Title of Invention	"A PROCESS FOR THE PREPARATION OF NOVEL DERIVATISED MACROPOROUS BEADED CROSSLINKED POLYMER USEFUL AS AFFINITY CHROMATOGRAPHY SUPPORTS."
Abstract	A process for the preparation of novel derivatised macroporous beaded copolymer useful as affinity chromatography supports by suspending macroporous beaded copolymers in a solution containing a ligand selected from amine in an organic solvent, shaking the suspension at an rpm in the range of 75 to 200, at a temperature in the range of 20 to 30°C for a period ranging upto 48 hours, isolating the beads from the reaction mixture by conventional methods, washing with water and drying the beads under vacuum.

Title of Invention

"A PROCESS FOR THE PREPARATION OF NOVEL DERIVATISED MACROPOROUS BEADED CROSSLINKED POLYMER USEFUL AS AFFINITY CHROMATOGRAPHY SUPPORTS."

Abstract

A process for the preparation of novel derivatised macroporous beaded copolymer useful as affinity chromatography supports by suspending macroporous beaded copolymers in a solution containing a ligand selected from amine in an organic solvent, shaking the suspension at an rpm in the range of 75 to 200, at a temperature in the range of 20 to 30°C for a period ranging upto 48 hours, isolating the beads from the reaction mixture by conventional methods, washing with water and drying the beads under vacuum.

