Title of Invention	"A PROCESS FOR PRODUCING A SUPPORT FOR EXPANDED BED CHROMATOGRAPHY FOR PROTEIN PURIFICATIONS."
Abstract	A process for the preparation of cross-linked cellulose absorbent beads which comprises and characterised by the step of polymerization of cellulose or its derivative suspended in a solvent and in the presence of a cross linking agent and a catalyst at a temperature of 30-100°C and at atmospheric pressure.

Full Text

FIELD OF INVENTION This invention relates to a process for producing cross linked cellulose absorbent beads and which may advantageously be used for various applications such as a support for expanded bed chromatography for protein purification. BACKGROUD Use of solid liquid fluidized beds for absorption and chromatographic separations in biotechnology is a recent development. Solid liquid fluidised bed or also known as absorption or Expanded Bed Adsorption of proteins has important advantages. The most important is that the crude mixture to be passed through the bed need not be clarified of the solid suspensions. Thus, a whole cell broth or a homogenised cell broth (for intracellular products) may be directly passed through the expanded bed of absorbent without the danger of clogging or choking the bed. In a conventional packed bed or fixed bed adsorption, an unclarified crude will block and choke the bed resulting in extremely high pressure drops and damage to the adsorbent and the equipment. Expanded bed adsorption thus provides with three very useful concepts towards improving downstream purification, namely: 1) a number of pretreatment operations like filtration, centrifugation, membrane separations are bypassed. This results in saving considerable losses of the products as well as savings in time and capital investments. 2) simple adsorption of incorporation of some kind of affinity interactions of the products with the solid matrix results in large crude volume reductions for further processing. In addition a degree of purification is 'achieved so that the number of subsequent purification steps are reduced, thereby further improving process yields. 3) since the flow rates possible in expended bed operations are high, quick purifications can be achieved on preparative as well as laboratory scale, if the adsorbent matrix is suitable. Expended bed adsorption thus combines the specificity of chromatography and the volume reduction and solids removal operations of filtration, centrifugation and membrane separation. However for achieving the advantages possibly offered by solid liquid fluidised bed adsorption, the mode of fluidisation has to necessarily meet certain requirements. A vertical column packed with absorbent particles phase shows bed expansion when velocity of the liquid flowing upwards exceeds a minimum fluidization velocity. In the fluidized state the adsorbent particles are 'freely' suspended in flowing liquid, their net gravity force downwards being balanced by frictional force with the passing liquid upwards. A fluidized bed may show turbulence or steady flow in the bed. Turbulent flow in a fluidized bed is associated with considerable liquid phase as well as particle phase back mixing. This back mixing results in mixing of liquid packets having different concentrations of products as also mixing of adsorbent particles with different amount of absorbed products. Such mixing in the bed considerably lowers the performance of the adsorption process providing lesser number of equivalent equilibrium stages in a given bed height. Different methods have been employed to control and minimize the extents of liquid and particle mixing a fluidized beds. The simplest approach has been to employ a designer particle phase. Use of a particle size distribution, instead of using monosize particles has been shown to result in a more stable bed expansion wherein both liquid phase and particle phase mixing are substantially restricted. In addition one has to operate within a certain particles Reynolds number range to achieve the desired mode of fluidization. A stabilized fluidized bed with reduced particle phase and liquid mixing has been called an 'expanded bed'. There are very few adsorbents available in the market that can be used for large and/or preparative scale adsorptive separation of bio-products. Supports based on normal polystyrene copolymers, unless surface modified, are not suitable for most protein separations on account of their highly hydrophobic nature. Hydrophilic xerogel type adsorbents such as those based on polyacrylamide and, cross-linked agarose, dextran or cellulose are used for adsorptive purification of proteins/enzymes or or other bio-active product. Sepharose, Sephadex from Amersham Pharmacia Biotechnologies, Uppsala, Sweden, BioGel range from BioRad laboratories, USA are some examples of such matrices. However these adsorbents are generally not suitable for operation at high linear velocities of pressure tends to copress and deform the adsorbents at velocities exceeding about 100 cm/hr resulting in choking of the adsorbent column. Larger particle sizes can be used to mitigate this effect but at the expense of a greatly increased intraparticle mass transfer resistance that leads to reduction in efficiency and dynamic capacity of the adsorbent. Next generation more rigid adsorbents were then developed for high flow rate separations. Sepharose CL, Sephacel, and Sepharose Fast Flow are some examples of the matrices that have been used for large/preparative scale operations, and are products of Amersham pharmacia Biotechnologies, Uppsala, Sweden. These products, and similar products from BioRad Laboratories, USA; Millipore, USA and Toya Sada, Japan (TSK gel and TOYOPEARL range) may permit high mobile phase velocities (upto 300 cm/h) in adsorbent beds. Preparative scale adsorbents available from Perseptive Biosystems, USA (POROS range) are rigid, macroporous beads based on hydrophilised polystrene-divinylbenzene and also allow high linear flow rates without getting compressed. A relatively recent development in the field of purification of bio-active products has been use of fluidized bed technique for fast and large-scale purifications. Such methods when used with adsorbent having properties that results in low axial mixing of liquid and adsorbent in the fluidized bed, has been termed as expended bed adsorportion or expended bed chromatography. Expended bed or fluidized bed technique requires adsorbent beads that in addition to being porous and rigid are heavier on account of their larger size and/or higher density. Normal adsorbents like those cited above are unsuitable for expended bed operation due to their low particle size and/or their low density. Composite adsorbents made from agarose and having higher density and large particle size (100-300 um) have been specially developed for expended bed operations. Another approach adopted has been to develop matrices that have an inherent high density; for example, porous glass and solid perfluoro-polymer matrices. However, agarose based adsorbents have the disadvantage of high cost and fragility under rough operating conditions. On the other hand, porous glass is also fragile to be handled safely over long periods, while perflurocarbons have low adsorption capacity and require complicated chemistry of derivatization for converting them into hydrophilic adsorbents suitable for protein binding. Commercial adsorbents suitable for fluidized bed or expanded bed separations are far fewer than those suitable for packed bed adsorbents. STREMLINE™ range of adsorbents introduced by Amersham Pharmacia Biotechnologies, Uppsala, Sweden have been designed and are marked to suit industrial implementation of the expanded bed techniques. STREAMLINE range, one of the four marketed adsorbent products for expended bed protein purifications, is based on composite of cross-linked agarose coated over a quartz core. Another adsorbent Mimo from Upfront Chromatography AS, Denmark, is also based on cross-linked agarose on core glass particles. The major disadvantages of these adsorbents. are their high cost and fragility under rough conditions (like repeated particle-particle abrasion, stirring or pumping) on account of their being wet gel matrices. Other adsorbents that have been recently introduced into the market for expanded bed operations are PROSEP™ from Bioprocessing Ltd., UK (based on porous glass beads) and HyperD from Biosepra (based on a mineral oxide). These adsorbents are sold as pre-designed ion-exchanger, immobilized metal affinity, hydrophobic interaction or other specific affinity supports and thus are not amenable to surface modifications for use as user tailored affinity adsorbents. OBJECTS OF THE INVENTION A primary object of this invention is to propose cross linked cellulose beads that may, for example, be employed as a chromatographic support having desirable properties for expended bed protein adsorption. Another object of this invention is to propose cross linked cellulose beads be used for protein purification for expanded bed protein adsorption employing cellulose as the base material. Yet another object of this invention is to propose cross linked cellulose beads that may be employed as an adsorbent for conventional packed bed as well as expanded/fluidized bed operations. Still another object of this invention is to propose a chromatographic support having desirable properties for expanded bed protein adsorption with protein extracts and also amenable to surface modifications for specific protein adsorption. Yet another object of this invention is to propose a purification of important proteins of plant/animal origin on the developed adsorbent in expended bed operation. DESCRIPTION OF INVENTION According to this invention there is provided a process for the preparation of cross-linked cellulose absorbent beads which comprises and characterised by the step of polymerization of cellulose or its derivative suspended in a solvent and in the presence of a cross linking agent and a catalyst at a temperature of 30-100°C and at atmospheric pressure. Further according to this invention there is provided cross-linked cellulose adsorbent beads having a pore size ranging from 1-3 (nm, a pore volume of about 60% v/v, a particle size ranging from 100-300 um, a bulk density of 1200-1600 kg/m3 in water, and a surface hydroxyl group density in the range 5-100 µmole/ml beads. In accordance with this invention, the beads are prepared by polymerization of cellulose ethers or esters, alone or in combination with each other. Polymerization is carried out in an organic solvent using a cross linker in the presence of catalyst. Typical conditions include : polymerization temperature between 30-100 degree celcius, at atmospheric pressure. Cellulose ethers or esters, out of which alkyl celluloses such as methyl cellulose, ethyl cellulose, hydroxy methyl cellulose, hydroxypropyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose and sodium carboxymethyl cellulose are the preferred polymers. The other polymers which may also be used for the process are polyethylene glycol, and such other polymers generally used, including modified polymethacrylate copolymers (including methyl methacrylate copolymers sold under the trade name "Eudragit"). Various grades of these polymers including mixtures, may also be used in the instant invention to develop beads according to the requirements. The amount of the polymers employed will depend upon the properties desired in the polymer beads obtained. The dispersing solvent being selected on basic of the solubility of the polymer employed. Preferred solvents for use in the present process include chloroform, carbon tetrachloride, benzene, toluene, hexane or any other alkanes. The amount of the dispersing solvent depends upon the viscosity of the reaction mixture required. The cross-linkers include diisocyanates or diepoxides. The crosslinkers used include diisocyanates from 1, 6 - diisocyanatohexane; dicyclohexylmethane 4, 4'- diisocyanate; 1,6- diisocyanoto - 2, 2, 4 (or 2, 4, 4) trimehtylhexane; 5 - isocyanato - 1- isocyanatimethyl) - 1, 3, 3 trimethylcyclohexane; trans -1,4- diisocyanato - cyclohexane; 1,3-diisocyanato methylbenzene; 1, 5 - diisocyanato - naphthalene; 1, 3 -diisocyanatobenzene; 1,4- diisocyanatobenzene; 2, 4 - diisocyanato - 1 - methylbenzene and 1, 3 - diisocyanato -methylbenzene or diepoxides from vinylcyclohexene dioxide, butadiene dioxide and diglycidyl other. The amount of crosslinker employed depends on the temperature of polymerisation, nature of the crosslinker, molecular weight of the polymer desired and the rate of the reaction desired. Thus the amount will vary depending on the product desired. The catalyst include the amines like butyl, pentyl, hexyl, heptyl, triethyl and octyl amine or organometallic compounds like dimethyl tin dichloride, di - n - butyltin sulphide. In a typical process cellulose or one of its derivatives or a mixture of the polymers is completely dried free of moisture by washing with dry acetone by repeated suspension and filtration steps. The cellulose derivative is then suspended in a dry organic solvent. To this mixture, a catalyst is added and the resulting mixture is agitated in a glass reactor for a few minutes. Thereafter a crosslinker is added and the mixture is further rotated gently at a constant temperature of 30 -100 degree celcius. The mixture is then cooled suitably and carefully added to a large dispersion vessel containing distilled water at room temperature. The dispersion vessel can be a stirred vessel provided with a turbine agitator rotating at a constant speed between 50-500 rotation per minute. The suspension of the polymerised slurry is stirred for 30 min to 300 min and the water is drained from the vessel while the viscous mass is retained tin the vessel. Thereafter fresh water is added to the vessel and the suspension stirred further for another 30 to 300 min or till the beads are fairly hardened. The suspension of beads is then carefully filtered. The beads are then recovered and re-suspended in water in a similar manner 4-10 times and are thereafter finally harvested and suspended in acetone or alcohol (ethyl, methyl, or propyl alcohol) for 24 hours at 10 - 35 degree celcius. The beads are then re-suspended in water repeatedly till free from organic solvent. The prepared beads are stored in 0.1% sodium azide in distilled water. The beads thus prepared are found to be hard, rigid beads. Shape and size of beads are found to be a strong function of the parameters that include the polymerization process conditions as well as the suspension and hardening conditions. EXAMPLE 1 A glass reactor equipped with a four blade turbine agitator and containing a cellulose derivative like ethyl methyl cellulose 6.0g dried free of moisture was charged with an organic solvent xylene (300 ml). The resulting reaction mixture was heated to a temperature of about 55 degree Celsius and agitated at about 100 rpm. To this solution a catalyst hexyl amine (75%) is added and the reaction further agitated till a homogenous reaction mixture is obtained. To this mixture a cross-linker like diisocyanate for example dicyclohexylmethane 4, 4' - diisocyanate (100%) is added and the reaction mixture further agitated at 55 degree celcius for 2 hours. The reactant mixture is then cooled and is then dispersed in water and stirred at 1000 rpm for about 30 min. the beads are separated and repeatedly suspended in distilled or deionised water followed by same procedure repeated in acetone, till the beads harden sufficiently. The prepared CELBEADS are collected in a sieve. The CELBEADS thus obtained were characterised. The various characterization tests carried out include mercury porosimetry or SEC analysis, image analysis, porosity measurements, bed expansion studies, density determination, surface hydroxyl content determination etc. the above preparation process gave CELBEADS with the following properties; Porosity of CELBEADS : 55% v/v Sphericity of CELBEADS : 0.9 Particle size of CELBEADS : Maximum : 200µm Minimum : 100µm Density of CELBEADS : 1500 kg/m3 Hydroxyl content of CELBEADS : 10 nmoles/ml EXAMPLE 2 A glass reactor equipped with a four blade turbine agitator and containing a cellulose derivative like ethyl methyl cellulose (100g) dried free of moisture was charged with an organic solvent xylene (300 ml). The resulting reaction mixture was heated to a temperature of about 55 degree celsius and agitated at about 100 rpm. To this solution a catalyst hexyl amine (75%) is added and the reaction further agitated till a homogenous reaction mixture is obtained. To this mixture a cross-linker like diisocyanate for example dicyclohexylmethane 4, 4' - diisocyanate (100%) is added and the reaction mixture further agitated at 55 degree celcius for 2 hours. The reactant mixture is then cooled and is then dispersed in water and stirred at 1000 rpm for about 30 min. the beads are separated and repeatedly suspended in distilled or deionised water followed by same procedure repeated in acetone, till the beads harden sufficiently. The prepared CELBEADS are collected in a sieve. The CELBEADS thus obtained were characterised. The various characterization tests carried out include mercury porosimetry or SEC analysis, image analysis, porosity measurements, bed expansion studies, density determination, surface hydroxyl content determination etc. the bove preparation process gave CELBEADS with the following properties; Porosity of CELBEADS : 43 % v/v Sphericity of CELBEADS: 0.82 Particle size of CELBEADS: Maximum: 200 |am Minimum: 300 µm Density of CELBEADS: 1300 kg/m3 Hydroxyl content of CELBEADS: 10 nmoles/ml Example 3 The procedure employed was similar to Example 1 except that the amount of crosslinker taken was increased to 150%. The rest of the procedure of preparation of beads followed the same procedure as discussed in example 1 and 2. The various characterization testes carried out include mercury porosimetry or SEC analysis, image analysis, porosity measurements, bed expansion studies, density determination, surface hydroxyl content determination etc. The above preparation process gave CELBEADS with the following properties; Porosity of CELBEADS: 50% v/v Sphericity of CELBEADS: 0.8 Particle size of CELBEADS: Maximum: 200 µm Minimum: 150 µm Density of CELBEADS: 1350 kg/m3 Hydroxyl content of CELBEADS: 8 nmoles/ml Example 4 The procedure employed was similar to Example 1 except that a mixture of polymers were employed like ethyl methyl cellulose and sodium salt of carboxymethyl cellulose and the reaction temperature increased from 55 to 75 degree celcius. The rest of the procedure of formation of beads followed the same procedure as discussed above. The various characterization tests carried out include mercury porosimetry or SEC analysis, image analysis, porosity measurements, bed expansion studies, density determination, surface hydroxyl content determination etc. The above preparation process gave CELBEADS with the following properties; Porosity of CELBEADS: 0.50% v/v sphericity of CELBEADS: 0.85 particle size of CELBEADS: Maximum: 150 µm Minimum: 100 µm Density of CELBEADS: 1600 kg/m3 Hydroxyl content of CELBEADS: 11 |amoles/ml WE CLAIM; 1. A process for the preparation of cross-linked cellulose absorbent beads which comprises and characterised by the step of polymerization of cellulose or its derivative suspended in a solvent and in the presence of a cross linking agent and a catalyst at a temperature of 30-100°C and at atmospheric pressure. 2. A process as claimed in claim 1 wherein said cellulose or its derivative is selected from a group consisting of cellulose, methyl cellulose, cellulose triacetate, sodium carboxymethyl cellulose, hydroxyl propyl cellulose, ethyl cellulose, hydroxy ethyl methyl cellulose and ethyl methyl cellulose. 3. A process as claimed in claim 1 wherein cellulose or derivatives are polyethylene glycol, and such other polymers generally used, are modified polymethacrylate copolymers and methyl methacrylate copolymers. 4. A process as claimed in claim 1 wherein said polymer is a mixture of two or more cellulose polymers of different molecular weights. 5. A process as claimed in claim 1 wherein said organic solvent is such as carbon tetrachloride, chloroform, benzene, toluene, hexane, or any other alkane. 6. A process as claimed in claim 1 wherein said catalyst is selected from butyl, pentyl, hexyl, heptyl, octyl, triethyl amines, or diamines like tetramethyl butane or organometallic compounds like dimethyl tin dichloride, di-n- butyl tin sulphide, singularly or in any combination. 7. A process as claimed in claim 1 wherein said crosslinkers used are diisocyanates from 1,6-diisocyanatohexane; dicyclohexylmethane 4,4'-diisocyanate; l,6-diisocyanoto-2, 2,4 (or 2,4,4) trimehtylhexane; 5-isocyanato-1 - isocyanatomethyl_-l,3,3-trimethylcyclohexane; trans-1, 4- diisocyanato-cyclohexane; 1,3-diisocyanato methylbenzene, 1,5-diisocyanato-naphthalene; 1,3-diisocyanatobenzene; 1,4- diisocyanatobenzene; 2-4-diisocyanato-1 -methylbenzene and 1,3-diisocyanato-methylbenzene or diepoxides from vinylcyclohexane dioxide, butadiene dioxide and diglycidyl ether. 8. A process as claimed in claim 1 wherein cross linked cellulose adsorbent beads have a pore size ranging from 1-3 µm, a pore volume of about 60% v/v, a particle size ranging from 100-300 µm, a bulk density of 1200-1600 kg/m3 in water, and a surface hydroxyl group density in the range 5- 100 µmole/ml beads. 9. A process for the preparation of cross linked cellulose adsorbent beads substantially as herein described.

Documents:

222-del-2000-abstract.pdf

222-del-2000-claims.pdf

222-del-2000-correspondence-others.pdf

222-del-2000-correspondence-po.pdf

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

222-del-2000-form-1.pdf

222-del-2000-form-19.pdf

222-del-2000-form-2.pdf

222-del-2000-form-26.pdf

222-del-2000-form-3.pdf

222-del-2000-form-5.pdf

222-del-2000-petition-others.pdf