Title of Invention	"A PROCESS FOR THE PREPARATION OF BEADED COPOLYMER AS AFFINITY CHROMATOGRAPHY SUPPORTS"
Abstract	A process for the preparation of novel derivatised macroporous beaded crosslinked copolymers : The present invention provides a process for the preparation of novel derivatised macroporous beaded crosslinked copolymers by suspending macroporous beaded crosslinked allyl glycidyl ether copolymers in an inorganic buffer solution having a particular molarity and pH , adding to the suspension an antibiotic ligand solution adjusting pH in the range of 8.0 to 9.0, at a temperature of 20 to 30°C , suspending the obtained ligand linked copolymer beads in a basic solution having pH in the range of 10.0 to 11, collecting the beads by filtration and preserving the beaded macroporuous copolymers in a buffer solution .

Title of Invention

"A PROCESS FOR THE PREPARATION OF BEADED COPOLYMER AS AFFINITY CHROMATOGRAPHY SUPPORTS"

Abstract

A process for the preparation of novel derivatised macroporous beaded crosslinked copolymers : The present invention provides a process for the preparation of novel derivatised macroporous beaded crosslinked copolymers by suspending macroporous beaded crosslinked allyl glycidyl ether copolymers in an inorganic buffer solution having a particular molarity and pH , adding to the suspension an antibiotic ligand solution adjusting pH in the range of 8.0 to 9.0, at a temperature of 20 to 30°C , suspending the obtained ligand linked copolymer beads in a basic solution having pH in the range of 10.0 to 11, collecting the beads by filtration and preserving the beaded macroporuous copolymers in a buffer solution .

Full Text	This invention relates to a process for the preparation of noveldesivatised macroporous beaded crosslinked polymers that are useful iit the preparation as 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 enzymes such as penicillinase. Penicillinase hydrolyses the cyclic amide bond present in penicillins to form corresponding penicilloic acid. The penicilloic acid thus formed does not exhibit any antimicrobial activity. This property enables the use of penicillinase in Sterility Test of sterile penicillin preparations and as a marker enzyme in Enzyme Linked Immunosorbant Assay (ELISA). The substrates, inhibitors or their analogues are used as ligands for purification of penicillinase by affinity chromatography. The use of affinity supports prepared by derivatisation of gels particularly agarose gels are known for purification of penicillinase. The Antimicrobial Agents and Chemotherapy, Vol 15, p. 229 (1979) describes the use of 6-amino penicillanic acid or 7-amino desacetoxycephalosporanic acid bound to agarose. Current Chemotherapy and Immunotherapy. Vol 1, p. 751, (1982) describes the use of cefotaxime bound to Sepharose 4E. Yet another publication in Journal of Chromatography, Vol 448, p. 109, (1988) describes the use of N-acetyl-D-penicillamine coupled to Sepharose 4B. German Patent No. DD 260,292 (1988) describes the use of aminobenzylpenicillin coupled to macroporous carriers of organic, inorganic or bioorganic material. The use of aforesaid affinity supports for purification of penicillinase 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. NF 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: uniform bead size, resistant to attrition, stable bed volume at varying conditions of pH and ionic strength and presence of built-in glycidyl functional group for binding of ligand molecules. Thus, the macroporous beaded crosslinked copolymers described and claimed in our copending application No. NF 307/00 can be derivatised and used as a support in affinity chromatography. Ampicillin, amoxycillin, cephalexin, cephadroxyl, cephalosporin C, 6-amino penicillanic acid. 7-amino des acetoxy cephalosporanic acid are either substrate, substrate analogues or product analogues of penicillinase. 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 beta-lactam moieties on these derivatised affinity supports are labile and prone to degradation by hydrolysis of cyclic amide bond with time, resulting into chemically nonhomogenous structures. In order to maintain structural uniformity, the beta-lactam moiety is chemically hydrolysed to generate the corresponding acid moiety. The main object of the present invention to provide a process for the preparation of novel macroporous beaded crosslinked copolymers. Another object is to provide a process for the preparation of affinity chromatography supports useful for the purification of penicillinase Yet another object is to provide a process for the preparation of affinity chromatography supports useful for the purification of penicillinase wherein toxic chemicals are not used. Accordingly, the present invention provides a process for the preparation of novel desivatised macroporous beaded crosslinked copolymers which comprises suspending macroporous beaded crosslinked allyl glycidyl ether copolymers in a buffer solution having molarity in the range of 0.05 to 0.2 and pH in the range of 7 to 8, adding to the suspension the ligand solution, adjusting pH in the range of 8.0 to 9.0, shaking the mixture at a temperature in the range of 20 to 30°C for a period upto 48 hours, decanting the supernatant from the reaction mixture, suspending the ligand linked copolymer beads in a base solution having pH in the range of 10.0 to 11, shaking the mixture at a temperature in the range of 20 to 30°C for a period upto 2 hours, collecting the beads by filtration and if required preserving the beaded macroporous beaded copolymers in a suitable buffer. In an embodiment of the invention, the ligand chosen for making the ligand solution is an antibiotic and is selected from ampicillin, amoxycillin, cephalexin, cephadroxyl, cephalosporin C, 6-amino penicillanic acid, 7-amino des acetoxy cephalosporanic acid and the ratio of the copolymer and ligand may be in the range of 1:1 to 1: 20. In another embodiment of the invention, the base solution used is such as sodium hydroxide, potassium hydroxide, ammonium hydroxide. In yet another embodiment of the invention, the buffer solution used may be such as phosphate, tris chloride and its molar concentration in the range of 0.05 to 0.2 and pH in the range of 7 to 8. In still another embodiment of the invention, the novel beaded crosslinked copolymer beads is preserved by suspending the beads in buffer solution such as phosphate, tris chloride having molarity in the range of 0.05 to 0.2 and pH in the range of 7.0. to 8.0 The process of the present invention is described with reference to the following examples, which are given 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 allyl glycidyl ether copolymer prepared as described in Example 4 was suspended in 1000 ml of phosphate buffer solution having molarity of 0.1 and pH of 7.5. To this suspension 41 g of ampicillin sodium dissolved in 2000 ml of phosphate buffer solution having molarity of 0.1 and pH of 7.5 was added. The pH of the suspension was made to 8.5 by adding sodium hydroxide solution. The suspension was incubated on a rotary shaker at 100 rpm, 25°C for 48 hours. The supernatant was decanted. 100 ml of aqueous solution of sodium hydroxide having pH of 10.5 was added to the beads and the suspension was incubated on a rotary shaker at 100 rpm, 25°C for 2 hours. The beads were collected by filtration on a Buchner funnel, washed thrice with 200 ml of distilled water and suspended in 50 ml of phosphate buffer solution having molarity of 0.05 and pH of 7.5. The derivatised macroporous beaded crosslinked allyl glycidyl ether copolymer beads were stored at 4°C as suspension in phosphate buffer solution having molarity of 0.05, pH of 7.5 and containing sodium azide at concentration of 0.2% w/v as preservative. Example 8 10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 4 was suspended in 1000 ml of phosphate buffer solution having molarity of 0.2 and pH of 7.8. To this suspension 43 g of amoxycillin trihydrate dissolved in 2000 ml of phosphate buffer solution having molarity of 0.1 and pH of 7.5 was added. The pH of the suspension was made to 8.5 by adding sodium hydroxide solution. The suspension was incubated on a rotary shaker at 100 rpm, 25°C for 48 hours. The supernatant was decanted. 100 ml of aqueous solution of sodium hydroxide having pH of 10.5 was added to the beads and the suspension was incubated on a rotary shaker at 100 rpm, 25°C for 2 hours. The beads were collected by filtration on a Buchner funnel, washed thrice with 200 ml of distilled water and suspended in 50 ml of phosphate buffer solution having molarity of 0.05 and pH of 7.5. The derivatised macroporous beaded crosslinked copolymer beads were stored at 4°C as suspension in phosphate buffer solution having molarity of 0.1, pH of 7.5 and containing sodium azide at concentration of 0.2% w/v as preservative. Example 9 10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 4 was suspended in 1000 ml of tris chloride buffer solution having molarity of 0.15 and pH of 8.0. To this suspension 41.44 g of cephadroxyl dissolved in 15000 ml of phosphate buffer solution having molarity of 0.1 and pH of 7.5 was added. The pH of the suspension was made to 8.5 by adding potassium hydroxide solution. The suspension was incubated on a rotary shaker at 150 rpm, 25°C for 40 hours. The supernatant was decanted. 100 ml of aqueous solution of potassium hydroxide having pH of 10.5 was added to the beads and the suspension was incubated on a rotary shaker at 100 rpm, 25°C for 2 hours. The beads were collected by filtration on a Buchner funnel, washed thrice with 200 ml of distilled water and suspended in 50 ml of tris chloride buffer solution having molarity of 0.05 and pH of 7.5. The derivatised macroporous beaded crosslinked copolymer beads were stored at 4°C as suspension in phosphate buffer solution having molarity of 0.05, pH of 7.5 and containing sodium azide at concentration of 0.2% w/v as preservative. Example 10 10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 4 was suspended in 1000 ml of phosphate buffer solution having molarity of 0.1 and pH of 7.5. To this suspension 48 g of cephalosporin C dissolved in 2000 ml of phosphate buffer solution having molarity of 0.1 and pH of 7.5 was added. The pH of the suspension was made to 8.5 by adding ammonium hydroxide solution. The suspension was incubated on a rotary shaker at 200 rpm, 200C for 48 hours. The supernatant was decanted. 100 ml of aqueous solution of ammonium hydroxide having pH of 10.5 was added to the beads and the suspension was incubated on a rotary shaker at 200 rpm, 25°C for 2 hours. The beads were collected by filtration on a Buchner funnel, washed thrice with 200 ml of distilled water and suspended in 50 ml of phosphate buffer solution having molarity of 0.05 and pH of 7.5. The derivatised macroporous beaded crosslinked copolymer beads were stored at 4°C as suspension in phosphate buffer solution having molarity of 0.05, pH of 7.5 and containing sodium azide at concentration of 0.2% w/v as preservative. Example 11 10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 4 was suspended in 1000 ml of tris chloride buffer solution having molarity of 0.1 and pH of 7.5. To this suspension 25.36 g of 6-amino penicillanic acid dissolved in 1000 ml of phosphate buffer solution having molarity of 0.1 and pH of 7.5 was added. The pH of the suspension was made to 8.5 by adding sodium hydroxide solution. The suspension was incubated on a rotary shaker at 100 rpm, 25°C for 48 hours. The supernatant was decanted. 100 ml of aqueous solution of sodium hydroxide having pH of 10.5 was added to the beads and the suspension was incubated on a rotary shaker at 100 rpm, 25°C for 2 hours. The beads were collected by filtration on a Buchner funnel, washed thrice with 200 ml of distilled water and suspended in 50 ml of phosphate buffer solution having molarity of 0.05 and pH of 7.5. The derivatised macroporous beaded crosslinked copolymer beads were stored at 4°C as suspension in phosphate buffer solution having molarity of 0.05, pH of 7.5 and containing sodium azide at concentration of 0.2% w/v as preservative. Example 12 10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 4 was suspended in 1000 ml of phosphate buffer solution having molarity of 0.1 and pH of 7.5. To this suspension 25.16 g of 7-amino desacetoxy cephalosporanic acid dissolved in 2500 ml of phosphate buffer solution having molarity of 0.1 and pH of 7.5 was added. The pH of the suspension was made to 8.5 by adding ammonium hydroxide solution. The suspension was incubated on a rotary shaker at 100 rpm, 25°C for 48 hours. The supernatant was decanted. 100 ml of aqueous solution of ammonium hydroxide having pH of 10.5 was added to the beads and the suspension was incubated on a rotary shaker at 100 rpm, 25°C for 2 hours. The beads were collected by filtration on a Buchner funnel, washed thrice with 200 ml of distilled water and suspended in 50 ml of phosphate buffer solution having molarity of 0.05 and pH of 7.5. The derivatised macroporous beaded crosslinked copolymer beads were stored at 4°C as suspension in phosphate buffer solution having molarity of 0.05, pH of 7.5 and containing sodium azide at concentration of 0.2% w/v as preservative. Example 13 10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 4 was suspended in 1000 ml of phosphate buffer solution having molarity of 0.1 and pH of 7.5. To this suspension 42.6 g of cephalexin dissolved in 1400 ml of phosphate buffer solution having molarity of 0.1 and pH of 7.5 was added. The pH of the suspension was made to 8.5 by adding sodium hydroxide solution. The suspension was incubated on a rotary shaker at 100 rpm, 25°C for 48 hours. The supernatant was decanted. 100 ml of aqueous solution of sodium hydroxide having pH of 10.5 was added to the beads and the suspension was incubated on a rotary shaker at 100 rpm, 25°C for 2 hours. The beads were collected by filtration on a Buchner funnel, washed thrice with 200 ml of distilled water and suspended in 50 ml of phosphate buffer solution having molarity of 0.05 and pH of 7.5. The derivatised macroporous beaded crosslinked copolymer beads were stored at 4°C as suspension in phosphate buffer solution having molarity of 0.05, pH of 7.5 and containing sodium azide at concentration of 0.2% w/v as preservative. Example 14 10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 2 was suspended in 1000 ml of phosphate buffer solution having molarity of 0.1 and pH of 7.5. To this suspension 40.6 g of cephalexin dissolved in 1400 ml of phosphate buffer solution having molarity of 0.1 and pH of 7.5 was added. The pH of the suspension was made to 8.5 by adding sodium hydroxide solution. The suspension was incubated on a rotary shaker at 100 rpm, 25oC for 48 hours. The supernatant was decanted. 100 ml of aqueous solution of sodium hydroxide having pH of 10.5 was added to the beads and the suspension was incubated on a rotary shaker at 100 rpm, 25°C for 2 hours. The beads were collected by filtration on a Buchner funnel, washed thrice with 200 ml of distilled water and suspended in 50 ml of phosphate buffer solution having molarity of 0.05 and pH of 7.5. The derivatised macroporous beaded crosslinked copolymer beads were stored at 4°C as suspension in phosphate buffer solution having molarity of 0.05, pH of 7.5 and containing sodium azide at concentration of 0.2% w/v as preservative. Example 15: 10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 3 was suspended in 1000 ml of phosphate buffer solution having molarity of 0.1 and pH of 7.5. To this suspension 40.6 g of cephalexin dissolved in 1400 ml of phosphate buffer solution having molarity of 0.1 and pH of 7.5 was added. The pH of the suspension was made to 8.5 by adding sodium hydroxide solution. The suspension was incubated on a rotary shaker at 100 rpm, 25oC for 48 hours. The supernatant was decanted. 100 ml of aqueous solution of sodium hydroxide having pH of 10.5 was added to the beads and the suspension was incubated on a rotary shaker at 100 rpm, 25°C for 2 hours. The beads were collected by filtration on a Buchner funnel, washed thrice with 200 ml of distilled water and suspended in 50 ml of phosphate buffer solution having molarity of 0.05 and pH of 7.5. The derivatised macroporous beaded crosslinked copolymer beads were stored at 4°C as suspension in phosphate buffer solution having molarity of 0.05, pH of 7.5 and containing sodium azide at concentration of 0.2% w/v as preservative. Example 16 10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 5 was suspended in 1000 ml of phosphate buffer solution having molarity of 0.1 and pH of 7.5. To this suspension 40.6 g of cephalexin dissolved in 50 ml of phosphate buffer solution having molarity of 0.1 and pH of 7.5 was added. The pH of the suspension was made to 8.5 by adding sodium hydroxide solution. The suspension was incubated on a rotary shaker at 100 rpm, 25°C for 48 hours. The supernatant was decanted. 100 ml of aqueous solution of sodium hydroxide having pH of 10.5 was added to the beads and the suspension was incubated on a rotary shaker at 100 rpm, 25°C for 2 hours. The beads were collected by filtration on a Buchner funnel, washed thrice with 200 ml of distilled water and suspended in 50 ml of phosphate buffer solution having molarity of 0.05 and pH of 7.5. The derivatised macroporous beaded crosslinked copolymer beads were stored at 4°C as suspension in phosphate buffer solution having molarity of 0.05, pH of 7.5 and containing sodium azide at concentration of 0.2% w/v as preservative. Advantages of the invention The invention provides a process for derivatisation of macroporous beaded crosslinked copolymers useful for purification of enzymes such as penicillinase by affinity chromatography. Preparation of the affinity chromatography supports by the process of this invention offers the following advantages: i) The process does not involve the use of toxic chemicals like cyanogen bromide. ii) The macroporous beaded crosslinked 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 operation procedure is simple and easy to scale-up. We claim: \. A process for the preparation of noveldesivatised macroporous beaded crosslinked copolymers which comprises suspending macroporous beaded crosslinked allyl glycidyl ether copolymers in an inorganic buffer solution having molarity in the range of 0.05 to 0.2 and pH in the range of 7 to 8, adding to the suspension the ligand solution, adjusting pH in the range of 8.0 to 9.0, shaking the mixture at a temperature in the range of 20 to 30°C for a period upto 48 hours, decanting the supernatant from the reaction mixture, suspending the ligand linked copolymer beads in a basic solution having pH in the range of 10.0 to 11, shaking the mixture at a temperature in the range of 20 to 30°C for a period upto 2 hours, collecting the beads by filtration and if required preserving the beaded macroporous beaded copolymers in a suitable buffer. 2. A process as claimed in claim 1, wherein, the ligand chosen for making the ligand solution is an antibiotic such as ampicillin, amoxycillin, cephalexin, cephadroxyl, cephalosporin C, 6-amino penicillanic acid, 7-amino des acetoxy cephalosporanic acid and the ratio of the copolymer and ligand is in the range of 1:1 to 1:20. 3. A process as claimed in claims 1 and 2, wherein, the base solution used is such as sodium hydroxide, potassium hydroxide, ammonium hydroxide. 4. A process as claimed in claims 1 to 3, wherein, the buffer solution used is phosphate, tris chloride and its molar concentration in the range of 0.05 to 0.2 and pH in the range of 7.0 to 8.0. 5. A process as claimed in claims 1 to 4, wherein, the novel beaded crosslinked copolymer beads is preserved by suspending the beads in buffer solution such as phosphate, tris chloride having molarity in the range of 0.05 to 0.2 and pH in the range of 7 to 8. 6. A process for the preparation of novel desivatised macroprocess baded crosslimted copolymer useful as macaffinity chromatography supports as herein described with reference to the Examples 7 to 16.

Full Text

This invention relates to a process for the preparation of noveldesivatised
macroporous
beaded crosslinked polymers that are useful iit the preparation as 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 enzymes such as penicillinase.
Penicillinase hydrolyses the cyclic amide bond present in penicillins to form corresponding penicilloic acid. The penicilloic acid thus formed does not exhibit any antimicrobial activity. This property enables the use of penicillinase in Sterility Test of sterile penicillin preparations and as a marker enzyme in Enzyme Linked Immunosorbant Assay (ELISA). The substrates, inhibitors or their analogues are used as ligands for purification of penicillinase by affinity chromatography.
The use of affinity supports prepared by derivatisation of gels particularly agarose gels are known for purification of penicillinase. The Antimicrobial Agents and Chemotherapy, Vol 15, p. 229 (1979) describes the use of 6-amino penicillanic acid or 7-amino desacetoxycephalosporanic acid bound to agarose. Current Chemotherapy and Immunotherapy. Vol 1, p. 751, (1982) describes the use of cefotaxime bound to Sepharose 4E. Yet another publication in Journal of Chromatography, Vol 448, p. 109, (1988) describes the use of N-acetyl-D-penicillamine coupled to Sepharose 4B. German Patent No. DD 260,292 (1988) describes the use of aminobenzylpenicillin coupled to macroporous carriers of organic, inorganic or bioorganic material.
The use of aforesaid affinity supports for purification of penicillinase 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. NF 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: uniform bead size, resistant to attrition, stable bed volume at varying conditions of pH and ionic strength and presence of built-in glycidyl functional group for binding of ligand molecules. Thus, the macroporous beaded crosslinked copolymers described and claimed in our copending application No. NF 307/00 can be derivatised and used as a support in affinity chromatography.
Ampicillin, amoxycillin, cephalexin, cephadroxyl, cephalosporin C, 6-amino penicillanic acid. 7-amino des acetoxy cephalosporanic acid are either substrate, substrate analogues or product analogues of penicillinase. 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 beta-lactam moieties on these derivatised affinity supports are labile and prone to degradation by hydrolysis of cyclic amide bond with time, resulting into chemically nonhomogenous structures. In order to maintain structural uniformity, the beta-lactam moiety is chemically hydrolysed to

generate the corresponding acid moiety.
The main object of the present invention to provide a process for the preparation of novel macroporous beaded crosslinked copolymers.
Another object is to provide a process for the preparation of affinity chromatography supports useful for the purification of penicillinase
Yet another object is to provide a process for the preparation of affinity chromatography supports useful for the purification of penicillinase wherein toxic chemicals are not used.
Accordingly, the present invention provides a process for the preparation of novel desivatised macroporous beaded crosslinked copolymers which comprises suspending macroporous
beaded crosslinked allyl glycidyl ether copolymers in a buffer solution having molarity in the range of 0.05 to 0.2 and pH in the range of 7 to 8, adding to the suspension the ligand solution, adjusting pH in the range of 8.0 to 9.0, shaking the mixture at a temperature in the range of 20 to 30°C for a period upto 48 hours, decanting the supernatant from the reaction mixture, suspending the ligand linked copolymer beads in a base solution having pH in the range of 10.0 to 11, shaking the mixture at a temperature in the range of 20 to 30°C for a period upto 2 hours, collecting the beads by filtration and if required preserving the beaded macroporous beaded copolymers in a suitable buffer.
In an embodiment of the invention, the ligand chosen for making the ligand solution is an antibiotic and is selected from ampicillin, amoxycillin, cephalexin,

cephadroxyl, cephalosporin C, 6-amino penicillanic acid, 7-amino des acetoxy cephalosporanic acid and the ratio of the copolymer and ligand may be in the range of 1:1 to 1: 20.
In another embodiment of the invention, the base solution used is such as sodium hydroxide, potassium hydroxide, ammonium hydroxide.
In yet another embodiment of the invention, the buffer solution used may be such as phosphate, tris chloride and its molar concentration in the range of 0.05 to 0.2 and pH in the range of 7 to 8.
In still another embodiment of the invention, the novel beaded crosslinked copolymer beads is preserved by suspending the beads in buffer solution such as phosphate, tris chloride having molarity in the range of 0.05 to 0.2 and pH in the range of 7.0. to 8.0
The process of the present invention is described with reference to the following examples, which are given 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 allyl glycidyl ether copolymer prepared as described in Example 4 was suspended in 1000 ml of phosphate buffer solution having molarity of 0.1 and pH of 7.5. To this suspension 41 g of ampicillin sodium dissolved in 2000 ml of phosphate buffer solution having molarity of 0.1 and pH of 7.5 was added. The pH of the suspension was made to 8.5 by adding sodium hydroxide solution. The suspension was incubated on a rotary shaker at 100 rpm, 25°C for 48 hours. The supernatant was decanted. 100 ml of aqueous solution of sodium hydroxide having pH of 10.5 was added to the beads and the suspension was incubated on a rotary shaker at 100 rpm, 25°C for 2 hours. The beads were collected by filtration on a Buchner funnel, washed thrice with 200 ml of distilled water and suspended in 50 ml of phosphate buffer solution having molarity of 0.05 and pH of 7.5. The derivatised macroporous beaded crosslinked allyl glycidyl ether copolymer beads were stored at 4°C as suspension in phosphate buffer solution having molarity of 0.05, pH of 7.5 and containing sodium azide at concentration of 0.2% w/v as preservative.
Example 8
10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 4 was suspended in 1000 ml of phosphate buffer solution having molarity of 0.2 and pH of 7.8. To this suspension 43 g of amoxycillin trihydrate dissolved in 2000 ml of phosphate buffer solution having molarity of 0.1 and pH of 7.5 was added. The pH of the suspension was made to 8.5 by adding sodium hydroxide solution. The suspension was
incubated on a rotary shaker at 100 rpm, 25°C for 48 hours. The supernatant was decanted. 100 ml of aqueous solution of sodium hydroxide having pH of 10.5 was added to the beads and the suspension was incubated on a rotary shaker at 100 rpm, 25°C for 2 hours. The beads were collected by filtration on a Buchner funnel, washed thrice with 200 ml of distilled water and suspended in 50 ml of phosphate buffer solution having molarity of 0.05 and pH of 7.5. The derivatised macroporous beaded crosslinked copolymer beads were stored at 4°C as suspension in phosphate buffer solution having molarity of 0.1, pH of 7.5 and containing sodium azide at concentration of 0.2% w/v as preservative.
Example 9
10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 4 was suspended in 1000 ml of tris chloride buffer solution having molarity of 0.15 and pH of 8.0. To this suspension 41.44 g of cephadroxyl dissolved in 15000 ml of phosphate buffer solution having molarity of 0.1 and pH of 7.5 was added. The pH of the suspension was made to 8.5 by adding potassium hydroxide solution. The suspension was incubated on a rotary shaker at 150 rpm, 25°C for 40 hours. The supernatant was decanted. 100 ml of aqueous solution of potassium hydroxide having pH of 10.5 was added to the beads and the suspension was incubated on a rotary shaker at 100 rpm, 25°C for 2 hours. The beads were collected by filtration on a Buchner funnel, washed thrice with 200 ml of distilled water and suspended in 50 ml of tris chloride buffer solution having molarity of 0.05 and pH of 7.5. The derivatised macroporous beaded crosslinked copolymer beads were stored at 4°C as suspension in phosphate buffer solution having molarity of 0.05, pH of 7.5 and containing sodium azide at concentration of 0.2% w/v as
preservative.
Example 10
10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 4 was suspended in 1000 ml of phosphate buffer solution having molarity of 0.1 and pH of 7.5. To this suspension 48 g of cephalosporin C dissolved in 2000 ml of phosphate buffer solution having molarity of 0.1 and pH of 7.5 was added. The pH of the suspension was made to 8.5 by adding ammonium hydroxide solution. The suspension was incubated on a rotary shaker at 200 rpm, 200C for 48 hours. The supernatant was decanted. 100 ml of aqueous solution of ammonium hydroxide having pH of 10.5 was added to the beads and the suspension was incubated on a rotary shaker at 200 rpm, 25°C for 2 hours. The beads were collected by filtration on a Buchner funnel, washed thrice with 200 ml of distilled water and suspended in 50 ml of phosphate buffer solution having molarity of 0.05 and pH of 7.5. The derivatised macroporous beaded crosslinked copolymer beads were stored at 4°C as suspension in phosphate buffer solution having molarity of 0.05, pH of 7.5 and containing sodium azide at concentration of 0.2% w/v as preservative.
Example 11
10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 4 was suspended in 1000 ml of tris chloride buffer solution having molarity of 0.1 and pH of 7.5. To this suspension 25.36 g of 6-amino penicillanic acid dissolved in 1000 ml of phosphate buffer solution having molarity of 0.1 and pH of 7.5 was added. The pH of the suspension was made to 8.5 by adding sodium hydroxide solution. The

suspension was incubated on a rotary shaker at 100 rpm, 25°C for 48 hours. The supernatant was decanted. 100 ml of aqueous solution of sodium hydroxide having pH of 10.5 was added to the beads and the suspension was incubated on a rotary shaker at 100 rpm, 25°C for 2 hours. The beads were collected by filtration on a Buchner funnel, washed thrice with 200 ml of distilled water and suspended in 50 ml of phosphate buffer solution having molarity of 0.05 and pH of 7.5. The derivatised macroporous beaded crosslinked copolymer beads were stored at 4°C as suspension in phosphate buffer solution having molarity of 0.05, pH of 7.5 and containing sodium azide at concentration of 0.2% w/v as preservative.
Example 12
10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 4 was suspended in 1000 ml of phosphate buffer solution having molarity of 0.1 and pH of 7.5. To this suspension 25.16 g of 7-amino desacetoxy cephalosporanic acid dissolved in 2500 ml of phosphate buffer solution having molarity of 0.1 and pH of 7.5 was added. The pH of the suspension was made to 8.5 by adding ammonium hydroxide solution. The suspension was incubated on a rotary shaker at 100 rpm, 25°C for 48 hours. The supernatant was decanted. 100 ml of aqueous solution of ammonium hydroxide having pH of 10.5 was added to the beads and the suspension was incubated on a rotary shaker at 100 rpm, 25°C for 2 hours. The beads were collected by filtration on a Buchner funnel, washed thrice with 200 ml of distilled water and suspended in 50 ml of phosphate buffer solution having molarity of 0.05 and pH of 7.5. The derivatised macroporous beaded crosslinked copolymer beads were stored at 4°C as suspension in phosphate buffer solution having molarity of 0.05, pH of 7.5 and containing sodium azide at concentration
of 0.2% w/v as preservative.
Example 13
10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 4 was suspended in 1000 ml of phosphate buffer solution having molarity of 0.1 and pH of 7.5. To this suspension 42.6 g of cephalexin dissolved in 1400 ml of phosphate buffer solution having molarity of 0.1 and pH of 7.5 was added. The pH of the suspension was made to 8.5 by adding sodium hydroxide solution. The suspension was incubated on a rotary shaker at 100 rpm, 25°C for 48 hours. The supernatant was decanted. 100 ml of aqueous solution of sodium hydroxide having pH of 10.5 was added to the beads and the suspension was incubated on a rotary shaker at 100 rpm, 25°C for 2 hours. The beads were collected by filtration on a Buchner funnel, washed thrice with 200 ml of distilled water and suspended in 50 ml of phosphate buffer solution having molarity of 0.05 and pH of 7.5. The derivatised macroporous beaded crosslinked copolymer beads were stored at 4°C as suspension in phosphate buffer solution having molarity of 0.05, pH of 7.5 and containing sodium azide at concentration of 0.2% w/v as preservative.
Example 14
10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 2 was suspended in 1000 ml of phosphate buffer solution having molarity of 0.1 and pH of 7.5. To this suspension 40.6 g of cephalexin dissolved in 1400 ml of phosphate buffer solution having molarity of 0.1 and pH of 7.5 was added. The pH of the suspension was made to 8.5 by adding sodium hydroxide solution. The suspension was
incubated on a rotary shaker at 100 rpm, 25oC for 48 hours. The supernatant was decanted. 100 ml of aqueous solution of sodium hydroxide having pH of 10.5 was added to the beads and the suspension was incubated on a rotary shaker at 100 rpm, 25°C for 2 hours. The beads were collected by filtration on a Buchner funnel, washed thrice with 200 ml of distilled water and suspended in 50 ml of phosphate buffer solution having molarity of 0.05 and pH of 7.5. The derivatised macroporous beaded crosslinked copolymer beads were stored at 4°C as suspension in phosphate buffer solution having molarity of 0.05, pH of 7.5 and containing sodium azide at concentration of 0.2% w/v as preservative.
Example 15:
10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 3 was suspended in 1000 ml of phosphate buffer solution having molarity of 0.1 and pH of 7.5. To this suspension 40.6 g of cephalexin dissolved in 1400 ml of phosphate buffer solution having molarity of 0.1 and pH of 7.5 was added. The pH of the suspension was made to 8.5 by adding sodium hydroxide solution. The suspension was incubated on a rotary shaker at 100 rpm, 25oC for 48 hours. The supernatant was decanted. 100 ml of aqueous solution of sodium hydroxide having pH of 10.5 was added to the beads and the suspension was incubated on a rotary shaker at 100 rpm, 25°C for 2 hours. The beads were collected by filtration on a Buchner funnel, washed thrice with 200 ml of distilled water and suspended in 50 ml of phosphate buffer solution having molarity of 0.05 and pH of 7.5. The derivatised macroporous beaded crosslinked copolymer beads were stored at 4°C as suspension in phosphate buffer solution having molarity of 0.05, pH of 7.5 and containing sodium azide at concentration of 0.2% w/v as

preservative.
Example 16
10 g of the macroporous beaded crosslinked copolymer prepared as described in Example 5 was suspended in 1000 ml of phosphate buffer solution having molarity of 0.1 and pH of 7.5. To this suspension 40.6 g of cephalexin dissolved in 50 ml of phosphate buffer solution having molarity of 0.1 and pH of 7.5 was added. The pH of the suspension was made to 8.5 by adding sodium hydroxide solution. The suspension was incubated on a rotary shaker at 100 rpm, 25°C for 48 hours. The supernatant was decanted. 100 ml of aqueous solution of sodium hydroxide having pH of 10.5 was added to the beads and the suspension was incubated on a rotary shaker at 100 rpm, 25°C for 2 hours. The beads were collected by filtration on a Buchner funnel, washed thrice with 200 ml of distilled water and suspended in 50 ml of phosphate buffer solution having molarity of 0.05 and pH of 7.5. The derivatised macroporous beaded crosslinked copolymer beads were stored at 4°C as suspension in phosphate buffer solution having molarity of 0.05, pH of 7.5 and containing sodium azide at concentration of 0.2% w/v as preservative.
Advantages of the invention
The invention provides a process for derivatisation of macroporous beaded crosslinked copolymers useful for purification of enzymes such as penicillinase by affinity chromatography. Preparation of the affinity chromatography supports by the process of this invention offers the following advantages:
i) The process does not involve the use of toxic chemicals like cyanogen bromide.
ii) The macroporous beaded crosslinked 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 operation procedure is simple and easy to scale-up.

We claim:
\. A process for the preparation of noveldesivatised macroporous beaded crosslinked copolymers which comprises suspending macroporous beaded crosslinked allyl glycidyl ether copolymers in an inorganic buffer solution having molarity in the range of 0.05 to 0.2 and pH in the range of 7 to 8, adding to the suspension the ligand solution, adjusting pH in the range of 8.0 to 9.0, shaking the mixture at a temperature in the range of 20 to 30°C for a period upto 48 hours, decanting the supernatant from the reaction mixture, suspending the ligand linked copolymer beads in a basic solution having pH in the range of 10.0 to 11, shaking the mixture at a temperature in the range of 20 to 30°C for a period upto 2 hours, collecting the beads by filtration and if required preserving the beaded macroporous beaded copolymers in a suitable buffer.
2. A process as claimed in claim 1, wherein, the ligand chosen for making the ligand
solution is an antibiotic such as ampicillin, amoxycillin, cephalexin, cephadroxyl,
cephalosporin C, 6-amino penicillanic acid, 7-amino des acetoxy
cephalosporanic acid and the ratio of the copolymer and ligand is in the range of
1:1 to 1:20.
3. A process as claimed in claims 1 and 2, wherein, the base solution used is such as
sodium hydroxide, potassium hydroxide, ammonium hydroxide.

4. A process as claimed in claims 1 to 3, wherein, the buffer solution used is phosphate, tris chloride and its molar concentration in the range of 0.05 to 0.2 and pH in the range of 7.0 to 8.0.

5.

A process as claimed in claims 1 to 4, wherein, the novel beaded crosslinked copolymer beads is preserved by suspending the beads in buffer solution such as phosphate, tris chloride having molarity in the range of 0.05 to 0.2 and pH in the range of 7 to 8.

6. A process for the preparation of novel desivatised macroprocess baded crosslimted
copolymer useful as macaffinity chromatography supports as herein described with reference to the Examples 7 to 16.

Documents:

1063-del-2000-abstract.pdf

1063-del-2000-claims.pdf

1063-del-2000-correspondence-others.pdf

1063-del-2000-correspondence-po.pdf

1063-del-2000-description (complete).pdf

1063-del-2000-form-1.pdf

1063-del-2000-form-19.pdf

1063-del-2000-form-2.pdf

1063-del-2000-form-3.pdf

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Patent Number

217160

Indian Patent Application Number

1063/DEL/2000

PG Journal Number

13/2008

Publication Date

31-Mar-2008

Grant Date

25-Mar-2008

Date of Filing

24-Nov-2000

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG, NEW DELHI-110 001, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	SURENDRA PONRATHNAM	NATIONAL CHEMICAL LABORATORY, PUNE 411 008, MAHARASHTRA
2	BHAGWAT SHAMRAO DESHPANDE	HINDUSTAN ANTIBIOTICS, PIMPRI, PUNE, MAHARASHTRA, 411018,
3	VARSA BHIKOBA GHADGE	NATIONAL CHEMICAL LABORATORY, PUNE 411 008, MAHARASHTRA
4	CHELANATTU KHIZHAKKE MADATH RAMAN RAJAN	NATIONAL CHEMICAL LABORATORY, PUNE 411 008, MAHARASHTRA
5	VAYALOMBRON KANDYAN SUDHAKARAN	HINDUSTAN ANTIBIOTICS, PIMPRI, PUNE, MAHARASHTRA, 411018,
6	JAIPRAKASH GANPATRAO SHEWALE	HINDUSTAN ANTIBIOTICS, PIMPRI, PUNE, MAHARASHTRA, 411018,

PCT International Classification Number

C08F 016/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA