated according to the nature and the amount of solvent carried by the fibrous element.
  • the impregnation is carried out by soaking in a bath of activating and / or grafting solution, as the case may be.
  • This embodiment allows longer contact times between the fibrous element and the activation or grafting solution, as the case may be.
  • the product obtained by the process of the invention is a fibrous element grafted with an oligosaccharide derivative, said fibrous element being intended to be used to remove antibodies, in particular anti-A and / or anti-B antibodies from the blood or a blood component such as a plasma.
  • the oligosaccharide derivative is covalently linked to the fibrous element.
  • the grafting rate of said fibrous element which corresponds to the concentration of oligosaccharide grafted for a given mass of fibrous element is between 0.05 and 10 mg / g, in particular between 0.2 and 5 mg / g, more particularly between 0.3 and 2 mg / g.
  • the grafting rate is determined by analysis of the constituent monosaccharides after total hydrolysis of the oligosaccharide, according to the method described in document WO2016 / 177967.
  • the fibrous element is grafted with an oligosaccharide derivative capable of binding with an anti-A antibody or with an oligosaccharide derivative capable of binding with an anti-B antibody.
  • the fibrous element is grafted with an oligosaccharide derivative capable of binding with an anti-A antibody and with an oligosaccharide derivative capable of binding with an anti-B antibody.
  • the fibrous element is a porous element used in filters to leukocyte the blood or the blood components and is capable of removing leukocytes and anti-A and / or anti-B antibodies from the blood or a blood component, such as plasma.
  • the fibrous element grafted with an oligosaccharide derivative according to the method of the invention is intended to be arranged in a filtration unit for the removal of antibodies from the blood or of a blood component.
  • said filtration unit comprises at least one fibrous element grafted with an oligosaccharide derivative according to the method of the invention.
  • the fibrous element is formed from at least one non-woven layer of meltblown fibers. More particularly, the fibrous element consists of a stack of several layers of nonwoven such as layers of nonwoven of fibers blown in the molten state. Each of the layers is grafted with an oligosaccharide derivative according to the process of the invention.
  • the filtration unit comprises an outer casing provided with at least one inlet port and at least one outlet port, the casing containing a fibrous element interposed between said orifices, said fibrous element being grafted with an oligosaccharide derivative according to the process of the invention.
  • the outer casing of the filtration unit is flexible, rigid or semi-rigid.
  • Said pocket system includes:
  • a filtration unit as described above comprising a fibrous element grafted according to the method of the invention
  • a bag for collecting the filtrate said bag being connected, via a tube, to an outlet orifice of the filtration unit.
  • the inlet opening of the filtration unit is connected, by means of a tube, to a connector intended to be connected to a bag containing the blood or the blood component to be filtered.
  • the inlet of the filtration unit is connected during manufacture, by means of a tube, to a bag intended to contain the blood or the blood component to be filtered.
  • the bag system comprising the filtration unit is sterilized, in particular by steam, ethylene oxide, gamma irradiation or beta irradiation.
  • the filtration process includes the following steps: passing the blood or the blood component through a filtration unit as described above and comprising a fibrous element grafted according to the method of the invention, - collect the filtrate in a filtrate collection bag.
  • the blood or blood component to be filtered is in particular plasma, and more particularly fresh frozen plasma, leukoreduced or not.
  • fresh frozen plasma is meant a labile blood product prepared either from whole blood or from plasma collected by apheresis, frozen at a temperature and within a time period compatible with the maintenance of the biological activity of the coagulation factors.
  • the plasma, leukoreduced or not is filtered during its preparation, that is to say before freezing.
  • PBT Polybutylene terephthalate
  • O2 gas plasma
  • the PBT layers have an average pore size between 8 and 10 ⁇ m, a grammage between 40 and 60 g / m2, and a filtration surface of about 25 cm2.
  • the grafting of oligosaccharide derivatives is carried out by impregnating the layers of PBT previously treated by gas plasma, in a bath or by padding.
  • the grafting solution consists of an oligosaccharide derivative capable of binding to anti-B antibodies (tetra B and hexa B from Elictyl) in an ethanol solvent.
  • oligosaccharide derivatives are as follows:
  • Gal ⁇ 1-3 (Fuc ⁇ 1-2) Gal ⁇ 1-3GlcNAc ⁇ 1-3Gal ⁇ 1-4Glc ⁇ -NAc-Spacer1-NH2 (6GrB1-Nac-sp1-NH2) (hexa B)
  • Gal ⁇ 1-3 (Fuc ⁇ 1-2) Gal ⁇ 1-4Glc ⁇ -NAc-Spacer1-NH2 (GLY038-3-NAc-sp1-NH2) (Tétra B)
  • the PBT layers are immersed for 2 hours in the activation solution, then 2 hours in the grafting solution.
  • the grafted PBT layers are dried in the open air for at least 24 hours.
  • the running speed of the PBT coil is around 1 m / min.
  • the grafting rate of the oligosaccharide derivative is then determined on the PBT.
  • the grafting rate corresponds to the concentration of grafted oligosaccharide for a given mass of PBT layer. It is evaluated by analysis of the constituent monosaccharides after total hydrolysis of the oligosaccharide, according to the method described in document WO2016 / 177967.
  • a filtration unit (rigid case) includes 14 layers of PBT grafted with oligosaccharide derivatives.
  • the filtration unit is mounted with a bag for collecting the filtrate and then the whole is sterilized by steam or by beta irradiation.
  • fresh frozen plasma (FFP) from a group A donor (with anti-B antibodies) is leukoreduced by filtration through a Miniplas filter (Macopharma, France).
  • the leukoreduced plasma is then passed three times through the same filtration unit using a pump (25 ml / min).
  • the antibody titer is then determined by agglutination test.
  • Plasma samples (before and after filtration) are diluted in PBS buffer, by successive dilution by a factor of 2 (1/1, 1/2, .. ,, 1/64) and brought into contact with red blood cells from the group B.
  • the title returned corresponds to the last positive dilution.

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  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Organic Chemistry (AREA)
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Abstract

The invention relates to a method for grafting a fibrous element for eliminating antibodies from blood or a blood component, said fibrous element having carboxylic acid functions, said method involving impregnating a fibrous element having carboxylic acid functions with a grafting solution comprising at least one oligosaccharide derivative that can bind to at least one antibody, said oligosaccharide derivative having at least one amine function, such as to ensure covalent bridging by formation of an amide bond between the oligosaccharide derivative and said fibrous element, the grafting solution comprising said oligosaccharide derivative solubilised in an organic solvent.

Description

Procédé de greffage d’un élément fibreux pour l’élimination d’anticorps du sang ou d’un composant sanguinMethod for grafting a fibrous element for the removal of antibodies from the blood or of a blood component

L’invention concerne un procédé de greffage d’un élément fibreux pour l’élimination d’anticorps du sang ou d’un composant sanguin.The invention relates to a method of grafting a fibrous element for the removal of antibodies from the blood or of a blood component.

L’invention s’applique au domaine de la filtration du sang ou d’un composant sanguin, et plus généralement au domaine de la transfusion sanguine.The invention applies to the field of filtration of blood or a blood component, and more generally to the field of blood transfusion.

Le plasma est un constituant du sang dans lequel les cellules sanguines sont en suspension. Le plasma sanguin comprend essentiellement de l’eau, des protéines plasmatiques (notamment de l’albumine et des anticorps) et des facteurs de coagulation.Plasma is a constituent of the blood in which blood cells are suspended. Blood plasma consists mainly of water, plasma proteins (including albumin and antibodies) and coagulation factors.

Le plasma est utilisé en thérapeutique pour traiter notamment les hémorragies aigues, par exemple après un traumatisme. Le groupe sanguin du patient (A, B, AB ou O) doit être compatible avec celui du donneur afin d’éviter que les anticorps présents dans le plasma du donneur, à savoir les anticorps anti-A et/ou anti-B n’attaquent les globules rouges du receveur. En effet, le contact de ces anticorps avec les globules rouges du receveur pourrait conduire à provoquer l’agglutination et/ou l’hémolyse des globules rouges, ce qui pourrait entraîner la mort du receveur.Plasma is used in therapy to treat in particular acute hemorrhages, for example after trauma. The patient's blood group (A, B, AB or O) must be compatible with that of the donor in order to prevent the antibodies present in the donor's plasma, namely anti-A and / or anti-B antibodies attack the recipient's red blood cells. In fact, contact of these antibodies with the recipient's red blood cells could lead to agglutination and / or hemolysis of the red blood cells, which could lead to the death of the recipient.

Afin de préparer un plasma dit universel, c’est-à-dire compatible avec tous les groupes sanguins, il est connu de neutraliser et/ou éliminer les anticorps anti-A anti-B du plasma.In order to prepare a so-called universal plasma, that is to say compatible with all blood groups, it is known to neutralize and / or eliminate the anti-A anti-B antibodies from the plasma.

Ainsi, le document WO99/07390 propose un procédé pour obtenir un plasma universel par neutralisation des anticorps. Ce plasma universel est obtenu en mélangeant notamment entre 6 et 10 unités de plasma de groupe A, entre 1 et 3 unités de plasma de groupe B et entre 0 et 1,5 unités de plasma de groupe AB. Le plasma universel ainsi obtenu présente un titre d’anticorps inférieur à 16 pour une IgM anti A/anti B et inférieur à 64 pour une IgG anti A/anti B.Thus, the document WO99 / 07390 proposes a method for obtaining a universal plasma by neutralization of the antibodies. This universal plasma is obtained by mixing in particular between 6 and 10 units of group A plasma, between 1 and 3 units of group B plasma and between 0 and 1.5 units of group AB plasma. The universal plasma thus obtained has an antibody titer of less than 16 for an anti A / anti B IgM and less than 64 for an anti A / anti B IgG.

Le document WO2016/055647 propose également un procédé de préparation d’un plasma universel par mélange de plasmas non-universels, le procédé comprenant en outre l’élimination des anticorps anti-A et anti-B par chromatographie d’immunoaffinité ou par déplétion en batch, puis éventuellement la lyophilisation ou l’atomisation du plasma universel. L’étape de chromatographie d’immunoaffinité est réalisée notamment sur une colonne comprenant des billes de cellulose réticulée sur lesquelles sont greffées, par l’intermédiaire d’un espaceur, des trisaccharides correspondant à un épitope du groupe sanguin A et/ou B.Document WO2016 / 055647 also proposes a process for preparing a universal plasma by mixing non-universal plasmas, the process further comprising the elimination of anti-A and anti-B antibodies by immunoaffinity chromatography or by depletion in batch, then optionally lyophilization or atomization of the universal plasma. The immunoaffinity chromatography step is carried out in particular on a column comprising crosslinked cellulose beads onto which are grafted, by means of a spacer, trisaccharides corresponding to an epitope of blood group A and / or B.

Dans le document US7700746, il est décrit une colonne utilisée pour réduire la quantité d’anticorps anti-A et anti-B dans un plasma sanguin. La colonne comprend notamment des particules d’agarose réticulé et activé par le NHS (N-hydroxysuccinimide), liées par l’intermédiaire d’un espaceur, à un saccharide tel qu’un déterminant du groupe sanguin A ou B.Document US7700746 describes a column used to reduce the amount of anti-A and anti-B antibodies in a blood plasma. The column notably comprises particles of crosslinked agarose and activated by NHS (N-hydroxysuccinimide), linked via a spacer, to a saccharide such as a blood group determinant A or B.

Ces deux derniers documents utilisent des colonnes chromatographiques pour l’élimination des anticorps anti-A et/ou anti-B. D’autres supports pour l’élimination des anticorps anti-A et/ou anti-B ont été envisagés.These last two documents use chromatographic columns for the elimination of anti-A and / or anti-B antibodies. Other supports for the elimination of anti-A and / or anti-B antibodies have been considered.

Le document WO2016/191691 décrit une méthode pour préparer un produit sanguin universel par contact du sang total ou du plasma d’un seul donneur ou de plusieurs donneurs, avec de l’hydroxyapatite, des particules de carbone poreuses et une matrice associée chimiquement à un déterminant du groupe sanguin A ou B. La matrice peut être une bille, une feuille ou une membrane à fibres creuses.WO2016 / 191691 describes a method for preparing a universal blood product by contacting whole blood or the plasma of a single donor or several donors, with hydroxyapatite, porous carbon particles and a matrix chemically associated with a blood group determinant A or B. The matrix can be a bead, sheet or hollow fiber membrane.

Le document EP2556848 propose un matériau de séparation pour éliminer notamment les anticorps anti-A et/ou anti-B du sang total ou du plasma, ledit matériau étant de type saccharide-lieur-matrice. La matrice est sous forme d’une bille, membrane plane ou membrane à fibres creuses. La réaction de couplage fait intervenir notamment une matrice porteuse de fonctions amine, azide, carboxy, aldéhyde, diimide, acetylène ou époxy avec un saccharide fonctionnalisé avec fonction carboxyle, amine, acetylène, azide, diimide, ou hydroxy. La réaction de couplage est réalisée dans un solvant unique ou un mélange de solvants choisis parmi l’eau, les alcools, le DMSO, le DMF, le tBuOH, l’acétone ou le 1,4-dioxane. Lorsque la réaction de couplage est réalisée par liaison d’un carboxyle avec une amine pour former une liaison amide, celle-ci est réalisée avec un carbodiimide tel que l’EDC ((1-éthyl-3-(3-diméthylaminopropyl)carbodiimide) et un additif tel que le NHS (N-hydroxysuccinimide). Dans certains exemples particuliers, des oligosaccharides solubilisés dans une solution aqueuse comprenant un tampon phosphate ou un tampon MES sont couplés à des billes ou des membranes de fibres creuses, en présence d’un agent de couplage EDC ou diisopropylcarbodiimide (DIC). La présence importante d’eau dans ce procédé rend difficile la mise à l’échelle industrielle de cette méthode de couplage.Document EP2556848 proposes a separation material for eliminating in particular anti-A and / or anti-B antibodies from whole blood or from plasma, said material being of the saccharide-linker-matrix type. The matrix is in the form of a ball, flat membrane or hollow fiber membrane. The coupling reaction notably involves a matrix carrying amine, azide, carboxy, aldehyde, diimide, acetylene or epoxy functions with a functionalized saccharide with carboxyl function, amine, acetylene, azide, diimide, or hydroxy. The coupling reaction is carried out in a single solvent or a mixture of solvents chosen from water, alcohols, DMSO, DMF, tBuOH, acetone or 1,4-dioxane. When the coupling reaction is carried out by bonding a carboxyl with an amine to form an amide bond, this is carried out with a carbodiimide such as EDC ((1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) and an additive such as NHS (N-hydroxysuccinimide). In certain specific examples, oligosaccharides solubilized in an aqueous solution comprising a phosphate buffer or an MES buffer are coupled to balls or membranes of hollow fibers, in the presence of a EDC or diisopropylcarbodiimide (DIC) coupling agent The large presence of water in this process makes it difficult to scale up this coupling method on an industrial scale.

Le document WO2016/177967 propose un support de purification pour la capture d’anticorps anti-A et/ou anti-B comprenant une phase solide, qui peut être une particule ou une matrice fibreuse, sur laquelle sont greffées, par liaison covalente, des molécules d’oligosaccharides capables de se lier aux anticorps anti-A et/ou anti-B. La phase solide est porteuse de fonctions aldéhyde libres qui peuvent interagir avec les anticorps et stabiliser leur liaison d'affinité au support de purification. Par exemple, de la cellulose micronisée est activée par la création de fonctions aldéhyde. Une liaison covalente est ensuite réalisée entre une partie de ces fonctions aldéhydes et des fonctions amines de molécules d’oligosaccharides activées par ajout d’une molécule espaceur amine. Les molécules d’oligosaccharides sont solubilisées dans l’eau. Il est en outre indiqué que ce greffage à partir d’aldéhydes est plus facile à contrôler qu’un greffage epoxy amine et plus simple que celui sur un acide carboxylique faisant intervenir des molécules EDC et/ou NHS.Document WO2016 / 177967 provides a purification support for the capture of anti-A and / or anti-B antibodies comprising a solid phase, which may be a particle or a fibrous matrix, onto which are grafted, by covalent bond, oligosaccharide molecules capable of binding to anti-A and / or anti-B antibodies. The solid phase carries free aldehyde functions which can interact with the antibodies and stabilize their affinity binding to the purification support. For example, micronized cellulose is activated by the creation of aldehyde functions. A covalent bond is then produced between part of these aldehyde functions and amine functions of oligosaccharide molecules activated by the addition of an amine spacer molecule. The oligosaccharide molecules are dissolved in water. It is further indicated that this grafting from aldehydes is easier to control than an epoxy amine grafting and simpler than that to a carboxylic acid involving EDC and / or NHS molecules.

La réaction de couplage faisant intervenir un groupe carboxyle et une fonction amine avec l’EDC et/ou NHS est bien connue, l’EDC et/ou NHS étant utilisés pour faciliter la formation de la liaison amide. Cette réaction est réalisée généralement en deux étapes en milieux aqueux. La première étape consiste à activer l’acide carboxylique avec un carbodiimide (tel que l’EDC) pour former une O-acylurée. La deuxième étape consiste à faire réagir la O-acylurée avec l’amine pour former l’amide et l’urée en produit secondaire. Lors de la première étape, afin de stabiliser l’O-acylurée, sensible à l’hydrolyse, on utilise classiquement des additifs tels que le NHS qui va réagir avec l’O-acylurée pour former un ester actif plus stable.The coupling reaction involving a carboxyl group and an amine function with EDC and / or NHS is well known, EDC and / or NHS being used to facilitate the formation of the amide bond. This reaction is generally carried out in two stages in aqueous media. The first step is to activate the carboxylic acid with a carbodiimide (such as EDC) to form an O-acylurea. The second step is to react the O-acylurea with the amine to form the amide and urea as a secondary product. In the first step, in order to stabilize the O-acylurea, which is sensitive to hydrolysis, additives such as NHS are conventionally used which will react with the O-acylurea to form a more stable active ester.

Le pH est l’un des paramètres important de la réaction de couplage avec l’EDC/NHS. En effet, la première réaction d’activation de l’acide carboxylique avec l’EDC/NHS est plus efficace dans des conditions acides (pH 4,6) et est réalisée généralement en présence d’une solution tampon telle que la solution MES (acide (2-[morpholino]éthanesulfonique). La deuxième réaction de couplage à l’amine est optimale à pH physiologique (Thermo Scientific. "Crosslinking technical handbook." Thermo Fisher Scientific Inc., Waltham (2012)).One of the important parameters of the coupling reaction with EDC / NHS is pH. Indeed, the first activation reaction of the carboxylic acid with EDC / NHS is more effective under acidic conditions (pH 4.6) and is generally carried out in the presence of a buffer solution such as the MES solution ( (2- [morpholino] ethanesulfonic acid) The second amine coupling reaction is optimal at physiological pH (Thermo Scientific. "Crosslinking technical handbook." Thermo Fisher Scientific Inc., Waltham (2012)).

Ces conditions particulières de réaction rendent la mise à l’échelle industrielle difficile à réaliser. L’invention propose ainsi un procédé industriel de greffage d’oligosaccharides de déterminant de groupe sanguin A et/ou de déterminant de groupe sanguin B sur un élément fibreux.These particular reaction conditions make it difficult to scale up on an industrial scale. The invention thus provides an industrial process for grafting oligosaccharides of blood group A determinant and / or blood group B determinant onto a fibrous element.

A cet effet et selon un premier aspect, l’invention propose un procédé de greffage d’un élément fibreux pour l’élimination d’anticorps du sang ou d’un composant sanguin, ledit élément fibreux portant des fonctions acides carboxyliques, ledit procédé prévoyant d’imprégner un élément fibreux porteur de fonctions acides carboxyliques avec une solution de greffage comprenant au moins un dérivé d’oligosaccharide capable de se lier à un ou plusieurs anticorps, ledit dérivé d’oligosaccharide portant au moins une fonction amine, de sorte à assurer un pontage covalent par formation d’une liaison amide entre le dérivé d’oligosaccharide et ledit élément fibreux, la solution de greffage comprenant ledit dérivé d’oligosaccharide solubilisé dans un solvant organique.To this end and according to a first aspect, the invention provides a method of grafting a fibrous element for the elimination of antibodies from the blood or of a blood component, said fibrous element carrying carboxylic acid functions, said method providing impregnating a fibrous element carrying carboxylic acid functions with a grafting solution comprising at least one oligosaccharide derivative capable of binding to one or more antibodies, said oligosaccharide derivative carrying at least one amine function, so as to ensure covalent bridging by forming an amide bond between the oligosaccharide derivative and said fibrous element, the grafting solution comprising said oligosaccharide derivative solubilized in an organic solvent.

D'autres objets et avantages apparaîtront au cours de la description qui suit.Other objects and advantages will become apparent from the following description.

L’invention propose un procédé de greffage d’un élément fibreux pour l’élimination d’anticorps du sang ou d’un composant sanguin.The invention provides a method of grafting a fibrous element for the removal of antibodies from the blood or a blood component.

L’élément fibreux est apte à éliminer des anticorps du sang ou d’un composant sanguin, notamment les anticorps anti-A et/ou anti-B du plasma sanguin afin d’obtenir un plasma universel. L’élimination des anticorps est réalisée notamment par filtration, c’est-à-dire par passage du plasma sanguin au travers de l’élément fibreux.The fibrous element is capable of removing antibodies from the blood or from a blood component, in particular anti-A and / or anti-B antibodies from the blood plasma in order to obtain a universal plasma. The elimination of the antibodies is carried out in particular by filtration, that is to say by passage of the blood plasma through the fibrous element.

Par « plasma universel », on entend un plasma qui peut être transfusé à un patient, quel que soit son groupe sanguin, sans risque d’agglutination ou d’hémolyse des globules rouges du receveur. Par exemple, un donneur de groupe sanguin AB ne possède pas d’anticorps anti-A ni anti-B et son plasma est donc déjà universel."Universal plasma" means plasma that can be transfused to a patient, regardless of blood type, without the risk of agglutination or hemolysis of the recipient's red blood cells. For example, an AB blood donor does not have anti-A or anti-B antibodies and therefore their plasma is already universal.

En particulier, pour obtenir un plasma universel à partir d’un plasma d’un donneur de groupe sanguin A qui possède des anticorps anti-B, l’élément fibreux est apte à retenir les anticorps anti-B.In particular, to obtain a universal plasma from a plasma of a blood group A donor which has anti-B antibodies, the fibrous element is capable of retaining the anti-B antibodies.

Pour obtenir un plasma universel à partir d’un plasma d’un donneur de groupe sanguin B qui possède des anticorps anti-A, l’élément fibreux est apte à retenir les anticorps anti-A.To obtain a universal plasma from a plasma of a blood group B donor which has anti-A antibodies, the fibrous element is capable of retaining anti-A antibodies.

Pour obtenir un plasma universel à partir d’un plasma d’un donneur de groupe sanguin O qui possède des anticorps anti-A et anti-B, l’élément fibreux est apte à retenir les anticorps anti-A et anti-B.To obtain a universal plasma from a plasma of a blood group O donor which has anti-A and anti-B antibodies, the fibrous element is able to retain anti-A and anti-B antibodies.

Avantageusement, l’élément fibreux est apte à retenir les anticorps anti-A et anti-B pour obtenir un plasma universel à partir de n’importe quel plasma ou d’un plasma dont on ne connaît pas le groupe sanguin du donneur.Advantageously, the fibrous element is capable of retaining the anti-A and anti-B antibodies in order to obtain a universal plasma from any plasma or from a plasma of which the donor's blood group is not known.

L’obtention d’un plasma universel présente plusieurs avantages, parmi lesquels l’élimination du risque d’erreurs lié à l’incompatibilité des groupes sanguins entre le donneur et le receveur et la simplification logistique des hôpitaux.Obtaining universal plasma has several advantages, including the elimination of the risk of errors related to incompatibility of blood groups between donor and recipient and the logistical simplification of hospitals.

Dans le cadre de l’invention, le procédé permet le greffage de l’élément fibreux avec au moins un dérivé d’oligosaccharide capable de se lier à un ou plusieurs anticorps, notamment capable de se lier aux anticorps anti-A et/ou anti-B.In the context of the invention, the method allows the grafting of the fibrous element with at least one oligosaccharide derivative capable of binding to one or more antibodies, in particular capable of binding to anti-A and / or anti antibodies -B.

Le greffage du dérivé d’oligosaccharide et de l’élément fibreux est assuré par un pontage covalent par formation d’une liaison amide entre le dérivé d’oligosaccharide et l’élément fibreux.The grafting of the oligosaccharide derivative and of the fibrous element is ensured by covalent bridging by the formation of an amide bond between the oligosaccharide derivative and the fibrous element.

Pour ce faire, l’élément fibreux porte des fonctions acides carboxyliques et le dérivé d’oligosaccharide capable de se lier à un ou plusieurs anticorps porte au moins une fonction amine primaire.To do this, the fibrous element carries carboxylic acid functions and the oligosaccharide derivative capable of binding to one or more antibodies carries at least one primary amine function.

L’élément fibreux est en particulier un élément poreux utilisé dans les filtres à déleucocyter le sang ou les composants sanguins, tel que ceux décrits dans le document EP 1 336 417.The fibrous element is in particular a porous element used in filters for leukoreducing blood or blood components, such as those described in document EP 1 336 417.

Notamment, l’élément fibreux présente une taille moyenne de pores comprise entre 5 et 15 µm, notamment entre 8 e 10 µm.In particular, the fibrous element has an average pore size of between 5 and 15 μm, in particular between 8 and 10 μm.

En outre, l’élément fibreux comprend un grammage compris entre 20 et 80 g/m², notamment compris entre 40 et 60 g/m².In addition, the fibrous element comprises a basis weight between 20 and 80 g / m², in particular between 40 and 60 g / m².

Par exemple, l’élément fibreux est sous forme d’au moins une couche de non-tissé, notamment un non-tissé de fibres soufflées à l'état fondu.For example, the fibrous element is in the form of at least one layer of nonwoven, in particular a nonwoven of fibers blown in the molten state.

L’élément fibreux est réalisé en un matériau biocompatible avec le sang et les composants sanguins. Par exemple, l’élément fibreux est réalisé en polyester tel que le polybutylène téréphtalate ou le polyéthylène téréphtalate.The fibrous element is made of a material biocompatible with blood and blood components. For example, the fibrous element is made of polyester such as polybutylene terephthalate or polyethylene terephthalate.

Si l’élément fibreux ne porte pas, ou pas suffisamment, de fonctions acides carboxyliques, il est fonctionnalisé avant greffage.If the fibrous element does not carry, or not enough, carboxylic acid functions, it is functionalized before grafting.

Par exemple, l’élément fibreux est, avant l’étape de greffage, traité avec un plasma gazeux, notamment avec de l’oxygène, de l’argon ou un mélange azote/hydrogène, de sorte à augmenter le nombre de fonctions acides carboxyliques.For example, the fibrous element is, before the grafting step, treated with a gaseous plasma, in particular with oxygen, argon or a nitrogen / hydrogen mixture, so as to increase the number of carboxylic acid functions .

Le dérivé d’oligosaccharide est capable de se lier à un ou plusieurs anticorps anti-A et/ou anti-B, c’est-à-dire qu’il présente une affinité pour lesdits anticorps.The oligosaccharide derivative is capable of binding to one or more anti-A and / or anti-B antibodies, that is to say that it has an affinity for said antibodies.

Le dérivé d’oligosaccharide comprend notamment un oligosaccharide et une fonction amine, séparés par un bras de liaison, dit aussi espaceur.The oligosaccharide derivative notably comprises an oligosaccharide and an amine function, separated by a link arm, also called a spacer.

L’oligosaccharide est un oligosaccharide antigénique d’un déterminant d’un groupe sanguin. L’oligosaccharide est avantageusement un trisaccharide, tétrasaccharide, pentasaccharide ou hexasaccharide.The oligosaccharide is an antigenic oligosaccharide of a blood group determinant. The oligosaccharide is advantageously a trisaccharide, tetrasaccharide, pentasaccharide or hexasaccharide.

Des exemples de tels oligosaccharides antigéniques d’un déterminant d’un groupe sanguins sont :Examples of such antigenic oligosaccharides from a blood group determinant are:

Pour les oligosaccharides antigéniques du groupe A : GalNAcα1-3(Fucα1-2)Gal, GalNAcα-3(Fucα-2)Galβ-3GlcNAc, GalNAcα1-3(Fucα1-2)Galβ1-3GlcNAcβ1-3Gal, GalNAcα1-3(Fucα1-2)Galβ1-3GlcNAcβ1-3-Lac, GalNAcα1-3(Fucα1-2)Galβ1-4GlcNAc, GalNAcα1-3(Fucα1-2)Galβ1-4GlcNAcβ1-3Gal, GalNAcα1-3(Fucα1-2)Galβ1-4GlcNAcβ1-3-Lac, GalNAcα1-3(Fucα1-2)Galβ1-3GalNAcβ1-3Gal et GalNAcα1-3(Fucα1-2)Galβ1-4Glc.For group A antigenic oligosaccharides: GalNAcα1-3 (Fucα1-2) Gal, GalNAcα-3 (Fucα-2) Galβ-3GlcNAc, GalNAcα1-3 (Fucα1-2) Galβ1-3GlcNAcβ1-3Gal, GalNAcα1-3 (Fucα1- 2) Galβ1-3GlcNAcβ1-3-Lac, GalNAcα1-3 (Fucα1-2) Galβ1-4GlcNAc, GalNAcα1-3 (Fucα1-2) Galβ1-4GlcNAcβ1-3Gal, GalNAcα1-3 (Fucα1-2) Galβ1-4GlcNAcβ1-3- Lac, GalNAcα1-3 (Fucα1-2) Galβ1-3GalNAcβ1-3Gal and GalNAcα1-3 (Fucα1-2) Galβ1-4Glc.

Pour les oligosaccharides antigéniques du groupe B :For group B antigenic oligosaccharides:

Galα1-3(Fucα1-2)Galβ1-3GlcNAc, Galα1-3(Fucα1-2)Galβ1-3GlcNAcβ1-3Gal, Galα1-3(Fucα1-2)Galβ1-3GlcNAcβ1-3-Lac, Galα1-3(Fucα1-2)Galβ1-4GlcNAc, Galα1-3(Fucα1-2)Galβ1-4GlcNAcβ1-3Gal, Galα1-3(Fucα1-2)Galβ1-4GlcNAcβ1-3-Lac, Galα1-3(Fucα1-2)Galβ1-3GalNAcβ1-3Gal et Galα1-3(Fucα1-2)Galβ1-4Glc.Galα1-3 (Fucα1-2) Galβ1-3GlcNAc, Galα1-3 (Fucα1-2) Galβ1-3GlcNAcβ1-3Gal, Galα1-3 (Fucα1-2) Galβ1-3GlcNAcβ1-3-Lac, Galα1-3 (Fucα1-2) Galβ1-4GlcNAc, Galα1-3 (Fucα1-2) Galβ1-4GlcNAcβ1-3Gal, Galα1-3 (Fucα1-2) Galβ1-4GlcNAcβ1-3-Lac, Galα1-3 (Fucα1-2) Galβ1-3GalNAcβ1-3Gal and Galα1- 3 (Fucα1-2) Galβ1-4Glc.

Le bras de liaison entre l’oligosaccharide et la fonction amine comporte notamment une fonction triazole et/ou une liaison thiol et/ou une chaîne polyéthylène glycol et/ou une ou plusieurs chaînes alkylène.The link arm between the oligosaccharide and the amine function especially comprises a triazole function and / or a thiol bond and / or a polyethylene glycol chain and / or one or more alkylene chains.

De tels dérivés d’oligosaccharide sont notamment disponibles dans le commerce.Such oligosaccharide derivatives are in particular commercially available.

Le procédé de l’invention est destiné à être mis en œuvre à l’échelle industrielle, c’est-à-dire qu’il doit répondre aux contraintes de production industrielle, notamment en terme de temps de traitement, de coût et d’efficacité.The method of the invention is intended to be implemented on an industrial scale, that is to say it must meet the constraints of industrial production, in particular in terms of processing time, cost and efficiency.

A cet effet, le procédé de greffage mis au point par la demanderesse réduit, et même élimine, la présence d’eau dans le procédé. En effet, l’eau présente des inconvénients pour une utilisation industrielle puisque l’eau engendre un risque de moisissure et de contamination bactérienne. L’eau doit ainsi être purifiée ce qui complexifie le procédé. En outre, l’eau implique de longs temps de séchage, souvent supérieurs à 24 heures, qu’il convient de minimiser.To this end, the grafting process developed by the applicant reduces, and even eliminates, the presence of water in the process. In fact, water has drawbacks for industrial use since water creates the risk of mold and bacterial contamination. The water must therefore be purified, which complicates the process. In addition, water involves long drying times, often longer than 24 hours, which should be minimized.

Le procédé de greffage comprend l’imprégnation de l’élément fibreux portant des fonctions acides carboxyliques avec une solution de greffage comprenant ledit dérivé d’oligosaccharide capable de se lier à un ou plusieurs anticorps et portant au moins une fonction amine, la solution de greffage comprenant ledit dérivé d’oligosaccharide solubilisé dans un solvant organique.The grafting process comprises impregnating the fibrous element carrying carboxylic acid functions with a grafting solution comprising said oligosaccharide derivative capable of binding to one or more antibodies and carrying at least one amine function, the grafting solution comprising said oligosaccharide derivative solubilized in an organic solvent.

Par solvant organique, on entend une substance à base de carbone capable de dissoudre une autre substance.By organic solvent is meant a carbon-based substance capable of dissolving another substance.

L’eau est le solvant habituellement utilisé pour solubiliser des oligosaccharides. De façon surprenante et inattendue, la demanderesse a trouvé que le dérivé d’oligosaccharide est soluble dans un solvant organique.Water is the solvent usually used to dissolve oligosaccharides. Surprisingly and unexpectedly, the Applicant has found that the oligosaccharide derivative is soluble in an organic solvent.

Le solvant organique de la solution de greffage est par exemple un solvant alcoolique tel que le méthanol, l’éthanol ou l’isopropanol, ou une cétone tel que l’acétone ou la méthyléthylcétone, et notamment l’éthanol.The organic solvent of the grafting solution is for example an alcoholic solvent such as methanol, ethanol or isopropanol, or a ketone such as acetone or methyl ethyl ketone, and in particular ethanol.

Dans une réalisation, la solution de greffage ne contient pas d’eau ajoutée. Dans ce cas, la solution de greffage est constituée uniquement du dérivé d’oligosaccharide et du solvant organique, lequel est exempt d’eau ou contient une quantité minime d’eau, c’est-à-dire moins de 5% d’eau.In one embodiment, the grafting solution does not contain any added water. In this case, the grafting solution consists only of the oligosaccharide derivative and of the organic solvent, which is free of water or contains a minimal amount of water, i.e. less than 5% water .

Par exemple, lorsque le solvant est l’éthanol, l’éthanol est de l’éthanol pur ou de l’éthanol à 96%.For example, when the solvent is ethanol, the ethanol is pure ethanol or 96% ethanol.

Avantageusement, la solution de greffage est exempte d’eau et est constituée uniquement du dérivé d’oligosaccharide et d’un solvant organique exempt d’eau.Advantageously, the grafting solution is free of water and consists only of the oligosaccharide derivative and of an organic solvent free of water.

Par exemple, la solution de greffage est constituée uniquement du dérivé d’oligosaccharide et d’éthanol. L’utilisation de l’éthanol est avantageuse en ce que le temps de séchage de l’élément fibreux est réduit. En outre, l’éthanol a une plus grande affinité avec l’élément fibreux et fait plus gonfler les fibres que l’eau, rendant potentiellement les fonctions acides carboxyliques de l’élément fibreux plus accessibles à un réactif.For example, the grafting solution consists only of the oligosaccharide and ethanol derivative. The use of ethanol is advantageous in that the drying time of the fibrous element is reduced. In addition, ethanol has a greater affinity for the fibrous element and makes the fibers swell more than water, potentially making the carboxylic acid functions of the fibrous element more accessible to a reagent.

La concentration du dérivé d’oligosaccharide dans la solution de greffage dépend de la quantité de fonctions acides carboxyliques présentes sur l’élément fibreux.The concentration of the oligosaccharide derivative in the grafting solution depends on the amount of carboxylic acid functions present on the fibrous element.

Avantageusement, le rapport molaire entre les fonctions acides carboxyliques et les fonctions amines du dérivé d’oligosaccharide est compris entre 1:1 et 1:5, particulièrement de l’ordre de 1:1.Advantageously, the molar ratio between the carboxylic acid functions and the amine functions of the oligosaccharide derivative is between 1: 1 and 1: 5, particularly of the order of 1: 1.

Afin de faciliter la réaction d’amidation entre l’élément fibreux portant des fonctions carboxyliques et le dérivé d’oligosaccharide portant une fonction amine, les fonctions carboxyliques de l’élément fibreux sont activées, c’est-à-dire rendues plus réactives vis-à-vis de l’amine.In order to facilitate the amidation reaction between the fibrous element carrying carboxylic functions and the oligosaccharide derivative carrying an amine function, the carboxylic functions of the fibrous element are activated, that is to say made more reactive vis -with respect to the amine.

Selon une réalisation, l’activation des fonctions carboxyliques est réalisée par transformation des acides carboxyliques en O-acylurée ou esters actifs par un composé carbodiimides. L’O-acylurée est avantageusement stabilisée avec un composé succinimide pour former un ester actif stable.According to one embodiment, the activation of the carboxylic functions is carried out by transformation of the carboxylic acids into O-acylurea or active esters by a carbodiimide compound. The O-acylurea is advantageously stabilized with a succinimide compound to form a stable active ester.

Dans cette réalisation, l’élément fibreux est imprégné avec une solution d’activation comprenant un composé carbodiimide ou un mélange d’un composé carbodiimide avec un composé succinimide, de sorte à activer au moins une partie des fonctions acides carboxyliques.In this embodiment, the fibrous element is impregnated with an activation solution comprising a carbodiimide compound or a mixture of a carbodiimide compound with a succinimide compound, so as to activate at least part of the carboxylic acid functions.

Particulièrement, le composé carbodiiimide est choisi parmi le dicyclohexylcarbodiimide (DCC), le diisopropylcarbodiimide (DIC) et le 1-éthyl-3-(3-diméthylaminopropyl)carbodiimide (EDC).In particular, the carbodiiimide compound is chosen from dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC).

Particulièrement, le composé succinimide est le N-hydroxysuccinimide (NHS) ou le N-hydroxysulfosuccinimide (Sulfo-NHS).In particular, the succinimide compound is N-hydroxysuccinimide (NHS) or N-hydroxysulfosuccinimide (Sulfo-NHS).

Encore plus particulièrement, la solution d’activation comprend un mélange d’EDC et de NHS.Even more particularly, the activation solution comprises a mixture of EDC and NHS.

Le rapport molaire entre le composé carbodiimide et le composé succinimide est compris entre 5:1 et 1:5, particulièrement de l’ordre de 1:1. Plus particulièrement, le rapport molaire entre l’EDC et le NHS est de l’ordre de 1 :1.The molar ratio between the carbodiimide compound and the succinimide compound is between 5: 1 and 1: 5, particularly of the order of 1: 1. More specifically, the molar ratio between EDC and NHS is about 1: 1.

La quantité du composé carbodiimide et du composé succinimide dans la solution d’activation dépend de la quantité de fonctions acides carboxyliques sur l’élément fibreux.The amount of the carbodiimide compound and the succinimide compound in the activation solution depends on the amount of carboxylic acid functions on the fibrous element.

Avantageusement, le rapport molaire entre les fonctions acides carboxyliques et le composé carbodiimide est compris entre 1:1 et 5:1, particulièrement de l’ordre de 1:1,2. Plus particulièrement, le rapport molaire entre les fonctions acides carboxyliques de l’élément fibreux et l’EDC est de l’ordre de 1:1,2.Advantageously, the molar ratio between the carboxylic acid functions and the carbodiimide compound is between 1: 1 and 5: 1, particularly of the order of 1: 1.2. More particularly, the molar ratio between the carboxylic acid functions of the fibrous element and the EDC is of the order of 1: 1.2.

Encore plus particulièrement, le rapport molaire entre les fonctions acides carboxyliques, l’EDC et le NHS est de l’ordre de 1 :1,2 :1,2.Even more particularly, the molar ratio between the carboxylic acid functions, the EDC and the NHS is of the order of 1: 1.2: 1.2.

Selon une réalisation, la solution d’activation comprend le composé carbodiimide ou le mélange du composé carbodiimide avec le composé succinimide, solubilisé dans un solvant organique.According to one embodiment, the activation solution comprises the carbodiimide compound or the mixture of the carbodiimide compound with the succinimide compound, dissolved in an organic solvent.

Le solvant organique de la solution d’activation est par exemple un solvant alcoolique tel que le méthanol, l’éthanol ou l’isopropanol, ou une cétone tel que l’acétone ou la méthyléthylcétone, et notamment l’éthanol.The organic solvent of the activation solution is for example an alcoholic solvent such as methanol, ethanol or isopropanol, or a ketone such as acetone or methyl ethyl ketone, and in particular ethanol.

Avantageusement, le solvant organique de la solution d’activation est le même que le solvant organique de la solution de greffage, notamment l’éthanol.Advantageously, the organic solvent of the activation solution is the same as the organic solvent of the grafting solution, in particular ethanol.

En particulier, la solution d’activation ne contient pas d’eau ajoutée. Dans ce cas, la solution d’activation est constituée uniquement d’un composé carbodiimide ou d’un mélange d’un composé carbodiimide avec un composé succinimide solubilisé dans un solvant organique, lequel est exempt d’eau ou contient une quantité minime d’eau, c’est-à-dire moins de 5% d’eau.In particular, the activation solution does not contain any added water. In this case, the activation solution consists only of a carbodiimide compound or of a mixture of a carbodiimide compound with a succinimide compound solubilized in an organic solvent, which is free of water or contains a minimal amount of water, i.e. less than 5% water.

Par exemple, lorsque le solvant organique est l’éthanol, l’éthanol est de l’éthanol pur ou de l’éthanol à 96%.For example, when the organic solvent is ethanol, the ethanol is pure ethanol or 96% ethanol.

Avantageusement, la solution d’activation est exempte d’eau. La solution d’activation est constituée uniquement d’un composé carbodiimide ou d’un mélange d’un composé carbodiimide avec un composé succinimide, solubilisé dans un solvant organique exempt d’eau.Advantageously, the activation solution is free of water. The activation solution consists only of a carbodiimide compound or a mixture of a carbodiimide compound with a succinimide compound, dissolved in an organic solvent free of water.

Plus particulièrement, la solution d’activation est constituée uniquement d’EDC, de NHS et d’éthanol.More specifically, the activation solution consists only of EDC, NHS and ethanol.

En variante, la solution d’activation comprend un composé carbodiimide ou un mélange d’un composé carbodiimide avec un composé succinimide, solubilisé dans un mélange solvant organique/eau, tel que un mélange éthanol/eau.Alternatively, the activation solution comprises a carbodiimide compound or a mixture of a carbodiimide compound with a succinimide compound, dissolved in an organic solvent / water mixture, such as an ethanol / water mixture.

Par exemple, le rapport volumique en pourcentage du solvant organique/eau est compris entre 95/5 et 50/50, notamment 70/30.For example, the volume ratio in percentage of the organic solvent / water is between 95/5 and 50/50, in particular 70/30.

Dans cette variante, la solution d’activation contient avantageusement une solution tampon à pH acide (de 4 à 6), telle que la solution MES, afin de favoriser l’activation des fonctions carboxyliques.In this variant, the activation solution advantageously contains a buffer solution with an acidic pH (from 4 to 6), such as the MES solution, in order to promote the activation of the carboxylic functions.

Dans une réalisation particulière, l’imprégnation de l’élément fibreux porteur de fonctions acides carboxyliques avec la solution d’activation est réalisée préalablement à l’imprégnation dudit élément fibreux avec ladite solution de greffage.In a particular embodiment, the impregnation of the fibrous element carrying carboxylic acid functions with the activation solution is carried out prior to the impregnation of said fibrous element with said grafting solution.

Dans une variante de réalisation, la réaction de greffage est monotope, c’est-à-dire que l’activation des fonctions carboxyliques et la réaction d’amidation sont réalisées dans un seul mélange réactionnel. Dans ce cas, la solution d’activation et la solution de greffage forment ensemble une solution d’imprégnation qui comprend un composé carbodiimide ou un mélange d’un composé carbodiimide avec un composé succinimide et un dérivé d’oligosaccharide, solubilisés dans un solvant organique.In an alternative embodiment, the grafting reaction is monotopic, that is to say that the activation of the carboxylic functions and the amidation reaction are carried out in a single reaction mixture. In this case, the activation solution and the grafting solution together form an impregnation solution which comprises a carbodiimide compound or a mixture of a carbodiimide compound with a succinimide compound and an oligosaccharide derivative, dissolved in an organic solvent .

En particulier, la solution d’imprégnation ne contient pas d’eau ajoutée. Dans ce cas, la solution d’imprégnation est constituée uniquement d’un composé carbodiimide ou d’un mélange d’un composé carbodiimide avec un composé succinimide; d’un dérivé d’oligosaccharide ; et d’un solvant organique lequel est exempt d’eau ou contient une quantité minime d’eau, c’est-à-dire moins de 5% d’eau.In particular, the impregnation solution does not contain any added water. In this case, the impregnation solution consists only of a carbodiimide compound or of a mixture of a carbodiimide compound with a succinimide compound; an oligosaccharide derivative; and an organic solvent which is free of water or contains a minimal amount of water, i.e. less than 5% water.

Par exemple, lorsque le solvant organique est l’éthanol, l’éthanol est de l’éthanol pur ou de l’éthanol à 96%.For example, when the organic solvent is ethanol, the ethanol is pure ethanol or 96% ethanol.

Avantageusement, la solution d’imprégnation est exempte d’eau et comprend uniquement un composé carbodiimide ou un mélange d’un composé carbodiimide avec un composé succinimide; un dérivé d’oligosaccharide ; et un solvant organique exempt d’eau.Advantageously, the impregnation solution is free of water and comprises only a carbodiimide compound or a mixture of a carbodiimide compound with a succinimide compound; an oligosaccharide derivative; and a water-free organic solvent.

Par exemple, la solution d’imprégnation est constituée uniquement d’un dérivé d’oligosaccharide, d’EDC, de NHS et d’éthanol.For example, the impregnation solution consists only of an oligosaccharide derivative, EDC, NHS and ethanol.

Il existe différentes techniques d’imprégnation d’un élément fibreux avec une solution. Ces techniques comprennent le trempage, le foulardage ou la pulvérisation.There are different techniques for impregnating a fibrous element with a solution. These techniques include soaking, padding or spraying.

Selon une réalisation, l’imprégnation de l’élément fibreux avec la solution d’activation et/ou de greffage est réalisée par foulardage, permettant ainsi d’imprégner l’élément fibreux en profondeur.According to one embodiment, the impregnation of the fibrous element with the activation and / or grafting solution is carried out by padding, thus permitting the impregnation of the fibrous element in depth.

Dans cette réalisation, l’élément fibreux est trempé en défilement continu dans la solution d’activation et/ou de greffage, selon le cas. Le surplus de la solution d’activation et/ou de greffage sur l’élément fibreux est ensuite exprimé ou essoré en passant entre deux rouleaux dont la pression est comprise entre 1 et 5 bars. Puis l’élément fibreux est convoyé dans un four équipé d’une ventilation mécanique afin de le sécher par évaporation du solvant. La vitesse, comprise entre 1 et 10 m/min, est régulée en fonction de la nature et de la quantité de solvant emportée par l’élément fibreux.In this embodiment, the fibrous element is soaked in continuous scrolling in the activation and / or grafting solution, as the case may be. The surplus of the activation and / or grafting solution on the fibrous element is then expressed or wrung out by passing between two rollers whose pressure is between 1 and 5 bars. Then the fibrous element is conveyed into an oven equipped with mechanical ventilation in order to dry it by evaporation of the solvent. The speed, between 1 and 10 m / min, is regulated according to the nature and the amount of solvent carried by the fibrous element.

Selon une autre réalisation, l’imprégnation est réalisée par trempage dans un bain de solution d’activation et/ou de greffage, selon le cas. Cette réalisation permet des temps de contact plus long entre l’élément fibreux et la solution d’activation ou de greffage, selon le cas.According to another embodiment, the impregnation is carried out by soaking in a bath of activating and / or grafting solution, as the case may be. This embodiment allows longer contact times between the fibrous element and the activation or grafting solution, as the case may be.

Le produit obtenu par le procédé de l’invention est un élément fibreux greffé avec un dérivé d’oligosaccharide, ledit élément fibreux étant destiné à être utilisé pour éliminer des anticorps, notamment les anticorps anti-A et/ou anti-B du sang ou d’un composant sanguin tel qu’un plasma.The product obtained by the process of the invention is a fibrous element grafted with an oligosaccharide derivative, said fibrous element being intended to be used to remove antibodies, in particular anti-A and / or anti-B antibodies from the blood or a blood component such as a plasma.

En mettant en œuvre le procédé de l’invention, le dérivé d’oligosaccharide est lié de façon covalente à l’élément fibreux.By implementing the method of the invention, the oligosaccharide derivative is covalently linked to the fibrous element.

Le taux de greffage dudit élément fibreux, qui correspond à la concentration d’oligosaccharide greffé pour une masse donnée d’élément fibreux est compris entre 0,05 et 10 mg/g, en particulier entre 0,2 et 5 mg/g, plus particulièrement entre 0,3 et 2 mg/g.The grafting rate of said fibrous element, which corresponds to the concentration of oligosaccharide grafted for a given mass of fibrous element is between 0.05 and 10 mg / g, in particular between 0.2 and 5 mg / g, more particularly between 0.3 and 2 mg / g.

Le taux de greffage est déterminé par analyse des monosaccharides constitutifs après hydrolyse totale de l’oligosaccharide, selon la méthode décrite dans le document WO2016/177967.The grafting rate is determined by analysis of the constituent monosaccharides after total hydrolysis of the oligosaccharide, according to the method described in document WO2016 / 177967.

Selon un exemple particulier, l’élément fibreux est greffé avec un dérivé d’oligosaccharide capable de se lier avec un anticorps anti-A ou avec un dérivé d’oligosaccharide capable de se lier avec un anticorps anti-B. Dans une variante avantageuse, l’élément fibreux est greffé avec un dérivé d’oligosaccharide capable de se lier avec un anticorps anti-A et avec un dérivé d’oligosaccharide capable de se lier avec un anticorps anti-B.According to a particular example, the fibrous element is grafted with an oligosaccharide derivative capable of binding with an anti-A antibody or with an oligosaccharide derivative capable of binding with an anti-B antibody. In an advantageous variant, the fibrous element is grafted with an oligosaccharide derivative capable of binding with an anti-A antibody and with an oligosaccharide derivative capable of binding with an anti-B antibody.

Selon un autre exemple, l’élément fibreux est un élément poreux utilisé dans les filtres à déleucocyter le sang ou les composants sanguins et est apte à éliminer les leucocytes et les anticorps anti-A et/ou anti-B du sang ou d’un composant sanguin, tel que le plasma.According to another example, the fibrous element is a porous element used in filters to leukocyte the blood or the blood components and is capable of removing leukocytes and anti-A and / or anti-B antibodies from the blood or a blood component, such as plasma.

L’élément fibreux greffé avec un dérivé d’oligosaccharide selon le procédé de l’invention est destiné à être arrangé dans une unité de filtration pour l’élimination d’anticorps du sang ou d’un composant sanguin. Notamment, ladite unité de filtration comprend au moins un élément fibreux greffé avec un dérivé d’oligosaccharide selon le procédé de l’invention.The fibrous element grafted with an oligosaccharide derivative according to the method of the invention is intended to be arranged in a filtration unit for the removal of antibodies from the blood or of a blood component. In particular, said filtration unit comprises at least one fibrous element grafted with an oligosaccharide derivative according to the method of the invention.

Par exemple, l’élément fibreux est formé d’au moins une couche de non-tissé de fibres soufflées à l’état fondu. Plus particulièrement, l’élément fibreux est constitué d’un empilement de plusieurs couches de non-tissé tel que des couches de non-tissé de fibres soufflées à l’état fondu. Chacune des couches est greffée avec un dérivé d’oligosaccharide selon le procédé de l’invention.For example, the fibrous element is formed from at least one non-woven layer of meltblown fibers. More particularly, the fibrous element consists of a stack of several layers of nonwoven such as layers of nonwoven of fibers blown in the molten state. Each of the layers is grafted with an oligosaccharide derivative according to the process of the invention.

A titre illustratif, l’unité de filtration comprend une enveloppe extérieure munie d’au moins un orifice d’entrée et d’au moins un orifice de sortie, l’enveloppe renfermant un élément fibreux interposé entre lesdits orifices, ledit élément fibreux étant greffé avec un dérivé d’oligosaccharide selon le procédé de l’invention.By way of illustration, the filtration unit comprises an outer casing provided with at least one inlet port and at least one outlet port, the casing containing a fibrous element interposed between said orifices, said fibrous element being grafted with an oligosaccharide derivative according to the process of the invention.

L’enveloppe extérieure de l’unité de filtration est souple, rigide ou semi-rigide.The outer casing of the filtration unit is flexible, rigid or semi-rigid.

A titre illustratif, on décrit maintenant un système à poches intégrant une unité de filtration décrite pour l’élimination d’anticorps du sang ou d’un composant sanguin. Ledit système à poches comprend :By way of illustration, a bag system is now described incorporating a filtration unit described for the removal of antibodies from the blood or of a blood component. Said pocket system includes:

- une unité de filtration telle que décrite ci-dessus comprenant un élément fibreux greffé selon le procédé de l’invention, - a filtration unit as described above comprising a fibrous element grafted according to the method of the invention,

- une poche de recueil du filtrat, ladite poche étant reliée, par l’intermédiaire d’une tubulure, à un orifice de sortie de l’unité de filtration.- a bag for collecting the filtrate, said bag being connected, via a tube, to an outlet orifice of the filtration unit.

Selon une première réalisation, l’orifice d’entrée de l’unité de filtration est connecté, par l’intermédiaire d’une tubulure, à un connecteur destiné à être relié à une poche contenant le sang ou le composant sanguin à filtrer.According to a first embodiment, the inlet opening of the filtration unit is connected, by means of a tube, to a connector intended to be connected to a bag containing the blood or the blood component to be filtered.

Selon une autre variante, l’orifice d’entrée de l’unité de filtration est connecté de fabrication, par l’intermédiaire d’une tubulure, à une poche destinée à contenir le sang ou le composant sanguin à filtrer.According to another variant, the inlet of the filtration unit is connected during manufacture, by means of a tube, to a bag intended to contain the blood or the blood component to be filtered.

Avantageusement, le système à poches comprenant l’unité de filtration est stérilisé, notamment par vapeur, oxyde d’éthylène, irradiation gamma ou irradiation béta.Advantageously, the bag system comprising the filtration unit is sterilized, in particular by steam, ethylene oxide, gamma irradiation or beta irradiation.

On décrit maintenant un procédé de filtration du sang ou d’un composant sanguin pour retenir un anticorps à l’aide d’un système à poches décrit ci-dessus. Le procédé de filtration comprend les étapes suivantes :
- faire passer le sang ou le composant sanguin au travers d’une unité de filtration telle que décrite ci-dessus et comprenant un élément fibreux greffé selon le procédé de l’invention,
- recueillir le filtrat dans une poche de recueil du filtrat.A method of filtering blood or a blood component to retain an antibody using a bag system described above will now be described. The filtration process includes the following steps:
passing the blood or the blood component through a filtration unit as described above and comprising a fibrous element grafted according to the method of the invention,
- collect the filtrate in a filtrate collection bag.

Le sang ou le composant sanguin à filtrer est notamment du plasma, et plus particulièrement du plasma frais congelé, déleucocyté ou non.The blood or blood component to be filtered is in particular plasma, and more particularly fresh frozen plasma, leukoreduced or not.

On entend par plasma frais congelé, un produit sanguin labile préparé soit à partir de sang total, soit à partir de plasma recueilli par aphérèse, congelé à une température et dans des délais compatibles au maintien de l’activité biologique des facteurs de coagulation.By fresh frozen plasma is meant a labile blood product prepared either from whole blood or from plasma collected by apheresis, frozen at a temperature and within a time period compatible with the maintenance of the biological activity of the coagulation factors.

En variante, le plasma, déleucocyté ou non, est filtré au moment de sa préparation, c’est-à-dire avant congélation.As a variant, the plasma, leukoreduced or not, is filtered during its preparation, that is to say before freezing.

ExempleExample

1. Greffage des dérives d’oligosaccharides sur des feuilles de PBT1. Grafting of oligosaccharide derivatives on PBT sheets

Des couches de non-tissé de fibres soufflées à l’état fondu de polybutylène téréphtalate (PBT) ont été traitées de façon conventionnelle par plasma gazeux (O2). Les couches de PBT présente une taille moyenne de pores comprise entre 8 et 10 µm, un grammage compris entre 40 et 60 g/m², et une surface de filtration d’environ 25 cm².Polybutylene terephthalate (PBT) non-woven layers of meltblown fibers have been conventionally treated with gas plasma (O2). The PBT layers have an average pore size between 8 and 10 µm, a grammage between 40 and 60 g / m², and a filtration surface of about 25 cm².

Le greffage des dérivés d’oligosaccharides est réalisé par imprégnation des couches de PBT préalablement traitées par plasma gazeux, en bain ou par foulardage.The grafting of oligosaccharide derivatives is carried out by impregnating the layers of PBT previously treated by gas plasma, in a bath or by padding.

La solution de greffage est constituée d’un dérivé d’oligosaccharide capable de se lier aux anticorps anti-B (tétra B et héxa B provenant de Elictyl) dans un solvant éthanol.The grafting solution consists of an oligosaccharide derivative capable of binding to anti-B antibodies (tetra B and hexa B from Elictyl) in an ethanol solvent.

Les dérivés d’oligosaccharides sont les suivants : The oligosaccharide derivatives are as follows:

Galα1-3(Fucα1-2)Galβ1-3GlcNAcβ1-3Galβ1-4Glcβ-NAc-Spacer1-NH2 (6GrB1-Nac-sp1-NH2) (hexa B)Galα1-3 (Fucα1-2) Galβ1-3GlcNAcβ1-3Galβ1-4Glcβ-NAc-Spacer1-NH2 (6GrB1-Nac-sp1-NH2) (hexa B)

Galα1-3(Fucα1-2)Galβ1-4Glcβ-NAc-Spacer1-NH2 (GLY038-3-NAc-sp1-NH2) (Tétra B)Galα1-3 (Fucα1-2) Galβ1-4Glcβ-NAc-Spacer1-NH2 (GLY038-3-NAc-sp1-NH2) (Tétra B)

La solution d’activation est constituée d’un mélange d’EDC et NHS dans un solvant éthanol ou dans un mélange solvant éthanol/eau (70%/30% en volume) et tampon MES (pH = 5).The activation solution consists of a mixture of EDC and NHS in an ethanol solvent or in an ethanol / water solvent mixture (70% / 30% by volume) and MES buffer (pH = 5).

On considère que la quantité de fonctions acides carboxyliques dans les couches de PBT placée dans l’unité de filtration correspond à 1 équivalent molaire.It is considered that the amount of carboxylic acid functions in the PBT layers placed in the filtration unit corresponds to 1 molar equivalent.

On utilise pour 1 équivalent molaire de fonctions acides carboxyliques, 1 équivalent molaire de dérivé d’oligosaccharide, 1,2 équivalent molaire d’EDC et 1,2 équivalent molaire NHS.1 molar equivalent of carboxylic acid functions is used, 1 molar equivalent of oligosaccharide derivative, 1.2 molar equivalent of EDC and 1.2 molar equivalent NHS.

Pour l’imprégnation en bain, les couches de PBT sont plongées 2 heures dans la solution d’activation, puis 2 heures dans la solution de greffage. Les couches de PBT greffées sont séchées à l’air libre pendant au moins 24 heures.For bath impregnation, the PBT layers are immersed for 2 hours in the activation solution, then 2 hours in the grafting solution. The grafted PBT layers are dried in the open air for at least 24 hours.

Pour l’imprégnation par foulardage, la vitesse de défilement de la bobine de PBT est de l’ordre de 1 m/min.For pad impregnation, the running speed of the PBT coil is around 1 m / min.

On détermine ensuite le taux de greffage du dérivé d’oligosaccharide sur le PBT.The grafting rate of the oligosaccharide derivative is then determined on the PBT.

Le taux de greffage correspond à la concentration d’oligosaccharide greffé pour une masse donnée de couche de PBT. Il est évalué par analyse des monosaccharides constitutifs après hydrolyse totale de l’oligosaccharide, selon la méthode décrite dans le document WO2016/177967.The grafting rate corresponds to the concentration of grafted oligosaccharide for a given mass of PBT layer. It is evaluated by analysis of the constituent monosaccharides after total hydrolysis of the oligosaccharide, according to the method described in document WO2016 / 177967.

2. Fabrication d’une unité de filtration2. Manufacture of a filtration unit

Une unité de filtration (boitier rigide) comprend 14 couches de PBT greffées avec les dérivés d’oligosaccharide.A filtration unit (rigid case) includes 14 layers of PBT grafted with oligosaccharide derivatives.

3. Stérilisation3. Sterilization

L’unité de filtration est montée avec une poche de recueil du filtrat puis l’ensemble est stérilisé par vapeur ou par irradiation béta.The filtration unit is mounted with a bag for collecting the filtrate and then the whole is sterilized by steam or by beta irradiation.

3. Filtration du plasma3. Plasma filtration

Après décongélation à 37°C, du plasma frais congelé (FFP) d’un donneur de groupe A (ayant des anticorps anti-B) est déleucocyté par filtration au travers d’un filtre Miniplas (Macopharma, France). Le plasma déleucocyté est ensuite passé trois fois au travers d’une même unité de filtration à l’aide d’une pompe (25 ml/min).After thawing at 37 ° C, fresh frozen plasma (FFP) from a group A donor (with anti-B antibodies) is leukoreduced by filtration through a Miniplas filter (Macopharma, France). The leukoreduced plasma is then passed three times through the same filtration unit using a pump (25 ml / min).

On détermine ensuite le titre en anticorps par test d’agglutination.The antibody titer is then determined by agglutination test.

Des échantillons de plasma (avant et après filtration) sont dilués en tampon PBS, par dilution successive d'un facteur 2 (1/1, 1/2,..,, 1/ 64) et mis en contact avec des hématies du groupe B. Le titre rendu correspond à la dernière dilution positive.Plasma samples (before and after filtration) are diluted in PBS buffer, by successive dilution by a factor of 2 (1/1, 1/2, .. ,, 1/64) and brought into contact with red blood cells from the group B. The title returned corresponds to the last positive dilution.

4. Résultats4. Results

4.1 Greffage des couches de PBT4.1 Grafting of PBT layers

EssaiTrial ImprégnationImpregnation Oligo-saccharideOligosaccharide SolvantSolvent Solution tamponBuffer solution Taux de greffage (mg/g de support)Grafting rate (mg / g of support) stérilisationsterilization 11 BainBath Hexa BHexa B EthanolEthanol MESMY 1,41.4 -- 1bis1a Bain Bath Hexa BHexa B EthanolEthanol // 22 - - 44 Foulardagepadding Hexa BHexa B EthanolEthanol // 0,330.33 - - 55 Bain Bath Hexa BHexa B EthanolEthanol // 0,320.32 VapeurSteam 66 Bain Bath Hexa BHexa B EthanolEthanol // 0,320.32 BétaBeta 77 Foulardagepadding Hexa BHexa B Ethanol/eau Ethanol / water MESMY 0,290.29 -- 88 Foulardagepadding Tétra BTetra B Ethanol/eauEthanol / water MESMY 0,060.06 -- 99 BainBath Tétra BTetra B EthanolEthanol // 0,350.35

4.2 Rétention des anticorps anti-B4.2 Retention of anti-B antibodies

Pour certains essais, plusieurs filtrations ont été réalisées.For certain tests, several filtrations were carried out.

EssaiTrial Titre anticorps initialInitial antibody titer Titre anticorps finalFinal antibody titer Dilution moyenneAverage dilution 11 1/161/16 1/81/8 0,830.83 1/64 (marqué)1/64 (marked) 1/64 (léger)1/64 (light) 1/81/8 1/41/4 1bis1a 1/161/16 1/41/4 2,332.33 1/81/8 1/21/2 1/161/16 1/21/2 44 1/161/16 1/81/8 11 1/81/8 1/41/4 1/321/32 1/161/16 55 1/81/8 1/31/3 0,830.83 1/81/8 1/61/6 1/161/16 1/161/16 66 1/81/8 11 2,252.25 1/81/8 1/31/3 77 1/41/4 1/41/4 00 88 1/81/8 1/8 (léger)1/8 (light) 0,50.5 99 1/251/25 1/41/4 2,52.5

La filtration n’a pas d’impact sur les facteurs plasmatiques testés : Fibrinogène, Facteur V, Facteur VII, Facteur VIII et Facteur XI (résultats non montrés).Filtration has no impact on the plasma factors tested: Fibrinogen, Factor V, Factor VII, Factor VIII and Factor XI (results not shown).

Claims (18)

Procédé de greffage d’un élément fibreux pour l’élimination d’anticorps du sang ou d’un composant sanguin, ledit élément fibreux portant des fonctions acides carboxyliques, ledit procédé prévoyant d’imprégner un élément fibreux portant des fonctions acides carboxyliques avec une solution de greffage comprenant au moins un dérivé d’oligosaccharide capable de se lier à un ou plusieurs anticorps, ledit dérivé d’oligosaccharide portant au moins une fonction amine, de sorte à assurer un pontage covalent par formation d’une liaison amide entre le dérivé d’oligosaccharide et ledit élément fibreux, caractérisé en ce que la solution de greffage comprend ledit dérivé d’oligosaccharide solubilisé dans un solvant organique.A method of grafting a fibrous element for the removal of antibodies from the blood or of a blood component, said fibrous element carrying carboxylic acid functions, said method providing for impregnating a fibrous element carrying carboxylic acid functions with a solution grafting comprising at least one oligosaccharide derivative capable of binding to one or more antibodies, said oligosaccharide derivative carrying at least one amine function, so as to ensure covalent bridging by formation of an amide bond between the derivative d oligosaccharide and said fibrous element, characterized in that the grafting solution comprises said oligosaccharide derivative solubilized in an organic solvent. Procédé selon la revendication 1, caractérisé en ce qu’il prévoit en outre d’imprégner l’élément fibreux porteur de fonctions acides carboxyliques avec une solution d’activation comprenant un composé carbodiimide ou un mélange d’un composé carbodiimide avec un composé succinimide, de sorte à activer au moins une partie des fonctions acides carboxyliques.Process according to claim 1, characterized in that it further provides for impregnating the fibrous element carrying carboxylic acid functions with an activation solution comprising a carbodiimide compound or a mixture of a carbodiimide compound with a succinimide compound, so as to activate at least part of the carboxylic acid functions. Procédé selon la revendication 2, caractérisé en ce que l’imprégnation de l’élément fibreux porteur de fonctions acides carboxyliques avec la solution d’activation est réalisée préalablement à l’imprégnation dudit élément fibreux avec ladite solution de greffage.Method according to claim 2, characterized in that the impregnation of the fibrous element carrying carboxylic acid functions with the activating solution is carried out prior to the impregnation of said fibrous element with said grafting solution. Procédé selon l’une des revendications 2 ou 3, caractérisé en ce que le composé carbodiiimide est choisi parmi le dicyclohexylcarbodiimide (DCC), le diisopropylcarbodiimide (DIC) et le 1-éthyl-3-(3-diméthylaminopropyl)carbodiimide (EDC).Process according to either of Claims 2 and 3, characterized in that the carbodiiimide compound is chosen from dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC). Procédé selon l’une quelconque des revendications 2 à 4, caractérisé en ce que le composé succinimide est le N-hydroxysuccinimide (NHS) ou le N-hydroxysulfosuccinimide (Sulfo-NHS).Process according to any one of Claims 2 to 4, characterized in that the succinimide compound is N-hydroxysuccinimide (NHS) or N-hydroxysulfosuccinimide (Sulfo-NHS). Procédé selon l’une quelconque des revendications 2 à 5, caractérisé en ce que la solution d’activation comprend un composé carbodiimide ou un mélange d’un composé carbodiimide avec un composé succinimide solubilisé dans un solvant organique.Process according to any one of Claims 2 to 5, characterized in that the activation solution comprises a carbodiimide compound or a mixture of a carbodiimide compound with a succinimide compound dissolved in an organic solvent. Procédé selon la revendication 6, caractérisé en ce que le solvant organique de la solution d’activation est un solvant alcoolique tel que le méthanol, l’éthanol ou l’isopropanol, ou un solvant cétonique tel que l’acétone ou la méthyléthylcétone, et notamment l’éthanol.Process according to Claim 6, characterized in that the organic solvent of the activation solution is an alcoholic solvent such as methanol, ethanol or isopropanol, or a ketonic solvent such as acetone or methyl ethyl ketone, and especially ethanol. Procédé selon l’une des revendications 6 ou 7, caractérisé en ce que le solvant organique de la solution d’activation est le même que le solvant organique de la solution de greffage, notamment l’éthanol.Method according to one of claims 6 or 7, characterized in that the organic solvent of the activation solution is the same as the organic solvent of the grafting solution, in particular ethanol. Procédé selon l’une quelconque des revendications 2 à 8, caractérisé en ce que la solution d’activation ne contient pas d’eau ajoutée.Process according to any one of Claims 2 to 8, characterized in that the activation solution contains no added water. Procédé selon l’une quelconque des revendications 2 à 9, caractérisé en ce qu’il prévoit d’imprégner l’élément fibreux avec la solution d’activation par foulardage.Method according to any one of claims 2 to 9, characterized in that it provides for impregnating the fibrous element with the activation solution by padding. Procédé selon l’une quelconque des revendications 1 à 10, caractérisé en ce que le solvant organique de la solution de greffage est un solvant alcoolique tel que le méthanol, l’éthanol ou l’isopropanol, ou un solvant cétonique tel que l’acétone ou la méthyléthylcétone, et notamment l’éthanol.Process according to any one of Claims 1 to 10, characterized in that the organic solvent of the grafting solution is an alcoholic solvent such as methanol, ethanol or isopropanol, or a ketonic solvent such as acetone or methyl ethyl ketone, and in particular ethanol. Procédé selon l’une quelconque des revendications 1 à 11, caractérisé en ce que la solution de greffage ne contient pas d’eau ajoutée.Process according to any one of Claims 1 to 11, characterized in that the grafting solution contains no added water. Procédé selon l’une quelconque des revendications 1 à 12, caractérisé en ce que l’élément fibreux est sous forme d’au moins une couche de non-tissé notamment de fibres soufflées à l'état fondu.Method according to any one of Claims 1 to 12, characterized in that the fibrous element is in the form of at least one layer of nonwoven, in particular of meltblown fibers. Procédé selon l’une quelconque des revendications 1 à 13, caractérisé en ce que l’élément fibreux est réalisé en polyester tel que le polybutylène téréphtalate ou le polyéthylène téréphtalate.Process according to any one of Claims 1 to 13, characterized in that the fibrous element is made of polyester such as polybutylene terephthalate or polyethylene terephthalate. Procédé selon l’une quelconque des revendications 1 à 14, caractérisé en ce que le procédé prévoit, avant l’étape de greffage, de traiter l’élément fibreux avec un plasma gazeux, notamment avec de l’oxygène, de sorte à augmenter le nombre de fonctions acides carboxyliques.Method according to any one of Claims 1 to 14, characterized in that the method provides, before the grafting step, to treat the fibrous element with a gaseous plasma, in particular with oxygen, so as to increase the number of carboxylic acid functions. Procédé selon l’une quelconque des revendications 1 à 15, caractérisé en ce que le dérivé d’oligosaccharide comprend un oligosaccharide et une fonction amine, séparés par un bras de liaison, ledit bras de liaison comportant notamment une fonction triazole et/ou une liaison thiol et/ou une chaîne polyéthylène glycol et/ou une ou plusieurs chaînes alkylène.Process according to any one of Claims 1 to 15, characterized in that the oligosaccharide derivative comprises an oligosaccharide and an amine function, separated by a link arm, said link arm notably comprising a triazole function and / or a link thiol and / or a polyethylene glycol chain and / or one or more alkylene chains. Procédé selon la revendication 16, caractérisé en ce que l’oligosaccharide est un trisaccharide, tétrasaccharide, pentasaccharide ou hexasaccharide.Method according to claim 16, characterized in that the oligosaccharide is a trisaccharide, tetrasaccharide, pentasaccharide or hexasaccharide. Procédé selon l’une quelconque des revendications 1 à 17, caractérisé en ce qu’il prévoit d’imprégner l’élément fibreux avec la solution de greffage par foulardage.Method according to any one of claims 1 to 17, characterized in that it provides for impregnating the fibrous element with the padding grafting solution.
PCT/EP2019/067176 2018-06-27 2019-06-27 Method for grafting a fibrous element for eliminating antibodies from blood or a blood component Ceased WO2020002512A1 (en)

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