Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/34—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood group antigens
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography
  • B01D15/3804—Affinity chromatography
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/16—Extraction; Separation; Purification by chromatography
  • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/06—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies from serum
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/06—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies from serum
  • C07K16/065—Purification, fragmentation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/44—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man

Definitions

• the present invention is in the field of reference products used for the characterization of therapeutic concentrates of polyclonal immunoglobulins. It relates to a composition highly enriched in anti-A and / or anti-B polyclonal immunoglobulins, a process for the preparation of such a composition, as well as the use of the composition for the preparation of a positive control which is useful in a method of assaying the anti-A and / or anti-B activity of a human normal immunoglobulin concentrate, and using the composition for determining the blood group of a subject.
• Normal human immunoglobulins are used in the treatment of more and more pathologies. They are used as substitution therapy in primary (congenital deficiency) or secondary (chronic lymphocytic leukemia, myeloma, infections after bone marrow transplant, recurrent bacterial infections in children infected with VI H) deficiency deficiencies of antibodies.
• ITP idiopathic thrombocytopenic purpura
• NMM multifocal motor neuropathy
• IPDC chronic inflammatory demyelinating polyradiculoneuritis
• anti-A and anti-B immunoglobulins are present in too large proportions in normal human immunoglobulins, they are likely to cause accidental haemolysis, potentially severe, in treated patients carrying antigens A and / or B .
• IVIG normal human intravenous immunoglobulins
• TIA indirect antiglobulin test
• TIA has a high degree of variability and is also likely to underestimate the anti-A and anti-B activity of normal human immunoglobulins (Thorpe et al., Biologicals, 2005 Jun; 2): 111 -6).
• the TIA test is based on the use of an antiglobulin, that is to say immunoglobulins of animal origin specifically recognizing human immunoglobulins.
• an antiglobulin that is to say immunoglobulins of animal origin specifically recognizing human immunoglobulins.
• the ability of antiglobulin to agglutinate group A or B cells coated with anti-A or anti-B immunoglobulin is likely to be saturated by the concomitant presence of a high concentration of human immunoglobulins directed against other antigens than those of blood groups A and B.
• Thorpe et al. developed a direct agglutination method, without the use of antiglobulin, in which papain-treated A or B red blood cells are used. contact with serial half dilutions of a solution of normal human immunoglobulins at 5% (50 g / l), in a microtiter plate with V wells. After centrifugation, the plate is inclined at an angle of about 70 ° for about 4-5 minutes. In case of agglutination, the cell pellet remains stuck to the bottom of the V-well, forming a well rounded point. If not, the non-agglutinated pellet slides along the V-well wall, thereby generating a droplet-like form (Thorpe et al., Biologicals, 2005 Jun, 33 (2): 111 -6).
• a negative control (fraction 07/308) consisting of normal human immunoglobulins at 5% (50 g / l) purified from plasma of donors of all AB blood groups, the plasma of which does not contain anti-inflammatory immunoglobulins. A and anti-B.
• a positive control (fraction 07/306), consisting of normal human immunoglobulins at 5% (50 g / l) known to possess anti-A and anti-B activity above the average of normal human immunoglobulins, but not producing no agglutination of erythrocytes A or B at a dilution of 1/64 in the previously developed direct agglutination method.
• a limiting positive control (fraction 07/310), consisting of fraction 07/306, to which were added amounts of an anti-A murine monoclonal antibody (BRIC 145) and a murine anti-B monoclonal antibody (BGRL 1) as this control produces agglutination of erythrocytes A or B for a dilution of at most 1/64 in the previously developed direct agglutination method.
• Tests carried out with these controls have shown that they can make it possible to standardize the agglutination tests for anti-A and anti-B immunoglobulins, to verify that the tests used are sufficiently specific and sensitive, and to facilitate identification. batches of normal human immunoglobulins whose anti-A and anti-B activity exceeds that tolerated by the health authorities.
• IVIG must now be free of agglutination of A and B cells at 1/64 dilution of an initial concentration solution of 50 g / l (5%), in a particular direct agglutination test corresponding to that developed by Thorpe et al. (European Pharmacopoeia, Chapter 2.6.20 as revised by Supplement 7.2 of January 2011).
• the positive controls mentioned above have their drawbacks, and in particular the limiting positive control, supposed to possess anti-A and anti-B activity corresponding to the limit accepted by the health authorities. Indeed, as indicated by Thorpe et al., One of the main flaws of this limiting positive control is its limited availability. In particular, because of its insufficient availability, the use of this product is currently recommended only to check the anti-A and anti-B activity of normal human immunoglobulin concentrates with anti-A anti-A activity.
• the anti-A and anti-B immunoglobulins present in this limiting positive control are of murine and non-human origin, and are in addition monoclonal and non-polyclonal, unlike the anti-A and anti-B immunoglobulins present in the concentrates. normal human immunoglobulins. These two differences can lead to underestimating or overestimating the anti-A and anti-B activity of normal human immunoglobulins, which is undesirable in both cases.
• plasma fractionators In order to avoid accidental haemolysis without reducing donor pools that may be used, plasma fractionators have developed methods for removing anti-A and anti-B immunoglobulins from their normal human immunoglobulin concentrates.
• WO01 / 27623 and WO2007 / 077365 describe the use of different affinity chromatography supports specifically binding to anti-A and anti-B immunoglobulins to eliminate anti-A and anti-B immunoglobulins from biological compositions, especially from normal human immunoglobulin concentrates.
• the non-adsorbed fraction not comprising anti-A and anti-B immunoglobulins is recovered by percolation for further processing and conditioning.
• the anti-A anti-A affinity chromatography column is then regenerated by washes which are removed.
• the anti-A and anti-B immunoglobulin fraction adsorbed during the affinity chromatography step therefore corresponds to a lost fraction.
• the inventors have demonstrated that the anti-A and anti-B polyclonal immunoglobulin fraction adsorbed on the affinity chromatography column for the purpose of eliminating anti-A and anti-immunoglobulins.
• B and obtained by assembling two column wash fractions, can be used to prepare a new positive control useful for calibrating agglutination tests for anti-A and anti-B immunoglobulins and for reliably detecting batches.
• This new positive control has the advantage of being composed of the same anti-A and anti-B polyclonal human immunoglobulins as those which may be present in a variable amount in normal human immunoglobulin concentrates.
• this new positive control could be produced in sufficient quantities to allow its routine use as a limiting positive control in all agglutination tests for anti-human immunoglobulins.
• a and anti-B normal human immunoglobulin concentrates are provided.
• the present invention thus relates to a composition
• a composition comprising polyclonal human immunoglobulins, characterized in that at least 80% by weight of the polyclonal human immunoglobulins present in the composition are polyclonal human anti-A and / or anti-human immunoglobulins.
• the polyclonal human immunoglobulins represent at least 85% by weight of the total proteins of the composition.
• At least 80% by weight of the polyclonal human immunoglobulins present in the composition are anti-A polyclonal human immunoglobulins. In another advantageous embodiment, at least 80% by weight of the polyclonal human immunoglobulins present in the composition are anti-B polyclonal human immunoglobulins. In yet another advantageous embodiment, the composition comprises both anti-A polyclonal human immunoglobulins and anti-B polyclonal human immunoglobulins, and the ratio by weight polyclonal human anti-A immunoglobulins on anti-B polyclonal human immunoglobulins ( anti-A / anti-B) is between 1/10 and 10/1.
• the composition comprising both anti-A polyclonal human immunoglobulins and anti-B polyclonal human immunoglobulins is enriched in anti-A polyclonal human immunoglobulins, and therefore has a weight ratio of anti-A polyclonal human immunoglobulins.
• anti-B (anti-A / anti-B) polyclonal human immunoglobulins ranging from 2/1 to 10/1.
• the composition comprising both anti-A polyclonal human immunoglobulins and anti-B polyclonal human immunoglobulins is enriched in anti-B polyclonal human immunoglobulins, and therefore has a weight ratio of polyclonal anti-human immunoglobulins to A on anti-B (anti-A / anti-B) polyclonal human immunoglobulins comprised between 1/10 and 1/2.
• the composition comprising both anti-A polyclonal human immunoglobulins and anti-B polyclonal human immunoglobulins is balanced in both types of immunoglobulins, and thus has a weight ratio of polyclonal anti-human immunoglobulins to A on anti-B (anti-A / anti-B) polyclonal human immunoglobulins of between 3/10 and 7/10, advantageously between 4/1 and 6/10.
• the invention also relates to a process for the preparation of a composition according to the invention, comprising the following steps:
• the method advantageously comprises the following steps:
• step c) adsorbing the composition obtained in step c) on a substrate grafted with a ligand specific for anti-B polyclonal human immunoglobulins, and
• the method advantageously comprises the following steps:
• step c) adsorbing the composition obtained in step c) on a substrate grafted with a ligand specific for anti-A polyclonal human immunoglobulins, and
• the specific ligand of anti-A polyclonal human immunoglobulins is advantageously chosen from oligosaccharides representative of group A antigens of types 1, 2, 3 and 4.
• the specific ligand for anti-B polyclonal human immunoglobulins is advantageously chosen from oligosaccharides representative of group B antigens of type 1, 2, 3 and 4.
• the support used in step a) and / or in step d) is advantageously in the form:
• a polymeric membrane the membrane being grafted with the ligand or ligands of interest.
• the particles are advantageously incorporated into a gel or a resin, which is used as a matrix of an affinity chromatography column.
• the polymeric membrane can be included in an affinity chromatography column.
• the human plasma batch or the human plasma fraction enriched in polyclonal human immunoglobulins are then adsorbed on the affinity chromatography column, and the adsorbed fraction is eluted and recovered.
• the polymer of the particles or of the membrane is advantageously chosen from cellulose and its derivatives, agarose, dextran, polyacrylates, polystyrene, polyacrylamide, polymethacrylamide, copolymers of styrene and divinylbenzene, or mixtures of these polymers. .
• the ligand of interest is advantageously grafted on the polymer particles or on the polymeric membrane by means of a spacer.
