Title of Invention

AN IMPROVED PROCESS FOR THE PREPARATION OF MICRO CELLULOSE BEADS.

Abstract

A simple process for the preparation of micro cellulose beads (30 -50 micron) useful for the isolation and immobilization of biomolecules. Highly resistant to acid and alkali, autoclavable, non-compressible during column operation and having 100% water regain capacity beads were obtained. CMC (carboxy methyl cellulose) beads showed 45% protein binding capacity on dry weight basis. The ployethyleneimine cellulose beads showed 51'old purification of penicillin acylase from cell lysate in single step. The urease immobilized on ployethylencimine cellulose bead showed almost three years storage stability at 4°C and 91 % conversion in column reactor. The DFAE (dietyl amino ethyl) cellulose bead showed anchoring of the cell on the surface, indicating its utility as a microcarricr.

Full Text

The present invention relates to the process for the preparation of micro cellulose beads. More particularly it relates to the preparation of the said beads useful for isolation and immobilization of biomolccules and as a microcarrier. This finds application in pharmaceutical and biotech industries. Conventionally, in different biomolecule isolation and immobilization techniques, cross-linked dextran, agarose, polyacrylamide and other synthetic polymers has been used. However, these materials are generally too expensive to be used in industrial application since they are made from rather uncommon natural substances or they require carefully controlled process for preparation. Most of them are more or less hydrophobic, generally which make them less tolerable to biochemical system. On the other hand, cellulose is one of the most widespread natural biopolymeric substances. Billions of tons are created each year through photosynthesis, and mankind utilize more than 500 millions of tons in the form of wood, paper, textiles, films, plastics, coatings, fuel, etc (E. Olt and H. G. Tennet, in H. Oil, H. M. Spurlin and M. W. Grafilin (Eds.), Cellulose and Cellulose Derivatives, 2nd edn., Part I. Interscience Publishers, New York, 1954, p. 1.). Particularly interesting application of cellulose occur in the field of functional polymers, include adsorbent, ion-exchange resins, chemically reactive polymers, catalysts, redoxites and carriers of various biological functions useful in medicine, pharmacy and biotechnology. Unfortunately, its physical structure is much inferior by comparison with synthetic polymeric materials. Most problems related to the use of the old fibrillar and powdered cellulose have been solved by introducing a novel form (bead cellulose) which is both porous and spherical (O Neill and Reichardts; U.S. Pat. No 2, 543, 928: 1951). Since the first attempts by O Neill and Reichardts, a numbers of other process are invented. All the developed processes arc based on a common principle, which includes the dispersion of cellulose solutions in non-miscible liquid phase followed by regeneration of cellulose (G. Buschle-Diller, C. Fanter and F. Loth. Cellulose. 1995. 2, 179-203). The choice of the starting material and nature of dispersion and regeneration medium have a decisive effect on the properties of the product (H. Schlcichcr, F. Loth, and B. Lukanoff. Acta Polyinerica. 1989. 40, 170-177). Not only that, the chemicals used either for the preparation of starting material or dispersion and regeneration medium poses serious environmental problems. The processes patented so far can be divided into three groups on the basis of starting materials used as follows - 1. Viscose/Xanthate, where a highly toxic chemical carbondisulfied(CSi) is being used which can cause a marked psychic disturbance ranging from extreme irritability to mania with hallucinations, tremors, auditory and visual disturbances, weight loss, blood dyscrasias etc [U.S. Pat. No 2, 543, 928 (1951); Japanese Kokai Patent No. 73 43, 082 and 73 60, 753 (1973); U.S. Pat. No 4, 055, 510 (1977); Czech.Patent No. CS 2615 38 (1989)]. 2. Cellulose derivatives, where different toxic chemicals are being used for the dcrivatization of cellulose and the product looses its mechanical strength due to drastic chemical treatment given for the derivatization [German Offen. No. 2, 507, 551 (1975); Japanese Kokai Patent No. JP 63, 95, 237 (1988); Japanese Kokai Patent No. JP 01, 275, 641 151, 289 (1989); German (East) Patent No. DD 29 58 61 (1991)]. 3.Cellulose, where benzene which is a known carcinogen and may cause leukemia was used as a medium for dispersion and regeneration [French Patent No. Fr. 1, 575, 419 (1969): The cellulose beads were prepared either by pulverizing in air or by mixing at high speed (1250 - 2850 rpm) a solution of cellulose (2g) in concentrated NH4OH (180 ml) added with Cu (OH) 2 (1.2g) and CuCl (0.2g) to a solution of emulsifier (1.25g) in a solvent (500ml C6H6). The beads were added to a solution of cellulose regenerating agent (250 ml BzOH) in a solvent (3800 ml C6H6). and metal salts were removed before final recovery. U.S. Pat. No. 3, 597, 350 (1971): Emulphor EL (Castor oil felly acids elhylene oxide condensnlc) in C6H6 was mixed with powd. linlers cellulose, Cu (OH)2 and CuCl in NH4OH : HOBz in C6H6 was added, and the dispersion was stored overnight. The solids were treated with 8% NaOH and the beads were removed, H2O- washed and sieved and freed of Cu salts using HOAc followed by alk. ethylcne diaminetetraacetic acid to give beads]. Our process involves use of less toxic chemicals for dispersion and regeneration and natural form of cellulose as a starting material to obtained a product useful for isolation and immobilization of biomolcculcs and as a microcarricr. We have claimed a process for the preparation of micro cellulose beads using natural form of cellulose (long or short fiber, powder, linters, raw cellulose etc) as a starting material and toluene a less toxic chemical (The Merk Index) as a medium for dispersion and regeneration. In our process there is no need for pulverization of cellulose solution in air or mixing at high speed (1250 - 2850 rpm) nor the requirement of derivatized emulsifier such as Emulphor EL (Castor oil fatly acids ethylene oxide condensale) or no need to store dispersion overnight and its treatment with NaOH prior to recovery. The object of the present invention is to provide a process for the preparation of micro cellulose beads, using less toxic chemicals useful for the isolation and immobilization of biomolecules and as a microcarrier. Accordingly the present invention provides a process for the preparation of micro cellulose beads which comprises, making a solution of cellulose in a mixture of ammonia solution and a copper salts, dispersing this solution in a solvent containing an emulsifying agent and a surfactant, regeneration using a mixture of solvent and mild acid to obtain the beads, separating and wasliiug the beads with water to obtain the product. hi one of the embodiment of the present invention, the cellulose may be in the form of long or short fiber, powder, lintcrs etc, preferably raw cellulose. In another embodiment of the present invention, the solvent used for dispersion and regeneration may be any aromatic hydrocarbon preferably toluene. In still another embodiment the emulsifying agent may be any commercially available emulsifying agents exemplified by Emulphor EL, Cremophor EL, BASF Ludwigs hafen etc, preferably castor oil. In a feature of the present invention, the cellulose solution is prepared using ammonium hydroxide and cooper sulphate and dispersed in toluene containing castor oil and triton X100. The micro cellulose beads collected by solidifying the dispersion medium with the mixture of toluene and benzoic acid and washing the bead with water to obtained the product. . In a another feature of the present invention, the DEAE (diethyl aniino ethyl), CM (carboxy methyl cellulose), polyethyleneimine micro cellulose beads prepared and tested for the isolation, immobilization of biomoleculcs as hemoglobin, penicillin acylase, urea.se and as a microcarrier. The compatibility of the bead with acid, alkali and autoclave tested. The compressibility of the bead tested by running column and sequential drying to estimate water regain capacity. The process of the present invention is described by following examples, which are illustrative only and should not be construed to limit the scope of the present invention in any manner. Example 1: Solution of cellulose (2.5 g) in NH4OH (90ml) added with CuSO4 (6 g), NH4C1 (4 g), NaOH (1.7g) and CuCl2 (0.1%) added to a solution of toluene (0.6 L) containing castor oil (6.33%) and triton X100 (0.33%). This mixture added to the solution of toluene (1.5 L) and benzoic acid (9%). Beaded material collected (30 - 50 microns) by washing with water and 0.01 M EDTA and treated with cpichlorohydrin. 96% yield was obtained on the basis of dry weight. Example 2: Solution of cellulose (5 g) in NH4OH (180 ml) added with CuSO4 (12 g), NH4C1 (8 g), NaOH(3.4) and CuCl2 (0.1%) added to a solution of toluene (1.2 L) containing castor oil (6.33%) and triton X100 (0.33%). This mixture added to the solution of toluene (3.0 L) and benzoic acid (9%). Beaded material collected (30 - 50 microns) by washing with water and 0.01M EDTA and treated with epichlorohydrin. 96% yield was obtained on the basis of dry weight. Example: 3 Solution of cellulose (10 g) in NH4OH (360 ml) added with CuSO4 (24 g), NH4C1 (16 g), NaOH (6.8) and CuCl2. (0.1%) added to a solution of toluene (2.4 L) containing castor oil (6.33%) and triton XI00 (0.33%). This mixture added to the solution of toluene (6.0 L) and benzoic acid (9%). Beaded material collected (30 - 50 microns) by washing with water and 0.01M EDTA and treated with cpichlorohydrin. 96% yield was obtained on the basis of dry weight. Example 4: To prepare CMC (carboxy methyl cellulose) bead, bead obtained in example 3 was treated with trichloroaceticacid (10%) in the presence of 2M NaOH at 70°C for 20 min, the bead collected by washing with acetic acid (10%) and water and used to determine protein adsorption capacity. Example 5: To check the utility of CMC bead for protein adsorption, the bead obtained in example 4 was packed in a column (2cm x 10cm) and equilibrated with sodium acetate buffer pH 5. Hemoglobin prepared in sodium acetate buffer pH 5 was loaded on the column. 45% protein binding capacity was obtained. Example 6: To study the utility of the bead for biomacromolecule purification and immobilization, polyethyleneimine cellulose bead was prepared. The bead obtained in example 3 treated with polyethyleneimine (0.2%) and packed in a column (2cm x 10cm). The cell lysate containing penicillin acylase loaded on the column. Non specific protein gets adsorbed on the column and all penicillin acylase obtained in the filtrate. This results into the five¬fold purification of penicillin acylase. Example 7: The bead obtained in example 6 treated with polyethyleneimine (0.2%). The crude ureasc preparation was used to study enzyme immobilization. The crude urease preparation was loaded on the column and the binding capacity was estimated, significant urease binding was obtained (675 U/ml of bead). xample 8: The immobilized enzyme obtained in example 7 was packed in a column (2cm x 10cm) and treated as column reactor. The substrate urea (30-180 mM) was fed in the column, 91% conversion was obtained. The immobilized enzyme was stored at 4°C for 3 years and the storage stability was estimated. Almost 100% catalytic activity was obtained after the period of three years. Example 9: To check the utility of bead as a microcarrier, the DEAE (dietyl amino ethyl) cellulose bead was prepared by treating bead with 2N NaOH and diethylaminoethyl chloride hydrochloride (2%) at 70°C for 20 minute. Bead was washed with IN NaOH, 1 N HC1 and water and tested its utility as a microcarrier. The significant binding of the cell were obtained. Example 10: To check the compatibility, the micro cellulose bead obtained as per example 3 were subjected to IN HC1, and 2N NaOH treatment at 60 - 70°C for 20 -30 min. No any physical changes in the product were observed. To check its compatibility with the autoclave, the bead was autoclaved for 20 min at 120°C. No any physical changes in the product was observed. Example 11: To check the compressibility, the micro cellulose bead (example 3) was packed in a column (1 x 50 cm) and was run for the period of one month at room temperature at 80 ml/hr flow rale by gravity. No any change in the height of the column was observed. Example 12: To check the water regain capacity, the micro cellulose bead (example 3) was dried at different alcohol concentration (10 99%) at 60°C. The water was added in the dry beads and the water regain capacity was estimated by measuring wet weight. 100% water regain capacity was obtained. We claim: 1. A process for the preparation of micro cellulose beads which comprises, making a solution of cellulose in a mixture of ammonia solution and a copper salts, dispersing this solution in a solvent containing an emulsifying agent and a surfactant, solidifying the dispersion medium using a mixture of solvent and mild acid to obtain the beads, separating and washing the beads with water to obtain the product. 2. A process as claimed in claim 1 wherein the cellulose is in the form of long or short fiber, powder, linters etc, preferably raw cellulose. 3. A process as claimed in claim 1 to 2 wherein the solvent used for dispersion and solidification may be any aromatic hydrocarbon preferably toluene. 4. A process as claimed in claims 1 to 3 wherein the emulsifying agent may be any commercially available emulsifying agents exemplified by Emulphor EL, Cremophor EL, BASF Ludwigs hafen etc, preferably castor oil. 5. A process for the preparation of micro cellulose beads as substantially described herein before with reference to examples.

Documents:

214-del-2000-abstract.pdf

214-del-2000-claims.pdf

214-del-2000-correspondence-others.pdf

214-del-2000-correspondence-po.pdf

214-del-2000-description (complete).pdf

214-del-2000-form-1.pdf

214-del-2000-form-2.pdf

214-del-2000-form-4.pdf