EP3204019A2

EP3204019A2 - Method for preparing universal plasma

Info

Publication number: EP3204019A2
Application number: EP15784303.8A
Authority: EP
Inventors: Abdessatar Chtourou
Original assignee: LFB SA
Current assignee: LFB SA
Priority date: 2014-10-09
Filing date: 2015-10-09
Publication date: 2017-08-16
Also published as: US10357512B2; WO2016055647A2; FR3026950A1; TW201618798A; WO2016055647A3; EP3459552A1; US20170281679A1; CA2964037A1

Abstract

The present invention relates to a universal plasma, prepared from a mixture of plasmas from individual donors with blood types A, B, AB and/or O and compatible with all blood types, and the method for obtaining same.

Description

PROCESS FOR THE PREPARATION OF UNIVERSAL PLASMA

Technical area

The present invention relates to a universal plasma, virally secure, depleted of anti-A and anti-B antibodies, and compatible with all blood groups, and its method of obtaining.

Technological background

"Blood plasma" or "plasma" is the liquid component of the blood, in which the blood cells are in suspension. Plasma is likely to be separated from whole blood by centrifugation or filtration through a membrane. Blood plasma consists essentially of water, plasma proteins (mainly albumin and antibodies) and coagulation factors including fibrinogen.

Blood plasma is used therapeutically for the treatment of severe coagulopathies with collapse of all coagulation factors as well as for treatment of acute hemorrhages, with global deficit of coagulation factors.

Currently, the therapeutic plasma generally used is fresh frozen plasma (FFP) from donations qualified logically biochemical (immunohematological tests, search for HIV, HCV and HBV by amplification of nucleic acids) and which can be secured vis-à-vis quarantine pathogens or physicochemical treatment. Such a plasma requires storage at a temperature of less than or equal to -25 ° C, which implies that its administration can be carried out only after thawing under specific conditions. In addition, this type of plasma can not be stored for more than a year, which generates significant losses. Finally, this therapeutic plasma must be used with caution since its administration must respect the delivery rules based on the ABO compatibilities of the recipients. Beyond the use of compatible plasmas, the iso-group plasma transfusion must be preferred in order to avoid any risk of post-transfusion hemolysis by ABO incompatibility.

Because of the restrictions related to its use and its conservation and therefore its availability, such fresh frozen plasma is unsuitable for application in emergency conditions, especially in the hospital environment or in the field outside hospital (places accidents or disasters, areas difficult to access, areas with restricted or no access to cold chains, military operations). There is therefore a need for a plasma meeting the constraints of the hospital and non-hospital environment, capable of being stored at room temperature, free from any form of bacterial, viral or parasitic contamination and compatible with any recipient, regardless of its blood group ABO.

The application WO2012 / 022914 describes a process for obtaining a freeze-dried plasma derived from a plasma mixture of selected donors exclusively belonging to the blood group AB or to the blood groups A or B. The method described in the application WO2012 / 022914 can not be used with donors belonging to blood group O.

US20110008459 discloses a universal plasma obtained by mixing the plasma of blood group donors A and B and optionally AB to the exclusion of any plasma from blood group donors O, wherein the anti-A and anti-antibody titer. Free B is less than 16 for immunoglobulin M (IgM) and less than 64 for immunoglobulin G (IgG).

Summary of the invention

The inventors have developed a method of preparing a universal plasma whose characteristics meet the requirements of conservation and use still unsatisfied and this, regardless of the blood group to which this plasma belongs.

Thus, the invention relates to a method for preparing a universal plasma, comprising the following steps:

a) Non-universal unit plasma mixture obtained from a sample of donors,

b) Elimination of the anti-A and anti-B antibodies present in the plasma by immunoaffinity chromatography or by batch depletion, and

c) optionally lyophilization or atomization of the universal plasma from step b).

The method of the invention ultimately provides a "universal plasma" that can be administered to any patient regardless of blood group and without prior determination of the blood group of the patient. The universal plasma according to the invention meets the regulatory requirements to which the plasmas currently used in therapy are subjected, in particular the concentrations of coagulation factors are satisfactory (for example the concentration of factor VIII is greater than or equal to 0.5 IU / mL , the factor V concentration is greater than or equal to at 0.7 IU / mL, the factor XI concentration is greater than or equal to 0.5 IU / mL and the fibrinogen concentration is greater than or equal to 2g / L) and there is no activation of the factors coagulation.

In a particular embodiment, step b) is carried out by means of an immunoaffinity chromatography or a batch depletion on a support whose matrix is grafted with oligosaccharide groups that are antigenically similar to the blood groups A and / or B, for example according to the techniques described in application WO2007 / 077365 or using a matrix as described in applications FR 13 56635 or FR 13 56636.

In a particular embodiment, said donors belong to the blood group selected from blood group A, blood group B, blood group AB and / or blood group O.

In a particular embodiment, the process for preparing a universal plasma may further comprise a lyophilization, freeze drying or atomization step at the end of step b), making it possible to obtain a " freeze-dried universal plasma "," freeze-dried universal plasma "or" atomized universal plasma ", by means of a freeze-drying, freeze-drying or appropriate atomization step.

In a particular embodiment, the universal plasma resulting from step b) has an anti-A antibody content and / or a content of anti-B antibodies contained in the universal plasma that is negative at dilution 1 / 64 of the Coombs test, performed according to method 2.6.20 of the European Pharmacopoeia 07/2011: 20620.

In a particular embodiment, the process for preparing a universal plasma may further comprise a step of biological securing, by viral inactivation and / or ultrafiltration upstream of step a).

The present invention also relates to a universal plasma, preferably a universal plasma in which the anti-A antibody content and the anti-B antibody content is consistent with a Coombs test negative result at 1/64 dilution of the test. Coombs, carried out according to the method 2.6.20 of the European Pharmacopoeia 07/2011: 20620.

The present invention also relates to the use of such a universal plasma for the treatment of patients:

suffering from severe hemorrhages, especially traumatic origin, replacing multiple coagulation emergency when a concentrate of coagulation factors is not available,

suffering from traumatic or spontaneous hemorrhages under oral anticoagulants (ACO),

- suffering from severe vitamin K deficiency, or

with thrombotic thrombocytopenic purpura.

Other features and advantages of the present invention will appear on reading the detailed description which follows and the preferred embodiment of the invention, given by way of example.

detailed description

By "non-universal unit plasma" is meant plasma collected from a single human donor, regardless of blood group. This unit plasma can be prepared from whole blood or collected by apheresis. This non-universal unit plasma is a human plasma. Preferably, in the context of this invention, the non-universal unit plasmas constituting the plasma mixture of step a) of the process of the invention are collected by apheresis. Preferably, non-universal unit plasmas constituting the mixture of plasmas of step a) meet the same regulatory requirements to which plasmas currently used in therapy or for fractionation are subjected. They therefore comply with the requirements in force, particularly in terms of the rate of hemostasis factors. Donor individuals must therefore meet the regulatory eligibility criteria for plasma donation. Preferentially, the non-universal unit plasmas are obtained from male donors or free of anti-HLA antibodies. In addition, in the context of the present invention, the donors must have a normal hemostasis report characterized by a factor VIII level of at least 0.9 IU / ml.

By "human donor individual" is meant a human individual who can donate (free or paid) blood or blood components.

By "plasma mixture" or "pool of plasmas" is meant a mixture of non-universal unit human plasmas. In the context of the invention, the "pool of plasmas" can be a pool of therapeutic plasmas, directly usable in therapy or a pool of plasmas for fractionation. According to the invention, the pool of plasmas is advantageously depleted to secure the plasma against the risk of transmission of the prion or other infectious agents. Advantageously, the leukocyte-depleted plasma according to the invention has a limit content in leukocytes. residuals <1.0 x 10 ⁶ / liter. Preferably, a "plasma mixture" or "pool of plasmas" corresponds to a mixture of at least 2 non-universal unit human plasmas, preferably at least 5 non-universal unit human plasmas, more preferably at least 10 plasmas. non-universal unitary humans.

In one embodiment of the invention, a "plasma mixture" or "pool of plasmas" is a mixture of at least 100 different non-universal unit human plasmas. In another particular embodiment of the invention, a "plasma mixture" or "pool of plasmas" corresponds to a mixture of at least 1500 different non-universal unit human plasmas.

In a particular embodiment of the invention, a "plasma mixture" or "pool of plasmas" corresponds to a mixture of at least 2,000 different non-universal unit human plasmas. In another particular embodiment of the invention, a "plasma mixture" or "pool of plasmas" corresponds to a mixture of at least 10,000 different non-universal unit human plasmas.

In another particular embodiment of the invention, the number of non-universal unit plasmas used to constitute a "plasma mixture" or "pool of plasmas" is advantageously adjusted to guarantee the biological safety. Thus, the addition of additional biological safety steps makes it possible to increase the number of non-universal unit plasmas while minimizing the biological risk.

By "whole blood" is meant all the compounds and cells constituting the blood.

By "apheresis" is meant a technique of taking, from a donor, certain blood components. The components to be removed are separated by centrifugation and stored, while the non-withdrawn components are reinjected into the donor.

In a particular embodiment, the mixture of plasmas of step a) of the process according to the invention is obtained from the non-universal unit plasmas of at least 2 different donors, from at least 5 different donors, preferably from at least 10 different human donor individuals. In a particular embodiment, the mixture of non-universal unit plasmas of step a) of the process according to the invention is obtained from 100 different human donor individuals.

In another particular embodiment, the mixture of non-universal unit plasmas of step a) of the process according to the invention is obtained from 1500 different human donor individuals. Such a limitation as to the number of human donor individuals makes it possible to considerably reduce the residual infectious risk while benefiting from the advantages of the mixture: reduced immunogenicity, beneficial effect on the infectious risk due to the dilution or neutralization of the pathogenic agents, and obtaining a universal plasma for blood grouping. It also allows simplified traceability of donor individuals.

In another particular embodiment, the mixture of non-universal unit plasmas of step a) of the process according to the invention is obtained from at least 2000 human donor individuals, preferably from at least 5000 even more preferably from at least 10,000 different human donor individuals. In order not to limit the number of human donor individuals, additional appropriate measures can be put in place to characterize and secure non-universal unit plasmas: separation of at-risk donors, quarantine, search for the genome of parvovirus B19 or hepatitis A, hepatitis B or HIV by nucleic amplification, hepatitis C virus antibody test, additional viral elimination or inactivation step, or any other suitable biological safety measure.

Preferably, said human donor individuals belong to the blood groups A, B, AB or O, which makes it possible not to exclude categories of donors on the basis of their blood group, and therefore, to increase the volumes of source plasma that can be used according to the invention. By "human donor individual belonging to the blood group A, B, AB or O" is meant a donor individual having the phenotype A, B, AB or O, respectively. Membership in a particular blood group has no influence on the method of the invention. No prior selection of non-universal unit plasmas is performed upstream of said method.

The Applicant has found, surprisingly and unexpectedly, that the method according to the invention makes it possible to obtain a universal plasma from non-universal unit plasmas, irrespective of the blood group to which the donor individuals belong. In other words, the method of the invention makes it possible to obtain a universal plasma from non-universal unit plasmas from a sample of donor individuals belonging to the blood group A, B, AB or O. In a particular embodiment, the nonuniversal unit plasma mixture of step a) of the process according to the invention advantageously comes from donor individuals belonging to the blood groups A, B, AB and O.

By "blood group" is meant a blood classification based on the presence or absence of antigenic substances on the surface of red blood cells. These antigenic substances define the ABO system. The antigen A corresponds to the the presence of an N-acetyl-galactosamine grafted at the _3- position of the epitope (H chain). The B antigen corresponds to the presence of a galactose grafted αι_ ₃ position of the epitope (H chain). The ABO system dictates the compatibility rules of blood transfusion. Failure to comply with these rules may result in a hemolytic accident in the transfused individual. Since the plasma contains antibodies depending on the group in the ABO system, the red blood cells of the recipient should not have the corresponding antigens. Thus, a plasma comprising anti-A antibodies should not be administered to a patient belonging to blood group A, and vice versa.

By "anti-A" or "anti-blood group A" is meant any antibody recognizing the blood group A antigens.

By "anti-B" or "anti-blood group B" is meant any antibody recognizing antigens of blood group B.

In a particular embodiment of the invention, the anti-A and anti-B antibodies consist of immunoglobulins recognizing the antigens of the blood group A and / or B, in particular immunoglobulins G or M.

In another particular embodiment of the invention, the anti-A and anti-B antibodies consist of immunoglobulins G or M recognizing the antigens of the blood group A and / or B, in particular hemolysins or agglutinins. Anti-A and anti-B hemolysin antibodies are immunoglobulins G likely to be present in the plasma and to cause lysis of red blood cells.

By "universal plasma" is meant plasma usable for any recipient individual, regardless of blood group. Universal plasma advantageously makes it possible to overcome the ABO compatibility constraint or the ABO iso-group between donor and recipient individuals. It is thus possible to produce and keep only one type of plasma, universal, instead of the 4 types of plasma (A, B, AB and O) currently required to meet ABO compatibility. Universal plasma thus avoids wasted plasma due to poor expectations of needs according to the blood groups of patients. Such universal plasma advantageously reduces the space dedicated to plasma storage, and also eliminates the risk of transfusion not accidentally compatible. The "universal plasma" consists in particular of a plasma depleted of any antibody directed against the blood group A and / or blood group B antigens, in particular depleted of anti-A and / or anti-B antibodies.

Advantageously, the plasma obtained at the end of step b) of the process of the invention does not contain anti-A or anti-B antibodies or contains a residual level. of anti-A and / or anti-B inferior antibody with a negative result at 1/64 dilution of the Coombs test and is referred to as "universal plasma".

In a particular embodiment of the invention, the universal plasma advantageously has a negative result in the Coombs test at the 1/32 dilution, still more advantageously at the 1/16 dilution, even more advantageously at the dilution. 8 or lower.

"Coombs test" means the direct or indirect Coombs test as described in paragraph 2.6.20 of European Pharmacopoeia 01/2005: 20620 or Chapter 2.6.20 of the European Pharmacopoeia 01/2008: 20620, or the European Pharmacopoeia 07/2011: 20620. The indirect Coombs test consists of an indirect method using an antiglobulin as described, for example, in Chapter 2.6.20 of the European Pharmacopoeia 01/2005: 20620 or the European Pharmacopoeia 07/2011: 20620. The direct Coombs test consists of a direct haemagglutination method, using papain-treated cells, as a reference method for the detection of anti-A and anti-B antibodies using 3 standards (a positive control, a negative control and a reference preparation for limit test) as described for example in Chapter 2.6.20 of European Pharmacopoeia 01/2008: 20620 or European Pharmacopoeia 07/2011: 20620.

Step b) of said process concerns the removal of the anti-A and anti-B antibodies present in the plasma by immunoaffinity chromatography or by batch depletion. This step is essential to obtain a universal plasma directly administrable to the recipient regardless of the blood group ABO of the latter.

The plasma resulting from step a) is subjected to an immunoaffinity chromatographic step or a batch depletion step on a support grafted with antigenically similar groups to the blood groups A and / or B, preferably on a column filled with such a group. support. Preferably, the chromatographic or batch depletion support consists of a polymer matrix on which grafts or coupling arms are grafted, in turn being grafted, with oligosaccharides advantageously representing trisaccharides corresponding to the epitopes of the blood groups. A and B.

By "batch depletion" is meant an affinity purification method using a grafted support of specific ligands, the elution and washing fractions being separated from this support after incubation with the molecules of interest by centrifugation. to recover said medium.

The array of immunoaffinity chromatography or depletion in batch comprises polymer particles on which at least one oligosaccharide corresponding to a blood group A and / or group B epitope is grafted, said oligosaccharide being grafted to said particles via a spacer, characterized in that said spacer has the formula (I) -NH-R 1 -CO-NH-R ₂ -, wherein R 1 is a C 4 -C 6 alkyl group, R ₂ is a C3-C8 alkyl group, and said spacer is linked by its amine function to the polymer particle.

In a particular embodiment, the matrix comprises (i) polymer particles on which at least one oligosaccharide corresponding to a blood group A epitope is grafted, and / or (ii) polymer particles on which at least one oligosaccharide corresponding to a blood group epitope B is grafted. In a preferred embodiment, the matrix comprises (i) polymer particles on which at least one oligosaccharide corresponding to a blood group A epitope is grafted, and (ii) polymer particles on which at least one oligosaccharide corresponding to an epitope of blood group B is grafted.

In another embodiment, the matrix comprises polymer particles on which at least one oligosaccharide corresponding to a blood group A epitope, and at least one oligosaccharide corresponding to a blood group B epitope, are grafted.

In a particular embodiment, the matrix comprises a mixture of (i) polymer particles on which at least one oligosaccharide corresponding to a blood group A epitope is grafted, (ii) polymer particles on which at least one oligosaccharide corresponding to a blood group B epitope is grafted, and (iii) polymer particles on which both at least one oligosaccharide corresponding to a blood group A epitope, and at least one oligosaccharide corresponding to a blood group B epitope, are grafted. .

In particular, very good results are obtained by implementing such a support whose trisaccharides, corresponding to the blood group A epitope, have the structure N-acetylgalactosamine (Gal Ac) - Galactose (Gai) - Fucose (Fuc) , and those corresponding to the blood group B epitope, have the Galactose-Galactose-Fucose structure (Gal-Gal-Fuc).

In a preferred embodiment, crosslinked cellulose beads are mixed on which are grafted ligands which are trisaccharides corresponding to a blood group A epitope (N-acetylgalactosamine (GalNAc) -Galactose (Gal) -Fucose), and crosslinked cellulose beads on which are grafted ligands which are trisaccharides corresponding to an epitope of the blood group B (Galactose-Galactose-Fucose).

The trisaccharides are grafted to the beads by a spacer of formula: NH-C ₅ H ₁₀ -CO-NH-C ₃ H ₆ . The support :

The matrix of the invention comprises a support based on polymer particles, and is preferably in the form of a gel or a resin.

These polymer particles are preferably of spherical or oblong shape, it may be in particular beads. These polymer particles generally have an average size of approximately Ο, ΐ μιη to approximately ΙΟΟΟμιη, preferably from approximately 20 to approximately 500μιη, more preferably from approximately 50 to approximately 200μιη, more preferably from approximately 70μιη to approximately 120μιη of diameter. Preferably these particles are porous.

The polymer may be natural or non-natural, organic or inorganic, crosslinked or uncrosslinked. The polymer is preferably an organic polymer, preferably crosslinked.

In a preferred embodiment, the polymer is cellulose, and the particles are preferably porous cellulose beads. More preferably, it is crosslinked cellulose.

Other types of possible polymers include agarose, dextran, polyacrylates, polystyrene, polyacrylamide, polymethacrylamide, copolymers of styrene and divinylbenzene, or mixtures of these polymers. The ligands:

The ligands carried by the support according to the invention are oligosaccharides representing the antigens of the blood groups A and / or B, which are naturally monosaccharides.

More specifically, the oligosaccharide corresponding to a blood group A epitope and / or the oligosaccharide corresponding to a blood group B epitope carried by the matrix according to the invention are typically trisaccharides. The term "blood group epitope corresponding oligosaccharides" refers to antigenically identical or similar motifs to the antigenic determinants recognized by the anti-A and anti-B antibodies, respectively. The ligands carried by the support according to the invention are therefore specific for anti-A or anti-B antibodies.

Preferably, the oligosaccharide corresponding to a blood group A epitope, used as ligand in the invention, is a trisaccharide N-acetylgalactosamine (Gal Ac) -Galactose (Gal) -Fucose, more precisely N-acetylGalal (Fucai2) Gal, the spacer is bonded by the oxygen atom preferably bonded to the carbon in position 1 of the galactose. The oligosaccharide corresponding to a blood group B epitope, used as a ligand in the invention, is preferably a Galactose-Galactose-Fucose oligosaccharide, more specifically Galal 3 (Fucal ₂ ) Gal, the spacer is bound by the oxygen preferably bonded to the carbon at the 1-position of galactose.

Advantageously, the ligand density, that is to say the amount of ligands (i.e., oligosaccharides corresponding to a blood group epitope A or B) grafted, per volume of template in gel form, can be from about 0.2 to about 0.7 mg / ml of matrix, more preferably from about 0.3 to about 0.4 mg / ml of matrix. Preferably the density of oligosaccharides is about 0.3 mg / ml of matrix.

The spacer has the formula (I) -NH-R 1 -CO-NH-R ₂ -, in which:

R1 is a linear or branched C4-C6 alkyl group

R ₂ is a linear or branched C ₃ -C 8 alkyl group

said spacer being bound by its amine function (in bold above) to the polymer particle.

In a particular embodiment, R 1 represents a linear alkyl group, preferably R 1 is a C ₅ alkyl group and R ₂ is a linear alkyl group, preferably R ₂ is a C 3 alkyl group.

A polymer particle may carry one or more spacers.

The spacer makes it possible to reduce the steric hindrance and to increase the accessibility of the ligand to the anti-A and anti-B antibodies to be bound.

The spacer serves to immobilize, on a particle, either an oligosaccharide corresponding to a blood group A epitope, which is preferably a N-acetylGalal 3 (Fucal ₂ ) Gal trisaccharide as described above, or an oligosaccharide corresponding to a Blood-type B epitope, which is preferably a Gal-3 (Fucal ₂ ) Gal trisaccharide as described above.

In a preferred embodiment, the ligands (preferably trisaccharides as described above) are grafted to the polymer particles by a spacer of formula: NH-C ₅ H ₀ -CO-NH-C ₃ H ₆ .

Such a chromatographic or batch depletion support very advantageously represents a gel or resin as described in the applications WO2007 / 077365, FR 13 56635 or FR 13 56636 or a gel or a resin available on the market such as GLYCOSORB® ABO (Glycorex Transplantation AS - Sweden), the Sepharose 4B affinity matrix grafted with trisaccharides of blood groups A and B (Dextra Laboratories Limited - Great Britain), or any other equivalent matrix. This chromatographic support or batch depletion allows the simultaneous removal of the anti-A and anti-B antibodies in a single step of immunoaffinity chromatography or batch depletion. In a preferred embodiment, the polymer particles on which at least one oligosaccharide corresponding to a blood group A epitope is grafted, and the polymer particles on which at least one oligosaccharide corresponding to a blood group B epitope is grafted, can then be mixed, for example in a proportion of 25/75 to 75/25 (v / v), preferably about 50/50 (v / v). It is indeed possible to adjust the proportion of the two ligands in the column to the population of donors according to the distribution of blood groups thereof.

The polymer particles on which at least one oligosaccharide corresponding to a blood group A epitope is grafted, and the polymer particles on which at least one oligosaccharide corresponding to a blood group B epitope is grafted, may also, if appropriate, to be mixed with polymer particles bearing both at least one oligosaccharide corresponding to a blood group A epitope, and at least one oligosaccharide corresponding to a blood group epitope B.

Advantageously, the chosen matrix does not cause undesired activation of the coagulation factors present in the plasma to be treated.

Immuno affinity chromatography or batch depletion:

The matrix as defined herein is used in immunoaffinity chromatography or batch depletion binding anti-A and anti-B antibodies.

Depending on the matrix used, we will obtain

or a so-called "anti-A" matrix that is to say containing only polymer particles on which are grafted only oligosaccharides corresponding to a blood group A epitope

a so-called "anti-B" matrix, that is to say containing only polymer particles on which are grafted only oligosaccharides corresponding to a blood group B epitope

a matrix called "anti-A anti-B" that is to say containing both polymer particles on which are grafted at least one oligosaccharide corresponding to a blood group epitope A and polymer particles on which at least one oligosaccharide corresponding to a blood group B epitope is grafted. In a particular embodiment, the matrices have the following formulas:

support - spacer - ligand (trisaccharide).

In a particular embodiment, the matrices have the following formulas:

Matrix "anti-A":

support - spacer - trisaccharide corresponding to the epitope of the blood group

AT,

"Anti-B" matrix:

B

Matrix "anti-A and anti-B":

support - spacer - trisaccharide corresponding to the blood group A epitope and support - spacer - trisaccharide corresponding to the blood group B epitope

More particularly:

Matrix "anti-A":

(Polymer particle) - NH-C ₅ Hio-CO-NH-C 3 H ₆ - (N-acétylGalai_3 (Fucai_2) Gal)

"Anti-B" matrix:

(Polymer particle) - NH-C ₅ H 0 -CO-NH-C ₃ H ₆ - (Gal ₃₎ ((Fuc ai ₂ ) Gal) Matrix "anti-A and anti-B":

(Polymer Particle) - NH-C ₅ H 10 -CO-NH-C ₃ H 6 - (N-Acetyl Galal ₃ (Fuc 2) Gal) and (Polymer Particle) - NH-C ₅ H 10 -CO-NH-C ₃ H ₆ - (Galai_ ₃ ((Fuc ai_ ₂ ) Gal)

The matrix can be introduced into a chromatography column. On an industrial scale, the column may contain from 1 to 150 liters, advantageously from 1 to 100 liters, preferably from 1 to 50 liters. In the case of a pilot scale implementation, columns of 1 to 50 cm in height can be used, the diameter is then adapted to the column height used. The volume of matrix used in the column is adjusted according to the desired residual level of anti-A and / or anti-B antibodies relative to the initial level of anti-A and / or anti-B antibodies of the solution.

The anti-A and / or anti-B antibodies present in the plasma bind to the matrix, and the unadsorbed (plasma) product is recovered, depleted of anti-A and / or anti-B antibodies. The plasma is poured through the matrix at a rate and under conditions that allow binding of the antibodies to the ligands carried by the matrix. The dies are suitable for industrial use and can be advantageously re-used repeatedly, without degradation. Their regeneration can be carried out by procedures known to those skilled in the art, for example by treatment with sodium hydroxide (NaOH), for example 1M.

Advantageously, the regeneration steps are adapted to take account of the fouling of the column due to the passage of plasma.

Preferably, the pH of the plasma is adjusted to a pH greater than or equal to 5, advantageously greater than or equal to 6, advantageously to a pH of about 7.4 before chromatography or batch depletion.

The charge of the column is adapted to the desired residual level of anti-

A and / or anti-B. The specificity of such a matrix does not require prior conditioning of the plasma, that is to say that any fraction or plasma may be suitable.

Plasma percolation does not involve an elution mechanism. Therefore, the plasma is drilled through the column, possibly through a pump. This percolation allows the retention of anti-A and anti-B antibodies. The contact time between the ligands and the plasma is greater than or equal to 30 seconds, advantageously greater than or equal to one minute, preferably greater than or equal to 2 minutes.

Step b) thus makes it possible to eliminate the anti-A and B antibodies. Preferably, step b) makes it possible to reach a residual content of anti-A and anti-B antibodies such as the result of the Coombs test. is negative at 1/64 dilution, ultimately allowing the production of a universal plasma according to the method of the invention that can be used on any recipient, regardless of blood group.

In a preferred embodiment, the universal plasma from step b) has an anti-A antibody content and an anti-B antibody content that is negative in the Coombs test at 1/64 dilution. Preferably, the anti-A antibody content is such that the Coombs test result is negative at 1/32 dilution, preferably negative at 1/16 dilution, even more preferably negative at 1/8 or lower dilution. . Preferably, the anti-B antibody content is such that the result of the Coombs test is negative at 1/32 dilution, preferably negative at 1/16 dilution, even more preferably negative at 1/8 or lower dilution. .

In a particular embodiment, the plasma thus depleted of anti-A and anti-B antibodies can then be subjected to a lyophilization step (step c) or freeze-drying or atomization. The freeze-drying step is particularly delicate since it is necessary not to compromise the hemostatic properties of the pool of secure plasmas. It is then necessary to control the balance between a very low residual moisture content and the conservation of coagulation factors, which may be particularly sensitive to the aggressive lyophilization process. Preferably, the lyophilization step c) makes it possible to obtain a freeze-dried plasma having a moisture content of less than 3%, preferably less than 2%. Typically, the lyophilization of step c) comprises several phases: freezing, primary drying or sublimation, and secondary drying or final drying.

By "lyophilization" or "freeze-drying" is meant a low temperature dehydration operation which consists of removing by sublimation most of the water contained in a product. It allows long-term preservation by lowering the water activity of the product.

A conventional lyophilization process consists of three steps:

1) A freezing step: each formulation has a critical temperature for lyophilization. They must be cooled below this point (eutectic temperature) for optimal solidification.

There are two types of freezing depending on the composition of the solution to be frozen:

- A slow freezing rate makes it possible to obtain a frozen solution structure presenting crystals of large sizes

- A fast freezing rate makes it possible to obtain a frozen solution structure with fine crystals.

2) A primary drying step in which the pressure of the drying chamber (lyophilization) is lowered allowing the sublimation of the ice. This phase is carried out at low or high temperature depending on the temperature of collapse or melting of the product. Sublimation requires energy, the heat is transferred to the product through the shelves where the flasks rest and by radiation. This step allows the sublimation of water called "free". The structure of the frozen solution (and thus the rate of freezing) influences the sublimation rate, in particular by modifying the vapor pressure necessary for the sublimation phenomenon.

3) A secondary desiccation step (desorption): the temperature of the shelves is increased or maintained and the pressure of the chamber is lowered to allow the desorption of the so-called water "bound" to the product. This which aims to obtain a final amount of moisture suitable for the preservation of the product.

Lyophilization is a time and energy consuming process that can take several days or even weeks to complete (if the lyophilization cycle is not optimized). Development and optimization of lyophilization cycles are established from critical temperatures (T'g solidification temperature, Te collapse temperature, Tf melting temperature).

In a particular embodiment of the invention, the lyophilization comprises a preliminary precooling step, advantageously at -5 ° C.

In a particular embodiment of the invention, the lyophilization comprises a rapid freezing phase between -30 ° C and -60 ° C in which the water contained in the plasma is solidified. Typically, this freezing step has a duration of a few hours, with a step of gradually decreasing the temperature to reach the total solidification temperature of the plasma to be freeze-dried.

In particular, the rapid freezing phase can take place at -50 ° C. The freezing step is carried out with a ramp having a duration of between 15 minutes and 60 minutes, preferably of approximately 30 minutes and a plateau lasting between 100 and 600 minutes, preferably of approximately 300 minutes.

When all the plasma is frozen, then intervenes the sublimation phase, also called primary desiccation, which will cause the passage of water from the solid form to the vapor form, without passing through a liquid form. This step is done at low pressure and at a temperature below the initial melting temperature, ie at a temperature between -20 ° C and 20 ° C. Typically, the primary drying step lasts a few hours. The primary drying phase is generally prolonged, that is to say that the sublimation is continued so as to introduce a safety step in the process to ensure homogeneity of the product, especially in short cycles.

The secondary drying step is carried out under a pressure of less than 300 μΕ ^ Γ (30 Pa) and a temperature of between 10 and 15 ° C. Typically, the first step at 10 ° C has a ramp of a duration of between 20 and 120 minutes, preferably about 60 minutes and a plateau with a duration of between 2000 and 4000 minutes, preferably about 3000 minutes. The second stage at 15 ° C has a ramp of a duration of between 5 and 60 minutes, preferably of about 10 minutes and a plateau with a duration between 800 and 2000 minutes, preferably about 1200 minutes. When the primary drying phase is complete, the product obtained still contains captive residual water which corresponds to the water molecules remaining trapped on the surface of a product subjected to primary drying. The secondary drying or final drying phase allows the desorption of the said residual water to obtain a minimum final product moisture, at a level of less than or equal to 3% by weight, in particular less than or equal to 2% by weight, in order to the preservation of the product.

This final drying step is carried out at a temperature between 20 and 50 ° C under a reduced pressure.

In particular, the final drying is carried out at a temperature of between 30 and

35 ° C under a reduced pressure of 30 μΕ ^ Γ (about 3 Pa). Typically, the first stage at 35 ° C has a ramp with a duration of between 2000 and 15000 minutes, preferably 6000 minutes and a plateau with a duration of between 800 and 2000 minutes, preferably 1200 minutes. The second stage at 30 ° C has a ramp with a duration of between 2000 and 1000 minutes, preferably 480 minutes and a plateau lasting between 1200 and 2500 minutes, preferably 1800 minutes.

The primary and secondary drying phases have durations adapted to the critical temperature of the product to be lyophilized (plasma).

Advantageously, the lyophilization protocol used makes it possible to obtain a freeze-dried universal plasma having a moisture content of less than 3%, preferably less than 2%.

By "atomization" is meant a method of dehydrating a liquid (milk, serum, plasma, etc.) in the form of powder by passing through a flow of hot air.

The method of the invention, regardless of the presence or absence of a freeze-drying or freeze-drying or atomization stage may further comprise a step of biosafety upstream of step a) of said method. The non-universal unit plasmas constituting the mixture of plasmas of step a) of the process of the invention are biologically safe, for example viro-inactivated by physicochemical treatment. By "biological safety" is meant the suppression of the pathogenic effect of viral agents that may be present in the plasma. "Biological safety" may include "viral inactivation" or "viro- inactivation". This biological safety destroys the majority of pathogens such as bacteria, enveloped or non-enveloped viruses, or other unconventional pathogens (prions) or prevents their replication. In the context of this invention, biological security is preferentially done by physicochemical treatment of non-universal unit plasmas, prior to their mixing. Alternatively, this biological security can be done on the mixture of plasmas obtained in step a). Biological securing by physicochemical treatment can be a treatment using a photochemical agent (UV, IR ...) or solvent-detergent.

In a particular embodiment of the invention, the biological safety is carried out by addition of Amotosalem (Intercept®) and UV irradiation 5-10 min at 380-400 nm.

Preferably, this biosafety is effected by solvent / detergent treatment. Preferably the solvent / detergent used is a solvent / detergent whose removal of the plasma of interest is facilitated because of its properties, for example a dialysable solvent / detergent or a filterable solvent / detergent. Indeed, the use of dialysable and / or filterable solvent / detergent makes it easier to eliminate these products, unlike non-dialyable and / or non-filterable solvents / detergents (such as TNBP / Triton X-100®) which require dedicated disposal steps for example by extraction with vegetable oil or adsorption on hydrophobic chromatography. As the dialysable and / or filterable solvent / detergent, for example, HECAMEG® (6-O- (N-heptylcarbamoyl) -methyl-α-D-glucopyranoside) may be mentioned.

Advantageously, the biological security step is coupled, if necessary, to a step of eliminating the treatment, for example by dialysis, by diafiltration, by affinity filtering (Plasmaflex PLAS4®, Macopharma) or by using a dedicated device ( Solvent Removal® Remover by Pall Biosepra) or an adsorbent resin (SDR hyperD® resin marketed by Pall Life Sciences).

In a particular embodiment, the step of eliminating the treatment is carried out at a temperature of between 0 and 25 ° C., advantageously between 5 and 20 ° C., and even more advantageously between 10 and 15 ° C. Temperatures below 25 ° C., in particular below 20 ° C., advantageously make it possible to partially or totally limit the activation of coagulation factors.

In another particular embodiment of the invention, the biological securing step is carried out by any appropriate means known to those skilled in the art, in particular by methods of inactivation or viral elimination. By viral inactivation methods is meant especially heat treatment (pasteurization and / or dry heating), and / or irradiation (UVC, and / or Gamma).

Viral elimination methods include nano filtration which can also be used to remove an infectious agent, including viruses and viruses. NCTA. Nanofiltration generally refers to plasma filtration through a filter with a pore size of less than 80 nm. Available filters are, for example, BioEx® filters, Planova® 75 nm, Planova® 35 nm, Planova® 20 nm or Planova® 15 nm (Asahi corporation), Ultipor DV 50® or DV 20® (Pall Corporation), Virosart CPV ® (Sartorius), Viresolve NFR® or NFP® (Millipore). The nanofiltration can advantageously be carried out on a single filter or on several filters in series of identical or decreasing porosity, for example with a 35nm-35nm, 35nm-20nm, 20nm-20nm or 20nm-15nm sequence.

The removal of infectious agents can also be achieved by means of filtration in depth on filters. The available filters are, for example, regenerated cellulose composite filters, in which filtering aids may have been added (such as cellite, perlite or Kieselguhr earths) marketed by Cuno (Zeta + VR series® filters), Seitz® (P-series Depth Filter®) or Sartorius (Virosart CPV®, Sartoclear P depth filters®).

By "pathogen" is meant a contaminant viral, bacterial or unconventional pathogenic type (prion). The presence of such contaminants is unacceptable for the use of a therapeutic plasma.

By "biologically secure plasma" or "viro-inactivated plasma" is meant a plasma having undergone a biological security step, for example by inactivation, that is to say the actual destruction or the inhibition of the replication of agents. pathogens such as viral, bacterial or unconventional pathogenic contaminants. In a particular embodiment, the plasma mixture of step a) of the process of the invention is inactivated by solvent / detergent. Such a plasma inactivation technique is particularly advantageous since it allows the biological safety of the plasma by simple addition of solvent / detergent under conditions allowing the preservation of the integrity of the other constituents of the plasma. The operating conditions allowing the removal of pathogens using solvent / detergent while preserving the other constituents of the plasma are known to those skilled in the art. According to the invention, this pathogen elimination technique is preferably applied to each unit plasma collected by apheresis. In another embodiment, the pathogen removal technique is advantageously applied to the plasma pool.

In a particular embodiment of the invention, an additional step may be added to adjust the physicochemical properties and restore physicochemical properties similar or similar to those of an untreated plasma. In particular, the ionic balance, the pH, the osmolarity, must be maintained at physiological levels compatible with the administration to the patients. Except according to the methods used in the process according to the invention, certain parameters, such as the ionogram, can be slightly altered, especially for example during diafiltration steps. A step that makes it possible to restore the physico-chemical properties of the normal plasma, for example the ionogram, are therefore particularly advantageous.

The method of the invention may also comprise a sterilizing filtration step or clarifying filtration on a 0.45 μιη and / or 0.2 μιη filter, for example on a polypropylene filter or equivalent media such as the ProFile filter (Pall).

The method of the invention may further comprise an ultrafiltration step upstream of step a) of said method for removing bacteria and the solvent-detergent possibly used to inactivate the plasma. The unit plasmas constituting the plasma mixture of step a) of the process of the invention are ultrafiltered in order to eliminate the bacteria potentially present in the plasma. Ultrafiltration may advantageously include a dialysis step to remove the solvent-detergent used for plasma inactivation.

In the context of this invention, the elimination of bacteria is by ultrafiltration of the unit plasmas, prior to their mixing. Alternatively, this bacterial removal can be done on the mixture of plasmas obtained in step a).

According to the invention, this technique of eliminating bacteria by ultrafiltration is preferably applied to each unit plasma collected by apheresis. In another embodiment of the invention, the removal of the bacteria by ultrafiltration is applied to the plasma pool.

In a particular embodiment of the invention, the ultrafiltration step is carried out so as to totally or partially limit the activation of coagulation factors.

The present invention also relates to a universal plasma obtained according to the method described above.

The invention also relates to a universal lyophilized plasma compatible with all blood groups. Preferably, the freeze-dried universal plasma of the invention is characterized in that it comprises a mixture of plasmas collected from donor individuals belonging to the blood groups A, B, AB and / or O. This freeze-dried plasma has a level of moisture less than 3%, preferably less than 2%. It is further characterized in that it can be stored at a temperature ambient or in a refrigerated chamber at a temperature between + 2 ° C and + 30 ° C and for a duration greater than one year, preferably at least 2 years.

Preferably, the freeze-dried universal plasma of the invention is characterized in that it comprises an anti-A and / or anti-B antibody content such that the result of the Coombs test is negative at the 1/64 dilution. Preferably, the anti-A antibody content of the freeze-dried universal plasma of the invention is such that the result of the Coombs test is negative at 1/32 dilution, preferably negative at 1/16 dilution, more preferably still negative. at 1/8 dilution or lower. Preferably, the anti-B antibody content of the freeze-dried universal plasma of the invention is such that the result of the Coombs test is negative at 1/32 dilution, preferably negative at the 1/16 dilution, more preferably still negative. at a dilution of 1/8 or lower Preferably, this freeze-dried blood plasma is sterile.

Advantageously, the freeze-dried blood plasma is depleted of endotoxins.

The invention also relates to a process for preparing reconstituted plasma comprising the step of reconstituting the lyophilized plasma, biologically secure, and compatible with all blood groups in a recovery solvent. The reconstitution of the plasma thus makes it possible to obtain an injectable preparation that can be administered to any recipient under emergency conditions.

Typically, the reconstitution of the plasma is carried out in a recovery solvent volume of between 10 and 500 ml, preferably 100-300 ml. Typically, this reconstitution is carried out with a volume making it possible to obtain an iso-osmotic plasma.

Preferably, this recovery solvent is water and more preferably water for injection. Preferably, the reconstitution of the freeze-dried plasma for obtaining an injectable preparation takes place in a time of less than 6 minutes, preferably less than 3 minutes.

Also, the use of the plasma according to the invention is very advantageous and eliminates the time required for thawing during the use of fresh frozen plasma.

The invention also relates to a reconstituted plasma, biologically secure, and compatible with all blood groups and directly injectable. Such reconstituted plasma is likely to be administered to any individual, regardless of blood type. It is therefore highly adapted for use in emergency conditions, in particular for the treatment of hemorrhagic emergencies with coagulopathy, especially in an isolated situation with logistical conditions that do not make it possible to control a negative cold chain. The reconstituted plasma according to the invention also has the advantage of destroying most of the pathogens, which considerably reduces the potential spread of pathogens to the recipient individuals. This reconstituted plasma meets all the regulatory requirements to which plasmas used in therapy are subjected.

The reconstituted plasma of the invention is characterized in that the factor VIII concentration is greater than 0.5 IU / ml, preferably greater than 0.7 IU / ml.

The reconstituted plasma of the invention is characterized in that the Factor V concentration is greater than 0.15 IU / ml, and preferably between 0.7 and 1.2 IU / ml. International Units (IU) for coagulation factors express the plasma activity of the proteins to which this expression is applied. An international unit (IU) of these plasma proteins is the amount of this factor contained in one mL of normal human plasma.

The reconstituted plasma of the invention is characterized in that the fibrinogen concentration is greater than 1 g / l and more preferably between 2 and 4 g / l.

The reconstituted plasma of the invention is characterized in that it is sterile and pyrogen-free.

Of course, the present invention is not limited to the embodiments described and shown, but it is capable of many variants accessible to those skilled in the art.

figures

FIGS. 1A and 1B: these figures illustrate the respective results of the factor V (FIG. 1A) and factor VIII (FIG. 1B) assay present in the universal plasma obtained from human donor specimens belonging to the blood groups A, B, AB and O after immunoaffinity chromatography, in the starting plasma and the non-retained fractions (FRN 1-4).

FIGS. 2A and 2B: these figures illustrate the respective results of the factor V (FIG. 2A) and factor VIII (FIG. 2B) assay present in the universal plasma obtained from human donor specimens belonging to the blood group O after the immunoaffinity chromatography, in the starting plasma and the non-retained fractions (FRN 1-14). Figure 3 shows the results of time measurement of Quick (TP) and measurement of activated partial thromboplastin time (APTT) of universal plasma obtained from donor individuals blood group O after immunoaffinity chromatography, in baseline plasma and non-retained fractions (FR 1-14).

FIG. 4 shows the result of assaying total proteins present in the universal plasma obtained from human donor specimens belonging to the blood group O after the depletion in batch, in the starting plasma and the samples respectively after 10 minutes, 30 minutes, 1h and 2h incubation.

FIGS. 5A and 5B: these figures illustrate the respective results of assaying factor VIII (FIG. 5A) and factor V (FIG. 5B) present in universal plasma obtained from human donor specimens belonging to blood group O after depletion in batch, in the starting plasma and the samples respectively after 10 min, 30 min, 1h and 2h of incubation. FIG. 6 shows the results of measurement of rapid time and measurement of the activated partial thromboplastin time of universal plasma obtained from human donor specimens belonging to the blood group O after the depletion in batch, in the starting plasma and in the samples respectively. after 10 minutes, 30 minutes, 1 hour and 2 hours of incubation. EXAMPLES

Example 1 Preparation of Universal Plasma Obtained from Human Donor Specimens belonging to the Blood Groups A, B, AB and O Al Collection of Non-Universal Unit Plasmas

About 200 mL of plasma from apheresis is collected from human donors belonging to blood groups A, B, AB and O.

During collection of the plasma, deleucocytation of said non-universal unit plasmas is carried out by centrifugation twice at 1500 g for 10 min.

The non-universal unit plasmas are then subjected to a biosafety step by treatment with Amotosalem (Intercept®): 15 ml of amotosalem at 150 μΜ are added to each non-universal unit plasma which are then subjected to 5-10 min of irradiation. by UVA at a wavelength of 380-400nm. The residual Amotosalem and the photoproducts are then removed by filtration on an adsorbent filter to reach a residual Amotosalem <2μΜ level. These non-universal attenuated unit plasmas are then deep-frozen within 8 hours after the plasma collection. This step then allows the conservation of said plasmas at a temperature of -25 ° C.

15 minutes before mixing the plasmas, the plasmas to be thawed are placed in a water bath at 37 ° C.

B / Mixture of non-universal unit plasmas

The plasma mixture is prepared by mixing the non-universal unit plasmas obtained from human donor specimens belonging to the blood groups A, B, AB and O. For example, for the mixture, use is made of:

6 plasma pockets (1327 mL of plasma) obtained from 3 different donors belonging to the blood group A,

6 plasma pockets (1327 mL of plasma) obtained from 3 different donors belonging to blood group B, and

- 6 plasma pockets (1327 mL of plasma) obtained from 3 different donors belonging to the blood group AB, and

6 plasma pockets (1327 mL of plasma) obtained from 3 different donors belonging to the blood group O.

A mixture comprising 5308 ml of non-universal plasmas is thus obtained.

Removal step of the anti-A and anti-B antibodies present in the plasma.

The non-universal plasma mixture obtained in the preceding step is subjected to an industrial scale, at a stage of anti-A / anti-B affinity chromatography carried out on a column comprising a 50/50 (v / v) mixture of cross-linked cellulose beads on which are grafted trisaccharides corresponding to a blood group A epitope (N-acetylgalactosamine (Gal Ac) -Galactose (Gal) -Fucose), designated Iso A gel HyperCel®, and crosslinked cellulose beads on which are grafted trisaccharides corresponding to a blood group epitope B (Galactose-Galactose-Fucose), designated Iso B HyperCel® gel.

The trisaccharides are grafted to the beads by a spacer of formula:

-NH-C ₅ H 10 -CO-NH-C ₃ H ₆ - The matrix used has the following formula:

(Polymer Particle) - NH-C ₅ H 0 -CO-NH-C ₃ H ₆ - (N-Acetyl Galal ₃ (Fuc ₂ ) Gal) and (Polymer Particle) - NH-C ₅ Hi ₀ -CO-NH- C ₃ H ₆ - (Galal-3 (Fuc ai ₂ ) Gal) The density of grafted trisaccharides is 0.3 mg / ml of matrix gel. The plasma mixture was passed through a 5.5 ml column (comprising 2.75 ml of HyperCel® Iso A gel + 2.75 ml of HyperCel® Iso B gel).

Column format: D = 1 cm x 7 cm (column volume (VC) = 5.5 ml).

The column is equilibrated in phosphate buffered saline (PBS).

Contact time: 2.5 min

Charge: 165mL of plasma mixture at a flow rate of 2mL / min.

The effluent (4 fractions not retained) is recovered in fractions of approximately 45 mL. The collection is stopped at the end of the injection to avoid dilution of the plasma.

Dosage tests:

The residual anti-A and anti-B activity, corresponding to the percentage of anti-A and anti-B isoagglutinins present in the final plasma preparation relative to their concentration before the affinity chromatography step, was measured by flow cytometry (sensitive and precise technique), according to the technique described below.

The wells of a bottom plate V are saturated with 125 μl of PBS supplemented with 2% fetal calf serum (FCS) for 1 hour at 37 ° C. 100 μl of suspension of Group AB red blood cells are then added (PBS + 2% FCS) at a rate of 0.05% per well. The plates are centrifuged for 1 min. at 100g and the supernatant is removed.

100 μl of native plasma or non-retained fraction (FNR) +/- diluted in PBS + 2% FCS are added before incubation for 60 min. at 37 ° C.

6 washes are carried out by addition of 150 μΐ in PBS + 2% FCS.

100 μl of Anti IgG-PE secondary antibody in PBS + 2% FCS are then added and incubated for 20 minutes. at +4 ° C.

After 2 washes in PBS + 2% FCS, the sample is read by flow cytometry.

The mean fluorescence intensity (MFI) of the positive control has been reported as a function of plasma concentration (standard curve) for concentrations ranging from 0.23 g / L to 30 g / L. The results are expressed as the ratio between the slope of the sample and the slope of the positive standard. The equation of the standard curve is y = ax + b; where "a" is the slope value of the standard curve and "b" is the zero point corresponding to the background noise of the test. Since the sample equation is y '= a'x + b, and using the known MFI values of the sample (y') and the plasma concentration (χ '), the slope ratio has been calculated as [(IMF-b) / [IgG concentration]] / a. Results:

The residual anti-A and anti-B activity obtained is shown in Table 1 below.

Table 1

The results show a significant reduction in the residual anti-A and anti-B (IgG type) activity, which is between 6 and 21% after immunoaffinity.

Residual anti-A and anti-B activity was also measured by Coombs test according to the following method:

100 μl of suspension of AB red blood cells at 1% in physiological saline are deposited per well on a microplate and then centrifuged for 1 min. at 100g. The supernatant is removed and then 100 μl of plasma or non-retained fraction (FNR) +/- diluted in physiological saline is added. After incubation 30 min. at 37 ° C and centrifugation 1 min. at 100g, the agglutinates are read by gentle agitation.

The results are illustrated in Table 2 below.

Table 2 The results in Table 2 show that the level expressed in the Coombs direct test in fractions 1 to 4 is between 1/4 and 1/16.

The product thus obtained is then in conformity (at 1/64 dilution) to a negative result in the Coombs direct test.

A universal plasma depleted of anti-A and anti-B antibodies is thus obtained.

Content of coagulation factors present in universal plasma

The activity of the coagulation factors contained in the universal plasma resulting from the immunoaffinity chromatography step was tested. The procedures and results are presented below.

Determination of factor V

Factor V (FV) was assayed by the Zymutest Factor V test of Hyphen Biomed, France (ref: RK009A) as recommended by the manufacturer. The assay is a sandwich ELISA that uses anti-FV antibody attached to microwells. The citrated plasma is diluted in the buffer provided (1/100 etl / 200) and incubated for 2 h at 37 ° C. The plate is washed 5 times and incubated with an anti-FV secondary antibody coupled to horse radish peroxidase for 2h at 37 ° C. The plate is washed 5 times and the bound antibody is revealed for 5 min with a solution of 3,3 ', 5,5'-tetramethylbenzidine (TMB). The reaction is stopped by adding 0.45 M H ₂ SO _4. The optical density is read at 450 nm after 10 minutes of stabilization. A standard curve is established and two control plasmas with intermediate concentrations of FV are performed at each test.

