Immunoglobulin removing sorbent

FIELD: medicine; immunology.

SUBSTANCE: sorbent is offered to remove immunoglobulin from human blood plasma. This sorbent contains agarous matrix covalently combined with ligand. As a ligand at that it contains F(ab)2 fragments of specific affinely-purified polyclonal antibodies blocking human immunoglobulin G. Sorbent is actually biologically inert, biocompatible agarous matrix. Sorbent is characterized with higher sorptive capacity and safety of immunosorbents used practical purposes, specifically for therapeutic aphaeresis in comparison to well-known polyclonal bodies based sorbents.

EFFECT: considerable reduction of prospective immunological response of human body for foreign protein.

1 ex, 1 tbl

 

The invention relates to medicine, in particular to immunology, and serves to bind immunoglobulins of various subclasses from human blood plasma and other biological fluids, and can also be used in biochemistry and biotechnology to extract specific components from protein solutions.

There are a number of neurological, hematological, systemic autoimmune diseases, the development of which is accompanied by formation and accumulation in the blood of patient antibodies to various antigens of its own body. It is shown that autoantibodies are an important part of the pathological process in such diseases as systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura [1], syndromes Guillena-Barre or?, male, dilated cardiomyopathy [2, 3] the rejection of transplants, immune conflict in pregnancy, etc. In many cases, when drug therapy is not effective or cannot be applied, the removal of immunoglobulins (Ig apheresis) from the bloodstream of the patient can significantly improve the patient's quality of life and prognosis of disease[4, 5, 6, 7].

Any sorption column for medical purposes consists of three main components: a matrix is biologically inert biocompatible material; ligand molecules to the verge covalently attached to the matrix and give the sorbent specificity, and the body of the column, and places the sorbent.

For the columns used in the clinic, the most important parameters are the efficiency and specificity of removal of pathogenic component or components, as well as the safe use of columns in the procedure of therapeutic apheresis. The removal efficiency of pathogenic component is called reduction (%) concentration of this component for the chromatographic cycle or procedure. The efficiency of the sorption column is determined by the sorption capacity of the sorbent, the amount of gel in the column and the reusability of the column within one procedure.

Currently in clinical practice for Ig apheresis most widely used sorbents with immobilized polyclonal antibodies to human immunoglobulins (Ig Adspec"®, NPF "PACKARD, Russia; "Ig-Therasorb"®, "Miltenyi Biotec"®Germany) [7, 8], columns with immobilized protein A ("Immunosorba"® and "Prosorba"®, "Fresenius HemoCare, Germany). Recently created a sorbent from a synthetic Oligopeptide "Globaffin" "Affinna", Germany.

Column "Immunosorba"® contain 62.5 ml of agarose gel with immobilized protein A. the Sorption capacity of this sorbent is approximately 20 mg of IgG /ml of gel sorbent [9]. Speakers designed for repeated use during the procedure. Main weeks the STATCOM sorbent is the selective interaction of a protein And with different IgG subclasses [10], that significantly reduces clinical results in the treatment of several diseases, in particular DC [11]. The sorbent with the immobilized protein And reduced the concentration of IgM and IgA by 20-40%, which is two times less effective than immunosorbents based on antibodies [12].

Similar ligand - protein a used in the columns "Prosorba"®, "Fresenius HemoCare, Germany, containing 300 ml of sorbent is silica-based. The sorption capacity of the sorbent from the column "Prosorba"® is 7.1±1.8 mg IgG/ml gel [13]. In addition to the above disadvantages, defined immobilized protein A, in vitro and in clinical use was marked leakage of the ligand from the medium [14, 15]. The fact that the speakers are "Prosorba"® designed for single use and, therefore, do not regenerate and cannot be re-therapeutic use is another important drawback of such columns [15, 16]because of this fact significantly limits the effectiveness of the columns in the procedure of therapeutic apheresis.

Columns with immobilized peptide capable of binding immunoglobulins G "Globaffin" ("Fresenius, Germany), contains 67 ml agarose gel with immobilized peptide PGAM146. Column "Globaffin" removes a broad range of human immunoglobulins and designed for multiple applications. The sorption capacity when ranta an average of 17 mg IgG/ml gel [17]. Because the use of such columns only begins in the framework of clinical trials, in the literature there are practically no data on the advantages and disadvantages of such a sorbent.

The closest analogue of the invention is a sorbent-based agarose matrix with immobilized polyclonal sheep antibodies against human IgG, which is used in the columns "Ig-Therasorb"®, (Myltenyi Biotec GmbH, Germany)containing 150 ml of agarose gel with immobilized polyclonal sheep antibodies against human IgG. The sorption capacity of the sorbent by an average of 12 mg IgG/ml of gel. Speakers intended for repeated use and regeneration during therapeutic apheresis, bind all subclasses of immunoglobulin G, and partially immunoglobulin classes a and M. the Main disadvantage of this sorbent is the sorption capacity at 30% inferior to the sorbent with immobilized protein A (see table).

The prototype of the present invention is a sorbent used in columns "F Adspec"®, (NPF "PACKARD", Russia), containing 200 ml of agarose gel with immobilized polyclonal sheep antibodies to human IgG. The sorption capacity of 1 ml of gel is 10-12 mg protein antibodies [18]. Column effectively remove IgM and IgA [19], designed for mn is multiple use and can withstand up to 100 cycles of sorption desorption with the loss of sorption capacity, not exceeding 30%. Immunosorbents, containing as a ligand polyclonal sheep antibodies to human IgG, do not have the above disadvantages of the sorbents with immobilized protein A, namely, low binding IgG 3 subclass, weak interaction with IgM and IgA. However, they are inferior to the latter in its sorption capacity.

The objective of the invention is to increase the sorption capacity and safety of the immunosorbents used for practical purposes, in particular in the procedures of therapeutic apheresis. This task is solved by using as a ligand F(ab)2fragments of antibodies immobilized on a matrix in a concentration which provides the desired sorption capacity. In spite of the fame F(ab)2fragments of IgG with the 60-ies of the last century [20], in the literature there are no data on their use as ligands in the synthesis of immunosorbents, found practical application in the field of therapeutic apheresis. This is probably due to the fact that attempts to obtain sorbents with immobilized not modified F(ab)2fragments did not lead to the desired result was not possible to increase the sorption capacity of the sorbent [21]. Thus, the use of F(ab)2fragments of antibodies to receive immunosorbents used for practical purposes, is neo evenin for specialists in this field.

The proposed sorbent is a biologically inert, biocompatible agarose or cellulose matrix, covalently associated with the F(ab)2fragments specific, affinity-purified polyclonal sheep antibodies against immunoglobulin G man. Covalent bond between the ligand and the matrix is formed after activation of the matrix such reagents as brazian or metaperiodate sodium. As a ligand can be used F(ab)2fragments of affinity-purified polyclonal or monoclonal antibodies of different specificity. The main advantage of the proposed sorbent is significantly large compared to the prototype sorption capacity (15,8±1.0 mg IgG/ ml of gel sorbent with immobilized F(ab)2fragments of polyclonal antibodies against immunoglobulin G man and 11.2±0.8 mg of IgG/ml of gel for the prototype), preserving all the above advantages of immunosorbents with antibodies, such as high specificity, interaction with all classes and subclasses of immunoglobulins, the possibility of multiple use. In addition, the absence of ligand constant region of the heavy chains of immunoglobulins, causing interspecific differences immunoglobulins (izotopicheskie antigenic determinant), significantly reduces potentially available is th immunological response of the organism to a foreign protein, thus, increases safety applications immunosorbent assay for the purposes of therapeutic apheresis.

Comparison of the proposed sorbent with literature data for analogs and prototypes are shown in the table, there are private data comparison of sorption capacity of the proposed sorbent with commercially available analogues and the prototype.

The implementation of the invention

Example. Anticigarette sheep to human immunoglobulins were obtained by immunization of animals donor immunoglobulins G man, purified by ion exchange chromatography as described by the authors previously [18]. Specific antibodies sheep to human immunoglobulins were obtained by the method of affinity chromatography sorbent with immobilized antigen immunoglobulin G [18].

Fragments specific antibodies were obtained by the method of proteolytic cleavage of the drug polyclonal sheep antibodies to human IgG on a column with immobilized enzyme pepsin ("Pierce, USA). To obtain specific F(ab)2fragments of antibodies affinity-purified polyclonal antibodies sheep in citrate buffer pH 2.5 with a concentration of 10 mg/ml was passed through the column with 2 ml of sorbent with pepsin-agarose. The contact time of the solution of antibodies to the immobilized enzyme was 4 min. Number of antibodies, ispolzuemykh in the reaction of enzymatic degradation, varied from 0.1 to 7.0, the Study of antibody preparations after splitting using vertical electrophoresis in polyacrylamide gel (gradient 4-22%) under denaturing conditions showed the presence of a major band corresponding to F(ab)2(100 kDa)and a minor band with a molecular mass of from 10 to 50 kDa, corresponding to fragments splitting constant region (Fc fragment of the immunoglobulin molecule.

The secretion of immunoactive F(ab)2fragments of antibodies to human immunoglobulins was performed by the method of affinity chromatography on a column with immobilized on agarose matrix antigen immunoglobulin G man. Chromatography was carried out at pH values not below 4.5, the application time of fragmented antibodies on the column was 2 hours. The elution of the active F(ab)2fragments was performed citrate buffer pH of 2.5. The presence of active F(ab)2fragments were determined by ELISA. On the plate the first layer were adsorbed immunoglobulins G man, the second layer rastarivanie F(ab)2fragments of antibodies as detection antibodies used rabbit antibodies to sheep immunoglobulins conjugated to peroxidase.

Received eluate concentrated to 10-15 mg/ml and was immobilized on a pre-activated agarose matrix, for which the solution F(ab)2FR is mentov, cialisovernight against water (pH 4.0 to 4.5) was added 10-fold carbonate buffer pH of 10.0, and incubated for 2 hours with pre-activated with bromine cyan agarose matrix. The concentration of immobilized ligand was assessed by differential method according to the difference in the amount of protein taken for the reaction of immobilization and remaining in solution after incubation with the matrix.

Result.

The sorption capacity of the proposed sorbent, prototype and commercial analogues tested by the method of affinity chromatography from plasma of human blood, with the concentration of immunoglobulin G 15 mg/ml Chromatography was performed on 100 μl of the sorbent, the time of contact of the sorbent with the plasma was 1 hour, the volume of plasma 1 ml After incubation sorbents were washed in 5 ml of phosphate buffer, and bound peroxidase protein was suirable 2 ml 0,05 m citrate buffer pH of 2.5. The sorption capacity of the proposed sorbent (the amount of protein in the eluate with 1 ml of gel) ranged from 14,3 to 17.8 mg/ml. the Results of research on the sorption capacity of the proposed sorbent, analogs and prototypes are given in the table.

Specificity, i.e. the ability to specifically bind immunoglobulins G of a mixture of proteins (human blood plasma), confirmed by testing the eluate from the sorbent by electrophoresis in gradient of polyaki the amide gel under denaturing conditions. The eluate from the proposed sorbent contains not less than 90% of immunoglobulins G and contains almost no impurities other plasma proteins. 1 ml of the sorbent with the immobilized F(ab)2fragments of polyclonal antibodies to human immunoglobulins associates 6.9 mg IgM and 3.3 mg of IgA plasma source data level of immunoglobulins 2.6 and 2.8 mg/ml, respectively.

20 chromatographic cycles with the blood plasma of a person lead to the fall of the sorption capacity of the sorbent with the immobilized F(ab)2fragments of antibodies to immunoglobulins G man on 7%, while the fall sorption capacity for 100 cycles does not exceed 30%, which allows efficient use of the proposed sorbent repeatedly.

Table

Comparison of commercially available systems for the removal of immunoglobulins in vitro procedure, therapeutic apheresis (literature and own data).
Product nameManufacturerMatrixThe ligandThe volume of gel in the columnThe sorption capacityUse
Qty IgG columnQuantity of IgG per ml of gel
L is teratornis data Own results
Imunosorba®Fresenius AG, Germany (formerly Excorim, Sweden)the agaroseProtein And cell wall of Staphylococcus aureus62,5 ml1.2 g19,2 mg/ml17,2 mg/mlmany times
Prosorba®Fresenius AG, Germany (formerly Imre and Cypress, USA)silica gelProtein And cell wall of Staphylococcus aureus300 ml1.2 g7,0 mg/ml-once the
Globaffin®Fresenius AG, Germany (formerly Affina GmbH, Germany)the agaroseSynthetic Oligopeptide67 ml1.2 g17.9 mg/ml-many times
Ig-TheraSorb®Myltenyi Biotec GmbH, Germany (formerly Baxter and PlasmaSelect, Germany)the agarosePolyclonal sheep antibodies (Pcat) against human IgG150 ml2.0 g13,0 mg/mlof 12.7 mg/mlmany times
Ig Adspec®NPF "PACKARD, Russiathe agarosePcat to human IgG200 ml2.2 g11.0 mg/mlof 11.2 mg/mlm is agarrate
ImmunotecDeveloper - FSE RK NPC, Ministry of Manufacturer - SPF "PACKARD, Russiathe agaroseF(ab)2fragments of polyclonal antibodies100 ml1.5 g-15.8 mg/mlmany times

The list of information sources

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4. Muller J., Wallukat G., M. Dandel, Bieda H., K. Brandes, Spiegelsberger S., Nissen, E., Kunze R., review : firebreath R. // Immunoglobulin adsorption in patients with idiopathic dilated cardiomyopathy / Circulation. - 2000. - V.101. - P.385-391.

5. Moreso F., R. Poveda, Gil-Vernet s, Carreras L., Garcia-Osuna R., Grino J.M., J. Alsina // Therapeutic immunoadsorption in Goodpasture disease./Med. Clin. (Barc) - 1995 Jun. 10. - V.105 (2). - P.59-61.

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7. Konovalov GA, Belenkov YU.N., Zvezdin PV, Chebyshev A.N., Semin S.N., Kuznetsov, Y., Adam YU, Kipor YEAR, S. Pokrovsky. Apheresis immunoglobulin - a new approach to the treatment of severe forms of dilated cardiomyopathy. // Cardiology. - 2002. - So 6. - Pp.92-96.

8. Koll R.A. // Ig-Therasorb immunoadsorption for selective removal of human immuoglobulins in diseases associated with pathogenic antibodies of all classes and IgG subclasses, immune complexes, and fragments of immunoglobulins. / Ther. Apher. - May 1998. - V.2 (2). - P.147-152.

9. Bygren p, C. Freiburghaus, T. Lindholm, et. al. // Goodpasture''s syndrome treated with gets protein A immunoadsorption. / Lancet. - 1985. - V.2 (8467). - P.1295-1296.

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11. Staudt, A., Bohm, M., Knebel f, Grosse Y., Bischoff, C., Hummel, A., Dahm JB., Borges, A., Jochmann n, Wernecke KD., Wallukat G., Baumann g, Felix SB. Potential role of autoantibodies are belonging to the immunoglobulin G subclass 3 in cardiac dysfunction among patients with dilated cardiomyopathy. / Circulation. - 2002. - V.106 (19). - P.2448-2453.

12. Matic G., Hofmann D., Winkler R., Tiess M., Michelsen, A., Schneidewind JM., Hebestreit, G., Keysser m, Muller W., Kinze EM., Ramlow W // Removal of immunoglobulins by a protein A versus an antihuman immunoglobulin G-based system: evaluation of 602 sessions of extracorporeal immunoadsorption. / Artif Organs. - 2000. - V.24 (2). - P.103-107.

13. Snyder HW Jr., Henry DH., Messerschmidt GL., et al. // Minimal toxicity during protein A immunoadsorption treatment of malignant disease: an outpatient therapy. / J Clin Apher. - 1991. - V.6 (1). - P.1-10.

14. Ray PK., Besa E., Idiculla, A., Rhoads JE Jr., Bassett JG., Cooper DR., // Efficient removal of abnormal immunoglobulin G from plasma of a multiple myeloma patient. Description of a new method for treatment of the hyperviscosity syndrome. / Cancer. - 1980. - V.45 (10). - 2633-2638.

15. Euler HH., Schwab UM., Schroeder JO., Hasford J. // The Lupus Plasmapheresis Study Group: rationale and updated interim report. /Artif Organs. - 1996. - V.20 (4). - P.356-359.

16. Knobl, P., K. Derfler, Korninger L., Kapiotis, S., Jager, U., Maier-Dobersberger t, Horl W, Lechner K, Pabinger I. // Elimination of acquired factor VIII antibodies by extracorporal antibody-based immunoadsorption (Ig-Therasorb). / Thromb Haemost. - 1995. - V.74 (4). - P.1035-8.

17. Ronspeck W., Brinckmann R., R. Egner, et al. // Peptide based adsorbers for therapeutic immunoadsorption. / Ther Apher Dial. - 2003. - V.7 (1). - P.91-97.

18. Kiseleva E.A., Afanasieva O.I., Kosheleva N.A., Pokrovsky S.N. // Immunosobent for IgG apheresis: an in vitro study. / Transfus. Sci. - 1996. - V.17 (4). - P.519-525.

19. Konovalov GA, Topalov I.N., Histoplasma N.A., Chebyshev A.N., Medicine L.A., V. Shmyrev, Yastrebova N.E., Kiseleva E., I. Afanasyeva, Adam YU, S. Pokrovsky. The first experience of application of course Ig-apheresis in the treatment of patients with multiple sclerosis. // The Kremlin medicine. - 1999. No. 3. - P.48-52.

20. Nisonoff, A., Wissler F.., Lipman L.N. and Woernley D.L. // Separation of univalent fragments from the left-hand drive vehicles rabbit antibody molecule by reduction of disulfide bonds. / Arch. Biodtem. and Biophysics. - 1960. - V.89. - P.230.

21. Yarmush M.L., Lu X., Yarmush D.M. // Coupling of antibody-binding fragments to solid-phase supports: site-directed binding of F(ab)2fragments. / Journal of Biochemical Methods. - 1992. - V.25. - P.285-297.

Sorbent for the removal of immunoglobulins from human blood plasma containing agarose matrix, covalently linked to a ligand, characterized in that, as a ligand, it contains F(ab)2fragments of affinity-purified polyclonal sheep antibodies against immunoglobulin G man.



 

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