Title of Invention

A PROCESS FOR THE PREPARATION OF IMMUNOADSORBENT MATRIX FOR HAEMOPERFUSION

Abstract

This invention relates to a process for the preparation of an immunoadsorbent matrix comprising of preparing polyvinyl alcohol beads by partially cross linking polyvinyl alcohol with a cross linking agent; coupling phenylalanine onto the polyvinyl alcohol beads to obtain a COOH functionality of the phenylalanine polyvinyl alcohol beads; . activating as herein described said COOH functionality for immobilizing heparin onto said phenylalanine coupled beads.

Full Text

FIELD OF INVENTION This invention relates to a process for the preparation of immunoadsorbent matrix based on modified polyvinyl alcohol micro spheres and its use in columns for direct haemo-perfusion. BACKGROUND OF INVENTION Plasma exchange has become the most common and effective therapeutic treatment of various intractable disorders and neurological diseases. Unavailability and high price of plasma products, and risk of infections lead to the development of plasma treatment devices. Selective adsorption plasma treatment is now a well established clinical practice for the treatment of myasthenia gravis Quillain-Barre syndrome (GBS), systemic lupus erythematosus (8LE) etc., but is only marginally cheaper than plasma exchange procedure, and the columns are meant for plasma perfusion only. However, this can eliminate the dependence of plasma products on plasmapheresis thereby avoiding risk of infections- Though various immunoabsorbtion columns for plasma perfusion only sirs available, it is stil1 not known to have direct haemoperfusion columns. Further, it is not known to have devices that can simplify and reduce treatment cost further by achieving plasma separation and plasma treatment in a single device or direct haemo-perfusion- An apparatus and method is known in the art which draws blood through a blood pump and a column^ and functions as a plasma separator to cause a separation of the blood cells from the plasma. The plasma is then passed through an immunoadsorbent column so that IgG proteins get adsorbed in the adsorbent, and the treated plasma is then mixed with the removed blood cells and returned to the patient. The disadvantage of such a method and apparatus is that of substantial costs. Further, such a method is to be repeated once or twice and each time the column and plasma separator is to be replaced. Yet another method and apparatus known in the art is to withdraw the blood of a patient, which is then centrifuged to remove the plasma, which is then replaced by fresh blood plasma from a donor. A disadvantage of such a method and apparatus is that of infection in the fresh blood of a donor. Further, hemolysis can occur in the centrifuge. OBJECTS OF THE INVENTION An object of this invention is to propose a process for the preparation of an immunoadsorbent matrix based on polyvinyl alcohol microphones for direct haemo-perfusion. A further object of this invention is to propose a process for the preparation of an immunoadsorbent matrix based on polyvinyl alcohol which can specifically remove IgG proteins from blood and is highly blood compatible. A still further object of this invention is top propose an immunoadsorbent column based on the immunoadsorbent matrix for the specific removal of antibodies belonging to or consisting of immunoglobulin of the class B and circulating immune complexes. Yet another object of this invention is to propose an immunoadsorbent column which can be used for direct perfusion of human patient blood for the treatment of Its mediated disorders. Another object of this invention is to propose an immunoadsorbent column for blood perfusion which may be applied clinically as a less complex and inexpensive alternative to plasma treatment devices. Further objects and advantages of this invention wi11 be more apparent from the ensuing description. DESCRIPTION OF INVENTION According to this invention there is provided a process for the preparation of an immunoadsorbent matrix comprising in the steps offs a) preparing polyvinyl alcohol beads by partially cross linking polyvinyl alcohol with a cross linking agent; b) coupling phenylalanine onto the polyvinyl alcohol beads to obtain a COOH functionality of the phenylalanine polyvinyl alcohol beads; c) activating said COOH functionality for immobilizing heparin onto said phenylalanine coupled beads. In accordance with this invention the preparation of immunoadsorbent beads comprises in preparing partially crosslinked polyvinyl alcohol (PVA) beads by mixing an aqueous solution of PVA with glutaraldehyde and dispersing the mixture in an oil medium by stirring. To the dispersed PVA solution is added benzoyl chloride slowly dropwise. On completion of reaction9 the beads thus formed are subjected to the step of filtering, washing, drying and sieving to obtain the crosslinked PVA beads. The dry crosslinked beads &re subjected to swelling in an alkaline medium followed by filtering and then adding a mixture of epichlorohydrin and lp4-dioxan to the swollen beads and the reaction is performed by heating• The reaction mixture is cooled and then followed by washing and subjecting the washed beads to treatment with aminoacid solution with gentle stirring, and washing the beads to obtain aminoacids coupled PVA beads. The ammonia acid coupled beads is activated by exposing them to 1-ethyl- 3-(3"dimethyl amino propyl) carbodiimide in PBS followed by the step of exposing the activated beads to heparin solution, washing to remove unbound heparin, to obtain the immunoadsorbent beads, Hep-ph-PVA- The first step of the process consists in preparation of polyvinyl alcohol (PVA) beads. For this purpose, partially crosslinked PVA beads Sire prepared by acid catalysed reaction by mixing an aqueous solution of PVA of molecular weight 1,00,000 to 1,50,000 in 3 to 6 ml of glutaraldehyde and dispersed in an oil medium and subjected to the step of stirring. Upon PVA solution being completely dispersed, a catalyst is added dropwise. The catalyst by way of example, is benzoyl chloride. After completion of the reaction in a period of approximately 30 mins, the beads sure filtered, washed, dried and classified. The oil medium used in the cross linking of PVA beads is heavy and light liquid paraffin oil in a proportion for example 2:1, with sorbitan monooleate added thereto. Preferably, approximately 500 ml. oil solution contains about 355 ml. of heavy oil and 165 ml. of light oil with 0-4 ml. of sorbitan monooleate. The washing of the beads is done using organic solvents preferably petroleum ether followed by acetone and subsequently with distilled water The next step in the process comprises in phenylalanine coupling onto the PVA micro spheres is achieved according to the reaction scheme (Scheme I) shown in Fig. 1 of the accompanying drawing. Stable aminoacid ligands are coupled onto the beads by swelling. Known amounts of dry beads were swollen in an alkaline medium and filtered. A mixture of epichlorohydrin and organic solvent such as 1,4- dioxin was added into the swollen beads and the reaction o was performed at 100-120 C for one hour. The mixture is cooled to room temperature^ followed by through washing with acetone and distilled water to remove the excess reaction mixture and finally to neutrality. The phenylalanine is coupled to the activated beads by treating them with 250 mg% and above aminoacid solution o at 40-60 C with gentle stirring. The beads were washed free of uncoupled aminoacid. The possible reaction is depicted in Fig. 1. The beads are washed free of the uncoupled amino acid by acetone and distilled water and finally to neutrality. Immobilization of heparin onto the phenylalanine coupled beads is completed according to the reaction scheme (Scheme II) as shown in Fig. 2 of the accompanying drawings. The aminoacid coupled PVA beads (wet) are exposed to l-ethyl-3"(3"dimethyl iminopropyl) carbodiimide in PBS. The activated beads are washed with distilled water followed by exposure to 4-25 mX heparin solution in PBS to obtain adsorbents with different amount of bound heparin. The Hep-ph-PVA beads then washed with distilled water to remove unbound heparin. The two reactions ore carried out at room temperature with gentle stirring at a pH of about 8.5 and 5.5 respectively. In accordance with another embodiment of the present invention, a column is packed with the Hep-ph-PVA beads and used in haemo-perfusion for the specific removal of antibodies that belong to or consist of immunoglobulin of G, i.e. antibodies such as anti-Dap Ahab, anti-GBM and circulating immune complexes. Other objects and advantages of the present invention will be apparent from the accompanying examples and the comparative study depicted in the following tables. ME CLAIM A process for the preparation of an immunoadsorbent matrix comprising in the steps of a a) preparing polyvinyl alcohol beads by partially cross linking polyvinyl alcohol with a cross linking agent; b) coupling phenylalanine onto the polyvinyl alcohol beads to obtain a COOH functionality of the phenylalanine polyvinyl alcohol beads; c) activating said COOH functionality for immobilizing heparin onto said phenylalanine coupled beads. 2. A process as claimed in claim 1 comprising in the step of preparing partially crosslinked polyvinyl alcohol (PVA) beads by mixing an aqueous solution of PVA with glutaraldehyde and dispersing the mixture in oil medium by stirring, adding benzoyl chloride slowly dropwise to the dispersed PVA solution on completion of reaction subjecting the beads thus formed to the step of filtering, washing drying and sieving to obtain the crosslinked PVA beads. 3. A process as claimed in claim 1 comprising in the step of subjecting the dry crosslinked beads to swelling in an alkaline medium followed by filtering, adding a mixture of epichlorohydrin and dioxan to the swollen beads, heating to react, cooling the reaction mixture followed by washing and subjecting the washed beads to treatment with aminoacid solution with gentle stirring, and washing the beads to obtain aminoacid coupled PVA beads« 4. A process as claimed in claim 1 comprising in the step of subjecting the amino acid coupled beads to activation by exposing to l"ethyl-"3-(3-dimethyl amino propyl) carbodimide in PBS followed by the step of exposing the activated beads to heparin solution, washing to remove unbound heparin, to obtain the immunoadsorbent t beads, Hep-ph-PVA• 5« A process as claimed in claim 2 wherein said oil medium comprises heavy and light liquid paraffin oil with sorbitan monooleate. 6- The process as claimed in claim 2 wherein epichlorohydrin and 1,4-dioxan is present in a proportion- 7. The process as claimed in claim 2 wherein the swollen beads Are treated with epichlorohydrin and 1,4- dioxane at a temperature in the range of 100'-'120 C. 8. The process as claimed in claim 2 wherein the step of treatment with the aminoacid solution is carried o out at a temperature in the range of 40 to 60 C for about 4 hrs. 9. The process as claimed in claim 3 wherein for the step of exposure to heparin^ a 4-25 mgX heparin solution in PBS is used. 10. An immunoadsorbent column comprising a column packed with the immunoadsorbent beads, Hep-ph»PVA» 11. A process for the preparation of the immunoadsorbent beads substantially as herein described and i1lustrated.

Documents:

215-mas-1999-abstract.pdf

215-mas-1999-claims filed.pdf

215-mas-1999-claims granted.pdf

215-mas-1999-correspondnece-others.pdf

215-mas-1999-correspondnece-po.pdf

215-mas-1999-description(complete)filed.pdf

215-mas-1999-description(complete)granted.pdf

215-mas-1999-drawings.pdf

215-mas-1999-form 1.pdf

215-mas-1999-form 26.pdf

215-mas-1999-form 3.pdf

215-mas-1999-other document.pdf