Factor VIII assay

Factor VIII (F VIII) was assayed by the Asserachrom® VIII: Ag test obtained from

Stago, France (ref: 00280) as recommended by the manufacturer. The assay is a sandwich ELISA that uses anti-FVIII antibody attached to microwells. The citrated plasma is diluted in the buffer provided (1/10 and 1/20) and incubated for 2 hours at room temperature. The plate is washed 5 times and incubated with a secondary anti-FVIII antibody coupled to horseradish peroxidase for 2 hours at room temperature. The plate is washed 5 times and the bound antibody is revealed for 5 min with TMB solution. The reaction is stopped by adding 0.45 M H ₂ SO _4. The optical density is read at 450 nm after 10 minutes of stabilization. A standard curve is established and a control plasma with a known concentration of FVIII is made at each test.

Results: The respective factor V and factor VIII assay results are illustrated in FIGS. 1A and 1B.

Passage on an immunoaffinity chromatography column does not affect the factor V and factor VIII plasma levels. The universal plasma obtained has a factor V level greater than 5 μg / mL and a factor VIII level greater than 0.5 IU / mL.

Example 2 Preparation of Universal Plasma From Human Donor Specimens belonging to Blood Type O Using Immunoaffinity Chromatography

Al Collection of Non-Universal Unit Plasmas

About 500 mL of plasma from human donors belonging to blood group O are collected by apheresis.

During collection of the plasma, deleucocytation of said non-universal unit plasmas is carried out by centrifugation twice 1500 g for 10 min.

The non-universal unit plasmas are then subjected to a biosafety step by treatment with Amotosalem (Intercept®): 15 ml of amotosalem at 150 μΜ are added to each non-universal unit plasma which is then subjected to 5-10 min of irradiation. per UVA at a wavelength of 380-

400nm. The residual Amotosalem and the photoproducts are then removed by filtration on an adsorbent filter to reach a residual Amotosalem

2μΜ.

These non-universal attenuated unit plasmas are then deep-frozen within 8 hours after the plasma collection. This step then allows the conservation of said plasmas at a temperature of -25 ° C.

15 minutes before mixing the plasmas, the plasmas to be thawed are placed in a water bath at 37 ° C. B / Mixture of non-universal unit plasmas

The plasma mixture is prepared by mixing three non-universal unit plasmas of 75mL obtained from 3 human donor specimens belonging to the O blood groups.

A mixture comprising 225 ml of non-universal plasma is thus obtained.

Removal step of the anti-A and anti-B antibodies present in the plasma. The non-universal plasma mixture obtained in the preceding step is subjected to an industrial scale, at a stage of anti-A / anti-B affinity chromatography carried out on a column comprising a 50/50 (v / v) mixture of crosslinked cellulose beads on which are grafted trisaccharides corresponding to a blood group A epitope (N-acetylgalactosamine (GalNAc) -Galactose (Gal) -Fucose), designated Iso A gel HyperCel®, and crosslinked cellulose beads on which are grafted trisaccharides corresponding to a blood group B (Galactose-Galactose-Fucose) epitope, designated HyperCel® Iso B gel. The trisaccharides are grafted to the beads by a spacer of formula:

-NH-C ₅ H 0 -CO-NH-C ₃ H ₆ -.

The matrix used has the following formula:

(Polymer Particle) - NH-C ₅ H 0 -CO-NF-C ₃ F ₆ - (N-acetyl Galal ₃ (Fuc 2) 2 and (polymer particle) - NH-C ₅ H ₀ -CO-NH-C ₃ H ₆ - (Galal-3 (Fuc ai ₂ ) Gal).

The density of grafted trisaccharides is 0.3 mg per ml of matrix gel.

The plasma mixture was passed through a 5.5 ml column (comprising 2.75 ml of HyperCel® Iso A gel + 2.75 ml of HyperCel® Iso B gel).

Column format: D = 1 cm x 7 cm (column volume (VC) = 5.5 ml).

The column is equilibrated in PBS buffer.

Contact time: 5 min

Charge: 20mL of plasma mixture at a flow rate of 1mL / min. The effluent (14 fractions not retained) is recovered in fractions of 1 ml. The collection is stopped at the end of the injection to avoid dilution of the plasma.

Dosage tests:

The wells of a bottom plate V are saturated with 125 μl of PBS supplemented with 2% fetal calf serum (FCS) for 1 hour at 37 ° C. 100 μl of suspension of red blood cells AB are then added (PBS + 2% FCS) at a rate of 0.05% per well. The plates are centrifuged for 1 min. at 100g and the supernatant is removed. 100 μl of native plasma or non-retained fraction (FNR) +/- diluted in PBS + 2% FCS are added before incubation for 60 min. at 37 ° C.

6 washes are carried out by addition of 150 μΐ in PBS + 2% FCS.

100 μl of Anti IgM-PE or Anti IgG-PE secondary antibody in PBS + 2% FCS are then added and incubated for 20 minutes. at +4 ° C.

After 2 washes in PBS + 2% FCS, the sample is read on the cytometer.

The mean fluorescence intensity (MFI) of the positive control has been reported as a function of plasma concentration (standard curve) for concentrations ranging from 0.23 g / L to 30 g / L. The results are expressed as the ratio between the slope of the sample and the slope of the positive standard. The equation of the standard curve is y = ax + b; where "a" is the slope value of the standard curve and "b" is the zero point corresponding to the background noise of the test. Since the sample equation is y '= a'x + b, and using the known MFI values of the sample (y') and the plasma concentration (χ '), the slope ratio has been calculated as [(IMF-b) / [IgG concentration]] / a.

Results:

The residual anti-A and anti-B activity obtained is shown in Table 3:

Table 3

The results show a significant reduction in residual anti-A and anti-B activities, which are between 9 and 12% (IgM) and between 7 and 9% (IgG) after immunoaffinity. Immunoaffinity chromatography thus makes it possible to eliminate at least 80% of the IgG and IgM anti-A and anti-B types of a plasma, even when the initial level in the starting plasma is very low.

100 μl of suspension of 1% AB red blood cells in physiological saline are deposited per well on a microplate and then centrifuged for 1 min. at 100g. The supernatant is removed and then 100 μΐ of plasma or non-retained fraction (FNR) +/- diluted in water is added physiological. After incubation 30 min. at 37 ° C and centrifugation 1 min. at 100g, the agglutinates are read by gentle agitation.

The results are shown in Table 4 below:

Table 4

The results show that even with a very low initial rate measured in Coombs test (less than 1/4 in the initial plasma), immunoaffinity chromatography can reduce the level of anti-A and anti-B in plasma at a rate less than or equal to 1/1 measured in Coombs test.

A universal plasma depleted of anti-A and anti-B antibodies is thus obtained.

Content of coagulation factors present in universal plasma

The activity of the coagulation factors contained in the universal plasma resulting from the immunoaffinity chromatography step was tested. The results are presented below.

Determination of factor V

Factor V (FV) was determined according to the assay protocol described in the example

1.

Factor VIII assay Factor VIII (FVIII) was assayed according to the assay protocol described in Example 1.

Results:

The respective factor V and factor VIII assay results are shown in Figures 2A and 2B.

Passage on an immunoaffinity chromatography column does not affect the factor V and factor VIII plasma levels. There is only a slight decrease in the level of FVIII and FV on the first fractions (FNR1-2) which are diluted by the buffer contained in the dead volume of the column. The other fractions are not affected by this dilution phenomenon.

The universal plasma obtained has a factor V level greater than 4 μg / mL and a factor VIII level greater than 0.5 IU / mL. This is further confirmed by the Quick Time and Activated Partial Thromboplastin time (APTT) measurements according to the following protocols:

Quick time measurement (TP)

The time of Quick is measured by means of the kit Neoplastine CI (ref: 00605, Stago, France) on a STAR device (Stago) following the dedicated program. Briefly, citrated plasma is used pure. Neoplastine® (thromboplastin prepared from fresh rabbit brain tissue) is mixed with the 25 mM calcium solution and serves as an initiator of coagulation. After incubation at 37 ° C the plasma and coagulation inducer are mixed by the biorobot and the coagulation time is measured and compared to a series of dedicated controls (STA coag control; ref: 00678; Stago).

Activated Partial Thromboplastin Time (APTT) Measurement

The TCA is measured via the STA CK prest kit (ref: 00597, Stago, France) on a STAR device (Stago) following the dedicated program. Briefly, citrated plasma is used pure. Cephalin (a platelet substitute prepared from rabbit brain tissue) is mixed with the kaolin solution (5 mg / ml) and serves as an initiator of coagulation. After incubation at 37 ° C the plasma and coagulation inducer are mixed by the biorobot and the coagulation time is measured and compared to a series of dedicated controls (STA coag control; ref: 00679; Stago). Results:

The results are illustrated in Figure 3.

The undiluted fractions coagulate as the starting plasma. The passage on immunoaffinity chromatography does not affect the ability of plasma to coagulate (TP and TCA) except on the fractions (FNR3-4) which are diluted by the buffer contained in the dead volume of the column.

Example 3 Preparation of the Universal Plasma from Human Donor Individuals belonging to the Blood Group O Using Batch Depletion

The collection of non-universal unit plasmas is carried out according to the method described in Example 2.

The mixture of non-universal unit plasmas is obtained according to the method described in Example 2.

The elimination of the anti-A and anti-B antibodies present in the non-universal unit plasma mixture is carried out by the batch depletion according to the following method:

• take 4mL of Iso A HyperCel® gel and 4mL of HyperCel® Iso B gel and mix the 2 gels with gentle shaking

• take 5.5mL of the gel mixture obtained above for batch capture

• wash the gel mixture with 5CV (column volume) 2M NaCl solution,

• wash the gel mixture with 4 CV of 0.1 M phosphate solution at pH 8

• wash the gel mixture with 10CV of ultrapure water,

· Equilibrate the gel mixture with 5CV of PBS filtered by a 0.22μιη filter,

• add 20mL of plasma pool in the gel mixture and stir with room temperature

• Take respectively 1 ml of the gel-plasma mixture after 10 min, 30 min, 1 h, 2h of incubation, ie 4 fractions

· Recover plasma from these fractions by centrifugation at 2300g

5 min.

Dosage tests:

The amount of total protein present in the plasma recovered after the anti-A / anti-B depletion in batch is measured.

The result, shown in Figure 4, shows that the amount of total protein is only slightly decreased after the anti-A / anti-B depletion. This decline is not influenced by incubation time with HyperCel® Gel A and HyperCel® Gel B.

The amount of anti-A and anti-B antibodies present in the samples obtained after 10 min, 30 min, 60 min and 120 min of incubation in batch was respectively determined. The assay result is given in Table 5 below.

Table 5

These results show that IgG and IgM type anti-A and anti-B antibodies are eliminated mainly after 10 min of batch incubation.

Content of coagulation factors present in universal plasma

The factor VIII and factor V activity contained in the universal plasma resulting from the batch depletion step were tested. The results are presented below.

Factor V (FV) and factor VIII (FVIII) were assayed according to the assay protocols described in Example 1.

The results are illustrated in Figures 5A and 5B. The anti-A / anti-B depletion in batch does not affect the factor V and plasma factor VIII levels resulting from the batch depletion step.

The universal plasma obtained has a factor V level greater than 5 μg / mL and a factor VIII level greater than 0.6 IU / mL.

This is furthermore confirmed by the measures of time of Quick and activated partial thromboplastin time (TCA) according to the protocols described in Example 2.

The results are illustrated in Figure 6. These results show that the anti-A / anti-B depletion in batch does not affect the ability of the plasma to coagulate (TP and TCA).

Example 4 Lyophilization of the Universal Plasma The universal plasma resulting from the immunoaffinity chromatography step is distributed in 500 ml "type I" vials, such that each of the vials contains 215 ml of universal plasma.

The freeze-drying of the universal plasmas contained in each of the bottles previously obtained is carried out in a freeze-dryer of the type SMH 615, marketed by USIFROID. Each bottle is placed on a shelf. Freeze-drying is carried out under the particular conditions detailed below.

AI Pre-cooling

This step allows the freeze dryer shelves to cool down to a temperature of -5 ° C. This step avoids the degradation of coagulation factors that are heat-sensitive during the time of distribution. The batches are loaded as and when in the freeze dryer.

B / Freezing

Universal plasma is frozen at a temperature of -50 ° C. The product is maintained at this temperature for 240 minutes. The duration of the ramp is 30 minutes and the landing is 300 minutes.

Cl Vacuum

To allow sublimation, the lyophilizer is evacuated. The vacuum is carried out for 2 minutes at a pressure of 600 mbar (ie 0.6 × 10 ⁵ Pa).

D / Sublimation

This step is carried out at a temperature of between 10 and 15 ° C. and at a pressure of less than 300 μBar (ie 30 Pa).

The first stage at 10 ° C has a 60-minute ramp and a 3000-minute plateau.

The second stage with a temperature of 15 ° C has a ramp of 10 minutes and a bearing of 1200 minutes.

E / Secondary desiccation

This step is carried out at a temperature between 30 and 35 ° C under a pressure of 30 μBar (or 3Pa).

The first landing at 35 ° C has a 600-minute ramp and a 1200-minute landing. The second stage at 30 ° C has a ramp of 480 minutes and a bearing of 1800 minutes. F / Quality control of lyophilisate

This protocol makes it possible to obtain a lyophilized plasma having a relative humidity of less than 2%.

Example 5: Reconstitution of the universal plasma

Take a vial of 500 mL of universal plasma. 200 ml of water for injection is added. A reconstituted universal plasma is thus obtained.

After reconstitution, the product obtained must meet the following requirements:

reconstitution time less than 6 minutes; factor VIII concentration greater than or equal to 0.5 IU / L;

as agglutinins anti A and anti B less than 64;

The composition and characteristics of the reconstituted universal plasma are detailed in Table 6 below:

Table 6: Compositions and characteristics of reconstituted plasma The criteria measured are in accordance with regulatory requirements. The plasma obtained according to the process of the invention is therefore suitable for a therapeutic use.

Claims

1. A process for the preparation of a universal plasma, comprising the following steps: a) Mixing non-universal unit plasmas obtained from a sample of donor individuals,

b) Simultaneous elimination of the anti-A and anti-B antibodies present in the plasma by immunoaffinity chromatography or by batch depletion, and c) optionally lyophilization or atomization of the universal plasma from step b).

2. A process for preparing a universal plasma according to claim 1, wherein step a) is performed without any prior selection of donors.

3. Process for the preparation of a universal plasma according to claim 1, in which step b) is carried out by means of immunoaffinity chromatography or by batch depletion on a grafted support of oligosaccharide groups antigenically similar to the blood groups. A and B.

4. The process for preparing a universal plasma according to claim 3, wherein the oligosaccharide groups represent trisaccharides corresponding to the A and B blood group epitopes.

5. A method for preparing a universal plasma according to claim 4 wherein the trissacharides, corresponding to the blood group A epitope, have the structure N-acetylgalactosamine (GalNAc) - Galactose (Gai) - Fucose (Fuc) and those corresponding to the epitope of the blood group B, have the Galactose-Galactose-Fucose structure.

6. A method for preparing a universal plasma according to one of claims 1 to 5, wherein said donor individuals belong to the blood group selected by blood group A, blood group B, blood group AB and / or group blood O.

7. A method for preparing a universal plasma according to one of claims 1 to 5, wherein said donor individuals belong to blood group A, blood group B, blood group AB and blood group O.

8. A method for preparing a universal plasma according to one of claims 1 to 5, wherein the universal plasma from step b) has a content of anti-A and anti-B antibodies contained in the plasma. universal as the result of the Coombs test is negative at 1/64 dilution measured according to method 2.6.20 of the European Pharmacopoeia 07/2011.

9. A method for preparing a universal plasma according to one of claims 1 to 8, further comprising a step of biological securing.

The process for preparing a universal plasma according to claim 9, wherein the biosafety step is carried out by virus inactivation using a solvent / detergent, preferably using a dialysable or filterable solvent / detergent.

11. A method for preparing a universal plasma according to one of claims 1 to 10, further comprising an ultrafiltration step.

12. A method for preparing a universal plasma according to claim 11, characterized in that the ultrafiltration step is associated with a dialysis step.

13. Universal plasma obtainable by the process according to any one of claims 1 to 12.

14. Universal plasma in which the anti-A antibody content and the anti-B antibody content are in accordance with a negative result in the Coombs test at the 1/64 dilution measured according to the method 2.6.20 of the European Pharmacopoeia 07 / 2011: 20620).

15. Plasma according to claim 14 characterized in that the universal plasma is obtained from donor individuals belonging to the blood groups A, B, AB and O.

16. Universal plasma for use in the treatment of patients suffering from severe haemorrhage, particularly of traumatic origin, as a replacement for multiple coagulation factors, in an emergency situation when a clotting factor concentrate is not present. available, in cases of severe vitamin K deficiency, or in patients with thrombotic thrombocytopenic purpura.