• the invention further relates to a composition obtainable by a preparation process according to the invention.
• the invention also relates to the use of a composition according to the invention as a positive control in a method for assaying the anti-A or anti-B activity of a composition comprising polyclonal human immunoglobulins.
• the invention also relates to the use of a composition according to the invention for the preparation of a positive control and / or a limiting positive control for use in a method for assaying the anti-A activity or anti-B composition comprising polyclonal human immunoglobulins.
• the invention also relates to a method for preparing a positive control for use in a method for assaying the anti-A and / or anti-B activity of a composition comprising polyclonal human immunoglobulins, comprising the steps of :
• composition comprising polyclonal human immunoglobulins having anti-A activity and / or anti-B activity below a given limit value in said assay method, and
• the invention also relates to a process for the preparation of a below-limit positive control, a limiting positive control or a positive control above the limit for use in a method for assaying anti-tumor activity. And / or anti-B of a composition comprising polyclonal human immunoglobulins, comprising the following steps:
• composition comprising polyclonal human immunoglobulins having anti-A activity and anti-B activity below a given limit value in said assay method
• composition according to the invention in which at least 80% by weight of the polyclonal human immunoglobulins present in the composition according to the invention are anti-A polyclonal human immunoglobulins and / or a composition according to the invention in which at least 80% by weight of the polyclonal human immunoglobulins present in the composition according to the invention are anti-human polyclonal immunoglobulins.
• -B to obtain different compositions enriched in anti-A and / or anti-B polyclonal human immunoglobulins,
• step b) the assay by said assay method of the anti-A and / or anti-B activity of the compositions enriched in anti-A and / or anti-B polyclonal human immunoglobulins obtained in step b), and
• composition enriched in anti-A and / or anti-B polyclonal human immunoglobulins having anti-A and / or anti-B activity in said lower assay method to obtain a positive control below the limit
• a limit positive control to obtain a limit positive control
• a positive control greater than the limit to the said limit value.
• the invention further relates to a positive control (sub-limit positive control, limiting positive control, or positive control greater than the limit) for use in a method for assaying for anti-A and / or anti- B of a composition comprising polyclonal human immunoglobulins, obtainable by one of the methods according to the invention for the preparation of a positive control described above.
• a positive control sub-limit positive control, limiting positive control, or positive control greater than the limit
• the invention finally relates to the use of a composition characterized in that at least 80% by weight of the polyclonal human immunoglobulins present in the composition are anti-A and / or anti-B polyclonal human immunoglobulins or a composition obtainable by the process according to the invention in a test for determining the blood group of a subject.
• the inventors have demonstrated that the fraction of anti-A and / or anti-B polyclonal immunoglobulins adsorbed on the affinity chromatography column for the purpose of eliminating anti-A immunoglobulins and / or anti-B may be used to prepare a new positive control useful for calibrating agglutination tests for anti-A and / or anti-B immunoglobulins and for reliably detecting batches of normal human immunoglobulins with anti-B activity. A and / or anti-B higher than the limits accepted by the health authorities.
• This new positive control has the advantage of being composed of the same polyclonal human anti-A and / or anti-B immunoglobulins as those which may be present in variable amounts in normal human immunoglobulin concentrates. Moreover, in view of the growing amount of normal human immunoglobulins produced each year, this new positive control could be produced in sufficient quantities to allow its systematic use as a limiting positive control in all agglutination tests for anti - A and / or anti - B immunoglobulins of normal human immunoglobulin concentrates.
• Antibody or "immunoglobulin” means a molecule comprising at least one binding domain to a given antigen and a constant domain comprising an Fc fragment capable of binding to the FcR receptors.
• polyclonal human immunoglobulin is meant a composition of human immunoglobulins directed against a number of distinct antigens, and comprising, for each recognized antigen, a plurality of distinct immunoglobulins capable of recognizing said antigen, generally at several distinct epitopes.
• Such polyclonal human immunoglobulins are generally purified from the plasma of one or preferably more than one donor (this is referred to as a pool of donors). Therapeutic normal human immunoglobulins are thus purified from plasma pools of generally at least 1000 donors.
• anti-A polyclonal human immunoglobulin or "anti-A immunoglobulin” is meant polyclonal human immunoglobulins recognizing blood group A antigens.
• Bood group A antigens or “A antigens” are characterized by the presence of a trisaccharide comprising an N-acetylgalactosamine (abbreviated herein as “GalNAc”) linked to a galactose (abbreviated herein as “Gaal”), itself bound to a fucose (abbreviated herein as “ Fuc "), according to the following sequence: GalNAcal -3 (Fuca1 -2) Gal.
• GalNAc N-acetylgalactosamine
• Fuc fucose
• This trisaccharide can itself be attached by its central galactose to other sugars, the number and the assembly of which varies according to the type of antigen A, as indicated in Table 1 below for type A antigens. 1 to 4, and the presence or absence as well as the nature of a Lewis antigen.
• Table 1 Structure of different types of antigen of the blood group A.
• GalNAc N-acetylgalactosamine
• Fuc fucose
• Gal galactose
• GlcNAc N-acetylglucosamine
• G le glucose
• R support (oligosaccharide, glycoprotein, glycolipid). The group A trisaccharide is indicated in bold.
• anti-B polyclonal human immunoglobulin or "anti-B immunoglobulin” is meant polyclonal human immunoglobulins recognizing blood group B antigens.
• Bood group B antigens or “B antigens” are characterized by the presence of a trisaccharide comprising a first galactose linked to a second galactose, itself linked to a fucose, according to the following sequence: Galal - 3 (Fuca1 -2) Gal.
• This trisaccharide can itself be attached by its central galactose to other sugars, the number and the assembly of which varies according to the type of antigen B, as indicated in Table 2 below for type B antigens. 1 to 4, and the presence or absence as well as the nature of a Lewis antigen.
• Table 2 Structure of different types of antigen of blood group B.
• GalNAc N-acetylgalactosamine
• Fuc fucose
• Gal galactose
• GlcNAc N-acetylglucosamine
• G le glucose
• R support (oligosaccharide, glycoprotein, glycolipid). The group B trisaccharide is indicated in bold.
• Fraction of human plasma enriched in polyclonal human immunoglobulins means any fraction of human plasma that can be obtained by fractionation of human plasma and the percentage by weight of polyclonal immunoglobulins relative to the total proteins of the fraction is greater than to that of human plasma. Such fractions are advantageously obtained by fractionation of plasma pools of at least 1000 donors.
• They may in particular include any part or subpart of the plasma which has been the subject of one or more purification steps and in particular, the supernatant of cryoprecipitated plasma, plasma cryoprecipitate (delivered or not in suspension), the fractions I at V obtained by ethanolic fractionation (according to the method of Cohn or Kistler & Nitschmann), the supernatant and the precipitate obtained after precipitation with caprylic acid and / or caprylate, the filtrates, or any fraction enriched with immunoglobulins (eluates of chromatographies and / or non-adsorbed fractions) by chromatographic separation, as described in particular in WO99 / 64462 and WO02 / 092632, and more particularly in WO02 / 092632.
• oligosaccharide representing blood group A antigens is meant any oligosaccharide comprising the trisaccharide characteristic of blood group A antigens as defined above.
• GalNAcal -3 Fuca1 -2) Gal.
• Such an oligosaccharide may further comprise other sugars present in the antigens blood group A defined above.
• such an oligosaccharide may also be chosen from tetrasaccharides, pentasaccharides and hexasaccharides derived from the group A, type 1, 2, 3 or 4 antigens described above and comprising the characteristic trisaccharide GalNAcal -3 (Fuca1 -2) Gal:
• Type 1 GalNAcal -3 (Fuca1 -2) Galp1 -3GlcNAc,
• Type 3 or 4 GalNAcal -3 (Fuca1-2) Gai i -3GalNAc
• Type 1 GalNAcal -3 (Fuca1 -2) Gal1 -3GlcNAc p1 -3Gal,
• Type 2 GalNAcal -3 (Fuca1 -2) Gal 1 -4GlcNAc p1 -3Gal,
• Type 3 GalNAcal -3 (Fuca1 -2) Gal 1 -3GalNAc a1 -3Gal,
• Type 4 GalNAcal -3 (Fuca1 -2) Gal 1 -3GalNAc p1 -3Gal,
• Type 1 GalNAcal -3 (Fuca1 -2) Gal 1 -3GlcNAc p1 -3GalB1 -4Glc
• Type 2 GalNAcal -3 (Fuca1 -2) Gal1 -4GlcNAc p1 -3GalB1 -4Glc
• o Type 3 GalNAcal - 3 (Fuca1 -2) Gal 1 -3GalNAc a1 -3Gal61 -4GlcNAc
• Type 4 GalNAcal -3 (Fuca1 -2) Gal1 -3GalNAc p1 -3Gala1 -4Gal.
• the oligosaccharide may also comprise repeats of the characteristic GalNAcal -3 (Fuca1 -2) Gal trisaccharide, or tetrasaccharides, pentasaccharides and hexasaccharides derived from the type 1, 2, 3 or 4 Group A antigens described above.
• the specific ligand for immunoglobulins recognizing antigens of blood group A is the characteristic trisaccharide GalNAcal -3 (Fuca1-2) Gal.
• oligosaccharide representing antigens of blood group B is meant any oligosaccharide comprising the trisaccharide characteristic of antigens of blood group B as defined above. : Galal -3 (Fuca1 -2) Gal. Such an oligosaccharide may further comprise other sugars present in the blood group B antigens defined above.
• such an oligosaccharide may also be chosen from tetrasaccharides, pentasaccharides and hexasaccharides derived from group B, type 1, 2, 3 or 4 antigens described above and comprising the characteristic trisaccharide Galal -3 (Fuca1 -2) Gal:
• Type 1 Gala1 -3 (Fuca1 -2) Galp1 -3GlcNAc,
• Type 2 Gala1 -3 (Fuca1 -2) Galp1 -4GlcNAc,
• Type 3 or 4 Gala1 -3 (Fuca1-2) Galp1 -3GalNAc
• Type 3 Gala1 -3 (Fuca1 -2) Galp1 -3GalNAc a1 -3Gal61 -4GlcNAc
• o Type 4 Gala1 -3 (Fuca1 -2) Gal1 -3GalNAc p1 -3Gala1 -4Gal.
• the oligosaccharide may also comprise repeats of the characteristic Gala1-3 (Fuca1-2) Gal trisaccharide, or tetrasaccharides, pentasaccharides, and hexasaccharides derived from the type 1, 2, 3, or 4 B-group antigens described above.
• the specific ligand of immunoglobulins recognizing antigens of blood group B is the characteristic trisaccharide Galal -3 (Fuca1 -2) Gal.
• a reference to "a" specific ligand of anti-A or anti-B polyclonal human immunoglobulins includes the possibility of using either a single type of ligand specific for anti-A or anti-B polyclonal human immunoglobulins (that is to say that all the ligands grafted on the support have the same chemical structure), or several different types of ligands (that is to say of different chemical structure) specific (s) polyclonal human immunoglobulins anti -A or anti-B.
• each ligand of given chemical structure that can be used is necessarily grafted several times on the support, so that the polyclonal human anti-A or anti-B immunoglobulins can be retained by the support.
• One skilled in the art knows which ligand density should be used to allow optimal adsorption of anti-A or anti-B polyclonal human immunoglobulins on the support.
• positive control of a method for assaying anti-A and / or anti-B activity of a composition comprising polyclonal human immunoglobulins is meant a composition of polyclonal human immunoglobulins comprising polyclonal human anti-A immunoglobulins. and / or anti-B.
• Regulatory authorities require that normal human immunoglobulin compositions have anti-A activity and anti-B activity less than a limit value, set at a given time, but may vary over time and changes in regulatory authorities.
• the expression “positive control” covers three types of positive controls, referred to in the present description as “positive control less than the limit”, “positive control, respectively. limit “, and” positive control above the limit ", each of these positive controls being of interest in a method for assaying for anti-A and / or anti-B activity.
• lower-the-limit positive control of a method for assaying anti-A and / or anti-B activity of a composition comprising polyclonal human immunoglobulins is meant a positive control comprising an amount of polyclonal human immunoglobulins.
• anti-A and / or anti-B generating in the assay method an activity value below a predefined limit value.
• Such a limit value may in particular correspond to the maximum value of anti-A and / or anti-B activity accepted by the health authorities of a country for normal human immunoglobulins intended for administration to humans by any route of administration. administration, especially intravenously or intramuscularly or subcutaneously.
• limiting positive control of a method for assaying anti-A and / or anti-B activity of a composition comprising polyclonal human immunoglobulins is meant a positive control comprising a quantity of polyclonal human anti-A immunoglobulins. and / or anti-B generating in the assay method an activity value equal to a predefined limit value.
• Such a limit value may in particular correspond to the maximum value of anti-A and / or anti-B activity accepted by the health authorities of a country for normal human immunoglobulins intended for administration to humans by any route of administration. administration, especially intravenously or intramuscularly or subcutaneously.
• Such a limiting positive control is useful in particular to determine whether or not a therapeutic concentrate has an anti-A and / or anti-B activity lower than the maximum limit value accepted by the health authorities.
• anti-A and / or anti-B By “above-the-limit positive control" of a method for assaying anti-A and / or anti-B activity of a composition comprising polyclonal human immunoglobulins is meant a positive control comprising a quantity of polyclonal human immunoglobulins.
• anti-A and / or anti-B generating in the assay method an activity value higher than a predefined limit value.
• a limit value may in particular correspond to the maximum value of anti-A and / or anti-B activity accepted by the health authorities of a country for normal human immunoglobulins intended for administration to humans by any route of administration. administration, especially intravenously or intramuscularly or subcutaneously.
• Such a positive control above the limit is useful in particular to verify that the assay performed has proceeded satisfactorily, such a witness must always have a anti-A and / or anti-B activity greater than the chosen limit value. It can also be used to determine whether a therapeutic concentrate is close to the maximum limit value accepted by health authorities or well above that limit.
• Anti-A or Anti-B polyclonal human immunoglobulin compositions are useful in particular to verify that the assay performed has proceeded satisfactorily, such a witness must always have a anti-A and / or anti-B activity greater than the chosen limit value. It can also be used to determine whether a therapeutic concentrate is close to the maximum limit value accepted by health authorities or well above that limit.
• the present invention relates to a composition
• a composition comprising polyclonal human immunoglobulins, characterized in that at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, advantageously at least 85% at least 86%. %, at least 87%, more preferably at least 88%, at least 89%, still more preferably at least 90%, at least 91%, at least 92% at least 93%, at least 94%, or at least 95% %, at least 96%, at least 97%, at least 98%, or at least 99% by weight of the polyclonal human immunoglobulins present in the composition are polyclonal human anti-A and / or anti-B immunoglobulins.
• the weight percentage of the anti-A and / or anti-B polyclonal human immunoglobulins among total polyclonal human immunoglobulins of a composition comprising purified polyclonal human immunoglobulins can be measured by purifying the composition by affinity chromatography on a graft-donated column. ligands specific for anti-A and / or anti-B polyclonal human immunoglobulins, and relating the weight of immunoglobulins adsorbed on the column and the weight of total immunoglobulins.
• composition is not purified in immunoglobulins, a preliminary step of purification of total immunoglobulins then makes it possible to measure the percentage by weight of anti-A and / or anti-B polyclonal human immunoglobulins among the total polyclonal human immunoglobulins.
• compositions according to the invention are preferably purified, and the polyclonal human immunoglobulins advantageously represent at least 85%, advantageously at least 86%, at least 87%, more advantageously at least 88%, at least 89%, even more advantageously at least 90%, at least 91%, at least 92% at least 93%, at least 94% or at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% in at least weight of the total proteins of the composition.
• compositions according to the invention may comprise polyclonal human immunoglobulins of a single isotype (IgG, IgM, IgA, IgD, IgE, advantageously IgG or IgM, preferably IgG) or several isotypes.
• the polyclonal human immunoglobulins present in the compositions according to the invention are predominantly (at least 90%, advantageously at least 91%, at least 92%, at least 93%, at least 94% more preferably at least 95%, at least 96%, at least 97%, still more preferably at least 98%, at least 99% by weight, weight) IgG.
• the compositions according to the invention are advantageously enriched in IgG2 subclass immunoglobulins compared to normal human immunoglobulin concentrates.
• the IgG compositions according to the invention are advantageously characterized in that at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least less than 70%, at least 75%, or even at least 80% by weight of the polyclonal human immunoglobulins of IgG isotype present in the composition are immunoglobulins of subclass IgG2, advantageously measured by nephelemetry and / or by spectrography and / or by subclass ELISA kit (ie by nephelemetry, by spectrography, by subclass ELISA kit, or by several of these methods, for example by nephelemetry and by spectrography, or by each of these three methods).
• the IgG compositions according to the invention advantageously have a weight ratio of IgG 2 / IgG 1 of at least 0.8, at least 0.9, advantageously at least 1, at least 1.1, at least 1, 2, at least 1, 3, at least 1, 4, at least 1, 5, at least 1, 6, at least 1, 7, at least 1, 8, at least 1, 9 or at least 2 at least 2.5, at least 3, at least 3.1, at least 3.2, at least 3.3, at least 3.4, at least 3.5, at least 3.6, at least 3 , 7, at least 3.8, at least 3.9, or even at least 4, advantageously measured by nephelemetry and / or by spectrography and / or by subclass ELISA assay kit (ie by nephelometry, by spectrography, by kit Subclass ELISA, or by several of these methods, for example by nephelemetry and spectrography, or by each of these three methods).
• the polyclonal human immunoglobulins present in the compositions according to the invention are predominantly (at least 90%, advantageously at least 91%, at least 92%, at least 93%, at least 94%, more preferably at least 95%, at least 96%, at least 97%, still more preferably at least 98%, at least 99% by weight) IgM.
• At least 80%, at least 81%, at least 82%, at least 83%, at least 84%, preferably at least 85% at least 86%, at least 87%, more preferably at least 85%. less 88%, at least 89%, even more advantageously at least 90%, at least 91%, at least 92% at least 93%, at least 94%, or at least 95%, at least 96%, at least 97% %, at least 98%, or at least 99% by weight of the polyclonal human immunoglobulins present in the composition are anti-A polyclonal human immunoglobulins, as defined above.
• At least 80%, at least 81%, at least 82%, at least 83%, at least 84%, preferably at least 85% at least 86%, at least 87%, more preferably at least 88%, at least 89%, even more preferably at least 90%, at least 91%, at least 92% at least 93%, at least 94%, or at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by weight of the polyclonal human immunoglobulins present in the composition are anti-B polyclonal human immunoglobulins, as defined above.
• the composition comprises both anti-A polyclonal human immunoglobulins and anti-B polyclonal human immunoglobulins, and in that the weight ratio anti-A polyclonal human immunoglobulins on anti-human polyclonal immunoglobulins -B (anti-A / anti-B) is between 1/10 and 10/1.
• the composition comprising both anti-A polyclonal human immunoglobulins and anti-B polyclonal human immunoglobulins is enriched in anti-A polyclonal human immunoglobulins, and therefore has a weight ratio of anti-A polyclonal human immunoglobulins.
• anti-B (anti-A / anti-B) polyclonal human immunoglobulins ranging from 2/1 to 10/1.
• the composition comprising both anti-A polyclonal human immunoglobulins and anti-B polyclonal human immunoglobulins is enriched in anti-B polyclonal human immunoglobulins, and therefore has a weight ratio of polyclonal anti-human immunoglobulins to A on anti-B (anti-A / anti-B) polyclonal human immunoglobulins comprised between 1/10 and 1/2.
• the composition comprising both anti-A polyclonal human immunoglobulins and anti-B polyclonal human immunoglobulins is balanced in both types of immunoglobulins, and thus has a weight ratio of polyclonal anti-human immunoglobulins to A on anti-B (anti-A / anti-B) polyclonal human immunoglobulins of between 3/10 and 7/10, advantageously between 4/10 and 6/10.
• the composition enriched in anti-A polyclonal human immunoglobulins according to the invention may have an anti-A activity enriched by a factor of at least 4, advantageously at least 5. at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least minus 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 125, at least 150 at least 175, at least 200, at least 400, at least 400, at least 500, at least 500, at least 400, at least 400, at least minus 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, or at least 10000 in relation to a reference Im lyophil
• the composition enriched in anti-B polyclonal human immunoglobulins according to the invention may have an anti-B activity enriched by a factor of at least 4, advantageously at least 5. at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 125, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 700, at least 800, at least 900, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9,000, at least 10,000, at least 11,000, at least 12,000
• the composition may comprise both anti-A polyclonal human immunoglobulins and anti-B polyclonal human immunoglobulins and has a ratio (anti-A activity / anti-activity).
• -B between 1/10 and 10/1, in particular between 1/9 and 9/1, between 1/8 and 8/1, between 1/7 and 7/1, between 1/6 and 6/1, between 1/5 and 5/1, between 1/4 and 4/1, between 1/3 and 3/1, even between 1/2 and 2/1 or between 0.6 and 1, 5.
• the anti-A activity and the anti-B activity are determined using an activity assay method (in particular one of those described below, and in particular the flow cytometry method described here) and expressed in arbitrary units with respect to the same reference standard (standard EDQM ref Y0001688 or freeze-dried human normal immunoglobulin drug).
• the ratio (anti-A activity / anti-B activity) and the ratio (anti-B activity / anti-A activity) are calculated on the basis of anti-A and anti-B activity results obtained in tests performed in accordance with parallel, with the same method of activity assay (in particular one of those described below, and in particular the method of flow cytometry described here) and expressed in arbitrary units with respect to the same reference standard (standard EDQM ref Y0001688 or freeze-dried human normal immunoglobulin drug).
• compositions according to the invention are purified and are advantageously concentrated.
• the compositions according to the invention are concentrated according to any technique known to those skilled in the art, for example by using an ultrafiltration membrane, a centrifugation, a dialysis, or several of these steps.
• compositions according to the invention have a concentration sufficient to allow successive dilutions of the composition for use as a standard range in an appropriate assay method, for example the indirect Coombs test, the agglutination method. direct, flow cytometry, or by several of these methods (for example, the indirect Coombs test and the direct agglutination method, the indirect Coombs test and flow cytometry; direct agglutination method and flow cytometry; or each of these three methods).
• an appropriate assay method for example the indirect Coombs test, the agglutination method. direct, flow cytometry, or by several of these methods.
• the concentrated compositions according to the invention exhibit an indirect Coombs test result (described below) greater than 1/64, advantageously greater than 1/128, greater than 1/256, greater than 1/512, greater than 1/1024, greater than 1/2048, even greater than 1/4096, to allow successive dilutions of the composition to make it a standard range in the indirect Coombs test method.
• An indirect Coombs test result greater than 1 / N means that the result of the indirect Coombs test is negative at a dilution of the sample at 1 / N.
• the compositions according to the invention concentrate have a result with the direct agglutination method (described below) greater than 1/64, advantageously greater than 1/128, greater than 1/256 , greater than 1/512, greater than 1/1024, greater than 1/2048, or even greater than 1/4096, to allow successive dilutions of the composition to make a standard range in the direct agglutination method.
• a direct agglutination test result greater than 1 / N means that the result with the direct agglutination method is negative at a dilution of the sample at 1 / N.
• the compositions according to the invention concentrate a result with the flow cytometry method (described below) greater than 1/64, advantageously greater than 1/128, greater than 1/256 , greater than 1/512, greater than 1/1024, greater than 1/2048, or even greater than 1/4096, to allow successive dilutions of the composition to make a standard range in the flow cytometry method.
• flow cytometry test result greater than 1 / N it is meant that the result with the flow cytometry method is negative at a dilution of the sample at 1 / N.
• the concentrated compositions according to the invention exhibit a concentration of anti-A and / or anti-B polyclonal human immunoglobulins greater than 1 g / L, greater than 1.5 g / L, greater than 2 g / l. L, greater than 5 g / L, greater than 10 g / L, greater than 15 g / L, greater than 20 g / L, or even greater than 50 g / L.
• compositions according to the invention can be obtained from more or less purified fractions of human plasma comprising polyclonal immunoglobulins by various purification methods.
• the present invention thus relates to a method for preparing a composition according to the invention, comprising the following steps: a) adsorption of a batch of human plasma or a fraction of human plasma enriched with human immunoglobulins polyclonal on a support grafted by a specific ligand of anti-A polyclonal human immunoglobulins and / or a ligand specific for anti-B polyclonal human immunoglobulins,
• the present invention therefore relates to a process for preparing a composition according to the invention, comprising the following steps:
• the present invention therefore relates to a method for preparing a composition according to the invention, comprising the following steps:
• compositions according to the invention is based on a step of specific adsorption of anti-A and / or anti-B polyclonal human immunoglobulins on a substrate grafted with a specific ligand of these immunoglobulins, which are then eluted.
• a step of specific adsorption of anti-A and / or anti-B polyclonal human immunoglobulins on a substrate grafted with a specific ligand of these immunoglobulins which are then eluted.
• the process for the preparation of the compositions according to the invention is integrated in a more general method for the purification of therapeutic normal human immunoglobulins (recovery of fractions which have hitherto been eliminated) and is therefore implemented on a polyclonal human immunoglobulin fraction prepurified.
• This prepurified polyclonal human immunoglobulin fraction advantageously contains a polyclonal human immunoglobulin content of at least 80%, advantageously at least 81%, at least 82%, more preferably at least 83%, at least 84%, even more preferably at at least 85%, at least 86%, at least 87% at least 88%, at least 89% or at least 90%, at least 91%, or at least 92% by weight of the total protein of the fraction.
• the fraction of prepurified polyclonal human immunoglobulins may in particular have been obtained by chromatographic separation, in particular according to the purification methods of polyclonal human immunoglobulins described in WO99 / 64462 and WO02 / 092632, and more particularly in WO02 / 092632.
• the prepurified polyclonal human immunoglobulin fraction is obtained by pre-purification by a step of precipitation of lipid contaminants from a blood plasma or an IgG-enriched blood plasma fraction, and a single chromatography step on an anion exchange resin carrier carried out at alkaline pH, with a selective elution of IgG in one step by an appropriate buffer at a pH of between 4 and 7.
• the prepurification by a step of precipitation of lipid contaminants consists of a step precipitation with caprylic acid.
• the pre-purified polyclonal human immunoglobulin fraction may also have undergone a biological security step (viral elimination and / or viral inactivation, in particular by a solvent-detergent treatment), a concentration step (in particular by ultrafiltration), and / or a sterilizing filtration step.
• a biological security step viral elimination and / or viral inactivation, in particular by a solvent-detergent treatment
• concentration step in particular by ultrafiltration
• sterilizing filtration step may also have undergone a biological security step (viral elimination and / or viral inactivation, in particular by a solvent-detergent treatment), a concentration step (in particular by ultrafiltration), and / or a sterilizing filtration step.
• the support may be any suitable carrier capable of being chosen by those skilled in the art for adsorbing polyclonal human anti-A and / or anti-B immunoglobulins.
• Such a support used in step a) is advantageously in the form:
• a polymeric membrane grafted with the ligand or ligands of interest.
• the support may thus in particular be in the form of particles grafted by the ligand or ligands of interest.
• the particles are advantageously of spherical or oblong shape, they may especially be balls. These particles generally have an average size of about 0.1 ⁇ to about 1000 ⁇ , preferably about 20 to about 50 ⁇ , more preferably about 50 to about 200 ⁇ , more preferably about 70 ⁇ . ⁇ at about 120 ⁇ in diameter. They may consist of polymer or inorganic materials (such as silica or glass for example).
• the particles are porous.
• the polymer may be natural or non-natural (synthetic or semisynthetic), organic or inorganic (preferably the polymer will be organic), crosslinked or uncrosslinked (preferably the polymer will be crosslinked).
• the polymer is a crosslinked organic polymer.
• the polymer may especially be chosen from cellulose and its derivatives, agarose, dextran, polyacrylates, polystyrene, polyacrylamide, polymethacrylamide, copolymers of styrene and divinylbenzene, or mixtures of these polymers.
• the polymer is cellulose, and the particles are preferably porous cellulose beads. More preferably, it is crosslinked cellulose.
• the support may also be in the form of a polymeric membrane, the membrane being grafted with the ligand or ligands of interest.
• the polymer of the membrane may be selected from the polymers mentioned above for the polymer particles.
• the particles are advantageously incorporated in a gel or a resin, which is used as a matrix of an affinity chromatography column.
• the polymeric membrane can be included in an affinity chromatography column.
• the human plasma batch or the human plasma fraction enriched in polyclonal human immunoglobulins are then adsorbed on the affinity chromatography column, and the adsorbed fraction is eluted and recovered.
• an affinity chromatography column is not essential, and other modes of adsorption, dissociation and harvesting can be used.
• the specific ligand for anti-A polyclonal human immunoglobulins may be any suitable molecule known to those skilled in the art and binding to anti-A polyclonal human immunoglobulins as defined above and not not binding to other immunoglobulins.
• a ligand is advantageously chosen from oligosaccharides representative of group A, type 1, 2, 3 and 4 antigens, and in particular from the following oligosaccharides:
• Trisaccharide GalNAca1-3 (Fuca1 -2) Gal;
• Type 1 GalNAca1 -3 (Fuca1 -2) Galp1 -3GlcNAc,
• Type 1 GalNAca1 -3 (Fuca1 -2) Galp1 -3GlcNAc p1 -3Gal,
• Type 2 GalNAca1 -3 (Fuca1 -2) Gal1 -4GlcNAc p1 -3Gal,
• Type 3 GalNAca1 -3 (Fuca1 -2) Gal 1 -3GalNAc a1 -3Gal,
• Type 4 GalNAca1 -3 (Fuca1 -2) Gal 1 -3GalNAc p1 -3Gal,
• Type 1 GalNAca1 -3 (Fuca1 -2) Galp1 -3GlcNAc p1 -3GalB1 -4Glc
• Type 2 GalNAca1 -3 (Fuca1 -2) Gal1 -4GlcNAc p1 -3GalB1 -4Glc
• o Type 3 GalNAca1 -3 (Fuca1 -2) Gal 1 -3GalNAc a1 -3Gal61 -4GlcNAc
• Type 4 GalNAcal -3 (Fuca1 -2) Gal61 -3GalNAc B1 -3Gala1 -4Gal.
• the specific ligand of anti-B polyclonal human immunoglobulins may be any appropriate molecule known to those skilled in the art and binding to anti-B polyclonal human immunoglobulins as defined above and not not binding to other immunoglobulins.
• a ligand is advantageously chosen from oligosaccharides representative of group B antigens of type 1, 2, 3 and 4, and in particular from the following oligosaccharides:
• Trisaccharide Gala1-3 (Fuca1 -2) Gal;
• Type 3 Gala1-3 (Fuca1 -2) Gal 1 -3GalNAc a1 -3Gal61 -4GlcNAc
• Type 4 Gala1-3 (Fuca1-2) Gal61 -3GalNAc 61 -3Gala1 -4Gal.
• the support may be grafted with a ligand specific for anti-A polyclonal human immunoglobulins and / or with a ligand specific for anti-B polyclonal human immunoglobulins.
• the support is grafted only by a ligand specific for anti-A polyclonal human immunoglobulins.
• the support is grafted only by a specific ligand of anti-B polyclonal human immunoglobulins.
• the support is grafted with a specific ligand specific for anti-A polyclonal human immunoglobulins and with a ligand specific for anti-B polyclonal human immunoglobulins.
• a mixture of supports grafted with a ligand specific for anti-A polyclonal human immunoglobulins and supports grafted with a ligand specific for anti-B polyclonal human immunoglobulins in respective proportions generally between 25/75 ( v / v) and 75/25 (v / v), and in particular 50/50 (v / v).
• particles grafted with the ligand (s) of interest when particles grafted with the ligand (s) of interest are used, a mixture of particles grafted with a specific ligand of the ligands can be used.
• the particles grafted with a specific ligand of the anti-A polyclonal human immunoglobulins and the particles grafted with a specific ligand of anti-B polyclonal human immunoglobulins are mixed in respective proportions generally between 25/75 (v / v).
• v / v 75/25 (v / v), and especially 50/50 (v / v).
• a support comprising particles grafted with both a ligand specific for anti-A polyclonal human immunoglobulins and with a ligand specific for anti-B polyclonal human immunoglobulins.
• a mixture it is also possible to use a mixture:
• the ligand of interest is advantageously grafted onto the polymer particles or on the polymeric membrane by means of a spacer, which makes it possible to reduce the steric hindrance and to make the trisaccharide characteristic of antigens A or B more accessible to immunoglobulins likely to adsorb on the support.
• a spacer may be any appropriate group known to those skilled in the art to allow the grafting of the ligand of interest and therefore in particular of oligosaccharides on a support of interest, in particular the polymers described above.
• the spacer typically comprises at least one C, O, N, or S atom, and will most often include at least one of the following chemical functions: ether (-O-), thioether (-S-), amino (-NH -), carboxy - (- COO- or -OCO-), amide (-CONH- or -HNOC-).
• the spacer may in particular have a structure chosen from: wherein each of X1 and X2 is independently selected from 0, S, and NH; and each of Ra, Rb, Rc, and Rd is independently selected from H, OH, and methyl.
• R1 is a linear or branched, preferably linear, C4-C6 alkyl group.
• R 1 is a C 5 alkyl group.
• R2 is a linear or branched, preferably linear, C3-C8 alkyl group.
• R2 is a C3 alkyl group.
• the ligands (which are preferably trisaccharides as described above) are grafted to the particles or to the membrane by a spacer according to the formula: (particle / membrane) -NH-C5Hio-
• the coupling between the particle or the membrane and the spacer, on the one hand, and the coupling between the spacer and the specific ligand of the anti-A polyclonal human immunoglobulins or the specific ligand of the anti-B polyclonal human immunoglobulins can be realized. by any appropriate chemical synthesis protocol known to those skilled in the art.
• the particle or membrane may carry an -NH-R1 -COOH arm.
• it is ⁇ -aminocaproic acid (where R 1 is a pentyl group).
• the particle can then be activated using bifunctional reagents such as epichlorohydrin, epibromohydrin, dibromo- and dichloropropanol, dibromobutane, ethylene glycol diglycidylether, butanediol diglycidylether, divinylsulfone, and the like. allyl glycidyl ether, and allyl bromide.
• the bifunctional reagent is capable of reacting with both the particles / membrane and the NH-R1 -COOH arm.
• the heterofunctional allyl compounds such as allyl bromide, are preferred bifunctional reagents and make it possible to obtain an activated matrix.
• ligands representing the blood group A and / or B antigens are then immobilized on the activated particle / membrane carrying the -NH-R1 -COOH arm via a linker group -NH-R2-, where R2 is a C3 alkyl group.
• -C8 linear or branched, preferably linear.
• the COOH function of the -NH-R1 -COOH arm carried by the particle / membrane is reacted with the NH 2 function of the NH 2 -R 2 -ligosaccharide ligand, by the use of a type of condensation agent.
• substrates grafted with a ligand specific for anti-A polyclonal human immunoglobulins which may be used in the context of the invention are the following: Glycosorb ABO A (Sepharose matrix on which is grafted the trisaccharide characteristic of the antigen A, Glycorex Transplantation AB, Lund, Sweden), AUotran A (trisaccharide characteristic of FF Sepharose-grafted polyadrylamide, Lectinity Corp), HyperCel IsoA (cross-linked cellulose particles grafted with the trisaccharide characteristic of the A antigen , Pall).
• Glycosorb ABO A Sepharose matrix on which is grafted the trisaccharide characteristic of the antigen A
• Glycorex Transplantation AB Glycorex Transplantation AB, Lund, Sweden
• AUotran A trisaccharide characteristic of FF Sepharose-grafted polyadrylamide, Lectinity Corp
• substrates grafted with a ligand specific for anti-B polyclonal human immunoglobulins which may be used in the context of the invention are the following: Glycosorb ABO B (Sepharose matrix on which is grafted the trisaccharide characteristic of antigen B, Glycorex Transplantation AB, Lund, Sweden), AUotran B (trisaccharide characteristic of B-grafted Sepharose FF by polyacrylamide, Lectinity Corp), HyperCel IsoB (crosslinked cellulose particles grafted with the trisaccharide characteristic of B antigen , Pall).
• Glycosorb ABO B Sepharose matrix on which is grafted the trisaccharide characteristic of antigen B, Glycorex Transplantation AB, Lund, Sweden
• AUotran B trisaccharide characteristic of B-grafted Sepharose FF by polyacrylamide, Lectinity Corp
• HyperCel IsoB crosslinked cellulose particles grafted with the tri
• a substrate ligated with a specific ligand of anti-A polyclonal human immunoglobulins and with a ligand specific for anti-B polyclonal human immunoglobulins a mixture of a polyclonal human immunoglobulin-specific ligand-specific support will generally be used.
• anti-A as described above and a specific ligand-grafted anti-B polyclonal human immunoglobulin support as described above.
• particles grafted with the ligand (s) of interest when particles grafted with the ligand (s) of interest are used, it is possible to use a mixture of particles grafted with a ligand specific for anti-A polyclonal human immunoglobulins and particles grafted with a ligand specific for anti-human polyclonal immunoglobulins. -B to prepare a gel and fill an affinity chromatography column. It is also possible to use one of the particles grafted with both a polyclonal anti-A human immunoglobulin specific ligand and with a specific anti-B polyclonal human immunoglobulin specific ligand to prepare a gel and fill an affinity chromatography column. In another embodiment, it is also possible to use a mixture:
• Particles grafted at the same time by a ligand specific for anti-A polyclonal human immunoglobulins and by a ligand specific for anti-B polyclonal human immunoglobulins and Particles grafted with a ligand specific for anti-A polyclonal human immunoglobulins and / or particles grafted with a ligand specific for anti-B polyclonal human immunoglobulins.
• the batch of human plasma or the fraction of human plasma enriched with polyclonal human immunoglobulins is adsorbed in step a) on the chromatography column under any condition appropriate known to those skilled in the art, including any condition recommended by the manufacturer of the chromatography medium, depending on the support chosen.
• Percolation of the batch of human plasma or the fraction of human plasma enriched in polyclonal human immunoglobulins on the column may in particular be implemented.
• the unadsorbed fraction is advantageously recovered for other subsequent uses.
• the adsorbed fraction is then dissociated and recovered using one or more washes of the column with one or more appropriate elution buffers.
• An acidic elution buffer for example a glycine-HCl buffer, pH between 2 and 4 and / or a basic elution buffer (for example a glycine-NaOH solution, pH between 10 and 12) may in particular be used.
• the composition thus obtained may, in addition, be subjected to one or more subsequent optional steps, such as: a step of neutralization of the composition (pH adjustment between 3 and 9, preferably between 4 and 5, one or more additional purification steps, a concentration step (for example by ultrafiltration), at least one inactivation step (solvent-detergent treatment for example) or viral elimination (nanofiltration for example), or a combination of several of these steps.
• the method according to the invention as described above makes it possible to obtain a composition of polyclonal human immunoglobulins recognizing, for at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, advantageously at least 85% at least 86%, at least 87%, more preferably at least 88%, at least 89%, even more preferably at least 90%, at least 91%, at least 92% at least 93%, at least at least 96%, or at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by weight of the polyclonal human immunoglobulins present in the composition of the blood group A and B antigens.
• step a) When the support used in step a) is grafted solely by a ligand specific for anti-A polyclonal human immunoglobulins, anti-A polyclonal human immunoglobulins are retained, and the composition obtained then comprises anti-A polyclonal human immunoglobulins.
• step a) when the support used in step a) is grafted solely by a specific ligand of anti-B polyclonal human immunoglobulins, the Anti-B polyclonal human immunoglobulins are retained, and the composition obtained then comprises anti-B polyclonal human immunoglobulins.
• the purified composition then comprises a mixture of human immunoglobulins.
• polyclonal anti-A and anti-B polyclonal human immunoglobulins In polyclonal human immunoglobulins purified from plasma pools, the proportion of polyclonal human anti-A immunoglobulins and anti-B polyclonal human immunoglobulins depend on the initial donor population. In fact, differences in the percentage of distribution of the different blood groups exist according to the donor populations.
• the method advantageously comprises the following steps:
• step c) adsorbing the composition obtained in step c) on a substrate grafted with a ligand specific for anti-B polyclonal human immunoglobulins, and
• Steps a) to c) are identical to those described above for the preparation of a composition according to the invention in which at least 80% by weight of the polyclonal human immunoglobulins present in the composition are polyclonal human anti-A immunoglobulins. and / or anti-B.
• the conditions of adsorption, storage of the unadsorbed fraction and dissociation and harvest of the adsorbed fraction may be similar to those described above.
• Steps d) and e) are aimed at specifically isolating polyclonal human anti-A immunoglobulins.
• a substrate ligated with a ligand specific for anti-B polyclonal human immunoglobulins is used so that all the anti-B polyclonal human immunoglobulins are adsorbed onto the support.
• Anti-A polyclonal human immunoglobulins can thus be recovered directly (step e)), leading to the production of a composition specifically enriched in anti-A polyclonal human immunoglobulins.
• the conditions of step d) are adapted to allow the adsorption on the support of all anti-B polyclonal human immunoglobulins, for example by adjusting the gel volume and / or the residence time.
• the support used in step d) is an affinity chromatography column comprising as support a membrane or a particle gel grafted with a ligand specific for anti-B polyclonal human immunoglobulins
• the adsorption conditions may be similar to those described above for step a).
• the unadsorbed fraction is then directly recovered in step e).
• the method advantageously comprises the following steps:
• step c) adsorbing the composition obtained in step c) on a substrate grafted with a ligand specific for anti-A polyclonal human immunoglobulins, and
• the method advantageously comprises the following steps:
• step c) adsorbing the composition obtained in step c) on a substrate grafted with an immunoglobulin specific ligand recognizing the antigens of the blood group A, and
• Steps a) to c) are identical to those described above for the preparation of a composition according to the invention in which at least 80% by weight of the polyclonal human immunoglobulins present in the composition are polyclonal human anti-A immunoglobulins. or anti-B.
• the conditions for adsorption, storage of the unadsorbed fraction and dissociation and harvesting of the adsorbed fraction may be similar to those described above.
• Steps (d) and (e) are aimed at specifically isolating anti-B polyclonal human immunoglobulins.
• a substrate ligated with a ligand specific for anti-A polyclonal human immunoglobulins is used so that all the anti-A polyclonal human immunoglobulins are adsorbed on the support.
• Anti-B polyclonal human immunoglobulins can thus be recovered directly (step e)), resulting in a composition specifically enriched with anti-B polyclonal human immunoglobulins.
• the conditions of step d) are adapted to allow the adsorption on the support of all anti-B polyclonal human immunoglobulins, for example by adjusting the gel volume and / or the residence time.
• the support used in step d) is an affinity chromatography column comprising as support a membrane or a gel of particles grafted with a ligand specific for anti-A polyclonal human immunoglobulins
• the adsorption conditions may be similar to those described above for step a).
• the unadsorbed fraction is then directly recovered in step e).
• the method advantageously comprises the following steps:
• step c) adsorbing the composition obtained in step c) on a substrate grafted with a ligand specific for anti-B polyclonal human immunoglobulins, and
• the batch of human plasma or the fraction of human plasma enriched in polyclonal human immunoglobulins (fraction of pre-purified polyclonal human immunoglobulins) not adsorbed in step a) is used in a method of preparation of human polyvalent immunoglobulins.
• This particular embodiment of the invention advantageously allows the preparation at a time
• composition specifically enriched in anti - A polyclonal human immunoglobulins
• composition specifically enriched with anti - B polyclonal human immunoglobulins.
• the process for preparing a purified anti-A immunoglobulin composition therefore comprises the following steps: a) adsorption of a fraction of pre-purified polyclonal human immunoglobulins onto a support grafted with a specific ligand of anti-B polyclonal human immunoglobulins and by a ligand specific for anti-A polyclonal human immunoglobulins,
• step c) adsorbing the composition obtained in step c) on a substrate grafted with an immunoglobulin specific ligand recognizing the antigens of the blood group B, and
• the process for preparing a purified anti-A immunoglobulin composition thus comprises the following steps: a) adsorption of a pre-purified polyclonal human immunoglobulin fraction, in particular a fraction of human plasma enriched with polyclonal human immunoglobulins from an alkaline pH anion exchange chromatography, on a support ligated with a ligand specific for anti - B polyclonal human immunoglobulins and with a ligand specific for anti - A polyclonal human immunoglobulins,
• step c) adsorbing the composition obtained in step c) on a substrate grafted with an immunoglobulin specific ligand recognizing the antigens of the blood group B, and
• the process for preparing a purified anti-B immunoglobulin composition therefore comprises the following steps: a) adsorption of a prepurified polyclonal human immunoglobulin fraction on a grafted support by a specific ligand of anti-B polyclonal human immunoglobulins and by a ligand specific for anti-A polyclonal human immunoglobulins,
• step c) adsorbing the composition obtained in step c) on a substrate grafted with an immunoglobulin specific ligand recognizing the antigens of the blood group A, and
• the process for preparing a purified anti-B immunoglobulin composition therefore comprises the following steps: a) adsorption of a pre-purified polyclonal human immunoglobulin fraction, in particular a fraction of human plasma enriched with polyclonal human immunoglobulins from an alkaline pH anion exchange chromatography, on a support ligated with a ligand specific for anti - B polyclonal human immunoglobulins and with a ligand specific for anti - A polyclonal human immunoglobulins, b) recovering the non-adsorbed fraction for use in a process for purifying a polyvalent human immunoglobulin concentrate depleted in anti-A and anti-B polyclonal human immunoglobulins,
• step c) adsorbing the composition obtained in step c) on a substrate grafted with an immunoglobulin specific ligand recognizing the antigens of the blood group A, and
• the process for preparing a purified anti-A immunoglobulin composition and a purified anti-B immunoglobulin composition thus comprises the following steps:
• step c) adsorption of a part of the composition obtained in step c) on a substrate grafted with an immunoglobulin specific ligand recognizing the antigens of blood group B, and harvesting of the non-adsorbed fraction enriched in anti-A
• step c) adsorption of the other part of the composition obtained in step c) on a substrate grafted with a ligand specific for immunoglobulins recognizing antigens of blood group A, and harvesting of the non-adsorbed fraction enriched in anti-B.
• This particularly advantageous embodiment makes it possible to optimize the retained and non-retained fractions of the affinity chromatography using ligands specific for anti-B polyclonal human immunoglobulins and ligands specific for anti-A polyclonal human immunoglobulins.
• the supports and ligands used in steps a) and d) and / or e) can be selected from those described above. above for the general process for the preparation of a composition enriched in anti-A and anti-B immunoglobulins.
• the invention further relates to a composition obtainable by one of the preparation methods according to the invention as described above.
• compositions according to the invention are particularly useful for the preparation of a positive control (below-limit positive control, limiting positive control or positive control greater than the limit) useful in a method for assaying anti-A activity and or anti-B of a concentrate of normal human immunoglobulins, which does not have the disadvantages of the positive control and / or the positive limit control available to date (fractions 07/306 and 07/310), that is, that is to say, which comprises human and polyclonal and non-murine and monoclonal anti-A and / or anti-B immunoglobulins and which can be prepared in sufficient amounts.
• a positive control below-limit positive control, limiting positive control or positive control greater than the limit
• a concentrate of normal human immunoglobulins which does not have the disadvantages of the positive control and / or the positive limit control available to date (fractions 07/306 and 07/310), that is, that is to say, which comprises human and polyclonal and non-murine and monoclonal anti-A and / or anti
• the present invention relates to the use of a composition according to the invention as a positive control in a method for assaying the anti-A and / or anti-B activity of a composition comprising immunoglobulins. polyclonal human.
• the invention also relates to the use of a composition according to the invention for the preparation of a positive control (sub-limit positive control, limiting positive control or positive control greater than the limit) intended to be used in a method assay for the anti-A and / or anti-B activity of a composition comprising polyclonal human immunoglobulins.
• a positive control sub-limit positive control, limiting positive control or positive control greater than the limit
• the invention also relates to a method for preparing a positive control for use in a method for assaying the anti-A and / or anti-B activity of a composition comprising polyclonal human immunoglobulins, comprising the following steps :
• composition comprising polyclonal human immunoglobulins having anti-A activity and anti-B activity below a given limit value in said assay method, and
• the invention also relates to a method for preparing a positive control (below-limit positive control, limiting positive control, or positive control above the limit) for use in a method for assaying anti-A activity. and / or anti-B of a composition comprising polyclonal human immunoglobulins, comprising the following steps:
• composition comprising polyclonal human immunoglobulins having anti-A activity and anti-B activity below a given limit value in said assay method
• composition according to the invention in which at least 80% by weight of the polyclonal human immunoglobulins present in the composition according to the invention are polyclonal human anti-A immunoglobulins and / or composition according to the invention in which at least 80% by weight of the polyclonal human immunoglobulins present in the composition according to the invention are anti-B polyclonal human immunoglobulins, to obtain different compositions enriched in anti-A and / or anti-B polyclonal human immunoglobulins,
• step b) the assay by said assay method of the anti-A and / or anti-B activity of the compositions enriched in anti-A and / or anti-B polyclonal human immunoglobulins obtained in step b), and
• composition enriched in anti-A and / or anti-B polyclonal human immunoglobulins having anti-A and / or anti-B activity in said lower assay method to obtain a positive control below the limit
• equal to obtain a limit positive control
• greater to obtain a positive control greater than the limit
• a positive control (below-limit positive control, limiting positive control, or positive control above the limit) may be prepared by the above method for any method of assay for anti-A and / or anti-B activity. of interest, and for any limit value of interest.
• a positive control (below-limit positive control, limiting positive control, or positive control above the limit) may be prepared by the above method for either of the methods of assaying the activity.
• anti-A and / or anti-B which are generally used:
• TIA indirect antiglobulin test
• TIA indirect Coombs test
• Detection of the agglutination of red blood cells in the presence of anti-A and / or anti-B polyclonal human immunoglobulins can be made by various methods, more or less precise.
• agglutination is read visually by detecting the presence of a precipitate.
• the TIA test is the old test recommended by the health authorities for the determination of the anti-A and / or anti-B activity of normal human immunoglobulin therapeutic concentrates.
• papain treated A or B red blood cells are contacted with serial half dilutions of an immunoglobulin solution.
• 5% normal human (50 g / l) in a microtiter plate with V wells. After centrifugation, the plate is tilted at an angle of about 70 ° for about 4-5 minutes.
• the cell pellet remains stuck to the bottom of the V-well, forming a well rounded point. Otherwise, the non-agglutinated pellet slides along the V-well wall, thereby creating a droplet-like shape.
• the direct agglutination method is the new test recommended by the health authorities for the determination of the anti-A and / or anti-B activity of normal human immunoglobulin therapeutic concentrates.
• a cytometry test uses an F (ab ') 2 labeled with a fluorescent marker.
• the limit value for which the limiting positive control is prepared may be any limit value of anti-A and / or anti-B activity of interest. In particular, this may be the maximum value of anti-A and / or anti-B activity accepted by health authorities in a country or region of the world.
• this limit value can therefore be chosen advantageous.
• this limit value is subject to change by the health authorities depending on the monitoring of haemolysis accidents that may occur in the future on the basis of therapeutic concentrates meeting this limit value.
• the method described above can then be implemented again to prepare a new limit positive control, adapted to the new limit set by the health authorities.
• a range of sub-limit positive controls with increasing anti-A and / or anti-B activity but below the limit set by health authorities could help establish whether a therapeutic concentrate is close to the limit. the maximum limit accepted by health authorities or well below this limit.
• a range of positive controls above the limit with increasing anti-A and / or anti-B activities but all above the limit set by the health authorities could establish whether a therapeutic concentrate is close the maximum limit accepted by the health authorities or well above this limit.
• a composition comprising polyclonal human immunoglobulins having anti-A activity and anti-B activity below a given limit value in said assay method (starting composition) is provided, to which increasing amounts of a composition according to the invention in which at least 80% by weight of the polyclonal human immunoglobulins present in the composition according to the invention are anti-A polyclonal human immunoglobulins and / or a composition according to the invention.
• step b) in which at least 80% by weight of the polyclonal human immunoglobulins present in the composition according to the invention are anti-B polyclonal human immunoglobulins are added in step b) to obtain different compositions enriched in polyclonal human anti-A immunoglobulins and / or anti-B.
• the starting composition may be free of anti-A and / or anti-B polyclonal human immunoglobulins (for example by appropriate selection of the plasma donors used), or may comprise anti-A and / or anti-human polyclonal immunoglobulins.
• B provided that its anti-A activity and its anti-B activity is lower than the limit value chosen in the chosen assay method.
• such a starting composition can in particular be a composition of polyclonal human immunoglobulins purified and depleted in anti-A and anti-B polyclonal human immunoglobulins, obtained for example as described in WO2007 / 077365.
• this starting composition preferably has an immunoglobulin concentration equal to or greater than that recommended in the test for anti-A and anti-B activity required by the health authorities (50 g / L, ie 5% at this level). day).
• step b) increasing amounts of a composition according to the invention in which at least 80% by weight of the polyclonal human immunoglobulins present in the composition according to the invention are polyclonal human immunoglobulins anti-A and / or d a composition according to the invention in which at least 80% by weight of the polyclonal human immunoglobulins present in the composition according to the invention are anti-B polyclonal human immunoglobulins are added to obtain different compositions enriched to a greater or lesser extent with immunoglobulins polyclonal human anti-A and / or anti-B.
• step c) the anti-A and / or anti-B activity of the compositions enriched in anti-A and / or anti-B polyclonal human immunoglobulins obtained in step b) is tested by the chosen assay method. initially.
• the composition enriched in anti-A and / or anti-B polyclonal human immunoglobulins having anti-A and / or anti-B activity is selected in said lower assay method (to obtain a control positive lower than the limit), equal (to obtain a limit positive control), or greater (to obtain a positive control greater than the limit) to the limit value initially chosen.
• steps a) to c) may be implemented again until the composition having an anti-A and / or anti-B activity greater than the limit value initially selected is obtained.
• the steps a) c) can be implemented again until the composition having an anti-A and / or anti-B activity lower than the initially chosen limit value is obtained.
• steps a) to c) can be carried out. again until the composition having anti-A and / or anti-B activity is equal to the initially chosen limit value is obtained.
• compositions tested in step c) have a lower anti-A and / or anti-B activity, equal to or greater than the limit value initially chosen, those skilled in the art will be able to adapt the amounts of composition according to the invention to be added in step b) so as to be able to select the positive control of interest.
• the invention further relates to a positive control (sub-limit positive control, limiting positive control, or positive control greater than the limit) for use in a method for assaying for anti-A and / or anti- B of a composition comprising polyclonal human immunoglobulins, obtainable by the method according to the invention for the preparation of a positive control (below - limit positive control, limit positive control, or positive control above the limit) described above.
• a positive control sub-limit positive control, limiting positive control, or positive control greater than the limit
• compositions according to the invention in which at least 80% by weight of the polyclonal human immunoglobulins present in the composition are polyclonal human anti-A immunoglobulins may further be used in a blood group test of a subject.
• compositions according to the invention in which at least 80% by weight of the polyclonal human immunoglobulins present in the composition are anti-B polyclonal human immunoglobulins may further be used in a test for determination of the blood group of a subject .
• the compositions according to the invention are then brought into contact with the red blood cells of a subject whose blood group is to be determined, and the blood group is determined as follows:
• composition comprising at least 80% of anti-A polyclonal human immunoglobulins, but not by the composition comprising at least
• composition comprising at least 80% of anti-B polyclonal human immunoglobulins but not by the composition comprising at least 80% of anti-A polyclonal human immunoglobulins: the patient is in a group
• the patient is of group AB, no agglutination by any of the two compositions: the patient is of group O.
• compositions according to the invention will advantageously be supplemented by a search for specific antibodies in the serum of the patient.
• EXAMPLE 1 Preparation of a polyclonal human immunoglobulin composition, enriched with anti-A and anti-B polyclonal human immunoglobulins A first composition of polyclonal human immunoglobulins according to the invention, enriched with anti-A and anti-polyclonal human immunoglobulins B has been prepared.
• a purified polyclonal human immunoglobulin composition was prepared from a plasma pool according to the method described in WO02 / 092632.
• This purified polyclonal human immunoglobulin composition was then adsorbed onto a gel-filled 1 mL affinity chromatography column comprising a mixture of porous crosslinked cellulose beads grafted with the trisaccharide characteristic of group A antigens (column A). and porous crosslinked cellulose beads grafted with the trisaccharide characteristic of group B antigens (column B), in proportions of 50/50 respectively.
• a gel-filled 1 mL affinity chromatography column comprising a mixture of porous crosslinked cellulose beads grafted with the trisaccharide characteristic of group A antigens (column A). and porous crosslinked cellulose beads grafted with the trisaccharide characteristic of group B antigens (column B), in proportions of 50/50 respectively.
• the unadsorbed fraction is recovered for further processing to prepare a polyclonal human immunoglobulin therapeutic concentrate free of anti-A and anti-B polyclonal human immunoglobulins.
• fraction of interest in the context of the present invention is then obtained by assembling two elution fractions:
• composition was then analyzed by the usual technologies to determine the concentration of IgG, IgA and IgM, the levels of polymers, dimers, monomers and immunoglobulin fragments.
• the anti-A and anti-B activity of the composition was also analyzed by the method described in WO2007 / 077365 and compared to that of a freeze-dried normal human immunoglobulin.
• IgG 9.20 g / L (Subclasses: IgG1: 65%, IgG2: 30%, IgG3: 3%, IgG4: 2%)
• the anti-A activity and the anti-B activity are expressed in arbitrary units relative to a freeze-dried normal human immunoglobulin, a product considered as a reference set of 1.
• Normal human immunoglobulin lyophilized is a product human normal immunoglobulins, having a marketing authorization in France.
• the freeze-dried normal human immunoglobulin therefore has for the anti-A and anti-B a negative result in the Coombs direct test at 1/64 dilution as required by the regulatory authorities.
• composition obtained by the process according to the invention has an anti-A activity and an anti-B activity approximately 600 times greater than the polyvalent normal human immunoglobulins of lyophilized normal human immunoglobulin (therapeutic concentrate of polyclonal human immunoglobulins). ).
• Example 2 Preparation of polyclonal human immunoglobulin compositions, enriched with anti-A polyclonal human immunoglobulins or anti-B polyclonal human immunoglobulins The inventors then sought to separate polyclonal human anti-A immunoglobulins from anti-B polyclonal human immunoglobulins.
• Example 1 In order to separate anti-A polyclonal human immunoglobulins from anti-B polyclonal human immunoglobulins, two aliquots of the composition obtained in Example 1 were subjected to two distinct treatments: In order to obtain a purified composition of human immunoglobulins purified polyclonal anti-A, a 1 aliquot was adsorbed pay over a 1 ml_ affinity chromatography column filled with a gel of crosslinked porous cellulose beads grafted with the characteristic trisaccharide Group B antigens (column 1). The charge was 1.2 mg of polyclonal human anti-A and anti-B immunoglobulin purified per ml of gel. The contact time was set at 2 minutes.
• the unadsorbed fraction was recovered for further analysis.
• the unadsorbed fraction was recovered for further analysis.
• the passage of the composition obtained in Example 1 on column 1 makes it possible to obtain a purified composition of anti-A immunoglobulins by harvesting the unadsorbed fraction.
• the results shown in Table 3 show that all anti-A immunoglobulins are adsorbed on column 2, with no anti-A activity being detected in the unadsorbed fraction. It has only a very strong anti-B activity. Indeed, with respect to the anti-B activity of 638.7 AU of the composition obtained in Example 1, the non-adsorbed fraction on column 2 has an anti-B activity of 4248 AU, or about 6.7 more than the composition obtained in Example 1.
• the passage of the composition obtained in Example 1 on column 2 (grafted with the trisaccharide characteristic of antigens A) makes it possible to obtain a purified composition of anti-B immunoglobulin by harvesting the unadsorbed fraction.
• the purified composition of anti-A immunoglobulin is obtained by harvesting the unadsorbed fraction on a column carrying the trisaccharide characteristic of the B antigens.
• the operating conditions are optimized so that all the anti-B immunoglobulins bind to an anti-B immunoglobulin. trisaccharide carrier column characteristic of B antigens.
• the purified composition of anti-B immunoglobulin is obtained by harvesting the unadsorbed fraction at a column carrying the trisaccharide characteristic of the antigens A.
• the operating conditions are optimized so that all the anti-A immunoglobulins are bind to a column carrying the trisaccharide characteristic of antigens A.
• the purified compositions of anti-A immunoglobulin and / or anti-B immunoglobulin can be used as a positive control in a method for assaying the anti-A and / or anti-B activity of an immunoglobulin composition.
• polyclonal human polyclonal human.
• Example 3 Characterization of polyclonal human immunoglobulin compositions, enriched in anti-A polyclonal human immunoglobulins or anti-B polyclonal human immunoglobulins, obtained in Example 2
• composition of human immunoglobulin polyclonal anti-A and anti-B obtained by eluting the adsorbed fraction with a 1 st chromatographic step in Example 1 was concentrated by ultrafiltration on a membrane PELLICON 2, 30kDa, 0, 1 m 2 (Merck Millipore®).
• Anti-A polyclonal human immunoglobulin compositions or anti-B polyclonal human immunoglobulins were concentrated by ultrafiltration at using a 50 cm 2 PELLICON XL Biomax membrane, 30kDa (Merck Millipore®) and then reconcentrated 10 times by centrifugation on Amicon Ultra, Ultracel membrane (Merck Millipore®).
• the compositions are then characterized more finely with regard to the distribution of the various IgG subclasses, the IgG integrity (percentages of monomers, dimers, polymers, and fragments), and their activity with respect to group red blood cells.
• blood O (not carrying A antigens or B antigens), blood group A red blood cells (antigen A carriers, but no B antigens), and blood group B red blood cells (antigen carriers B, but no antigens A).
• IglV intravenously
• IgG subclasses The distribution of IgG subclasses was measured by three distinct methods: nephelemetry, mass spectrography, and electroluminescence (MSD).
• Nephelemetry The principle of nephelometric assay is based on the fluid phase reaction of an antigen with a specific antibody.
• the insoluble antibody-antigen complex formed causes turbidity, which is measured by a nephelometric technique, the principle of which is as follows: when a light beam passes through a turbid medium, part of the light is deviated from its trajectory (phenomenon of diffusion).
• the measurement by means of a photoreceptor of the light scattered at a certain angle with respect to the incident beam makes it possible to quantify the disorder by an electrical signal.
• the relationship between the antigen concentration and the electrical signal obtained for a constant antibody concentration is described by the HEIDELBERGER-KENDALL curve.
• the measurement domain is located in the ascending part of the curve corresponding to an excess of antibody.
• Nephelometric measurements were performed with a BNII nephelometer (SIEMENS) with Software Version 2.5 / F.
• Enzymatic proteolysis and glycolysis were obtained by incubating 10 ⁇ l of the sample at a concentration of 10 mg / ml in phosphate buffered saline at 100 ⁇ l of Ides and 100 ⁇ l of IgGZero enzymes for 1 hour at 37 ° C.
• the reduction of the digested IgGs was carried out by adding 35 ⁇ l of denaturation buffer (8M guanidine-HCl, 0.1M Tris-HCl, pH 7.5) and 5 ⁇ l of a 200 mM solution of DTT. The preparation was incubated at 50 ° C for 30 minutes.
• the separation of the Fc / 2 fragments was performed on an Acquity system (Waters, Milford, MA, USA) coupled to a UV detector and an electrospray mass spectrometer (Synapt G2S, Waters, Milford, MA, USA). About 20 ⁇ g of the sample was injected onto a Pursuit diphenyl column 150 mm ⁇ 2.0 mm (Agilent, Santa Clara, Calif., USA) equilibrated at 70 ° C. and operating at a flow rate of 200 ⁇ / min. .
• Electroluminescence detection uses markers that emit light when electrochemically stimulated. Background noise is minimal because the pacing mechanism (electricity) is decoupled from the signal (light). The markers are stable, non-radioactive and emit light at -620 nm, eliminating quenching problems. Few compounds interfere with electroluminescent markers. Multiple excitation cycles of each marker amplify the light signal level and increase the sensitivity.
• Meso Scale Discovery (MSD) technology was used, following the manufacturer's recommendations. This technology relies on the use of microplates with carbon electrodes integrated at the bottom of the wells.
• IgG Integrity Quantification of polymeric, dimeric, monomeric forms and possible IgG degradation products (fragments) were measured by two distinct methods: SDS-PAGE and High Performance Protein Exclusion Chromatography (HPSEC).
• SDS-PAGE The samples were analyzed by SDS-PAGE gel electrophoretic migration in a MULTIPHOR (GE) system. 2 ⁇ g of sample are added to 3 ⁇ of 250 mM Tris buffer, pH 7.5, 5% SDS (+1.5 ⁇ NuPAGE Reducing Agent (10x) reductant - Invitrogen Ref NP0004 for reducing conditions). After stirring, the sample is heated at 95 ° C for 3 minutes just prior to deposition on ExcelGel SDS Homogeneous 12.5% gel. The following migration method is used:
• the gel is contacted with the staining solution (50% methanol, 7% acetic acid, 0.1% CBB) with stirring for 30 minutes. min, then with the decolorization solution 1 (50% methanol, 7% acetic acid) with stirring for 5 min, then the decolorization solution 2 (5% methanol, 7% acetic acid) with stirring until a clear background is obtained and homogeneous.
• the staining solution 50% methanol, 7% acetic acid, 0.1% CBB
• the decolorization solution 1 50% methanol, 7% acetic acid
• the decolorization solution 2 5% methanol, 7% acetic acid
• the gel is contacted for 30 minutes with the fixing solution (250 ml of 40% ethanol, 10% acetic acid in PPI water), with moderate stirring on a stirrer. then for 30 minutes with the sensitizing solution (75 ml of absolute ethanol + 17 g of sodium acetate + 10 ml of sodium thiosulphate, qs 250 ml of PPI + 1 water, 25 ml of extemporaneous glutardialdehyde) with stirring , before being rinsed 3 x 5 min in PPI water.
• the fixing solution 250 ml of 40% ethanol, 10% acetic acid in PPI water
• the sensitizing solution 75 ml of absolute ethanol + 17 g of sodium acetate + 10 ml of sodium thiosulphate, qs 250 ml of PPI + 1 water, 25 ml of extemporaneous glutardialdehyde
• the gel is then brought into contact for 20 min with the AgNO 3 solution (10% AgNO 3 in 250 ml + 100 ⁇ l of extemporaneous formaldehyde) with stirring, before being rinsed for 2 x 1 min in PPI water.
• the gel is then brought into contact with the developing solution (6.25 g of sodium carbonate in 250 ml of PPI water + 50 ⁇ l of extemporaneous formaldehyde), and the coloration is allowed to develop (2 to 5 min maximum). with stirring.
• the development is stopped by removing the solution and putting the gel in contact with the EDTA solution (3.65 g of EDTA in 250 ml of PPI water) for 10 min minimum.
• HPSEC This technique allows to separate proteins according to their molecular size.
• a DIONEX Ultimate 3000 system with Superdex Tricorn 200 10/300 GL column (GE Healthcare Ref 17-5175-01) was used. The following analysis conditions were used:
• the assay of the anti-A or anti-B activity on erythrocytes A, B or O was carried out by the cytometry test using a F (ab ') 2 labeled with a fluorescent marker, as described in FIG. Example 2 of the application WO2007 / 077365.
• a suspension of erythrocytes at 40 ⁇ 10 6 red blood cells / ml is prepared in 1% PBS / BSA. 50 ⁇ l of this suspension is deposited in a 96-well round-bottomed microplate (2.10 6 red cells / well). Dilution ranges of standards (freeze-dried normal human immunoglobulin or reference Y0001688 of EDQM (European Directorate for the Quality of Medicines & HealthCare)) are carried out. 50 ⁇ l of a standard dilution or a test sample are added to the 50 ⁇ of red blood cells in the microplate, which is covered with an adhesive film and incubated for 2 hours at 37 ° C. ⁇ 2 ° C. with shaking.
• the activity is expressed in arbitrary units relative to the standard (reference Y0001688 of the EDQM or drug (freeze-dried normal human immunoglobulin)) reported in 1.
• the results are shown in Table 4 below, and show that the anti-A and anti-B polyclonal human immunoglobulins obtained in Example 1 after elution of the total polyclonal human immunoglobulin fraction adsorbed on a chromatography column.
• the trisaccharide carrier affinity characteristic of Group A antigens and the trisaccharide characteristic of Group B antigens are enriched in IgG2 with respect to the composition of polyclonal human immunoglobulins depleted in anti-A and anti-B polyclonal human immunoglobulins.
• anti-A polyclonal human immunoglobulin compositions or human immunoglobulins anti-B polyclonal fractions are characterized by lgG2 isotype enrichment.
• composition of immunoglobulins Composition of immunoglobulins

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