Full Text	This invention relates to a process for the preparation novejjmacroporous beaded crosslinked polymers useful as affinity chromatography supports. The invention more particularly relates to a process for the derivatisation of macroporous beaded crosslinked copolymers useful as affinity chromatography supports for purification of penicillin G acylase. Penicillin G acylase hydrolyses the linear amide bond present in the penicillins to form beta-lactam nucleus 6-amino penicillanic acid (6-APA) and corresponding side chain acid. The enzyme does not hydrolyse the cyclic amide bond, which forms the beta-lactam moiety. This property enables the use of penicillin G acylase in the production of 6-APA, which is an indespensible raw material for the preparation of semisynthetic penicillins such as ampicillin, amoxycillin, cloxacillin etc. Penicillin G acylase, therefore, is a biocatalyst of commercial importance. The enzyme is produced by fermentation and purified prior to immobilization. The substrates, side chain acid amides or their analogues are used as ligands for purification of penicillin G acylase. Either affinity or hydrophobic supports prepared by derivatisation of gels particularly agarose gels are used in purification of penicillin G acylase. Applied Biochemistry and Biotechnology vol. 9, p. 421, 1984 describes a two step protocol involving hydrophobic chromatography on phenyl-Sepharose and ion exchange chromatography on DEAE-cellulose. Purification of penicillion G acylase on Sepharose(agarose) activated with cyanogen bromide and derivatised with ampicillin, amoxycillin, phenylglycine, norleucine, octylamine, aniline, benzyl amine, phenylethyl amine, leucine, m-hydroxy aniline, p-hydroxy aniline, 2,4-dinitro aniline, tyrosine and tryptophan is dercribed in Hindustan Antibiotics Bulletin, vol. 31, p. 25, 1989; Biotechnology Letters, vol. 9, p. 539, 1987 and Applied Biochemistry and Biotechnology vol. 9, p. 421, 1984. The use of aforesaid supports for purification of penicillin G acylase suffers from one or more of the following disadvantages: activation by using toxic chemicals like cyanogen bromide, bed compression, lower flow rates and attrition if used in batch mode. In our copending application No. 307/00 we have described and claimed a process for the preparation of macroporous beaded crosslinked copolymers. The copolymers have the following properties that are desired for a support for use in affinity chromatography such as: Presence of built-in glycidyl functional groups for binding of ligand molecules; Stable bed volume at varying conditions of pH and ionic strength; Uniform bead size and Resistance to attrition. Thus, the macroporous beaded crosslinked copolymers described and claimed in our copending application No. 307/00 can be derivatised and used as a support in affinity chromatography. Butylamine, hexylamine, octylamine, dodecylamine, phenylethylamine and benzylamine are either analogues of side chain of penicillin or are structures that possess degree of hydrophobicity in the range equivalent to the hydrophobicity of side chain moiety of penicillins. Primary amino group present in these molecules can react with the glycidyl functional group on the macroporous beaded crosslinked copolymers to form affinity supports. The main object of the present invention is to provide a process for the preparation of novel macroporous beaded copolymers. Another object is to provide a process for the preparation of affinity chromatography supports useful for the purification of penicillin G acylase wherein toxic chemicals are not used. Yet another object is to provide a process for the preparation of affinity chromatography supports useful for the purification of penicillin G acylase. Accordingly, the present invention provides a process for the preparation of novel derivatised macroporous beaded copolymer useful as affinity chromatography supports which comprises suspending macroporous beaded copolymers in a solution containing a ligand selected from amine in an organic solvent, shaking the suspension at an rpm in the range of 75 to 200, at a temperature in the range of 20 to 30°C for a period ranging upto 48 hours, isolating the beads from the reaction mixture by conventional methods, washing with water and drying the beads under vacuum. In an embodiment of the invention, the macroporous beaded crosslinked allyl glycidyl ether copolymers were prepared as described and claimed in our copending application No.NF-307/00. In another embodiment of the invention, the ligand may be selected from butylamine, hexylamine, octylamine, dodecylamine, phenylethylamine, benzylamine and the ratio of the copolymer and the ligand may be in the range of 1:1 to 1:20. In yet another embodiment of the invention, the solvent for dissolution of the ligand may be selected from methanol, ethanol, isopropanol. The process of the present invention is described with reference to the following Examples, which are by way of illustration only and should not be construed to limit the scope of the invention, in any manner. Examples for the preparation of macroporous beaded crosslinked copolymers Example 1 In an inert atmosphere of nitrogen, 1.5 grams of poly vinyl pyrrolidone was dissolved in 150 ml of distilled water in a 250 ml capacity glass reactor. 9.72 grams of allyl glycidyl ether, 8.38 grams of pentaerythritol triacrylate and 22.5 ml of cyclohexanol were added to the aqueous solution of poly vinyl pyrrolidone. The suspension was stirred at 300 rpm. 0.6 gram of azo bis isobutyronitrile was added to this suspension and the resultant polymerization reaction mixture was heated with agitation at a temperature of 70°C for 3 hours. The copolymer separated out as beads during the polymerization. The copolymer beads were isolated by filtration, washed with distilled water, followed by washing by methanol and dried in a vacuum oven at 40°C. The yield of copolymer obtained was 17.10 grams. Example 2 In an inert atmosphere of nitrogen, 1.5 grams of poly vinyl pyrrolidone was dissolved in 150 ml of distilled water in a 250 ml capacity glass reactor. 4.30 grams of allyl glycidyl ether. 14.50 grams of pentaerythritol trimethacrylate and 22.0 ml of cyclohexanol were added to the aqueous solution of poly vinyl pyrrolidone. The suspension was stirred at 300 rpm. 0.6 gram of azo bis isobutyronitrile was added to this suspension and the resultant polymerization reaction mixture was heated with agitation at a temperature of 70°C for 3 hours. The copolymer separated out as beads during the polymerization. The copolymer beads were isolated by filtration, washed with distilled water, followed by washing by methanol and dried in a vacuum oven at 40°C. The yield of copolymer obtained was 14.5 grams. Example 3 In an inert atmosphere of nitrogen, 2.0 grams of poly vinyl acetate was dissolved in 150 ml of distilled water in a 250 ml capacity glass reactor. 8.6 grams of allyl glycidyl ether, 7.8 grams of divinyl benzene and 23 ml of cyclohexanol were added to the aqueous solution of poly vinyl pyrrolidone. The suspension was stirred at 300 rpm. 0.8 gram of azo bis isobutyronitrile was added to this suspension and the resultant polymerization reaction mixture was heated with agitation at a temperature of 70°C for 3 hours. The copolymer separated out as beads during the polymerization. The copolymer beads were isolated by filtration, washed with distilled water, followed by washing by ethanol and dried in a vacuum oven at 50°C. The yield of copolymer obtained was 14.25 grams. Example 4 In an inert atmosphere of nitrogen, 1.8 grams of poly vinyl alcohol was dissolved in 150 ml of distilled water in a 250 ml capacity glass reactor. 9.0 grams of allyl glycidyl ether, 8 grams of ethylene glycol dimethacrylate and 43.5 ml of cyclohexanol were added to the aqueous solution of poly vinyl pyrrolidone. The suspension was stirred at 300 rpm. 0.6 gram of methyl ethyl ketone peroxide was added to this suspension and the resultant polymerization reaction mixture was heated with agitation at a temperature of 60°C for 4 hours. The copolymer separated out as beads during the polymerization. The copolymer beads were isolated by filtration, washed with distilled water, followed by washing by ethanol and dried in a vacuum oven at 40°C. The yield of copolymer obtained was 15.25 grams. Example 5 In an inert atmosphere of nitrogen, 1.5 grams of poly vinyl pyrrolidone was dissolved in 150 ml of distilled water in a 250 ml capacity glass reactor. 8.5 grams of allyl glycidyl ether, 8.25 grams of trimethylol propane triacrylate and 43.5 ml of cyclohexanol were added to the aqueous solution of poly vinyl pyrrolidone. The suspension was stirred at 300 rpm. 0.5 gram of benzoyl peroxide was added to this suspension and the resultant polymerization reaction mixture was heated with agitation at a temperature of 70°C for 3 hours. The copolymer separated out as beads during the polymerization. The copolymer beads were isolated by filtration, washed with distilled water, followed by washing by methanol and dried in a vacuum oven at 40°C. The yield of copolymer obtained was 15.35 grams. Example 6 In an inert atmosphere of nitrogen, 2.25 grams of sodium salt of poly acrylic acid was dissolved in 150 ml of distilled water in a 250 ml capacity glass reactor. 9.0 grams of allyl glycidyl ether, 8.75 grams of trimethylol propane trimethacrylate and 43.5 ml of cyclohexanol were added to the aqueous solution of poly vinyl pyrrolidone. The suspension was stirred at 300 rpm. 0.6 gram of benzoyl peroxide was added to this suspension and the resultant polymerization reaction mixture was heated with agitation at a temperature of 80°C for 2 hours. The copolymer separated out as beads during the polymerization. The copolymer beads were isolated by filtration, washed with distilled water, followed by washing by ethanol and dried in a vacuum oven at 50°C. The yield of copolymer obtained was 15.75 grams. Examples for the derivatisation of macroporous beaded crosslinked copolymers Example 7 10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 4 was suspended in 60 ml of ligand solution containing 34.4 g of butylamine dissolved in ethanol. The suspension was incubated in a stoppered 250-ml capacity conical flask on a rotary shaker at 100 rpm, 25°C for 48 hours. The beads were collected by filtration on a Buchner funnel, washed with 200 ml of distilled water and dried at 40°C under vacuum. The derivatised macroporous beaded crosslinked copolymer beads were stored at 4°C. Example 8 10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 4 was suspended in 80 ml of ligand solution containing 23.8 g of hexylamine dissolved in methanol. The suspension was incubated in a stoppered 250 ml capacity conical flask on a rotary shaker at 100 rpm, 25°C for 48 hours. The beads were collected by filtration on a Buchner funnel, washed with 200 ml of distilled water and dried at 40°C under vacuum. The derivatised macroporous beaded crosslinked copolymer beads were stored at 4°C. Example 9 10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 4 was suspended in 60 ml of ligand solution containing 30.4 g of octylamine dissolved in ethanol. The suspension was incubated in a stoppered 250 ml capacity conical flask on a rotary shaker at 150 rpm, 20°C for 48 hours. The beads were collected by filtration on a Buchner funnel, washed with 200 ml of distilled water and dried at 40°C under vacuum. The derivatised macroporous beaded crosslinked copolymer beads were stored at 4°C. Example 10 10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 4 was suspended in 100 ml of ligand solution containing 43.6 g of dodecylamine dissolved in methanol. The suspension was incubated in a stoppered 250 ml capacity conical flask on a rotary shaker at 200 rpm, 25°C for 48 hours. The beads were collected by filtration on a Buchner funnel, washed with 200 ml of distilled water and dried at 40°C under vacuum. The derivatised macroporous beaded crosslinked copolymer beads were stored at 4°C. Example 11 10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 4 was suspended in 60 ml of ligand solution containing 28.5 g of phenylethylamine dissolved in isopropanol. The suspension was incubated in a stoppered 250 ml capacity conical flask on a rotary shaker at 75 rpm, 30°C for 48 hours. The beads were collected by filtration on a Buchner funnel, washed with 200 ml of distilled water and dried at 40°C under vacuum. The derivatised macroporous beaded crosslinked copolymer beads were stored at 4°C. Example 12 10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 4 was suspended in 60 ml of ligand solution containing 25.2 g of benzylamine dissolved in ethanol. The suspension was incubated in a stoppered 250 ml capacity conical flask on a rotary shaker at 75 rpm, 30°C for 36 hours. The beads were collected by filtration on a Buchner funnel, washed with 200 ml of distilled water and dried at 40°C under vacuum. The derivatised macroporous beaded crosslinked copolymer beads were stored at4°C. Example 13 10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 1 was suspended in 60 ml of ligand solution containing 25.2 g of benzylamine dissolved in ethanol. The suspension was incubated in a stoppered 250 ml capacity conical flask on a rotary shaker at 100 rpm, 25°C for 48 hours. The beads were collected by filtration on a Buchner funnel, washed with 200 ml of distilled water and dried at 40°C under vacuum. The derivatised macroporous beaded crosslinked copolymer beads were stored at 4°C. Example 14 10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 3 was suspended in 80 ml of ligand solution containing 65.2 g of benzylamine dissolved in ethanol. The suspension was incubated in a stoppered 250 ml capacity conical flask on a rotary shaker at 100 rpm, 25°C for 48 hours. The beads were collected by filtration on a Buchner funnel, washed with 200 ml of distilled water and dried at 40°C under vacuum. The derivatised macroporous beaded crosslinked copolymer beads were stored at4°C. Example 15 10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 6 was suspended in 60 ml of ligand solution containing 25.2 g of benzylamine dissolved in ethanol. The suspension was incubated in a stoppered 250 ml capacity conical flask on a rotary shaker at 100 rpm, 25°C for 48 hours. The beads were collected by filtration on a Buchner runnel, washed with 200 ml of distilled water and dried at 40°C under vacuum. The derivatised macroporous beaded crosslinked copolymer beads were stored at 4°C. Advantages of the invention The invention provides a process for derivatisation of macroporous beaded crosslinked copolymers useful for affinity chromatography of penicillin G acylase. The preparation of the affinity chromatography supports offers the following advantages: i) The process does not involve the use of toxic chemicals like cyanogen bromide. ii) The macroporous beaded crosslinked ether copolymers are rigid crosslinked copolymers and hence bed compression at varying pH and ionic strength does not occur. iii) The beads are resistant for attrition thereby enables its use in a batch mode on large scale. iv) The operational procedure is simple and easy to scale-up. We claim : 1. A process for the preparation of novel derivatised macroporous beaded copolymer useful as affinity chromatography supports which comprises suspending macroporous beaded copolymers in a solution containing a ligand selected from amine in an organic solvent, shaking the suspension at an rpm in the range of 75 to 200, at a temperature in the range of 20 to 30°C for a period ranging upto 48 hours, isolating the beads from the reaction mixture by conventional methods, washing with water and drying the beads under vacuum. 2. A process as claimed in claim 1 wherein the ligand is selected from butylamine, hexylamine, octylamine, dodecylamine, phenylethylamine, benzylamine and the ratio of the copolymer and the ligand used is in the range of 1:1 to 1:20. 3. A process as claimed in claim 1-2 wherein the organic solvent used for dissolution of ligand is selected from methanol, ethanol, isopropanol. 4. A process for the preparation of novel derivatised macroporous beaded copolymer useful as affinity chromatography supports substantially as herein described with reference to the examples.

