Title of Invention	A PROCESS FOR THE PREPARATION OF IMPROVED POLYMERIC SUPPORT USEFUL FOR PURIFICATION AND IMMOBILIZATION OF PROTEINS
Abstract	A process for the preparation of improved polymer support which comprises, contacting in an inert atmosphere, a macroporous polymer containing hydroxy groups with a isocyanate in a protic solvent, in the presence of a catalyst and stirring the mixture at an rpm in the range of 150 to 200, at a temperature in the range of 25 to 50°C, for a period ranging from 12 to 48 hours, washing the product with the aprotic solvent to remove the unreacted isocyanale, separating the solids by conventional methods, washing with an alkanol and drying the product under reduced pressure at a temperature in the range of 40 to 60°C to get macroporous beads..

Title of Invention

A PROCESS FOR THE PREPARATION OF IMPROVED POLYMERIC SUPPORT USEFUL FOR PURIFICATION AND IMMOBILIZATION OF PROTEINS

Abstract

A process for the preparation of improved polymer support which comprises, contacting in an inert atmosphere, a macroporous polymer containing hydroxy groups with a isocyanate in a protic solvent, in the presence of a catalyst and stirring the mixture at an rpm in the range of 150 to 200, at a temperature in the range of 25 to 50°C, for a period ranging from 12 to 48 hours, washing the product with the aprotic solvent to remove the unreacted isocyanale, separating the solids by conventional methods, washing with an alkanol and drying the product under reduced pressure at a temperature in the range of 40 to 60°C to get macroporous beads..

Full Text	This invention relates to a process for the preparation of 'improved polymeric supports useful lor the purification and immobili/alion of proteins. More particularly, it relates to a process for the preparation of macroporous copolymers with isocyanate functional groups prepared by the modification of macroporous polymers containing hydroxy functional groups. The macroporous copolymers with highly reactive isocyanate functional groups prepared by the process of this invention can be used to immobilize commercially important enzymes under ambient conditions. Alternatively, the macroporous copolymers with isocyanate functional groups prepared by the process of this invention can be derivatised to attach suitable ligands that have affinity for proteins. These modified polymer supports can then be used in the purification of proteins such as cyclodcxlrin glycosyl transferase, amylase, lactase by using conventional techniques such as expanded bed column chromatography or packed bed chromatography. Purification of proteins involves a series of steps - such as clarification, concentration and separation. The operational difficulties encountered are high viscosity of the crude fermentation broth, particulate matter fouling of the membranes and loss encountered in each step in handling etc. affects the high cost of process economics. Conventional purification of proteins involves the use of suitable support as alumina, polymer or membranes. The polymer support use involves the additional step of crosslinking the base polymer with a crosslinking agent such as gluraraldchyde elc. The affinity of the proteins to the support varies and affects the process economies to a great extent. The polymeric supports prepared as per the process of this invention can be suitably derivnlizcd to have affinity towards a particular protein and used directly for the purification of the protein. The primary requirements for such applications are - 1. The support material should contain functional groups that have high affinity/ion exchange capacity to selectively bind proteins from mixed stream of crude fermentation broth feed. 2. The support material should have sufficient mechanical strength so that during the processing stage, there is no damage. 3. The support materials have densities high enough to allow stable bed expansion during operation. 4. The matrix material should be stable to variation in operating pH range during use. The main object of the present invention is to provide a process for the preparation of improved polymeric macroporous support useful for purification and immobililzation of proteins. Yet another object of the present invention is to provide a process for the preparation of macroporpous polymer support with isocyanale functional groups that has sufficient mechanical strength and resistance to variations in pH. Still another object of the present invention is to provide a process for the preparation of macroporous polymer matrix with multifunctional groups that can be used repeated for purification of enzyme by suitable derivatization method to effect purification of the proteins using the affinity of the proteins to the support. Accordingly, the present invention provides a process for the preparation of improved polymer support .characterized in that contacting in an inert atmosphere, a ^ macroporous polymer containing hydroxy groups with a isocyanate in a protic solvent, in the presence of a catalyst and stirring the mixture at an rpm in the range of 150 to 200, at a temperature in the range of 25 to 50°C, for a period ranging from 12 to 48 hours, washing the product with the aprotic solvent to remove the unreacted isocyanate, separating the solids by conventional methods, washing with an alkanol and drying the obtained product under reduced pressure at a temperature in the range of 40 to 60°C. In an embodiment of the invention, the macroporous polymer containing hydroxyl groups may be a copolymcr of hydroxy ethyl methacrylate - ethylene glycol dimethacrylate prepared as described and claimed in our copending application no. In another embodiment of the invention, the inert gas used is such as nitrogen orargon. In yet another embodiment of the present invention, the disocyanate used may be such as toluene isocyanate, polymeric methylene isocyanatc, isophornonc isocyanate In still another embodiment of the present invention, the aprolic solvent used may be such as 1,4-dioxan, dimethy sulfoxide, dimethyl formamide, dimethyl acetamide. In still another embodiment of the invention, the catalyst used may be such as dibutyl tin laurate, cobalt naphthanate. In yet another embodiment of the invention, the alkanol used may be such as isopropanol, methanol, ethanol. The process of the present invention is described with reference to following examples, which are illustrative only and should not be construed to limit the scope of this invention in any manner. As an illustration, the method for the preparation of macroporous polymer is given in Example 1 which describes the procedure for the preparation of the macroporous polymer as described and claimed in our copending application number EXAMPLE 1 21.8 ml of 2-hydroxy ethyl mclliacrylate, 8.2 nil of clhylcnc glycol dimethacrylate, 45.0 ml of cyclohexanol were stirred with 250 ml of distilled water and polymerized using 2.5 grams of polyvinyl pyrroliclone and 1.2 grams of azo bis isobutyronitrile for 3 hours at 70°C. The rpm of the reaction was maintained at 300. The beads generated at the end of the reaction were washed thoroughly with water to remove traces of unreacted monomers and methanol before drying at 40C. The beads were seived before use. The yield obtained was 28 grams. EX AMPLE 2 In an inert atmosphere of nitrogen, 1 gram of macroporous polymer (HEMA- EGDM), 8.75 ml of Toluene isocyanate and 80 ml 1,4 dioxan were mixed. 10 µL of dibutyltin dilaurate was added and the reactants were gently stirred at a rpm of 200 and maintained at a temperature of 25°C for 48 hours. The reactants were decanted, washed with dioxan and the product isolated by filtration, washing with methanol, followed by drying at 40°C. EXAMPLE 3 In an inert atmosphere of nitrogen, 1 gram of macroporous polymer (HEMA-EGDM), 6.0 ml of hexamethylene isocyanate and 80 ml 1,4-dioxan were mixed. 15 µL of dibutyltin dilaurate was added and the reactants were gently stirred at an rpm of 200 and maintained at a temperature of 30UC for 40 hours. The reactants were decanted, washed with 1.4-dioxan and the product isolated by filtration, washing with methanol, followed by drying at 50°C. Example 4 In an inert atmosphere of argon, 1 gram of macroporous polymer (HEMA-EGDM), 2.5 ml of polymeric methylenc isocyanate (PMDI) and 80 ml dimethyl sulfoxide were mixed. 10 µL of dibutyltin dilaurate was added and the reactants were gently stirred at an rpm of 200 and maintained at a temperature of 25°C for 48 hours. The reactanls were decanted, washed with dimethyl sulfoxide and the product isolated by filtration, washing with ethanol, followed by drying at 60°C. EXAMPLES In an inert atmosphere of nitrogen, 1 gram of macroporous polymer (ITEMA- EGDM), 8.8 ml of Isophornone isocyanale (IPDI) and 80 ml dimethyl formamide were mixed. 12 u.L of dibutyltin dilaurate was added and the reactants were gently stirred at an rpm of 200 and maintained at a temperature of 25°C for 48 hours. The reactants were decanted, washed with dimethyl formamide and the product isolated by filtration, washing with isopropanol, followed by drying at 60"C. Example 6 In an inert atmosphere of argon, 1 gram of macroporous polymer (HEMA-EGDM), 8.75 ml of Tolune isocyanate and 80 ml 1,4-dioxan were mixed. 15 µL of cobalt naphthanate was added and the reaelants were gently stirred at an rpm of 200 and maintained at a temperature of 25°C for 48 hours. The reactants were decanted, washed with 1,4-dioxan and the product isolated by filtration, washing with melhanol, followed by drying at 60°C. EXAMPLE7 In an inert atmosphere of nitrogen, 1 gram of macroporous polymer (HEMA-EGDM), 8.75 ml of Toluene isocyanate and 80 ml dimethyl sulfoxide were mixed. 10 µL of dibutyltin dilaurate was added and the reactants were gently stirred at an rpm of 200 and maintained at a temperature of 25°C for 48 hours. The reactants were decanted, washed with dimethyl sulfoxide and the product isolated by filtration, washing with melhanol, followed by drying at 60°C. EXAMPLE 8 In an inert atmosphere of argon, 1 gram of macroporous polymer (HEMA-EGDM), 8.75 ml of Toluene isocyanate and 80 ml dimethyl acetamide were mixed. 12 µL of dibutyltin dilaurate was then added and the reactants were gently stirred at a rpm of 200 and maintained at a temperature of 50°C for 12 hours. The reactanls were decanted, washed with dimethyl acetamide and the product isolated by filtration, washing with melhanol, followed by drying at 6()"C. EXAMPLE 9 In an inert atmosphere of nitrogen, 1 gram of macroporous polymer (HEMA-EGDM), 8.75 ml of toluene isocyanate and 80 ml dimethyl sulfoxide were mixed. 10 µL of dibutyltin dilaurate was added and the reactants were gently stirred at an rpm of 200 and maintained at a temperature of 45°C for 24 hours. The reactants were decanted, washed with dimethyl sulfoxide and the product isolated by filtration, washing with melhanol, followed by drying at 60°C. Advantages of the present invention: 1. The new improved macroporous copolymers with isocyanate functional groups synthesised by the process of this invention are not biodegradable ensuring enhanced shelf life. 2. The new improved macroporous copolymers with isocyanate functional groups obtained by the process of this invention can be used for the purification of proteins after suitable derivatization. 3. The process of this invention is amenable for scale up operation. 4. The new improved macroporous copolymers with isocyanale functional groups prepared by the process of this invention have adequate mechanical strength We Claim: 1. A process for the preparation of improved polymer support which comprises, contacting in an inert atmosphere, a macroporous polymer containing hydroxy groups with a isocyanate in a protic solvent, in the presence of a catalyst and stirring the mixture at an rpm in the range of 150 to 200, at a temperature in the range of 25 to 50°C, for a period ranging from 12 to 48 hours, washing the product with the aprotic solvent to remove the unreacted isocyanate, separating the solids by conventional methods, washing with an alkanol and drying the product under reduced pressure at a temperature in the range of 40 to 60°C to get macroporous beads.. 2. A process as claimed in claim 1 wherein, the macroporous polymer containing hydroxy group used may be a copolymer of hydroxy ethyl methacrylale'- ethylene glycol dimethacrylate prepared as described and claimed in our copending application number 375/DEL/94. 3. A process as claimed in claims 1 and 2 wherein, the inert gas used is nitrogen or argon. 4. A process as claimed in claims 1 to 3, wherein, the aprotic solvent used is such as 1,4-dioxan, dimethy sulfoxide, dimethyl formamide, dimethyl acetamide. 5. A process as claimed in claims 1 to 4, wherein, the catalyst used is such as dibutyl tin dilaurate, cobalt naphthanate. 6. A process as claimed in claims 1 to 5, wherein, the alkanol used is such as isopropanol, methanol, ethanol. 7. A process for the preparation of improved polymeric supports useful for the purification and immobilization of proteins as substantially described with reference to the examples.

Full Text

This invention relates to a process for the preparation of 'improved polymeric supports useful lor the purification and immobili/alion of proteins. More particularly, it relates to a process for the preparation of macroporous copolymers with isocyanate functional groups prepared by the modification of macroporous polymers containing hydroxy functional groups. The macroporous copolymers with highly reactive isocyanate functional groups prepared by the process of this invention can be used to immobilize commercially important enzymes under ambient conditions.
Alternatively, the macroporous copolymers with isocyanate functional groups prepared by the process of this invention can be derivatised to attach suitable ligands that have affinity for proteins. These modified polymer supports can then be used in the purification of proteins such as cyclodcxlrin glycosyl transferase, amylase, lactase by using conventional techniques such as expanded bed column chromatography or packed bed chromatography.
Purification of proteins involves a series of steps - such as clarification, concentration and separation. The operational difficulties encountered are high viscosity of the crude fermentation broth, particulate matter fouling of the membranes and loss encountered in each step in handling etc. affects the high cost of process economics.
Conventional purification of proteins involves the use of suitable support as alumina, polymer or membranes. The polymer support use involves the additional step of crosslinking the base polymer with a crosslinking agent such as

gluraraldchyde elc. The affinity of the proteins to the support varies and affects the process economies to a great extent.
The polymeric supports prepared as per the process of this invention can be suitably derivnlizcd to have affinity towards a particular protein and used directly for the purification of the protein.
The primary requirements for such applications are -
1. The support material should contain functional groups that have high
affinity/ion exchange capacity to selectively bind proteins from
mixed stream of crude fermentation broth feed.
2. The support material should have sufficient mechanical strength so
that during the processing stage, there is no damage.
3. The support materials have densities high enough to allow stable bed
expansion during operation.
4. The matrix material should be stable to variation in operating pH
range during use.
The main object of the present invention is to provide a process for the preparation of improved polymeric macroporous support useful for purification and immobililzation of proteins.

Yet another object of the present invention is to provide a process for the preparation of macroporpous polymer support with isocyanale functional groups that has sufficient mechanical strength and resistance to variations in pH.
Still another object of the present invention is to provide a process for the preparation of macroporous polymer matrix with multifunctional groups that can be used repeated for purification of enzyme by suitable derivatization method to effect purification of the proteins using the affinity of the proteins to the support.
Accordingly, the present invention provides a process for the preparation of
improved polymer support .characterized in that contacting in an inert atmosphere, a
^
macroporous polymer containing hydroxy groups with a isocyanate in a protic solvent, in the presence of a catalyst and stirring the mixture at an rpm in the range of 150 to 200, at a temperature in the range of 25 to 50°C, for a period ranging from 12 to 48 hours, washing the product with the aprotic solvent to remove the unreacted

isocyanate, separating the solids by conventional methods, washing with an alkanol
and drying the obtained product under reduced pressure at a temperature in the range of 40 to

60°C.
In an embodiment of the invention, the macroporous polymer containing hydroxyl groups may be a copolymcr of hydroxy ethyl methacrylate - ethylene glycol dimethacrylate prepared as described and claimed in our copending application no.

In another embodiment of the invention, the inert gas used is such as nitrogen
orargon.

In yet another embodiment of the present invention, the disocyanate used
may be such as toluene isocyanate, polymeric methylene isocyanatc, isophornonc isocyanate
In still another embodiment of the present invention, the aprolic solvent used may be such as 1,4-dioxan, dimethy sulfoxide, dimethyl formamide, dimethyl acetamide.
In still another embodiment of the invention, the catalyst used may be such as dibutyl tin laurate, cobalt naphthanate.
In yet another embodiment of the invention, the alkanol used may be such as isopropanol, methanol, ethanol.
The process of the present invention is described with reference to following examples, which are illustrative only and should not be construed to limit the scope of this invention in any manner.
As an illustration, the method for the preparation of macroporous polymer is given in Example 1 which describes the procedure for the preparation of the macroporous polymer as described and claimed in our copending application number

EXAMPLE 1
21.8 ml of 2-hydroxy ethyl mclliacrylate, 8.2 nil of clhylcnc glycol dimethacrylate, 45.0 ml of cyclohexanol were stirred with 250 ml of distilled water and polymerized using 2.5 grams of polyvinyl pyrroliclone and 1.2 grams of azo bis isobutyronitrile for 3 hours at 70°C. The rpm of the reaction was maintained at 300. The beads generated at the end of the reaction were washed thoroughly with water to
remove traces of unreacted monomers and methanol before drying at 40C. The beads
were seived before use. The yield obtained was 28 grams.
EX AMPLE 2
In an inert atmosphere of nitrogen, 1 gram of macroporous polymer (HEMA-
EGDM), 8.75 ml of Toluene isocyanate and 80 ml 1,4 dioxan were mixed. 10 µL of dibutyltin dilaurate was added and the reactants were gently stirred at a rpm of 200 and maintained at a temperature of 25°C for 48 hours. The reactants were decanted, washed with dioxan and the product isolated by filtration, washing with methanol, followed by drying at 40°C.
EXAMPLE 3
In an inert atmosphere of nitrogen, 1 gram of macroporous polymer (HEMA-EGDM), 6.0 ml of hexamethylene isocyanate and 80 ml 1,4-dioxan were mixed. 15 µL of dibutyltin dilaurate was added and the reactants were gently stirred at an rpm of 200 and maintained at a temperature of 30UC for 40 hours. The reactants were decanted, washed with 1.4-dioxan and the product isolated by filtration, washing with methanol, followed by drying at 50°C.

Example 4
In an inert atmosphere of argon, 1 gram of macroporous polymer (HEMA-EGDM), 2.5 ml of polymeric methylenc isocyanate (PMDI) and 80 ml dimethyl sulfoxide were mixed. 10 µL of dibutyltin dilaurate was added and the reactants were gently stirred at an rpm of 200 and maintained at a temperature of 25°C for 48 hours. The reactanls were decanted, washed with dimethyl sulfoxide and the product isolated by filtration, washing with ethanol, followed by drying at 60°C.
EXAMPLES
In an inert atmosphere of nitrogen, 1 gram of macroporous polymer (ITEMA-
EGDM), 8.8 ml of Isophornone isocyanale (IPDI) and 80 ml dimethyl formamide were mixed. 12 u.L of dibutyltin dilaurate was added and the reactants were gently stirred at an rpm of 200 and maintained at a temperature of 25°C for 48 hours. The reactants were decanted, washed with dimethyl formamide and the product isolated by filtration, washing with isopropanol, followed by drying at 60"C.
Example 6
In an inert atmosphere of argon, 1 gram of macroporous polymer (HEMA-EGDM), 8.75 ml of Tolune isocyanate and 80 ml 1,4-dioxan were mixed. 15 µL of cobalt naphthanate was added and the reaelants were gently stirred at an rpm of 200 and maintained at a temperature of 25°C for 48 hours. The reactants were decanted, washed with 1,4-dioxan and the product isolated by filtration, washing with melhanol, followed by drying at 60°C.

EXAMPLE7
In an inert atmosphere of nitrogen, 1 gram of macroporous polymer (HEMA-EGDM), 8.75 ml of Toluene isocyanate and 80 ml dimethyl sulfoxide were mixed. 10 µL of dibutyltin dilaurate was added and the reactants were gently stirred at an rpm of 200 and maintained at a temperature of 25°C for 48 hours. The reactants were decanted, washed with dimethyl sulfoxide and the product isolated by filtration, washing with melhanol, followed by drying at 60°C.
EXAMPLE 8
In an inert atmosphere of argon, 1 gram of macroporous polymer (HEMA-EGDM), 8.75 ml of Toluene isocyanate and 80 ml dimethyl acetamide were mixed. 12 µL of dibutyltin dilaurate was then added and the reactants were gently stirred at a rpm of 200 and maintained at a temperature of 50°C for 12 hours. The reactanls were decanted, washed with dimethyl acetamide and the product isolated by filtration, washing with melhanol, followed by drying at 6()"C.
EXAMPLE 9
In an inert atmosphere of nitrogen, 1 gram of macroporous polymer (HEMA-EGDM), 8.75 ml of toluene isocyanate and 80 ml dimethyl sulfoxide were mixed. 10 µL of dibutyltin dilaurate was added and the reactants were gently stirred at an rpm of 200 and maintained at a temperature of 45°C for 24 hours. The reactants were decanted, washed with dimethyl sulfoxide and the product isolated by filtration, washing with melhanol, followed by drying at 60°C.

Advantages of the present invention:
1. The new improved macroporous copolymers with isocyanate functional groups
synthesised by the process of this invention are not biodegradable ensuring
enhanced shelf life.
2. The new improved macroporous copolymers with isocyanate functional groups
obtained by the process of this invention can be used for the purification of
proteins after suitable derivatization.
3. The process of this invention is amenable for scale up operation.
4. The new improved macroporous copolymers with isocyanale functional groups
prepared by the process of this invention have adequate mechanical strength

We Claim:
1. A process for the preparation of improved polymer support which comprises,
contacting in an inert atmosphere, a macroporous polymer containing hydroxy
groups with a isocyanate in a protic solvent, in the presence of a catalyst and
stirring the mixture at an rpm in the range of 150 to 200, at a temperature in the
range of 25 to 50°C, for a period ranging from 12 to 48 hours, washing the
product with the aprotic solvent to remove the unreacted isocyanate, separating
the solids by conventional methods, washing with an alkanol and drying the
product under reduced pressure at a temperature in the range of 40 to 60°C to get
macroporous beads..
2. A process as claimed in claim 1 wherein, the macroporous polymer containing
hydroxy group used may be a copolymer of hydroxy ethyl methacrylale'- ethylene
glycol dimethacrylate prepared as described and claimed in our copending
application number 375/DEL/94.

3. A process as claimed in claims 1 and 2 wherein, the inert gas used is nitrogen or
argon.
4. A process as claimed in claims 1 to 3, wherein, the aprotic solvent used is such as
1,4-dioxan, dimethy sulfoxide, dimethyl formamide, dimethyl acetamide.
5. A process as claimed in claims 1 to 4, wherein, the catalyst used is such as dibutyl
tin dilaurate, cobalt naphthanate.

6. A process as claimed in claims 1 to 5, wherein, the alkanol used is such as
isopropanol, methanol, ethanol.
7. A process for the preparation of improved polymeric supports useful for the
purification and immobilization of proteins as substantially described with
reference to the examples.

Documents:

1034-del-2000-abstract.pdf

1034-del-2000-claims.pdf

1034-del-2000-correspondence-others.pdf

1034-del-2000-correspondence-po.pdf

1034-del-2000-description (complete).pdf

1034-del-2000-form-1.pdf

1034-del-2000-form-19.pdf

1034-del-2000-form-2.pdf

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Patent Number

242318

Indian Patent Application Number

1034/DEL/2000

PG Journal Number

35/2010

Publication Date

27-Aug-2010

Grant Date

23-Aug-2010

Date of Filing

17-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	DEBAPRASD NAYAK	NATIONAL CHEMICAL LABORATORY, PUNE-411008, MAHARASHTRA, INDIA.
2	OMPRAKASH SRINIVAS YEMUL	NATIONAL CHEMICAL LABORATORY, PUNE-411008, MAHARASHTRA, INDIA.
3	ANANT PATKAR	NATIONAL CHEMICAL LABORATORY, PUNE-411008, MAHARASHTRA, INDIA.
4	SURENDRA PONRATHNAM	NATIONAL CHEMICAL LABORATORY, PUNE-411008, MAHARASHTRA, INDIA.
5	CHELANATTU KHIZHAKKE MADATH RAMAN RAJAN	NATIONAL CHEMICAL LABORATORY, PUNE-411008, MAHARASHTRA, INDIA.
6	ARIKA KOTHA	NATIONAL CHEMICAL LABORATORY, PUNE-411008, MAHARASHTRA, INDIA

PCT International Classification Number

C08F 120/70

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA