GB2196007A

GB2196007A - Immobilised biologically active material

Info

Publication number: GB2196007A
Application number: GB08623664A
Authority: GB
Inventors: Terry Daniels; David Anthony Horton; Richard Stuart Williams; John Frederick Kennedy; Charles Andrew White; Andrew Slade
Original assignee: Bip Chemicals Ltd
Current assignee: Bip Chemicals Ltd
Priority date: 1986-10-02
Filing date: 1986-10-02
Publication date: 1988-04-20
Also published as: GB8623664D0

Abstract

Biologically active materials, including proteins (for example, enzymes), cells, antigens, antibodies, and hormones, are immobilised on a polymer support which comprises the reaction product of a polyepoxide (such as the diepoxide obtained from interaction of epichlorhydrin and 2,2-di[p-hydroxyphenyl]-propane) with a polyester which is obtained by the condensation of A. a hydroxy-terminated diester of a diol (eg ethylene glycol) and a dicarboxylic acid (eg succinic acid) with B. a polycarboxylic acid having at least 3 carboxylic acid groups (eg trimellitic acid) or an anhydride thereof. The reaction product may be activated (optionally after conversion of any carboxyl groups to acid chloride groups) by treatment first with 1,6-diaminohexane and then with glutaraldehyde.

Description

SPECIFICATION Polymer supports for the immobilisation of biologically active materials This invention relates to polymer supports for the immobilisation of biologically active materials, such as proteins (including glycoproteins), microbial cells, antigens, antibodies and hormones.

For certain purposes it is desirable to fix a biologically active material (for example, an enzyme) onto an inert, water-insoluble polymer as supporting framework, for which purpose cellulose, cross-linked agarose or dextran, and polyacrylamide are very commonly employed. Ordinarily the object of the procedure, which is usually known as 'immobilisation', is to obtain the active material in a form in which it can exert its influence on substances in a liquid phase brought into contact with it, but yet remain physically distinct and easily separable from that phase. This may be convenient for assay purposes, or-on a larger scale-to enable recovery of the active material, or re-use of it without having to go through expensive recovery procedures, or to enable continuous use with minimum loss.A well known example is the production of high fructose-content syrup from glucose syrup using the enzyme glucose isomerase immobilised with a cellulose ion-exchange polymer.

The present invention involves the use of certain polyester reaction products as polymer supports for the immobilisation of biologically active materials. These are the product of reaction of a polyepoxide with a polyester which is obtained by the condensation of A. a hydroxy-terminated diester of a diol (eg ethylene glycol) and a dicarboxylic acid (eg succinic acid), with B. a polycarboxylic acid having at least 3 carboxylic groups (eg trimellitic acid, that is to say benzene 1,2,4-tricarboxylic acid) or an anhydride thereof.

The diols and dicarboxylic acids for component A preferably contain from 2 to 8 carbon atoms in the molecule. Examples apart from the ethylene glycol and succinic acid already mentioned are: propane-1,2-diol, propane-1,3-diol, butanediol (1,2; 1,3; or 1.4), diethylene glycol and triethylene glycol; and maleic, glutaric, adipic, pimelic, terephthalic and isophthalic acids.

Component B preferably contains from 4 to 12 carbon atoms in the molecule. Examples in addition to the trimellitic acid already mentioned are: the anhydride of that acid; pyromellitic acid (benzene-1,2,4,5-tetracarboxylic acid) or the dianhydride thereof; hemimellitic acid (benzene1,2,3-tricarboxylic acid) or its anhydride; and prehnitic acid (benzene-1,2,3,5-tetracarboxylic acid) or its anhydride.

Preferably the molar proportion of components B:A in the polyester is not less than 1.1:1.

Usually a diepoxide will be employed as the polyepoxide, and a preferred diepoxide is that obtained by the condensation of epichlorhydrin with bisphenol A, that is, 2,2-di(p-hydroxyphenyl)propane, a commercially available material. The molar proportion of polyepoxide: polyester employed is such as to leave unreacted a significant proportion of the carboxylic acid groups of the polyester, so that these groups are left available for amide-formation by reaction with an amino-group of a polyamine in the known procedure for activation of a carboxyl-group-containing polymer prior to immobilisation of a biologically active material thereon. In its preferred version, as illustrated in Example 1 later in this specification, the activation procedure entails successive reactions, first with a diamine, then with a dialdehyde.

For use, the immediate product of the polyepoxide/polyester reaction requires to be heated above its temperature of formation (suitably for 1/2 hour at 180"C) in order to stabilise it against swelling by solvents. If the eventual polymer support/biologically active material complex is to be used in particulate form, as in a fluidised bed reactor or a packed column, stabilisation can be done at once. If, however, the polymer support is to take the form of a coating on a metal grid, or on the interior of a tube (eg a glass reaction coil) or on the exterior of column packing pieces (eg glass or ceramic bits), then the coating operation has first to be performed, as by dipping or spraying, whereupon the heating necessary to crosslink the deposited coating can be carried out.

If desired, the free carboxyl groups remaining in the product of the polyepoxide/polyester reaction product can be converted to acid halide groups (eg -COCI), as illustrated in Example 5 later.

The polymer supports of the invention can be used to immobilise a wide variety of biologically active materials, for example enzymes such as glucoamylase and a-amylase, or cells such as saccharomyces bayanas yeast, to give material of acceptable activity and operational stability (half-life).

The invention is further illustrated by the following Examples.

EXAMPLE 1 (i) The hydroxy-terminated diester

of ethylene glycol and maleic acid was prepared by heating ethylene glycol (2 moles) with maleic anhydride (1 mole) and distilling off the water formed on esterification. Heating was stopped after 98% of the water theoretically generable had been collected.

(ii) To the light-brown viscous residue of diester (204g; 1 mole) contained in a flask fitted with thermometer, agitator and reflux condenser there was added powdered pyromellitic dianhydride (2739; 1.25 mole)

and the mixture was heated from 20"C to 150"C during 1 hour. The dark-brown, which became brittle on cooling, had a measured acid value of 292 mg KOH/gram. The theoretical value for the polyester

is 355 mg KOH/gram.

(iii) The polyester (31.sag; 0.0137 mole) was ground to a powder passing a 500 ,um sieve and was mixed with the diepoxide commercially available under the trade mark Lopox 500 (8.39; 0.00914 mole; ground to pass a 500 ,um sieve) having the structure

and with colloidal zinc oxide (6.09; secondary crosslinking agent). The mixture was heated at 120-1500C for 10 minutes to melt-blend the components. The resulting mass was cooled, broken into small pieces, and then hammer-milled. The powder thus obtained was sieved through a 250 ,um sieve.

Part of the sieved material was heated at 180"C for 1/2 hour to crosslink it by reaction of the epoxy groups of the diepoxide with the carboxyl groups of the polyester; the remainder was used to coat a metal grid.

The crosslinked material This (a brittle solid) was ground to a fine powder and sieved through a 500 ,um sieve. The powder passing through the sieve was then activated by the known aminoalkane/glutaraldehyde procedure.

An aqueous solution of 1,6-diaminohexane (3%, 2.5 ml, 45"C) was added to the fine powder (500 mg). After 1/2 hour the material, now containing-CO.NH.(CH2)6NH2 groups where previously were COOH groups, was washed with distilled water (5 x 10 ml) and then treated with an aqueous solution of glutaraldehyde (1%, 2.5 ml, temperature 45"C, pH8.6) to converted CO.NH(CH2)6NH2 groups to--CO.NH(CH,),N CH(CH2)3CHO.

After 1/2 hour the product was washed with distilled water (4 x 10 ml) and then with sodium acetate buffer (0.02 M; pH4.5).

Formation of coating on metal grid The powder passing a 250 FLm sieve was applied to a degreased stainless steel grid (aperture 2x2mm, defined by strip 2mm wide) by means of an electrostatic spray gun of conventional type operating at -70kv. The coating thus formed was cured by heating the grid in an oven at 1 80C for 1/2 hour. The coating on the grid was activated by the procedure described above for powdered cross-linked polyester/polyepoxide reaction products, the grids being immersed successively in aqueous 1,6-diaminohexane solution and aqueous glutaraldehyde solution.

Coupling to a protein A solution of glucoamylase (10400 units, assessed according to the ability to hydrolyse starch, as in Cabral, Kennedy and Novais, Enzyme Microb Technol., 4 (1982) 337) in acetate buffer (0.02M, pH 4.5, room temperature; 4 ml for each 500 mg of powder or coating) in the presence of glucose (0.4 grams/100 ml) to protect the glucoamylase was added and allowed to remain in contact with the powder or coating for 2 hours at room temperature. The treated material was then washed free from unbound enzyme and assessed by standard methods for initial activity and operational stability. Results were: Powder. Initial activity = 5.2 units/gram Half-life = 122 hours Coated grid.Initial activity = 1.9 units/gram EXAMPLE 2 Following generally the procedure of Example 1 (it), the hydroxy-terminated diester of 1(i) was converted by treatment with 1.25 moles of powdered trimellitic anhydride

to a polyester

which was a brittle solid of measured acid value 240 mg KOH per gram. The theoretical value is 229.

The polyester was then reacted as in Example 1 (iii) with the diepoxide Lopox 500 to form a condensation product. Cross-linked powder and coated stainless steel grids were prepared from this, and these were activated and the activated forms were coupled to glucoamylase as in Example 1. Assay results were: Powder. Initial activity = 15 units/gram half-life = 156 hours Coated grid. Initial activity = 6.1 units/gram EXAMPLE 3 Following generally the procedure of Example 1, the following were prepared.

a. From succinic anhydride, the hydroxyterminated succinate diester corresponding to the maleate diester of Example 1(1).

b. From the product of a, by reaction with pyromellitic dianhydride (1.26 moles), a polyester of measured acid value 303 (theoretical value 354).

c. From the product of b (309) by reaction with the diepoxide Lopox 500 (7.99) in the presence of colloidal zinc oxide (6.0g), an epoxy condensate.

d. From the product of c, cross linked powder and stainless steel grids.

Activation of the product of d, followed by coupling with glucoamylase, gave products assaying thus: Powder. Initial activity = 13.3 units/gram Half-life = 133 hours Coated grid. Initial activity = 8.9 units/gram EXAMPLE 4 Following generally the procedure of Example 3, the following were prepared: a. From the hydroxy-terminated succinate diester and tri-mellitic anhydride, a polyester whose measured acid value was 204 (theoretical value, 227).

b. From the product of a and the diepoxide Lopox 500, in the presence of zinc oxide, an epoxy condensate.

c. From the product of b, crosslinked powder and coated metal grids.

Activation of the products of c, followed by coupling to glucoamylase, gave these assay results: Powder. Initial activity = 10.3 units/gram Half-life = 141 hours Coated grid. Initial activity = 7.7 units/gram EXAMPLE 5 a. The polyepoxide/polyester product of Example 1(iii) (containing free carboxylic acid groups) was suspended in hexane, and thionyl chloride was added in tenfold excess with stirring and moderate heating. After 2 hours the acid chloride formed was filtered off, washed with hexane to remove hydrogen chloride; and dried in a vacuum oven at 40"C. The product was sieved through a 500 ,um sieve, activated as previously described, and coupled to glucoamylase Assay results were: Initial activity = 4 units/gram Half-life = 31 hours B.A stainless steel grid was sprayed with the polyepoxide/polyester product of Example 1 (iii), stoved at 180"C for 1/2 hour, and then immersed for 24 hours in a solution of thionyl chloride (tenfold excess of theoretical) in hexane. After purification as in a, followed by activation and coupling with glucoamylase, an initial activity of 2.7 units/gram was observed.

EXAMPLE 6 Following the procedures of Example 5(a) and (b) but using instead the polyepoxide/polyester condensate of Example 2 for conversion to the acid chloride, powders and spray coated grids were prepared, activated, coupled with glucoamylase and assayed. Results were: Powder. Initial activity = 14.7 unit/gram Half life = 38 hours Coated grid. Initial activity = 9.9 units/gram EXAMPLE 7 Using the succinate-based polyepoxide/polyester condensates of Examples 3 and 4 for conversion to the acid chloride, powders and spray-coated grids were prepared, activated, coupled with glucoamylase and assayed. Results were: Product derived from Example 3 Powder. Initial activity = 9.7 unit/gram Half life = 34 hours Coated grid. Initial activity = 6.6 units/gram Product derived from Example 4 Powder. Initial activity = 2.4 units/gram Half life = 38 hours Coated grid. Initial activity = 1.3 units/gram

Claims

1. Polymer support for the immobilisation of biologically active material, said polymer being the product of reaction of a polyepoxide with a polyester which is obtained by the condensation of A. a hydroxy-terminated diester of a diol and a dicarboxylic acid with B. a polycarboxylic acid having at least 3 carboxylic acid groups, or an anhydride thereof.

2. Polymer support according to Claim 1, in which the diol and dicarboxylic acid components of A each contains from 2 to 8 carbon atoms.

3. Polymer support according to Claim 2, in which the diol is ethylene glycol, diethylene glycol, triethylene glycol, a propane diol or a butane diol.

4. Polymer support according to Claim 2 in which the dicarboxylic acid is maleic, succinic, glutaric, adipic, pimelic, terephthalic or isophthalic acid.

5. Polymer support according to any of Claims 1 to 4, in which the polycarboxylic acid or anhydride forming component B contains from 4 to 12 carbon atoms.

6. Polymer support according to Claim 5, in which component B is trimellitic acid, pyromellitic acid, or hemimellitic acid, or anhydride of such acid.

7. Polymer support according to any of Claims 1 to 6, in which the molar proportion of components B:A is not less than 1.1:1.

8. Polymer support according to any of Claims 1 to 7, in which the polyepoxide is the reaction product of epichlorphydrin with 2,2-di-(p-hydroxyphenyl) propane.

9. Polymer support according. to any of Claims 1 to 8, in the form of the acid chloride.

10. Polymer support according to any of Claims 1 to 9, in the activated form resulting from successive reactions with a diamine and a dialdehyde.

11. Biologically active material immobilised on a polymer support and in accordance with any of Claims 1-10, substantially as described with reference to the Examples herein.

11. Polymer support according to any of Claims 1-10, substantially as described with reference to the Examples herein.

12. Biologicallly active material immobilised on a polymer support according to any preceding claim.

CLAIMS Amendments to the claims have been filed, and have the following effect: Claims 1 to 12 above have been deleted or textually amended.

New or textually amended claims have been filed as follows:

1. Biologically active material immobilised on a polymer support, said polymer being the product of reaction of a polyepoxide with a polyester which is obtained by the condensation of A. a hydroxy-terminated diester of a diol and a dicarboxylic acid with B. a polycarboxylic. acid having at least 3 carboxylic acid groups, or an an hydride thereof.

2. Material according to Claim 1, in which the diol and dicarboxylic acid components of A each contains from 2 to 8 carbon atoms.

3. Material according to Claim 2, in which the diol is ethylene glycol, diethylene glycol, triethylene glycol, a propane diol or a butane diol.

4. Material according to Claim 2, in which the dicarboxylic acid is maleic, succinic, glutaric, adipic, pimelic, terephthalic or isophthalic acid.

5. Material according to any of Claims 1 to 4, in which the polycarboxylic acid or anhydride forming component B contains from 4 to 12 carbon atoms.

6. Material according to Claim 5, in which component B is trimellitic acid, pyromellitic acid, or hemimellitic acid, or anhydride of such acid.

7. Material according to any of Claims 1 to 6, in which the molar proportion of components B:A is not less than 1.1:1.

8. Material according to any of Claims 1 to 7, in which the polyepoxide is the reaction product of epichlorhydrin with 2,2-di(p-hydroxyphenyl) propane.

9. Material according to any of Claims 1 to 8, the polymer being in the form of the acid chloride.

10. Material according to any of Claims 1 to 9, the polymer being in the activated form resulting from successive reactions with a diamine and a dialdehyde.