Title of Invention	A PROCESS FOR THE PREPARATION OF CELLULOSE BASED POROUS MEMBRANE SUITABLE FOR THE SEPARATION OF BIOMOLECULES
Abstract	The present invention relates to a process for the preparation of cellulose based porous membrane suitable for the separation of biomolecules, which comprises casting the membrane from a solution comprising a mixture of cellulose based water insoluble polymer, water soluble polymer, amine, two water miscible solvent and quenching the cast membrane in water and then recovering the product membrane having high flux. This process finds application in bioseparation, bioconversion or related operations of biotech industries.

Title of Invention

A PROCESS FOR THE PREPARATION OF CELLULOSE BASED POROUS MEMBRANE SUITABLE FOR THE SEPARATION OF BIOMOLECULES

Abstract

The present invention relates to a process for the preparation of cellulose based porous membrane suitable for the separation of biomolecules, which comprises casting the membrane from a solution comprising a mixture of cellulose based water insoluble polymer, water soluble polymer, amine, two water miscible solvent and quenching the cast membrane in water and then recovering the product membrane having high flux. This process finds application in bioseparation, bioconversion or related operations of biotech industries.

Full Text	Field of the invention The present invention relates to a process for the preparation of cellulose based porous membrane useful for the separation of biomolecules. Background of the invention Separation of biomolecules using membranes has recently emerged as an alternative to column chromatography. An advantage of using membranes is that high flow rates at low pressure drops can be achieved, which improve the washing, elution, and regeneration processes and decrease the probability of deactivation of the biomolecules by shortening their exposure to an unfavorable medium. In general, two approaches have been employed to prepare membranes suitable for the separation of biomolecules. In the most common method, porous membranes are prepared from polyethylene, polypropylene, nylon, polysulfone, and glass. However, these membranes are usually hydrophobic and relatively inert, and hence require modifications. Some of the membranes may require amplification of the number of active groups. To overcome these drawbacks, a second approach has been employed wherein membranes are prepared that have preincorporated functional groups. However, the problems with this type of membranes include hydrophobicity (poly glycidyl methacrylate-co-ethylene dimethacrylate membrane), brittleness, and solubility in acids (cellulose acetate membrane). Another drawback with both of the above methods is that the pore size of the membrane cannot be easily controlled. Materials commonly used for making porous membranes are polycarbonates, polyamides (nylon 6, nylon 6,6, nylon 610, nylon 13), polysulfones, cellulose derivatives (for example, cellulose, cellulose dictate, cellulose triacetate, cellulose nitrate), polyacrylonitrile and copolymers, polypropylene, polytetrafluoroethylene, alumina, silica, carbon, polyvinylidene fluoride, high and low density polyethylene, polypropylene, polystyrene, polyvinyl chloride, acrylonitrile-butadiene-styrene terpolymers, styrene-acrylonitrile and styrene-butadiene copolymers, polyvinylacetate, polyvinylidene chloride, ethylenevinylacetate copolymers, ethylene-acrylic copolymers, polymethylacrylates, and oxidation polymers such as polyphonylencoxide. However, in comparison to synthetic polymers, use of cellulose appears to be more environments friendly. As with the increased loss of crude oil, renewed interest has been expressed in cellulose globally. Cellulose membranes could be superior to many of the synthetic membranes if they could be produced in a manner, which is economically and environmentally superior to the techniques now available. This would reduce the consumption of non-renewable petrochemical resources. Cellulose for many years has been converted into membranes using either cellulose or its derivatives such as cellulose diacetate, cellulose triacetate, and cellulose nitrate. More recently (2005), U.S. Pat. No. 6,929,884 described a method for manufacture of cellulose-based films which slowly releases solids, it was prepared by dissolving microcrystalline cellulose in a solvent such as an alkali metal salt in a polar solvent such as dimethylacetamide (DMAC), dispersing the solid in the solution and gelling the solution with water to form a film. Another recent patent (2004) filed by Sartorius Ag, Germany (DE 202004008615 U1) described a method for the preparation of cellulose microporous membranes by dispersing cellulose triacetate in methyl formate 3 h at 20°C, keeping 90 min at -40C°, heating up to room temp, adding a dispersion of cellulose diacetate in methyl formate (ratio cellulose diacetate/triacetate 1:1), coating onto glass substrate, drying 90 min under N2 to obtained a membrane. However, the involvement of cooling and heating operations may pose extra energy requirement as well as solvent exposure to the surrounding. Secondly, the chemical composition of the membrane may impose restriction on the development of chemically active membrane. A typical process is described in U.S. Pat. No. 3,412,184 which, involves casting a solution of a cellulose ester as a thin film, evaporating a portion of the casting solvent, leaching with an organic solvent, optionally immersing in a hot water bath to subject the film to a heating step, and then recovering the product membrane. A more advanced, continuous process for the production of cellulose ester membranes is described in U.S. Pat. No 3,792,135. This process involves the following steps, (1) coating a film of a "dope" of a cellulose ester on a web; (2) permitting solvent to evaporate into the atmosphere, to cause incipient formation of a skin or "active" layer; and (3) immersing the film in a hot aqueous bath, to gel the film in the form of an asymmetric membrane. The operation such as immersion of the film in hot water bath and solvent evaporation in to the atmosphere may cause serious concern for environmental point of view as well as overall product cost. In biosepration, the membrane should have the properties like hydrophilicity, excellent film-forming ability, good mechanical properties, and high chemical reactivity. In comparison to other cellulose derivatives, ethyl cellulose appears to be more suitable for this purpose because of its better chemical stability. However, there is very negligible literary evidence where ethyl cellulose based membrane have been used in biosepration. The present invention has given major thrust in the modification of membrane casting solution, which may help in the development of a chemically more reactive novel membrane. Prior art do not show any attempt where a major thrust has been given on the modification of casting solution to obtained more chemically reactive cellulose based porous membrane. U.S. Pat. No. 4,840,819 described a method for preparing composite membrane for enhanced gas separation. Where, the ethyl cellulose solution was prepared by dissolving 1.0% ethyl cellulose in isopropanol. Ravindra, et. al., used ethyl cellulose membrane for the separation of hydrazine where approximately 15 wt % clear solution of ethyl cellulose in toluene was prepared and cast on a clean glass plate as a dense membrane. The cast film was evaporated to dryness in open air at room temperature and the film was vacuum dried for a period of 5 h at ambient temperature to remove any traces of solvent. (J. Polymer Science: Part B: Polymer Physics, 1999,. 37, 1969-1980). The membrane in accordance with the present invention possess the advantages such as higher flux, good hydrophilicity, excellent film-forming ability, good mechanical properties, and high chemical reactivity as casting solution comprises chemically reactive molecules which helps in the enhancement of these properties. The membrane preparation process does not involve heating, cooling and highly hazardous chemicals. Another advantage is of high flux after binding of chemically reactive molecules, which require in bioseparation, this property make it suitable for commercial exploitation. As it is a charged membrane, it can be used without any further amplification of the number of active groups, in various applications such as bioseparation without major reduction in flux. It can also be modified easily to alter the charged either positively or negatively without major reduction in flux. This could be used in an application such as membrane bioreactors, chromatographic separation, affinity chromatography etc., for removing specific biological species from complex mixtures. Objectives of the invention The main objective of the present invention is to provide a process for the preparation of cellulose based porous membrane useful for the separation of biomolecules. Yet another object is to provide a possess for the preparation of cellulose based porous membrane having higher flux, good hydrophilicity, excellent film-forming ability, good mechanical properties, and high chemical reactivity as casting solution comprises chemically reactive molecules which helps in the enhancement of these properties. Still another object is to provide a possess for the preparation of cellulose based porous membrane, which does not involve heating, cooling and highly hazardous chemicals. Summary of the invention Accordingly the present invention provides a process for the preparation of cellulose based porous membrane useful for the separation of biomolecules, which comprises preparing a reaction mixture of cellulose based water insoluble polymer, a water soluble polymer, an amine and two water miscible solvents, spreading the mixture on a substrate to obtain the coated substrate and submerging the above said coated substrate in water, to obtain the membrane. In an embodiment of the present invention, the water insoluble polymer is ethyl cellulose. In another embodiment of the present invention, the water soluble polymer is polyethylene glycol. In still another embodiment of the present invention, the amine is triethylamine. In yet another embodiment of the present invention, one of the water miscible solvent is ethyl alcohol. In yet another embodiment of the present invention, one of the water miscible solvent is 1,4-dioxan. In yet another embodiment of the present invention, the concentration of ethyl cellulose is in the range of 5 to 10% wt/vol. In yet another embodiment of the present invention, the concentration of ethyl cellulose is preferably in the range of 5.5 to 6% wt/vol. In yet another embodiment of the present invention, the concentration of polyethylene glycol is in the range of 1 to 5% wt/vol having average Mn 8,500-11,500. In yet another embodiment of the present invention, the concentration of polyethylene glycol is preferably in the range of 1-2% wt/vol having average Mn 8,500-11,500. In yet another embodiment of the present invention, the concentration of triethylamine is in the range of 0.5 to 5% vol/vol. In yet another embodiment of the present invention, the concentration of triethylamine is preferably in the range of 4 -5% vol/vol. In yet another embodiment of the present invention, the concentration of ethyl alcohol is in the range of 5 to 50% vol/vol. In yet another embodiment of the present invention, the concentration of ethyl alcohol is preferably in the range of 20-25% vol/vol. In yet another embodiment of the present invention, the concentration of 1,4-dioxan is in the range of 45 to 99% vol/vol. In yet another embodiment of the present invention,the concentration of 1,4-dioxan is preferably in the range of 76-77% vol/vol. Brief description of the drawings Figure 1: SEM of the membrane Figure 2: Attenuated total reflectance Fourier Transform Infrared (ATR/FT-IR) spectrometry of the membrane. Figure 3: A representative biomolecule separation pattern obtained on SDS- PAGE Detailed description of the invention Separation of biomolecules using membranes has recently emerged as an alternative to column chromatography. An advantage of using membranes is that high flow rates at low pressure drops can be achieved, which improve the washing, elution, and regeneration processes and decrease the probability of deactivation of the biomolecules by shortening their exposure to an unfavorable medium. The efficient separation of biomolecule is mainly depends on the preparation of membrane. The present invention has given major thrust in the modification of membrane casting solution, which may help in the development of a chemically more reactive novel membrane. Prior art do not show any attempt where a major thrust has been given on the modification of casting solution to obtained more chemically reactive cellulose based porous membrane. The present invention relates to a process for the preparation of cellulose based porous membrane suitable for the separation of biomolecules, which comprises casting the membrane from a solution comprising a mixture of cellulose based water insoluble polymer such as ethyl cellulose, water soluble polymer such as polyethylene glycol, amine such as triethylamine, two water miscible solvent such as ethyl alcohol and 1, 4-dioxan. In the present invention, different water insoluble cellulose based polymer was tested for their film formation properties using different solvent. The effect of various concentration of selected polymer and other additives such as water insoluble polymer, water soluble polymer, amine, water miscible solvent were tested for their film formation, protein adsorption and water permeation properties. The selection of concentration of all the ingredients of the cast solution was decided on the basis of protein adsorption. The characterization of the developed membrane was carried out using different techniques such as bubble point, SEM, infrared spectra etc. In another feature, the suitability of the developed membrane for the separation/immobilization of biomolecules was checked by activation of the membrane using different chemicals commonly used in the binding of ligands suitable for separation/immobilization of biomolecules such as acid, base, bisoxarine, epichlorohydine etc. and their effect on flux and protein adsorption checked. In still another feature, the utilization of the charged available on the developed membrane without any chemical modification for the adsorption and separation of the biomolecules were tested on the basis of adsorption and separation of blood proteins at different pH. 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 To select most suitable water insoluble polymer for the preparation of the membrane different polymers were dissolve and thin smear was prepared on the glass plate (Table 1) and after immersing the plate in water, 1N NaOH and 1N HCI the selection was done on the basis of film quality. Table 1: Effect of different polymer on the film formation (Table Removed) Example 2 To select the most suitable concentration, different concentration of ethyl cellulose was dissolved in 1, 4-dioxan and thin smear was prepared on the glass plate and film quality was checked (Table 2) as described in example 1. Table 2: Effect of ethyl cellulose concentration on film formation (Table Removed) Example 3 To check the effect of water soluble polymer on the flux of the membrane, different concentration of polyethylene glycol (PEG: average Mn 8,500 -11,500) and 9% ethyl cellulose was dissolved in 1,4-dioxan and membrane was prepared on the glass plate by spreading cast solution using glass rod and recovering the membrane by immersing the plate in water. The water flux of the membrane was measured at 0.2 bar by using stirred cell (13.4 sq.cm) after treating the membrane with 0.1N NaOH. Table 3 shows the results. Table 3: Effect of different concentration of PEG on the flux of the membrane (Table Removed) Example 4 To check the effect lower concentration of ethyl cellulose on the flux of the membrane, different lower concentration of ethyl cellulose and 2% polyethylene glycol (PEG) was dissolved in 1,4-dioxan and membrane was prepared on the glass plate by spreading cast solution using glass rod and recovering the membrane by immersing the plate in water. The water flux of the membrane was measured at 0.2 bar by using stirred cell (13.4 sq.cm) after treating the membrane with 0.1N NaOH. Table 4 shows the results. Table 4: Effect of lower concentration of ethyl cellulose on flux (Table Removed) Example 5 To optimize the concentration of ingredients of the membrane cast solution, the membrane cast solution was prepared using different concentration of ethyl alcohol (5 -50%), 1,4-dioxan (45 - 99%), amine such as trietylamine (0.5 - 5%), 5.5% ethyl cellulose, 1% polyethylene glycol (PEG) and membrane was prepared on the glass plate by spreading cast solution using glass rod and recovering the membrane by immersing the plate in water. The water flux of the membrane was measured at 0.2 bar by using stirred cell (13.4 sq. cm). The membrane cast solution prepared using ethyl alcohol (20%), 1,4-dioxan (76%), amine such as trietylamine (4%), ethyl cellulose (5.5%), polyethylene glycol (PEG) (1%) showed good membrane quality, and flux. Example 6 To know the effect of different ingredients of the membrane cast solution on the blood protein adsorption to the membrane, the membrane was cast using solution made by different chemical ingredients (membrane cast solution type I, II, III) on the glass plate by spreading cast solution using glass rod and recovering the membrane by immersing the plate in water. The blood protein adsorption was checked using the following procedure. Adsorption procedure: 1) Membrane was fixed in a stirred cell (13.4 sq.cm) and washed with distilled water followed by equilibration with 10 ml of adsorption buffer (0.1M Phosphate buffer pH 7.4) for 15 mins. 2) Then diluted normal horse serum (NHS) was loaded on the membrane in a stirred cell and stirred for 15 mins. Dilution of the NHS was done using adsorption buffer. 3) Three washes were given for three times sequentially with 5 ml adsorption buffer by stirring 5 mins each. 4) This was followed by elution with 5 ml elution buffer (1M NaCI + 0.1M Phosphate buffer pH 7.4) after stirring for10 mins. 5) Lastly the membrane was washed with 5 ml 0.1 N NaOH after stirring for10 mins. Estimation of the protein concentration from the samples collected at each step was carried out using Lowry method (Lowry, O. H., Rosebrough, N. J., Farr, A. L, Randall, R. J., 1951. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193, 265-275). Table 6 shows the results, the membrane cast solution type III showed higher adsorption of the blood protein and best membrane quality. The adsorption of the protein to the membrane indicates the presence of charge on the membrane, which can be utilized for the separation of biomolecules. Table 6: Effect of different membrane cast solution on membrane quality and protein adsorption (Table Removed) PEG - Polyethylene glycol (average Mn 8,500 - 11,500); TRI - Triethylamine Example 7 To check the chemical and physical structure of the membrane, membrane was cast using membrane cast solution type III (Table 6, Example 6) on the glass plate by spreading cast solution using glass rod and recovering the membrane by immersing the plate in water. Sequential drying of the membrane was carried out using 10% to 50% ethyl alcohol followed by air drying for approximate 40 mins and hot oven drying at 70°c for approximate 3 hr. Scanning electron microscopy (SEM) was employed to investigate the morphology of the membranes. Figure 1 show that most of the pores are distributed uniformly. Figure 2 shows the attenuated total reflectance fourier transform infrared (ATR/FT-IR) spectra. The spectra reveal the chemical structure of the membrane, which is different form the membrane so far developed. It shows several characteristic peaks, the peak at 3475 cm"1 corresponds to the -OH groups of closed ring structure of the polymer unit. The peak at 2869 cm"1 corresponds to C-H bond whereas the peak at 1374 cm"1 corresponds to C-N bond. The sharp peak at 1074 cm"1 corresponds to -C-0 (stretch). This indicates the suitability of the developed membrane for the separation of biomolecules. Example 8 To check the suitability of the membrane for the binding of different affinity ligands, the membrane was treated with NaOH at 60°C in the presence of bisoxirane and epichlorohydrin, which primarily used for the activation of membrane in ligands binding. The membrane was cast using membrane cast solution type III (Table 6, Example 6) on the glass plate by spreading cast solution using glass rod and recovering the membrane by immersing the plate in water. The membrane was treated with different chemical treatment (l&ll, Table 7) and the blood protein adsorption was checked using stirred cell (13.4 sq.cm) as per the procedure given in the Example 6. The flux (LMH) of the membrane was measured using water prior to adsorption of protein (Initial flux) and after the completion of the experiment (End flux). Table 7 shows the results, which indicates the membrane stability at different chemical treatment and the reduction in flux suggest the binding of the proteins. These qualities are very much required for the ligand binding, which commonly used in the biomolecule separation. Table 7: Effect of different chemical treatment on the membrane (Table Removed) LMH - Liters/Square Meter/Hour; Bis- Bisoxirane; BH - Sodium borohydride; EPI - Epichlorohydrin; % Adsorption = 100 - [Protein concentration in wash x 100 / Protein concentration in feed]. Example 9 To check the suitability of the membrane for the separation of biomolecules without any chemical modification/treatment of the membrane, the membrane was cast using membrane cast solution type III (Table 6, Example 6) on the glass plate by spreading cast solution using glass rod and recovering the membrane by immersing the plate in water. The blood protein separation was checked using the following procedure and protein estimation was carried out as per the method given in the Example 6. Procedure: 1) Membrane was fixed in a stirred cell (13.4 sq cm) and washed with distilled water followed by equilibration with 10 ml of adsorption buffer for 15 mins. 2) To this membrane feed containing mixture of blood protein was loaded and stirred for 15 mins in cell. 3) Three washes were given for three times 5 ml 5 mins each. 4) This was followed by elution with 5 ml with different elution buffer of a particular pH with or without salt. 5) Lastly the membrane was regenerated with 0.1N NaOH or 0.1N HCI depending on the adsorption pH. Table I to VII shows the results, which clearly indicate the presence of charge on the membrane without any activation or chemical modification. As the adsorption pH change, the adsorption and elution pattern of the blood proteins changed. The sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the eluted samples indicates that in most of the cases (Table I, II, III, VI & VII), albumin was selectively desorbed (Figure 3). This clearly indicates the suitability of the developed membrane for the separation of the biomolecules. Table I to VII shows the adsorption, elution profile of the blood proteins obtained with the membrane, which shows the selective separation of albumin. (Table Removed) Eluent I- 0.1M sodium acetated buffer pH 4.5; Eluent II - 150mM sodium acetate buffer pH 4.0; Eluent III 1M NaCI in 0.1 M sodium acetated buffer pH 4.5; Eluent IV - 0.1 N NaOH; ND - Not detected (Table Removed) Eluent I- 50mM Tris HCI buffer pH 8.0; Eluent II - 2M NaCI in 50mM Tris HCI buffer pH 8.0; Eluent III -0.1 N NaOH; ND - Not detected Eluent I- 0.1 M sodium acetated buffer pH 4.5; Eluent II - 2M NaCI in 0.1 M sodium acetated buffer pH 4.5; Eluent III - 0.1N NaOH; ND - Not detected IV (Table Removed) Eluent I- 0.2M Glycine NaOH buffer pH 10.0; Eluent II - 2M NaCI in 0.2M Glycine NaOH buffer pH 10.0; Eluent III - 0.1 N HCI; ND - Not detected V (Table Removed) Eluent I- 50 mM phosphate buffer pH 8.0; Eluent II - 2M NaCI in 50 mM Phosphate buffer pH 8.0; Eluent III - 0.1 N NaOH; ND - Not detected VI (Table Removed) Eluent I- 50 mM Phosphate buffer pH 8.0; Eluent II - 2M NaCI in 50 mM Phosphate buffer pH 8.0; Eluent III - 0.1N HCI; ND - Not detected VII (Table Removed) Eluent I- 50mM phosphate buffer pH 6.0; Eluent II - 50mM phosphate buffer pH 7.0; Eluent III - 0.1N HCI; ND - Not detected Advantages of the invention The membranes in accordance with the present invention possess the advantages such as high flux, good hydrophilicity, excellent film-forming ability, good mechanical properties, and high chemical reactivity as casting solution comprises chemically reactive molecules which help in the enhancement of these properties. The membrane preparation process does not involve heating and highly hazardous chemicals. Another advantage is of high flux after binding of chemically reactive molecules, which require in biosepration, this property make it suitable for commercial exploitation. As it is a charged membrane, it can be used without any further amplification of the number of active groups, in various applications such as bioseparation. It can also be modified easily to alter the charged either positively or negatively without major reduction in flux. This could be used in an application such as membrane bioreactors, chromatographic separation, affinity chromatography etc., for removing specific biological species from complex mixtures. This process finds application in bioseparation, bioconversion or related operations of biotech industries. We claim 1. A process for the preparation of cellulose based porous membrane useful for the separation of biomolecules, which comprises preparing a reaction mixture of cellulose based water insoluble polymer, a water soluble polymer, an amine and two water miscible solvents, spreading the mixture on a substrate to obtain the coated substrate and submerging the above said coated substrate in water, to obtain the membrane. 2. A process as claimed in claim 1 wherein the water insoluble polymer is ethyl cellulose. 3. A process as claimed in claim 1 wherein the water soluble polymer is polyethylene glycol. 4. A process as claimed in claim 1 wherein the amine is triethylamine. 5. A process as claimed in claim 1 wherein one of the water miscible solvent is ethyl alcohol. 6. A process as claimed in claim 1 wherein one of the water miscible solvent is 1,4-dioxan. 7. A process as claimed in claim 1 wherein the substrate used is a glass plate. 8. A process as claimed in claim 1 wherein the concentration of ethyl cellulose is in the range of 5 to 10% wt/vol. 9. A process as claimed in claim 1 wherein the concentration of ethyl cellulose is preferably in the range of 5.5 to 6% wt/vol. 10. A process as claimed in claim 1 wherein the concentration of polyethylene glycol is in the range of 1 to 5% wt/vol having average Mn 8,500-11,500. 11. A process as claimed in claim 1 wherein the concentration of polyethylene glycol is preferably in the range of 1-2% wt/vol having average Mn 8,500 - 11,500. 12. A process as claimed in claim 1 wherein the concentration of triethylamine is in the range of 0.5 to 5% vol/vol. 13. A process as claimed in claim 1 wherein the concentration of triethylamine is preferably in the range of 4 -5% vol/vol. 14. A process as claimed in claim 1 wherein the concentration of ethyl alcohol is in the range of 5 to 50% vol/vol. 15. A process as claimed in claim 1 wherein the concentration of ethyl alcohol is preferably in the range of 20-21% vol/vol. 16. A process as claimed in claim 1 wherein concentration of 1,4-dioxan is in the range of 45 to 99% vol/vol. 17. A process as claimed in claim 1 wherein the concentration of 1,4- dioxan is preferably in the range of 76-77% vol/vol. 18. A process for the preparation of cellulose based porous membrane useful for the separation of biomolecules, substantially described herein with reference to the examples.

Full Text

Field of the invention
The present invention relates to a process for the preparation of cellulose based porous membrane useful for the separation of biomolecules.
Background of the invention
Separation of biomolecules using membranes has recently emerged as an alternative to column chromatography. An advantage of using membranes is that high flow rates at low pressure drops can be achieved, which improve the washing, elution, and regeneration processes and decrease the probability of deactivation of the biomolecules by shortening their exposure to an unfavorable medium. In general, two approaches have been employed to prepare membranes suitable for the separation of biomolecules. In the most common method, porous membranes are prepared from polyethylene, polypropylene, nylon, polysulfone, and glass. However, these membranes are usually hydrophobic and relatively inert, and hence require modifications. Some of the membranes may require amplification of the number of active groups. To overcome these drawbacks, a second approach has been employed wherein membranes are prepared that have preincorporated functional groups. However, the problems with this type of membranes include hydrophobicity (poly glycidyl methacrylate-co-ethylene dimethacrylate membrane), brittleness, and solubility in acids (cellulose acetate membrane). Another drawback with both of the above methods is that the pore size of the membrane cannot be easily controlled. Materials commonly used for making porous membranes are polycarbonates, polyamides (nylon 6, nylon 6,6, nylon 610, nylon 13), polysulfones, cellulose derivatives (for example, cellulose, cellulose dictate, cellulose triacetate, cellulose nitrate), polyacrylonitrile and copolymers, polypropylene, polytetrafluoroethylene, alumina, silica, carbon, polyvinylidene fluoride, high and low density polyethylene, polypropylene, polystyrene, polyvinyl chloride, acrylonitrile-butadiene-styrene terpolymers, styrene-acrylonitrile and styrene-butadiene copolymers, polyvinylacetate, polyvinylidene chloride, ethylenevinylacetate copolymers, ethylene-acrylic copolymers, polymethylacrylates, and oxidation polymers such as polyphonylencoxide. However, in comparison to synthetic polymers, use of cellulose appears to be more environments friendly. As with the increased
loss of crude oil, renewed interest has been expressed in cellulose globally. Cellulose membranes could be superior to many of the synthetic membranes if they could be produced in a manner, which is economically and environmentally superior to the techniques now available. This would reduce the consumption of non-renewable petrochemical resources. Cellulose for many years has been converted into membranes using either cellulose or its derivatives such as cellulose diacetate, cellulose triacetate, and cellulose nitrate. More recently (2005), U.S. Pat. No. 6,929,884 described a method for manufacture of cellulose-based films which slowly releases solids, it was prepared by dissolving microcrystalline cellulose in a solvent such as an alkali metal salt in a polar solvent such as dimethylacetamide (DMAC), dispersing the solid in the solution and gelling the solution with water to form a film. Another recent patent (2004) filed by Sartorius Ag, Germany (DE 202004008615 U1) described a method for the preparation of cellulose microporous membranes by dispersing cellulose triacetate in methyl formate 3 h at 20°C, keeping 90 min at -40C°, heating up to room temp, adding a dispersion of cellulose diacetate in methyl formate (ratio cellulose diacetate/triacetate 1:1), coating onto glass substrate, drying 90 min under N2 to obtained a membrane. However, the involvement of cooling and heating operations may pose extra energy requirement as well as solvent exposure to the surrounding. Secondly, the chemical composition of the membrane may impose restriction on the development of chemically active membrane. A typical process is described in U.S. Pat. No. 3,412,184 which, involves casting a solution of a cellulose ester as a thin film, evaporating a portion of the casting solvent, leaching with an organic solvent, optionally immersing in a hot water bath to subject the film to a heating step, and then recovering the product membrane. A more advanced, continuous process for the production of cellulose ester membranes is described in U.S. Pat. No 3,792,135. This process involves the following steps, (1) coating a film of a "dope" of a cellulose ester on a web; (2) permitting solvent to evaporate into the atmosphere, to cause incipient formation of a skin or "active" layer; and (3) immersing the film in a hot aqueous bath, to gel the film in the form of an asymmetric membrane. The operation such as immersion of the film in hot water bath and solvent evaporation in to the atmosphere may cause serious
concern for environmental point of view as well as overall product cost. In biosepration, the membrane should have the properties like hydrophilicity, excellent film-forming ability, good mechanical properties, and high chemical reactivity. In comparison to other cellulose derivatives, ethyl cellulose appears to be more suitable for this purpose because of its better chemical stability. However, there is very negligible literary evidence where ethyl cellulose based membrane have been used in biosepration. The present invention has given major thrust in the modification of membrane casting solution, which may help in the development of a chemically more reactive novel membrane. Prior art do not show any attempt where a major thrust has been given on the modification of casting solution to obtained more chemically reactive cellulose based porous membrane. U.S. Pat. No. 4,840,819 described a method for preparing composite membrane for enhanced gas separation. Where, the ethyl cellulose solution was prepared by dissolving 1.0% ethyl cellulose in isopropanol. Ravindra, et. al., used ethyl cellulose membrane for the separation of hydrazine where approximately 15 wt % clear solution of ethyl cellulose in toluene was prepared and cast on a clean glass plate as a dense membrane. The cast film was evaporated to dryness in open air at room temperature and the film was vacuum dried for a period of 5 h at ambient temperature to remove any traces of solvent. (J. Polymer Science: Part B: Polymer Physics, 1999,. 37, 1969-1980). The membrane in accordance with the present invention possess the advantages such as higher flux, good hydrophilicity, excellent film-forming ability, good mechanical properties, and high chemical reactivity as casting solution comprises chemically reactive molecules which helps in the enhancement of these properties. The membrane preparation process does not involve heating, cooling and highly hazardous chemicals. Another advantage is of high flux after binding of chemically reactive molecules, which require in bioseparation, this property make it suitable for commercial exploitation. As it is a charged membrane, it can be used without any further amplification of the number of active groups, in various applications such as bioseparation without major reduction in flux. It can also be modified easily to alter the charged either positively or negatively without major reduction in flux. This could be used in an application such as
membrane bioreactors, chromatographic separation, affinity chromatography etc., for removing specific biological species from complex mixtures.
Objectives of the invention
The main objective of the present invention is to provide a process for the preparation of cellulose based porous membrane useful for the separation of biomolecules.
Yet another object is to provide a possess for the preparation of cellulose based porous membrane having higher flux, good hydrophilicity, excellent film-forming ability, good mechanical properties, and high chemical reactivity as casting solution comprises chemically reactive molecules which helps in the enhancement of these properties.
Still another object is to provide a possess for the preparation of cellulose based porous membrane, which does not involve heating, cooling and highly hazardous chemicals.
Summary of the invention
Accordingly the present invention provides a process for the preparation of cellulose based porous membrane useful for the separation of biomolecules, which comprises preparing a reaction mixture of cellulose based water insoluble polymer, a water soluble polymer, an amine and two water miscible solvents, spreading the mixture on a substrate to obtain the coated substrate and submerging the above said coated substrate in water, to obtain the membrane.
In an embodiment of the present invention, the water insoluble polymer is ethyl cellulose.
In another embodiment of the present invention, the water soluble polymer is polyethylene glycol.
In still another embodiment of the present invention, the amine is triethylamine.
In yet another embodiment of the present invention, one of the water miscible solvent is ethyl alcohol.
In yet another embodiment of the present invention, one of the water miscible solvent is 1,4-dioxan.
In yet another embodiment of the present invention, the concentration of ethyl cellulose is in the range of 5 to 10% wt/vol.
In yet another embodiment of the present invention, the concentration of ethyl cellulose is preferably in the range of 5.5 to 6% wt/vol.
In yet another embodiment of the present invention, the concentration of polyethylene glycol is in the range of 1 to 5% wt/vol having average Mn 8,500-11,500.
In yet another embodiment of the present invention, the concentration of polyethylene glycol is preferably in the range of 1-2% wt/vol having average Mn 8,500-11,500.
In yet another embodiment of the present invention, the concentration of triethylamine is in the range of 0.5 to 5% vol/vol.
In yet another embodiment of the present invention, the concentration of triethylamine is preferably in the range of 4 -5% vol/vol.
In yet another embodiment of the present invention, the concentration of ethyl alcohol is in the range of 5 to 50% vol/vol.
In yet another embodiment of the present invention, the concentration of ethyl alcohol is preferably in the range of 20-25% vol/vol.
In yet another embodiment of the present invention, the concentration of 1,4-dioxan is in the range of 45 to 99% vol/vol.
In yet another embodiment of the present invention,the concentration of 1,4-dioxan is preferably in the range of 76-77% vol/vol.
Brief description of the drawings
Figure 1: SEM of the membrane
Figure 2: Attenuated total reflectance Fourier Transform Infrared (ATR/FT-IR)
spectrometry of the membrane.
Figure 3: A representative biomolecule separation pattern obtained on SDS-
PAGE
Detailed description of the invention
Separation of biomolecules using membranes has recently emerged as an alternative to column chromatography. An advantage of using membranes
is that high flow rates at low pressure drops can be achieved, which improve the washing, elution, and regeneration processes and decrease the probability of deactivation of the biomolecules by shortening their exposure to an unfavorable medium. The efficient separation of biomolecule is mainly depends on the preparation of membrane. The present invention has given major thrust in the modification of membrane casting solution, which may help in the development of a chemically more reactive novel membrane. Prior art do not show any attempt where a major thrust has been given on the modification of casting solution to obtained more chemically reactive cellulose based porous membrane. The present invention relates to a process for the preparation of cellulose based porous membrane suitable for the separation of biomolecules, which comprises casting the membrane from a solution comprising a mixture of cellulose based water insoluble polymer such as ethyl cellulose, water soluble polymer such as polyethylene glycol, amine such as triethylamine, two water miscible solvent such as ethyl alcohol and 1, 4-dioxan.
In the present invention, different water insoluble cellulose based polymer was tested for their film formation properties using different solvent. The effect of various concentration of selected polymer and other additives such as water insoluble polymer, water soluble polymer, amine, water miscible solvent were tested for their film formation, protein adsorption and water permeation properties. The selection of concentration of all the ingredients of the cast solution was decided on the basis of protein adsorption. The characterization of the developed membrane was carried out using different techniques such as bubble point, SEM, infrared spectra etc.
In another feature, the suitability of the developed membrane for the separation/immobilization of biomolecules was checked by activation of the membrane using different chemicals commonly used in the binding of ligands suitable for separation/immobilization of biomolecules such as acid, base, bisoxarine, epichlorohydine etc. and their effect on flux and protein adsorption checked.
In still another feature, the utilization of the charged available on the developed membrane without any chemical modification for the adsorption
and separation of the biomolecules were tested on the basis of adsorption and separation of blood proteins at different pH.
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
To select most suitable water insoluble polymer for the preparation of the membrane different polymers were dissolve and thin smear was prepared on the glass plate (Table 1) and after immersing the plate in water, 1N NaOH and 1N HCI the selection was done on the basis of film quality. Table 1: Effect of different polymer on the film formation
(Table Removed)
Example 2
To select the most suitable concentration, different concentration of ethyl cellulose was dissolved in 1, 4-dioxan and thin smear was prepared on the glass plate and film quality was checked (Table 2) as described in example 1. Table 2: Effect of ethyl cellulose concentration on film formation

(Table Removed)
Example 3
To check the effect of water soluble polymer on the flux of the membrane, different concentration of polyethylene glycol (PEG: average Mn 8,500 -11,500) and 9% ethyl cellulose was dissolved in 1,4-dioxan and membrane was prepared on the glass plate by spreading cast solution using glass rod and recovering the membrane by immersing the plate in water. The water flux of the membrane was measured at 0.2 bar by using stirred cell (13.4 sq.cm) after treating the membrane with 0.1N NaOH. Table 3 shows the results. Table 3: Effect of different concentration of PEG on the flux of the membrane
(Table Removed)
Example 4
To check the effect lower concentration of ethyl cellulose on the flux of the membrane, different lower concentration of ethyl cellulose and 2% polyethylene glycol (PEG) was dissolved in 1,4-dioxan and membrane was prepared on the glass plate by spreading cast solution using glass rod and recovering the membrane by immersing the plate in water. The water flux of the membrane was measured at 0.2 bar by using stirred cell (13.4 sq.cm) after treating the membrane with 0.1N NaOH. Table 4 shows the results. Table 4: Effect of lower concentration of ethyl cellulose on flux
(Table Removed)
Example 5
To optimize the concentration of ingredients of the membrane cast solution, the membrane cast solution was prepared using different concentration of ethyl alcohol (5 -50%), 1,4-dioxan (45 - 99%), amine such as trietylamine (0.5 - 5%), 5.5% ethyl cellulose, 1% polyethylene glycol (PEG) and membrane was prepared on the glass plate by spreading cast solution using glass rod and recovering the membrane by immersing the plate in water. The water flux of the membrane was measured at 0.2 bar by using stirred cell (13.4 sq. cm). The membrane cast solution prepared using ethyl alcohol (20%), 1,4-dioxan (76%), amine such as trietylamine (4%), ethyl cellulose (5.5%), polyethylene glycol (PEG) (1%) showed good membrane quality, and flux.
Example 6
To know the effect of different ingredients of the membrane cast solution on the blood protein adsorption to the membrane, the membrane was cast using solution made by different chemical ingredients (membrane cast solution type I, II, III) on the glass plate by spreading cast solution using glass rod and recovering the membrane by immersing the plate in water. The blood protein adsorption was checked using the following procedure. Adsorption procedure:
1) Membrane was fixed in a stirred cell (13.4 sq.cm) and washed with distilled water followed by equilibration with 10 ml of adsorption buffer (0.1M Phosphate buffer pH 7.4) for 15 mins.
2) Then diluted normal horse serum (NHS) was loaded on the membrane in a stirred cell and stirred for 15 mins. Dilution of the NHS was done using adsorption buffer.
3) Three washes were given for three times sequentially with 5 ml adsorption buffer by stirring 5 mins each.
4) This was followed by elution with 5 ml elution buffer (1M NaCI + 0.1M Phosphate buffer pH 7.4) after stirring for10 mins.
5) Lastly the membrane was washed with 5 ml 0.1 N NaOH after stirring for10 mins. Estimation of the protein concentration from the samples collected at
each step was carried out using Lowry method (Lowry, O. H., Rosebrough, N. J., Farr, A. L, Randall, R. J., 1951. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193, 265-275). Table 6 shows the results, the membrane cast solution type III showed higher adsorption of the blood protein and best membrane quality. The adsorption of the protein to the membrane indicates the presence of charge on the membrane, which can be utilized for the separation of biomolecules.
Table 6: Effect of different membrane cast solution on membrane quality and protein adsorption
(Table Removed)
PEG - Polyethylene glycol (average Mn 8,500 - 11,500); TRI - Triethylamine
Example 7
To check the chemical and physical structure of the membrane, membrane was cast using membrane cast solution type III (Table 6, Example 6) on the glass plate by spreading cast solution using glass rod and recovering the membrane by immersing the plate in water. Sequential drying of the membrane was carried out using 10% to 50% ethyl alcohol followed by air drying for approximate 40 mins and hot oven drying at 70°c for approximate 3
hr. Scanning electron microscopy (SEM) was employed to investigate the morphology of the membranes. Figure 1 show that most of the pores are distributed uniformly. Figure 2 shows the attenuated total reflectance fourier transform infrared (ATR/FT-IR) spectra. The spectra reveal the chemical structure of the membrane, which is different form the membrane so far developed. It shows several characteristic peaks, the peak at 3475 cm"1 corresponds to the -OH groups of closed ring structure of the polymer unit. The peak at 2869 cm"1 corresponds to C-H bond whereas the peak at 1374 cm"1 corresponds to C-N bond. The sharp peak at 1074 cm"1 corresponds to -C-0 (stretch). This indicates the suitability of the developed membrane for the separation of biomolecules.
Example 8
To check the suitability of the membrane for the binding of different affinity ligands, the membrane was treated with NaOH at 60°C in the presence of bisoxirane and epichlorohydrin, which primarily used for the activation of membrane in ligands binding. The membrane was cast using membrane cast solution type III (Table 6, Example 6) on the glass plate by spreading cast solution using glass rod and recovering the membrane by immersing the plate in water. The membrane was treated with different chemical treatment (l&ll, Table 7) and the blood protein adsorption was checked using stirred cell (13.4 sq.cm) as per the procedure given in the Example 6. The flux (LMH) of the membrane was measured using water prior to adsorption of protein (Initial flux) and after the completion of the experiment (End flux). Table 7 shows the results, which indicates the membrane stability at different chemical treatment and the reduction in flux suggest the binding of the proteins. These qualities are very much required for the ligand binding, which commonly used in the biomolecule separation.
Table 7: Effect of different chemical treatment on the membrane
(Table Removed)
LMH - Liters/Square Meter/Hour; Bis- Bisoxirane; BH - Sodium borohydride; EPI - Epichlorohydrin; % Adsorption = 100 - [Protein concentration in wash x 100 / Protein concentration in feed].
Example 9
To check the suitability of the membrane for the separation of biomolecules without any chemical modification/treatment of the membrane, the membrane was cast using membrane cast solution type III (Table 6, Example 6) on the glass plate by spreading cast solution using glass rod and recovering the membrane by immersing the plate in water. The blood protein separation was checked using the following procedure and protein estimation was carried out as per the method given in the Example 6.
Procedure:
1) Membrane was fixed in a stirred cell (13.4 sq cm) and washed with distilled
water followed by equilibration with 10 ml of adsorption buffer for 15 mins.
2) To this membrane feed containing mixture of blood protein was loaded and stirred for 15 mins in cell.
3) Three washes were given for three times 5 ml 5 mins each.
4) This was followed by elution with 5 ml with different elution buffer of a
particular pH with or without salt.
5) Lastly the membrane was regenerated with 0.1N NaOH or 0.1N HCI depending on the adsorption pH.
Table I to VII shows the results, which clearly indicate the presence of charge on the membrane without any activation or chemical modification. As the adsorption pH change, the adsorption and elution pattern of the blood proteins changed. The sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the eluted samples indicates that in most of the cases (Table I, II, III, VI & VII), albumin was selectively desorbed (Figure 3). This clearly indicates the suitability of the developed membrane for the separation of the biomolecules.
Table I to VII shows the adsorption, elution profile of the blood proteins obtained with the membrane, which shows the selective separation of albumin.

(Table Removed)
Eluent I- 0.1M sodium acetated buffer pH 4.5; Eluent II - 150mM sodium acetate buffer pH 4.0; Eluent III 1M NaCI in 0.1 M sodium acetated buffer pH 4.5; Eluent IV - 0.1 N NaOH; ND - Not detected
(Table Removed)
Eluent I- 50mM Tris HCI buffer pH 8.0; Eluent II - 2M NaCI in 50mM Tris HCI buffer pH 8.0; Eluent III -0.1 N NaOH; ND - Not detected
Eluent I- 0.1 M sodium acetated buffer pH 4.5; Eluent II - 2M NaCI in 0.1 M sodium acetated buffer pH 4.5; Eluent III - 0.1N NaOH; ND - Not detected
IV
(Table Removed)
Eluent I- 0.2M Glycine NaOH buffer pH 10.0; Eluent II - 2M NaCI in 0.2M Glycine NaOH buffer pH 10.0; Eluent III - 0.1 N HCI; ND - Not detected
V
(Table Removed)

Eluent I- 50 mM phosphate buffer pH 8.0; Eluent II - 2M NaCI in 50 mM Phosphate buffer pH 8.0; Eluent III - 0.1 N NaOH; ND - Not detected
VI
(Table Removed)

Eluent I- 50 mM Phosphate buffer pH 8.0; Eluent II - 2M NaCI in 50 mM Phosphate buffer pH 8.0; Eluent III - 0.1N HCI; ND - Not detected
VII
(Table Removed)
Eluent I- 50mM phosphate buffer pH 6.0; Eluent II - 50mM phosphate buffer pH 7.0; Eluent III - 0.1N HCI; ND - Not detected
Advantages of the invention
The membranes in accordance with the present invention possess the advantages such as high flux, good hydrophilicity, excellent film-forming ability, good mechanical properties, and high chemical reactivity as casting solution comprises chemically reactive molecules which help in the enhancement of these properties. The membrane preparation process does not involve heating and highly hazardous chemicals.
Another advantage is of high flux after binding of chemically reactive molecules, which require in biosepration, this property make it suitable for commercial exploitation. As it is a charged membrane, it can be used without any further amplification of the number of active groups, in various applications such as bioseparation. It can also be modified easily to alter the charged either positively or negatively without major reduction in flux. This could be used in an application such as membrane bioreactors, chromatographic separation, affinity chromatography etc., for removing specific biological species from complex mixtures. This process finds application in bioseparation, bioconversion or related operations of biotech industries.

We claim
1. A process for the preparation of cellulose based porous membrane useful for the separation of biomolecules, which comprises preparing a reaction mixture of cellulose based water insoluble polymer, a water soluble polymer, an amine and two water miscible solvents, spreading the mixture on a substrate to obtain the coated substrate and submerging the above said coated substrate in water, to obtain the membrane.
2. A process as claimed in claim 1 wherein the water insoluble polymer is ethyl cellulose.
3. A process as claimed in claim 1 wherein the water soluble polymer is polyethylene glycol.
4. A process as claimed in claim 1 wherein the amine is triethylamine.
5. A process as claimed in claim 1 wherein one of the water miscible solvent is ethyl alcohol.
6. A process as claimed in claim 1 wherein one of the water miscible solvent is 1,4-dioxan.
7. A process as claimed in claim 1 wherein the substrate used is a glass plate.
8. A process as claimed in claim 1 wherein the concentration of ethyl cellulose is in the range of 5 to 10% wt/vol.
9. A process as claimed in claim 1 wherein the concentration of ethyl cellulose is preferably in the range of 5.5 to 6% wt/vol.
10. A process as claimed in claim 1 wherein the concentration of polyethylene glycol is in the range of 1 to 5% wt/vol having average Mn 8,500-11,500.
11. A process as claimed in claim 1 wherein the concentration of polyethylene glycol is preferably in the range of 1-2% wt/vol having average Mn 8,500 - 11,500.
12. A process as claimed in claim 1 wherein the concentration of triethylamine is in the range of 0.5 to 5% vol/vol.
13. A process as claimed in claim 1 wherein the concentration of triethylamine is preferably in the range of 4 -5% vol/vol.
14. A process as claimed in claim 1 wherein the concentration of ethyl
alcohol is in the range of 5 to 50% vol/vol.
15. A process as claimed in claim 1 wherein the concentration of ethyl
alcohol is preferably in the range of 20-21% vol/vol.
16. A process as claimed in claim 1 wherein concentration of 1,4-dioxan is
in the range of 45 to 99% vol/vol.
17. A process as claimed in claim 1 wherein the concentration of 1,4-
dioxan is preferably in the range of 76-77% vol/vol.
18. A process for the preparation of cellulose based porous membrane
useful for the separation of biomolecules, substantially described
herein with reference to the examples.

Documents:

849-DEL-2006-849-DEL-2006-849-DEL-2006-Claims-(03-06-2013).pdf

849-DEL-2006-849-DEL-2006-849-DEL-2006-Correspondence-Others-(03-06-2013).pdf

849-del-2006-abstract.pdf

849-del-2006-claims.pdf

849-del-2006-correspondence-others.pdf

849-del-2006-description (complete).pdf

849-del-2006-drawings.pdf

849-del-2006-form-1.pdf

849-del-2006-form-18.pdf

849-del-2006-form-2.pdf

849-del-2006-form-3.pdf

849-del-2006-form-5.pdf

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Patent Number

258360

Indian Patent Application Number

849/DEL/2006

PG Journal Number

01/2014

Publication Date

03-Jan-2014

Grant Date

02-Jan-2014

Date of Filing

28-Mar-2006

Name of Patentee

COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH

Applicant Address

ANUSANDHAN BHAWAN, RAFI MARG, NEW DELHI-110001, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	ADIKANE. HARSHAVARDHAN VISHAVANATH	NATIONAL CHEMICAL LABORATORY DR. HOMI BHABHA ROAD, PUNE-411008(MAHARASHTRA)INDIA.
2	THAKAR DNYANESHWAR MARUTI	NATIONAL CHEMICAL LABORATORY DR. HOMI BHABHA ROAD, PUNE-411008 (MAHARASHTRA)INDIA.
3	KHARUL ULHAS KANHAIYALAL	NATIONAL CHEMICAL LABORATORY DR. HOMI BHABHA ROAD, PUNE-411008 (MAHARASHTRA)INDIA.

PCT International Classification Number

B01D 67/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA