|Title of Invention
A PROCESS FOR THE PREPARATION OF IMMUNOADSORBENT MATRIX BASED ON MODIFIED POLYVINYL ALCOHOL MICROSPHERES
|This invention relates to an immunoadsorbent matrix provided with heparin immobilized onto phenylalanine coupled beads. The immunoadsorbent is useful in the columns for direct haemo-perfusion.
|EIELD OF INVENTION
This invention relates to an immunoadsorbent matrix based on modified polyvinyl alcohol microspheres and its use in columns for direct haemo-perfusion
BACKGROUND OF INVENIJON
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, ^nd risk of infections lead to the development of plasma treatment devices., Selective adsorption plasma treatment is now a wel1 established clinical practice for the treatment of myasthenia gravis5 GuilIain-Barre syndrome (GBS)* systemic lupus erythematosus (BLE) 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 immunoadsorption columns for plasma perfusion only are available, it is stil1 not known to havB direct haemoperfusion columns- Further 9 it is not known to have devices that can simplify and reduce treatment cost further by achieving plasma separation amd 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 immunoadsorhent 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
Yet another method and apparatus known in the
art is to withdraw the blood of a patient9 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.
QBJECTS OF THE UVJSS££IS
An object of this invention is to propose an immunoadsorbent matrix based on polyvinyl alcohol tnicropheres for direct haemo-perfusion.
A further object of this invention is to propose 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 to propose an immunoadsorbent column based on the immunoadsorbent matrix for the specific removal of antibodies belonging to or consisting of immunoglobulins of the class XG 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 IgG 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 will be more apparent from the ensuing description.
DESCRIPTION OF THE INVENTION
According to this invention there is provided a process for the preparation of an improved adsorbent matrix based on modified polyvinyl alcohol microshperes comprising of:
a) preparing crosslinked polyvinyl alcohol (PVA) as herein
b) beads formed by step (a) are subjected to the step of filtering, washing, drying and sieving tc get crosslinked PVA beads,
c) subjecting the beads to swelling in an alkaline medium followed by filtering, adding mixture of epi chlorohydrin and 1, 4-dioxan to swollen beads,
d) reaction mixture of step ( c) is cooled, washed and subjected to the treatment with aminoacid solution, stirring and washing the beads,
e) activating as herein described, the beads to obtain amino adsorbent matrix.
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 reaction, 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 are subjected to swelling in an alkaline medium followed by filtering and then adding a mixture of epichlorohydrin and 1,4-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 aminoacid coupled PVA beads. The aminoacid coupled beads is activated by exposing them to l-ehtyl-3-(3-demethyl amino proply) carbodimide in PBS followed by the step of exposing the activated beads to heparin solution, washing to remove unbound herparin, to botain 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 ar& prepared by acid catalysed reaction by mixing an aqueous solution of ^VA of molecular weight 1,00,000 to 1,50,000 in 3 to 6 ml of qlutaraldehyde 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 are 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 disti1 led water -
The next step in the process comprises in phenylalanine coupling onto the PVA microspheres is achieved according to the reaction scheme (Scheme I) shown in Fig. 1 of the accompanying drawing.
Stable aminoacid ligands ar& 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-
dioxan 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 mgX and above aminoacid solution
o at 40-60 C with gentle stirring - The beads were washed
free of uncoupled aminoacid * The possible reaction is depicated in Fig - 1.
The beads are washed free of the uncoupled
amino acid by acetone and distilled water and finally to neutrality-
Immobilisation 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) &r& exposed to l-ethyl-~3-(3~dimethyl aminopropyl )
carbodimide in PBS. The activated beads &r& washed with distilled water followed by exposure to 4-25 mgX heparin
solution in PBS to obtain adsorbents with different amount of bound heparin. The Hep-ph-PVA beads are then
washed with distilled water to remove unbound heparin.
The two reactions arB 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 immunoglobulins of G, i.e. antibodies such as anti-DNA,
AChRab, 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
1. A process for the preparation of an improved adsorbent matrix
based on modified polyvinyl alcohol microshperes comprising of:
a) preparing crosslinked polyvinyl alcohol (PVA) as herein described,
b) beads formed by step (a) are subjected to the step of filtering, washing, drying and sieving to get crosslinked PVA beads,
c) subjecting the beads to swelling in an alkaline
medium followed by filtering, adding mixture of epi
chlorohydrin and 1, 4-dioxan to swollen beads,
d) reaction mixture of step ( c) is cooled, washed and
subjected to the treatment with aminoacid solution,
stirring and washing the beads,
e) activating as herein described, the beads to obtain
amino adsorbent matrix.
2. A process as claimed in claim 1, wherein crosslinking is carried in oil medium preferably in paraffin oil.
3. A process as claimed in claim 1, wherein washing of beads is carried in organic solvent preferably petroleum ether followed by acetone and distilled water.
4. A process as claimed in claim 1, wherein reaction of step 1 ( c) is
carried at a temperature of 100°- I20°C.
5. A process for the preparation of an improved adsorbent matrix
based on modified polyvinyl alcohol microshperes substantially
as herein described and illustrated.
|Indian Patent Application Number
|PG Journal Number
|Date of Filing
|Name of Patentee
|CHANDRA P. SHARMA
|BIOMEDICAL TECHNOLOGY WING, SREE CHITRA TIRUNAL INSTITUTE FOR MEDICAL SCIENCES & TECHNOLOGY, POOJAPURA, THIRUVANATHAPURAM 695 012,
|PCT International Classification Number
|PCT International Application Number
|PCT International Filing date