Full Text

This invention relates to a process for the preparation novejjmacroporous beaded crosslinked polymers useful as affinity chromatography supports. The invention more particularly relates to a process for the derivatisation of macroporous beaded crosslinked copolymers useful as affinity chromatography supports for purification of penicillin G acylase.
Penicillin G acylase hydrolyses the linear amide bond present in the penicillins to form beta-lactam nucleus 6-amino penicillanic acid (6-APA) and corresponding side chain acid. The enzyme does not hydrolyse the cyclic amide bond, which forms the beta-lactam moiety. This property enables the use of penicillin G acylase in the production of 6-APA, which is an indespensible raw material for the preparation of semisynthetic penicillins such as ampicillin, amoxycillin, cloxacillin etc. Penicillin G acylase, therefore, is a biocatalyst of commercial importance.
The enzyme is produced by fermentation and purified prior to immobilization. The substrates, side chain acid amides or their analogues are used as ligands for purification of penicillin G acylase. Either affinity or hydrophobic supports prepared by derivatisation of gels particularly agarose gels are used in purification of penicillin G acylase. Applied Biochemistry and Biotechnology vol. 9, p. 421, 1984 describes a two step protocol involving hydrophobic chromatography on phenyl-Sepharose and ion exchange chromatography on DEAE-cellulose. Purification of penicillion G acylase on Sepharose(agarose) activated with cyanogen bromide and derivatised with ampicillin, amoxycillin, phenylglycine, norleucine, octylamine, aniline, benzyl amine, phenylethyl amine, leucine, m-hydroxy aniline, p-hydroxy aniline, 2,4-dinitro aniline, tyrosine and

tryptophan is dercribed in Hindustan Antibiotics Bulletin, vol. 31, p. 25, 1989; Biotechnology Letters, vol. 9, p. 539, 1987 and Applied Biochemistry and Biotechnology vol. 9, p. 421, 1984.
The use of aforesaid supports for purification of penicillin G acylase suffers from one or more of the following disadvantages: activation by using toxic chemicals like cyanogen bromide, bed compression, lower flow rates and attrition if used in batch mode.
In our copending application No. 307/00 we have described and claimed a process for the preparation of macroporous beaded crosslinked copolymers. The copolymers have the following properties that are desired for a support for use in affinity chromatography such as:
Presence of built-in glycidyl functional groups for binding of ligand molecules;
Stable bed volume at varying conditions of pH and ionic strength;
Uniform bead size and
Resistance to attrition.
Thus, the macroporous beaded crosslinked copolymers described and claimed in our copending application No. 307/00 can be derivatised and used as a support in affinity chromatography.
Butylamine, hexylamine, octylamine, dodecylamine, phenylethylamine and benzylamine are either analogues of side chain of penicillin or are structures that possess degree of hydrophobicity in the range equivalent to the hydrophobicity of side chain moiety of penicillins. Primary amino group present in these molecules can react with the

glycidyl functional group on the macroporous beaded crosslinked copolymers to form affinity supports.
The main object of the present invention is to provide a process for the preparation of novel macroporous beaded copolymers.
Another object is to provide a process for the preparation of affinity chromatography supports useful for the purification of penicillin G acylase wherein toxic chemicals are not used.
Yet another object is to provide a process for the preparation of affinity chromatography supports useful for the purification of penicillin G acylase.
Accordingly, the present invention provides a process for the preparation of novel derivatised macroporous beaded copolymer useful as affinity chromatography supports which comprises suspending macroporous beaded copolymers in a solution containing a ligand selected from amine in an organic solvent, shaking the suspension at an rpm in the range of 75 to 200, at a temperature in the range of 20 to 30°C for a period ranging upto 48 hours, isolating the beads from the reaction mixture by conventional methods, washing with water and drying the beads under vacuum.
In an embodiment of the invention, the macroporous beaded crosslinked allyl glycidyl ether copolymers were prepared as described and claimed in our copending application No.NF-307/00.
In another embodiment of the invention, the ligand
may be selected from butylamine, hexylamine,
octylamine, dodecylamine, phenylethylamine, benzylamine and

the ratio of the copolymer and the ligand may be in the range of 1:1 to 1:20.
In yet another embodiment of the invention, the solvent for dissolution of the ligand may be selected from methanol, ethanol, isopropanol.
The process of the present invention is described with reference to the following Examples, which are by way of illustration only and should not be construed to limit the scope of the invention, in any manner.
Examples for the preparation of macroporous beaded crosslinked copolymers
Example 1
In an inert atmosphere of nitrogen, 1.5 grams of poly vinyl pyrrolidone was dissolved in 150 ml of distilled water in a 250 ml capacity glass reactor. 9.72 grams of allyl glycidyl ether, 8.38 grams of pentaerythritol triacrylate and 22.5 ml of cyclohexanol were added to the aqueous solution of poly vinyl pyrrolidone. The suspension was stirred at 300 rpm. 0.6 gram of azo bis isobutyronitrile was added to this suspension and the resultant polymerization reaction mixture was heated with agitation at a temperature of 70°C for 3 hours. The copolymer separated out as beads during the polymerization. The copolymer beads were isolated by filtration, washed with distilled water, followed by washing by methanol and dried in a vacuum oven at 40°C. The yield of copolymer obtained was 17.10 grams.
Example 2
In an inert atmosphere of nitrogen, 1.5 grams of poly vinyl pyrrolidone was
dissolved in 150 ml of distilled water in a 250 ml capacity glass reactor. 4.30 grams of

allyl glycidyl ether. 14.50 grams of pentaerythritol trimethacrylate and 22.0 ml of cyclohexanol were added to the aqueous solution of poly vinyl pyrrolidone. The suspension was stirred at 300 rpm. 0.6 gram of azo bis isobutyronitrile was added to this suspension and the resultant polymerization reaction mixture was heated with agitation at a temperature of 70°C for 3 hours. The copolymer separated out as beads during the polymerization. The copolymer beads were isolated by filtration, washed with distilled water, followed by washing by methanol and dried in a vacuum oven at 40°C. The yield of copolymer obtained was 14.5 grams.
Example 3
In an inert atmosphere of nitrogen, 2.0 grams of poly vinyl acetate was dissolved in 150 ml of distilled water in a 250 ml capacity glass reactor. 8.6 grams of allyl glycidyl ether, 7.8 grams of divinyl benzene and 23 ml of cyclohexanol were added to the aqueous solution of poly vinyl pyrrolidone. The suspension was stirred at 300 rpm. 0.8 gram of azo bis isobutyronitrile was added to this suspension and the resultant polymerization reaction mixture was heated with agitation at a temperature of 70°C for 3 hours. The copolymer separated out as beads during the polymerization. The copolymer beads were isolated by filtration, washed with distilled water, followed by washing by ethanol and dried in a vacuum oven at 50°C. The yield of copolymer obtained was 14.25 grams.
Example 4
In an inert atmosphere of nitrogen, 1.8 grams of poly vinyl alcohol was dissolved in 150 ml of distilled water in a 250 ml capacity glass reactor. 9.0 grams of allyl glycidyl ether, 8 grams of ethylene glycol dimethacrylate and 43.5 ml of cyclohexanol were added

to the aqueous solution of poly vinyl pyrrolidone. The suspension was stirred at 300 rpm. 0.6 gram of methyl ethyl ketone peroxide was added to this suspension and the resultant polymerization reaction mixture was heated with agitation at a temperature of 60°C for 4 hours. The copolymer separated out as beads during the polymerization. The copolymer beads were isolated by filtration, washed with distilled water, followed by washing by ethanol and dried in a vacuum oven at 40°C. The yield of copolymer obtained was 15.25 grams.
Example 5
In an inert atmosphere of nitrogen, 1.5 grams of poly vinyl pyrrolidone was dissolved in 150 ml of distilled water in a 250 ml capacity glass reactor. 8.5 grams of allyl glycidyl ether, 8.25 grams of trimethylol propane triacrylate and 43.5 ml of cyclohexanol were added to the aqueous solution of poly vinyl pyrrolidone. The suspension was stirred at 300 rpm. 0.5 gram of benzoyl peroxide was added to this suspension and the resultant polymerization reaction mixture was heated with agitation at a temperature of 70°C for 3 hours. The copolymer separated out as beads during the polymerization. The copolymer beads were isolated by filtration, washed with distilled water, followed by washing by methanol and dried in a vacuum oven at 40°C. The yield of copolymer obtained was 15.35 grams.
Example 6
In an inert atmosphere of nitrogen, 2.25 grams of sodium salt of poly acrylic acid was dissolved in 150 ml of distilled water in a 250 ml capacity glass reactor. 9.0 grams of allyl glycidyl ether, 8.75 grams of trimethylol propane trimethacrylate and 43.5 ml of

cyclohexanol were added to the aqueous solution of poly vinyl pyrrolidone. The suspension was stirred at 300 rpm. 0.6 gram of benzoyl peroxide was added to this suspension and the resultant polymerization reaction mixture was heated with agitation at a temperature of 80°C for 2 hours. The copolymer separated out as beads during the polymerization. The copolymer beads were isolated by filtration, washed with distilled water, followed by washing by ethanol and dried in a vacuum oven at 50°C. The yield of copolymer obtained was 15.75 grams.
Examples for the derivatisation of macroporous beaded crosslinked copolymers
Example 7
10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 4 was suspended in 60 ml of ligand solution containing 34.4 g of butylamine dissolved in ethanol. The suspension was incubated in a stoppered 250-ml capacity conical flask on a rotary shaker at 100 rpm, 25°C for 48 hours. The beads were collected by filtration on a Buchner funnel, washed with 200 ml of distilled water and dried at 40°C under vacuum. The derivatised macroporous beaded crosslinked copolymer beads were stored at 4°C.
Example 8
10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 4 was suspended in 80 ml of ligand solution containing 23.8 g of hexylamine dissolved in methanol. The suspension was incubated in a stoppered 250 ml capacity conical flask on a rotary shaker at 100 rpm, 25°C for 48 hours. The beads were collected by filtration on a Buchner funnel, washed with 200 ml of distilled water and dried at 40°C

under vacuum. The derivatised macroporous beaded crosslinked copolymer beads were stored at 4°C.
Example 9
10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 4 was suspended in 60 ml of ligand solution containing 30.4 g of octylamine dissolved in ethanol. The suspension was incubated in a stoppered 250 ml capacity conical flask on a rotary shaker at 150 rpm, 20°C for 48 hours. The beads were collected by filtration on a Buchner funnel, washed with 200 ml of distilled water and dried at 40°C under vacuum. The derivatised macroporous beaded crosslinked copolymer beads were stored at 4°C.
Example 10
10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 4 was suspended in 100 ml of ligand solution containing 43.6 g of dodecylamine dissolved in methanol. The suspension was incubated in a stoppered 250 ml capacity conical flask on a rotary shaker at 200 rpm, 25°C for 48 hours. The beads were collected by filtration on a Buchner funnel, washed with 200 ml of distilled water and dried at 40°C under vacuum. The derivatised macroporous beaded crosslinked copolymer beads were stored at 4°C.
Example 11
10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 4 was suspended in 60 ml of ligand solution containing 28.5 g of phenylethylamine dissolved in isopropanol. The suspension was incubated in a stoppered

250 ml capacity conical flask on a rotary shaker at 75 rpm, 30°C for 48 hours. The beads were collected by filtration on a Buchner funnel, washed with 200 ml of distilled water and dried at 40°C under vacuum. The derivatised macroporous beaded crosslinked copolymer beads were stored at 4°C.
Example 12
10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 4 was suspended in 60 ml of ligand solution containing 25.2 g of benzylamine dissolved in ethanol. The suspension was incubated in a stoppered 250 ml capacity conical flask on a rotary shaker at 75 rpm, 30°C for 36 hours. The beads were collected by filtration on a Buchner funnel, washed with 200 ml of distilled water and dried at 40°C under vacuum. The derivatised macroporous beaded crosslinked copolymer beads were stored at4°C.
Example 13
10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 1 was suspended in 60 ml of ligand solution containing 25.2 g of benzylamine dissolved in ethanol. The suspension was incubated in a stoppered 250 ml capacity conical flask on a rotary shaker at 100 rpm, 25°C for 48 hours. The beads were collected by filtration on a Buchner funnel, washed with 200 ml of distilled water and dried at 40°C under vacuum. The derivatised macroporous beaded crosslinked copolymer beads were stored at 4°C.
Example 14
10 g of the macroporous beaded crosslinked copolymer prepared as described in

Example 3 was suspended in 80 ml of ligand solution containing 65.2 g of benzylamine dissolved in ethanol. The suspension was incubated in a stoppered 250 ml capacity conical flask on a rotary shaker at 100 rpm, 25°C for 48 hours. The beads were collected by filtration on a Buchner funnel, washed with 200 ml of distilled water and dried at 40°C under vacuum. The derivatised macroporous beaded crosslinked copolymer beads were stored at4°C.
Example 15
10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 6 was suspended in 60 ml of ligand solution containing 25.2 g of benzylamine dissolved in ethanol. The suspension was incubated in a stoppered 250 ml capacity conical flask on a rotary shaker at 100 rpm, 25°C for 48 hours. The beads were collected by filtration on a Buchner runnel, washed with 200 ml of distilled water and dried at 40°C under vacuum. The derivatised macroporous beaded crosslinked copolymer beads were stored at 4°C.
Advantages of the invention
The invention provides a process for derivatisation of macroporous beaded crosslinked copolymers useful for affinity chromatography of penicillin G acylase. The preparation of the affinity chromatography supports offers the following advantages:
i) The process does not involve the use of toxic chemicals like cyanogen bromide.

ii) The macroporous beaded crosslinked ether copolymers are rigid crosslinked copolymers and hence bed compression at varying pH and ionic strength does not occur.
iii) The beads are resistant for attrition thereby enables its use in a batch mode on large scale.
iv) The operational procedure is simple and easy to scale-up.

We claim :
1. A process for the preparation of novel derivatised macroporous beaded
copolymer useful as affinity chromatography supports which comprises
suspending macroporous beaded copolymers in a solution containing a
ligand selected from amine in an organic solvent, shaking the suspension
at an rpm in the range of 75 to 200, at a temperature in the range of 20 to
30°C for a period ranging upto 48 hours, isolating the beads from the
reaction mixture by conventional methods, washing with water and drying
the beads under vacuum.
2. A process as claimed in claim 1 wherein the ligand is selected from
butylamine, hexylamine, octylamine, dodecylamine, phenylethylamine,
benzylamine and the ratio of the copolymer and the ligand used is in the
range of 1:1 to 1:20.
3. A process as claimed in claim 1-2 wherein the organic solvent used for
dissolution of ligand is selected from methanol, ethanol, isopropanol.
4. A process for the preparation of novel derivatised macroporous beaded
copolymer useful as affinity chromatography supports substantially as
herein described with reference to the examples.

Documents:

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1061-del-2000-correspondence-others.pdf

1061-del-2000-correspondence-po.pdf

1061-del-2000-description (complete).pdf

1061-del-2000-form-1.pdf

1061-del-2000-form-19.pdf

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Patent Number

216785

Indian Patent Application Number

1061/DEL/2000

PG Journal Number

13/2008

Publication Date

31-Mar-2008

Grant Date

19-Mar-2008

Date of Filing

24-Nov-2000

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG, NEW DELHI-110001, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	CHELANATTU KHIZHAKKE MADATH RAMAN RAJAN	NATIONAL CHEMICAL LABORATORY, PUNE - 411008, MAHARASHTRA
2	VARSHA BHIKOBA GHADGE	NATIONAL CHEMICAL LABORATORY, PUNE - 411008, MAHARASHTRA
3	SURENDRA PONRATHNAM	NATIONAL CHEMICAL LABORATORY, PUNE - 411008, MAHARASHTRA
4	SALIM KASAM MUJAWAR	HINDUSTAN ANTIBIOTICS, PIMPRI, PUNE - 411018
5	JAIPRAKASH GANPATRAO SHEWALE	HINDUSTAN ANTIBIOTICS, PIMPRI, PUNE - 411018

PCT International Classification Number

C08L 5/54

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA