GB1571182A

GB1571182A - Carrier-bound biologically active substances and process for their manufacture

Info

Publication number: GB1571182A
Application number: GB48603/76A
Authority: GB
Original assignee: Behringwerke AG
Current assignee: Siemens Healthcare Diagnostics GmbH Germany
Priority date: 1975-11-22
Filing date: 1976-11-22
Publication date: 1980-07-09
Also published as: LU76243A1; ZA766942B; AU513890B2; SE7612998L; NL7612771A; IL50940A; AT358275B; DK522976A; IT1121742B; CA1069070A; AU1981776A; BE848601A; SE430066B; DE2552510B2; FR2332287A1; IL50940A0; JPS5265596A; DE2552510A1; ATA863176A; JPS6048524B2

Abstract

A compound made from a copolymer of vinylene glycol and a biologically active substance chemically bonded thereto is prepared by reacting a copolymer of vinylene carbonate with a biologically active substance. The cyclocarbonate groups are converted into hydroxyl groups before or after the reaction. The novel compounds are used for affinity chromatography, for carrying out immune reactions or for carrying out enzyme reactions.

Description

(54) CARRIER-BOUND BIOLOGICALLY ACTIVE SUBSTANCES AND PROCESS FOR THEIR MANUFACTURE (71) We, BEHRINGWERKE AKTIENGESELLSCHAFT, a Body Corporate organised according to the laws of the Federal Republic of Germany, of Marburg/Lahn, Federal Republic of Germany, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- The present invention relates to carrier-bound biologically active substances and to a process for their manufacture.

In recent years, in biochemistry and in biochemical methods of production there has been used, to an increasing extent, a technique which utilises the affinity of carrier (or matrix)-bound, biologically active substances for selective reactions.

One example of this technique is the formation of a specific complex of a carrierbound substance with a second substance present in admixture with other materials, whereby this substance can be eliminated from the mixture and if desired, subsequently isolated from the complex by desorption.

Another application is the use of carrier-bound enzymes, which have the advantages over free enzymes that they enable a substrate to be treated in a continuous process and that the reaction products obtained are free from enzymes.

Furthermore, in biochemical, enzymatic analyses, carrier-bound water-insoluble enzymes can be used repeatedly as reactants.

Since enzymes have an affinity not only for their substrates, but also for their specific inhibitors, the carrier-bound enzymes have proved to be especially useful for obtaining enzyme inhibitors, and conversely, inhibitors bound to waterinsoluble matrices enable the corresponding enzymes to be obtained.

When a so-called immune adsorbent, i.e. an antigen or antibody is bound to a water-insoluble matrix, the corresponding antibody or antigen can be isolated.

These techniques are called affinity chromatography.

Biologically active substances may be naturally occurring or synthetic, that is to say, non-naturally occurring, substances which act in vivo and/or in vitro, and include enzymes, enzyme activators, enzyme inhibitors, antigens and antibodies, vitamins and hormones. These biologically active substances both naturally occurring and non-naturally occurring, which are the active components of the carrier-bound biologically active compounds, are sometimes called "effectors" herein.

Most of the effectors so far described are essentially more stable when carrierbound than when in solution.

In general, suitable carrier materials or matrices are substances that are insoluble in aqueous systems and exhibit a non-specific adsorption that is as low as possible. To this effect, hydrophobic, hydrophilic and ionic interactions between the matrix and the substance with which the effector reacts should be as few as possible. Furthermore, bonding of the effector to substances which are not specific reactants thereof should also be excluded.

The matrices previously proposed as carriers for biologically active substances are on the one hand those which bind the effectors by physical adsorption, for example, polystyrene, active charcoal and glass beads, and, on the other hand, those which form a covalent bond with the effectors, for example, vinyl polymers, both homo- and copolymers, for example, polyacrylic acid, polyacrylic acid amides, amino-, carboxy- or sulfonyl-substituted polystyrene, cellulose and its derivatives, and natural and synthetic polypeptides and proteins. Because of the equilibrated interaction between the matrix and the effector, carbohydrates, especially cellulose, dextran, starch, agar and their derivatives, are widely used as matrices in aqueous systems, even though the carboxyl groups frequently contained in these naturally occurring substances are considered to be troublesome because of their non-specific affinities. Moreover, these substances have a relatively poor thermal and chemical stability.

Many of these disadvantages could be eliminated by using polyvinylene glycol, a synthetic polymer, as a matrix. Polyvinylene glycol, also called polyhydroxymethylene, is prepared by acid or basic hydrolysis from polyvinylene carbonate. Each carbon atom bears a hydroxyl group and a hydrogen atom. The uninterrupted -C-C-bond gives the carrier an especially high stability.

The present invention is based on the observation that vinylene glycol copolymers may advantageously be used as the carrier matrix.

The present invention accordingly provides a biologically active compound bound to a copolymer comprising vinylene glycol units, the copolymer preferably being water-insoluble.

The carrier matrix is a polyvinylene glycol copolymer preferably containing up to 45%, advantageously from 5 to 20%, of a comonomer. The biologically active substance is, for example, an enzyme, an enzyme activator or enzyme inhibitor, an antigen or antibody, another plasma protein, a blood group substance, a phythaemagglutinin, an antibiotic, a vitamin or hormone, a peptide or amino acid, or a non-naturally occurring biologically active substance.

The polymer carrier and the biologically active substance may be bound to each other either directly or via a side chain (a spacer), by a covalent bond.

It is advantageous to bind the biologically active compound via a side chain (spacer) when the molecular weights of its affinity partner is very different, or when in a biospecific process proteins having a very high molecular weight or those comprising several sub-units take part. The spacers or side-chains may be included in the polymer matrix by polymerisation, by addition or incorporation (linkage of a bi- or polyfunctional compound, for example, a tripeptide, to the matrix) or by incorporation of functional groups and stepwise prolongation.

By incorporating suitable comonomer units into polyvinylene carbonate by polymerization, the physical and chemical properties of the resulting polymer can be varied to a large extent, for example, the hydrophilic property or "swelling" of polyvinylene glycol can be modified by the incorporation of a hydrophobic monomer, for example, ethylene, vinyl chloride or styrene. By copolymerization with vinyl acetate monomers, the resulting polymer contains vinyl alcohol groups, which increase the swelling property in an aqueous medium.

Other comonomers, for example, diethylene glycol divinyl ether and divinyl benzene, enable cross-linking to occur and thus improve the mechanical properties of the carrier. By incorporating comonomers having electrophilic functions, for example, oxirane groups (epoxy groups) from vinylglycidyl ether comonomers, the matrix can be reacted directly with nucleophilic functions, for example, (-NH2 and -OH). Carboxyl groups can be introduced, for example, by means of acrylic acid ester comonomers which are hydrolysed when the copolymerization is complete. The introduction of -NH2 or -COOH groups enables activation by means of, for example, carbodiimides, isoxazolium salts and glutaraldehyde.

Copolymers of vinylene glycol and vinyl alcohol have a higher specific surface and so an increased binding capacity, for example, for proteins, on the surface of a polymer powder compared with a vinylene glycol homopolymer.

The invention also provides a process for the manufacture of a carrier-bound biologically active substance, which comprises A) reacting a copolymer of vinylene carbonate with a biologically active substance and converting the cyclocarbonate groups still present into hydroxy groups, or B) converting the cyclocarbonate groups of a copolymer of vinylene carbonate into hydroxy groups and a) reacting electrophilic groups contained in or introduced into the copolymer with a biologically active substance or b) reacting these hydroxy groups 1) either first with a compound containing an electrophilic group and then with a biologically active substance, 2) or directly with a biologically active substance carrying electrophilic groups.

The copolymer is preferably formed by co-polymerising vinylene carbonate and one or more of the following comonomers:

in which R1 represents a hydrogen atom or a methyl or ethyl group, R3 represents a methyl, ethyl or propyl group, and n is an integer of from 1 to 4.

In the polymerisation, there is preferably used up to 45% of the comonomer(s), advantageously from 5 to 20% thereof.

Generally, the chemical coupling of a biologically active substance to the carrier matrix is simple when the one component carries a nucleophilic function and the other carries an electrophilic function. Reactive, nucleophilic functions are especially amino, sulfhydryl and hydroxyl groups, which are generally already contained either in the matrix or in the reactant. Electrophilic functions generally have to be introduced. They may be introduced into the biologically active substance, but as many of these are proteins or peptides, they already contain at least one nucleophilic group, that is to say, the terminal amino group. It is therefore more general to introduce electrophilic groups into the carrier. This may be achieved either by copolymerising vinylene carbonate with a comonomer which contains an electrophilic group or by reacting the carrier with a compound, preferably a low molecular weight compound, carrying a reactive electrophilic group.

To introduce an electrophilic group into a reactant, a carboxyl group may be transformed into an acid halide, acid azide, acid anhydride, or imidazolide or may be activated directly with a carbodiimide. Other reactive electrophilic groups are isocyanate, isothiocyanate, and diazonium groups and also cyclic imidocarbonate esters. Especially suitable is the introduction of a reactive group by means of copolymerization, for example, the copolymerization of vinvlene carbonate with ethyl acrylate with subsequent hydrolysis of the polymer gives a matrix having -OH- and COOH-functions.

Carboxyl groups can be activated by means of a carbodiimide as mentioned above, whereupon an amide linkage may be formed with an effector having amino groups. Carboxyl groups can also form amide bonds by means of the isoxazolium salts as suggested by Woodward (cf Biopolymers 5 (1967) 577).

A further method of coupling biologically active compounds to monomers containing carboxyl groups is by means of N - ethoxycarbonyl - 2 - ethoxy 1,2 dihydroquinoline (EEDQ).

A carboxyl group can be esterified according to known methods, the ester then be converted into a hydrazide and the azide resulting therefrom can be linked via an amino group of an effector, for example, a peptide or protein, to the carrier matrix.

By the introduction of a monomer having an epoxy group, for example, an epoxyalkylvinyl ether, into the carrier by polymerization, and because of the very big differences in the rate of hydrolysis of cyclic carbonate and epoxy groups, there may readily be obtained a carrier matrix having reactive epoxy groups which can be reacted with nucleophilic functions, so the direct bonding of a biologically active substance having a nucleophilic function and/or the introduction (prolongation) of a side chain (spacer) having a nucleophilic function is possible.

When the comonomer in the matrix contains amino groups, which can be set free by following reactions, arylamino groups can be introduced by means of vinylsulfone derivatives containing arylamino groups or of sulfuric acid esters of p- hydroxyethyl sulfones. Then the arylamino groups can be diazotized according to known methods and bound to reactive groups of an effector, for example.

wherein "a" represents an integer of such a magnitude that the resulting molecule is insoluble.

In this way, a prolongation of a side chain (spacer) is possible.

A protein can also be bound via its amino group(s) by means of glutardialdehyde.

The linkage of an effector to the matrix after activation of hydroxyl or amino groups of the copolymer may be achieved by the use of a cyanohalide, preferably bromocyane and then reacting a biologically active effector containing amino groups via these activated groups.

An effector can also be bound to the polyvinylene glycol or polyvinylene glycol copolymer matrix by acylating the hydroxyl groups with bromoacetyl bromide, followed by alkylation of the amino group of the effector.

Similar courses of the reaction can be achieved, for example, by reacting hydroxyl groups of the carrier with a reactive triazine, one part of the reactive group of the triazine reacting with the polymer, another one with the amino groups of the effector.

Diazotizable aromatic amines which can react through a further reactive group with the hydroxyl groups of the carrier permit coupling with suitable activated amino acids, for example, the tyrosine or histidine radicals of a protein effector.

Vinylsulfone derivatives containing arylamino groups and sulfuric acid semi esters of ,5-hydroxylethyl sulfones can be reacted with hydroxyl groups of a carrier.

They allow the effector to be bound according to the above-mentioned diazotisation reaction.

Especially stable ether bonds are obtained by the reaction of the hydroxyl groups of a polyvinyleneglycol copolymer with an epoxide which does not form an ion and which contains at least two reactive groups, for example an epihalogenohydrin or a polyepoxide, for example epichlorohydrin or bisepoxide.

A further mode of modification is the introduction by polymerization of a comonomer into the polyvinylene carbonate chain for example, of vinylpyrrolidone units, followed by the partial reaction of the cyclocarbonate rings of the polyvinylene carbonate with an amine, for example, hexamethylenediamine, to give a polyvinylene carbonate copolymer substituted by a side chain or an effector through an urethane compound. The residual cyclocarbonate groups are then hydrolysed to hydroxyl groups:

As well as the above examples of methods for the production of a covalent bond between the carrier matrix and a protein effector or a non-protein effectors, there are further methods which lead to the reaction of the hydroxyl groups or of other groups of the copolymer with an effector, so giving rise to a covalent bond between them, for example, the known reaction with complex-forming metal compounds, for example, titanium compounds. All these methods are known to those skilled in the art.

When a low-molecular weight, biologically active compound is to be bound, it is advantageous to activate the effector rather than the carrier.

In principle, there can be used any method which is known in macromolecular chemistry for the modification of synthetic or natural macromolecular compounds.

The polyvinylene glycol copolymers are notable not only for their chemical and thermal stability but also for advantageous properties for technical procedures which makes them superior to the carrier matrices based on natural carbohydrates and previously deemed to be the best carriers, for example, the carriers of this invention can be prepared in the form of fibres, threads, foils or spherical particles, so that the most suitable form can be chosen in each case. In general the copolymers are preferably in the form of finely divided powders having a high specific surface.

A further advantage of the polyvinyleneglycol copolymers over the previously proposed carrier materials is the controllability on an industrial scale of the proportion of the surface accessible for the linkage of the effectors either directly or via side chains.

The carrier-bound biologically active substances of the present invention can be used for any of the methods for which carrier-bound effectors are suitable, for example, insolubilized enzymes are increasingly used for the determination of substrates in automatic analysers and as so-called enzyme electrodes. Carrierbound enzymes according to the present invention are particularly suitable for these and all enzymic reactions on a technical scale on account of their stability.

Carrier-bound, biologically active substances have found wide application in the field of affinity chromatography, for example, carrier bound, naturally occurring or non-naturally occurring enzyme inhibitors enable the high-grade purification of enzymes, while enzymes have proved to be particularly suitable effectors for isolating natural enzyme inhibitors from crude extracts. Carrier-bound water-insoluble antigens are used for the isolation of the corresponding antibodies which are thus obtained free from other serum constituents and antigens. By use of affinity chromatography, antibodies incapable of being precipitated and those which cannot be precipitated because of their low concentration in the serum can be isolated and be determined quantitatively. The relevant carrier-bound substances of the invention may be used for each of these purposes.

The invention also provides a pharmaceutical preparation which comprises a pharmaceutically useful, carrier-bound biologically active substance of the invention, in admixture or conjunction with a pharmaceutically suitable carrier.

The following Examples illustrate the invention.

EXAMPLES Manufacture of the Vinylene Carbonate Copolymers The vinylene carbonate used in the preparation of the copolymers (CP) was boiled under reflux, before its use, for an hour over sodium borohydride (100 parts by weight of vinylene carbonate to 2 parts by weight of NaBH4) under a pressure of 33 mm, distilled at 750/33 mm over a 50 cm long silver-sleeve column filled with Raschig glass rings and used as straight as possible for the copolymerization. Also, the comonomers used were purified before distillation.

EXAMPLE 1 Copolymer of Vinylene Glycol/Vinyl Alcohol From Saponified Copolymer of Vinylene Carbonate/Vinyl Acetate a) 0.05 part by weight of azobisisobutyronitrile are dissolved under nitrogen in 9 parts by weight of vinylene carbonate and 1 part by weight of vinylene acetate.

The monomer mixture is filled under nitrogen in a flattened screw cap aluminium tube (4 mm thick, 60 mm large, 150 mm long) (total volume of the mixture filled in: about 35 ml), sealed under N2 and hung into a 500C hot water bath during 48 hours.

Hard, brittle plastics plates are obtained which are precomminuted in a crushing mill. Then, the granules are heated under a pressure of 1--2 mm for 5 hours at 1200 C, to separate the part of the monomer which has not been polymerized. The demonomerized granules are further ground in a mill to get a grain size of < 0.1 mm, introduced into 500 parts by weight of 0.5 N sodium hydroxide solution, mixed at 20"C during 1 hour by means of a high-speed stirrer, whereupon the carbonate and acetyl groups are saponified. The water-insoluble CP-vinylene/glycoVvinyl alcohol is suction-filtered, thoroughly freed from inorganic salts with water and lyophilised.

Yield: 4.7 parts by weight of CP-vinylene glycol/vinyl alcohol having a specific surface measured according to BET of 34 m2g-'.

b) Process as described under la), using instead 7 parts by weight of vinylenecarbonate to 3 parts by weight of vinyl acetate.

Yield: 4.1 parts by weight of CP-vinylene glycoUvinyl alcohol having a specific surface of the lyophilised polymer powder of 62.5 m2g-l.

c) Comparison test as described under la), differing in that 10 parts by weight of vinylene carbonate and no comonomer are used.

Yield: 4.7 parts by weight of homopolymer of vinylene glycol having a specific surface of the lyophilised polymer powder of 20.5 m2g EXAMPLE 2 Copolymer of Vinylene Glycol/Vinylglycidyl Ether A mixture of 7 parts by weight of vinylene carbonate, 3 parts by weight of vinylglycidyl ether and 0.1 part by weight of azobisisobutyronitrile is heated to 50"C for 3 days under N2 in flattened aluminum tubes as described under la). As described under la) the plastic plates obtained are comminuted, demonomerized, ground to a grain size of < 0.1 mm, suspended in 500 parts by weight of ice cold 0.5 N NaOH using a high-speed stirrer during 30 minutes, the saponified polymer is immediately centrifuged off, suspended in ice-water, neutralised to pH 7 with 2 N H2SO4 while cooling with ice, suction-filtered, washed with ice-water and lyophilised.

Yield: 3.9 parts by weight of CP-vinylene glycol/vinylglycidyl ether containing 1.3 milliequivalent of oxirane groups/g. (determined according to "Praktikum der makromolekularen organischen Chemie", page 221, by Braun, D. Cherdron, H., Kern, W. Hiithig Verlag, Heidelberg 1966).

EXAMPLE 3 Aminosubstituted Copolymer-Carrier Material 3.9 parts by weight of CP-vinylene glycol/vinylglycidyl ether (of Example 2) are suspended in 100 parts by weight of H2O by means of an Ultra-Turrax (Trade Mark) stirrer and 10 ml of 30% ammonia are added. The mixture is stirred at 500C for again 10 hours by means of a small magnetic stirrer, suction-filtered and thoroughly washed with water and lyophilised.

Yield: 3.5 parts by weight of copolymer-carrier material containing 0.9 m of equivalent amino groups/g in the form of 3 - amino - 2 - hydroxy - propyl groups (obtained by reacting ammonia with glycidyl groups).

EXAMPLE 4 CP-vinylene Glycol/Acrylic Acid As described in Example la), 9 parts by weight of vinylene carbonate, 1 part by weight of acrylic acid ethyl ester and 0.05 part by weight of azobisisobutyronitrile are polymerized under N2, demonomerized and ground to a grain size of < 0.1 mm, saponified, suction-filtered, washed and lyophilised.

Yield: 5 parts by weight of hydrophilic polymer powder containing 1.1 milliequivalent of carboxyl groups per gramme of carrier having a specific surface according to BET of 27.2 m2g-1.

EXAMPLE 5 Polyvinylene Glycol Substituted by ev-aminohexyl Groups 5 parts by weight of CP-vinylene glycoVacrylic acid (Example 4) are suspended in 100 ml of H2O by means of the Ultra-Turrax stirrer, cooled to 50C, 2.5 g of N cyclohexyl - N' - (N - methylmorpholino) - ethyl - carbodiimide - p - toluene sulfonate are added, the mixture is stirred for 30 minutes at pH 5, and at 50C, filtered off and rapidly washed with water. The activated carriers is then immediately suspended in a solution of 5 g of hexamethylene diamine in 100 ml of water adjusted to pH 7.5 with 2 N HCI and cooled to 50C and further stirred under these conditions using a magnetic stirrer, suction-filtered, thoroughly washed with water and lyophilized.

Yield: 4.5 parts by weight of polyvinylene glycol substituted by o-aminohexyl groups containing 0.7 milliequivalent of aminogroups per gramme of carrier.

EXAMPLE 6 Reaction of CP-vinylene Glycol/Vinyl Alcohol of Example la) Having a Specific Surface According to BET of 34 m2g' a) with epichlorohydrin: 50 g of a CP-vinylene glycol/vinyl alcohol prepared according to Example la) are suspended in 1 liter of 2 N NaOH, 250 ml of epichlorohydrin are added to the suspension and the mixture is stirred at 5560 for 2 hours. After a short time, the pH of the suspension drops to 10--1 1. By adding NaOH this pH value is maintained for another hour. After a reaction time of 2 hours, the solid substance is suctionfiltered, washed with water, acetone and finally again with water.

b) with hexamethylene diamine: 50 g of hexamethylene diamine are dissolved in 1.5 1 of water and HC1 is added to reach pH 10. The CP-vinylene glycol/vinyl alcohol activated according to 6a is added to the solution and stirred at 50--550C for 6 hours. Then, the product is suction-filtered and washed with water until free of hexamethylene diamine.

c) with I - aminobenzene - 4 - ,B - hydroxyethylsulfonsulfuric acid ester: The product obtained according to 6b) is stirred at 550C and at pH 10 for an hour with 50 g of 1 - aminobenzene - - p - hydroxyethylsulfonsulfuric acid ester.

Then, the solid substance is filtered off, washed with water, acetone and again with water.

d) Diazotization: 10 g of the product obtained according to Example 6c) are washed with 200 ml of 0.1 N HCI on the suction filter and then suspended in 300 ml of 0.5 N HC1. 0.1 N NaNO2-solution is added to the suspension at 0--40C while stirring until a slight nitrite excess is stated by means of potassium iodide starch paper in the diazotisation. After 10 minutes, the mixture is suction-filtered and the residue is washed with ice water and then with 0.15 M sodium phosphate buffer at pH 7.5 at 04 C.

e) Covalent bond with protein: 0.8 g of albumin are dissolved in 350 ml of phosphate buffer at pH 7.5, cooled to 4"C and the product prepared under 6d) is added. The suspension is stirred at 4"C for 20 hours, filtered off and the solid substance is washed with 1 M NaC1 and phosphate-buffered sodium chloride solution (PBS) (aqueous 0.9% NaC1-solution having a content of 1/15 mol of Na2HPO4-KH2PO4 buffer of pH 7.2).

The filtrate and washing liquors are tested for albumin according to the method of the radial immuno diffusion. 60 mg of albumin are bound to 1 g of the carrier so prepared.

EXAMPLE 7 Reaction of CP-vinylene Glycol/Vinyl Alcohol of Example lb Having a Specific Surface According to BET of 62.5 m2g-' According to the reactions described under 6a to e, 80 mg of albumin can be bound to 1 g of activated carrier quantitatively.

EXAMPLE 8 Reaction of the Homopolymer Vinylene Glycol of Example lc Having a Specific Surface According to BET of 20.5 m2g-' According to the reactions described under 6a) to e) 45 mg of albumin can quantitatively be bound to I g of activated carrier.

EXAMPLE 9 Vinylene GlycoVVinylpyrrolidone Copolymer Substituted by w-aminohexyl Groups 0.05 part by weight of azobisisobutyronitrile are dissolved under nitrogen in 9 parts by weight of vinylene carbonate and 1 part by weight of vinylpyrrolidone and, as described in Example la), polymerized and worked up.

After demonomerization, the granules are dissolved to give a 12% by weight dimethylformamide solution and this solution is introduced through a nozzle into a methanol precipitation bath under a pressure of 15 atm gauge. A cqpolymer of vinylene carbonate and vinylpyrrolidone precipitates in fibrillate form, it is suctionfiltered, washed with methanol and resuspended in 200 parts by weight of methanol, 6 parts by weight of hexamethylene diamine, dissolved in 50 parts by weight of methanol are added, the suspension is stirred for 2 days at room temperature, suction-filtered and washed with methanol. The washed filter residue is then suspended in a solution of 10 parts by weight of sodium methylate in 300 parts by weight of 96% methanol during 4 days at room temperature, washed with methanol and then very thoroughly with water and lyophilised.

Yield: 5.0 parts by weight of a copolymer of vinylene glycol and vinylpyrrolidone substituted w-aminohexyl groups through urethane bonds containing 1.6 moles of equivalent NH2-groups/g of carrier.

EXAMPLE 10 Linkage of IgG (Immunoglobulin G) to the Copolymer Substituted by w-aminohexyl Groups of Example 9 by Means of Succinic Acid Anhydride and Water-Soluble Carbodiimide 5 parts by weight of the w-aminohexyl-substituted carrier prepared according to Example 9 are succinoylated at 100C and at pH 6 for 4 hours with 2.5 parts by weight of succinic acid anhydride which are suspended in 200 parts by weight of water. The pH is adjusted with 2 N NaOH. After washing the solid substance with water, the product is stirred with 1.25 parts by weight of N - cyclohexyl - N' - (N methyl - morpholino) - ethyl) - carbodiimide - p - toluene - sulfonate at pH 5 and 50C for 30 minutes, filtered off and rapidly washed with ice water.

0.5 part by weight of IgG are dissolved in 150 parts by weight of phosphate buffer of pH 7.5 and stirred with the activated carrier for 24 hours at 40C. After filtration, the product is washed with 1 M sodium chloride solution and with PBS.

75 mg of IgG are linked to 1 g of carrier in a covalent bond.

EXAMPLE 11 Copolymer of Vinylene Glycol/Diethylene Glycol Divinyl Ether 0.1 part by weight of azobisisobutyronitrile are dissolved under nitrogen in 9 parts by weight of vinylene carbonate and 1 part by weight of diethylene glycol divinyl ether and heated as described in the Example la) in flat aluminum tubes under N2 to 500C during 3 days and polymerized. After working-up and saponification as described under la), 3.5 parts by weight of the CP-vinylene glycol/diethylene glycol divinyl ether are obtained.

EXAMPLE 12 Copolymer of Vinylene Glycol/N - acryloylaminoacetaldehyde dimethyl Acetal a) 0.05 part by weight of azobisisobutyronitrile are dissolved under nitrogen in 9 parts by weight of vinylene carbonate and 1 part by weight of Nacryloylaminoacetaldehyde - dimethyl - acetal. The monomer mixture is polymerized as described under la), worked-up and saponified to give the 4.1 parts by weight of the copolymer of N - acryloyl - aminoacetaldehyde - dimethyl acetal and vinylene glycol.

10 parts by weight of the copolymer of N - acryloylaminoacetaldehyde dimethylacetal and vinylene glycol were stirred in 100 parts by weight of 1 N HCI for 4--5 hours. The activated carrier was washed with water and phosphate buffer of pH 7.5.

b) 0.5 g of albumin was dissolved in 200 ml of PBS and stirred with the product prepared under a) at 40C during 14 hours. After filtration the carrier-bound protein was washed with 1 M sodium chloride solution and with PBS.

1 g of the carrier so prepared binds 40 mg of albumin.

WHAT WE CLAIM IS: 1. A biologically active substance chemically bound to a copolymer carrier comprising vinylene glycol units.

2. A carrier-bound substance as claimed in Claim 1, in which the copolymer comprises at least 55% of monomer units of vinylene glycol and at most 45% of comonomer units.

3. A carrier-bound substance as claimed in Claim 2, wherein the copolymer comprises from 5 to 20% of comonomer units.

4. A carrier-bound substance as claimed in Claim 1 or Claim 2, wherein the copolymer comprises one or more monomers selected from the following general formulae

**WARNING** end of DESC field may overlap start of CLMS **.

Claims

**WARNING** start of CLMS field may overlap end of DESC **. parts by weight of vinylene carbonate and 1 part by weight of diethylene glycol divinyl ether and heated as described in the Example la) in flat aluminum tubes under N2 to 500C during 3 days and polymerized. After working-up and saponification as described under la), 3.5 parts by weight of the CP-vinylene glycol/diethylene glycol divinyl ether are obtained. EXAMPLE 12 Copolymer of Vinylene Glycol/N - acryloylaminoacetaldehyde dimethyl Acetal a) 0.05 part by weight of azobisisobutyronitrile are dissolved under nitrogen in 9 parts by weight of vinylene carbonate and 1 part by weight of Nacryloylaminoacetaldehyde - dimethyl - acetal. The monomer mixture is polymerized as described under la), worked-up and saponified to give the 4.1 parts by weight of the copolymer of N - acryloyl - aminoacetaldehyde - dimethyl acetal and vinylene glycol. 10 parts by weight of the copolymer of N - acryloylaminoacetaldehyde dimethylacetal and vinylene glycol were stirred in 100 parts by weight of 1 N HCI for 4--5 hours. The activated carrier was washed with water and phosphate buffer of pH 7.5. b) 0.5 g of albumin was dissolved in 200 ml of PBS and stirred with the product prepared under a) at 40C during 14 hours. After filtration the carrier-bound protein was washed with 1 M sodium chloride solution and with PBS.

1 g of the carrier so prepared binds 40 mg of albumin.

in which R, represents a hydrogen atom or a methyl or ethyl group, R3 represents a methyl, ethyl or propyl group, and n is an integer of from 1 to 4.'1 to 4.

5. A carrier bound substance as claimed in any one of Claims I to 4, which is water-insoluble.

6. A carrier-bound substance as claimed in Claim 1, wherein the copolymer is substantially as described in any one of the Examples herein.

7. A carrier-bound substance as claimed in any one of Claims 1 to 6, in which the biologically active substance is an enzyme, an enzyme activator, an enzyme inhibitor, an antigen, an antibody, a plasma protein, a blood group substance, a phythemagglutinin, an antibiotic, a vitamin, a hormone, a peptide, an amino acid or a non-naturally occurring biologically active substance.

8. A carrier-bound substance as claimed in Claim 1, substantially as described in any one of the Examples 6, 7, 8, 10 and 12 herein.

9. A process for the manufacture of a compound as claimed in Claim 1, which comprises A) reacting a copolymer of vinylene carbonate with a biologically active substance and converting the cyclocarbonate groups still present into hydroxy groups, or B) converting the cyclocarbonate groups of a copolymer of vinylene carbonate into hydroxy groups and a) reacting electrophilic groups contained in or introduced into the copolymer with a biologically active substance or b) reacting these hydroxy groups 1) either first with a compound containing an electrophilic group and then with a biologically active substance, 2) or directly with a biologically active substance carrying electrophilic groups.

10. A process as claimed in Claim 9, wherein the copolymer comprises at least 55% of vinylene glycol monomer units and at most 45% of comonomer units,

11. A process as claimed in Claim 10, wherein the copolymer comprises from 5 to 20% of comonomer units.

12. A process as claimed in any one of Claims 9 to 11, wherein the copolymer is produced by copolymerising vinylene carbonate and one or more of the monomers defined in Claim 4.

13. A process as claimed in any one of Claims 9 to 12, wherein the copolymer comprises comonomer units carrying a carboxyl group or a side chain carrying a carboxyl group.

14. A process as claimed in Claim 13, wherein the comonomer units are derived from an acrylate comonomer.

15. A process as claimed in Claim 14, wherein the acrylate is ethyl acrylate.

16. A process as claimed in any one of Claims 9 to 11, wherein the copolymer comprises comonomer units or side chains carrying an isocyanate, isothiocyanate or diazonium group, or a cyclic imidocarbonate ester group, an epoxy group or an amino group.

17. A process as claimed in Claim 9, wherein the polymer is produced by a process substantially as described in any one of Examples 1 to 6 and 9 to 12.

18. A process as claimed in Claim 9, carried out substantially as described in any one of Examples 6, 7, 8, 10 and 12 herein.

19. A carrier-bound biologically active substance as claimed in Claim 1, whenever produced by a process as claimed in any one of Claims 9 to 18.

20. A pharmaceutical preparation which comprises a carrier-bound biologically active substance as claimed in any one of Claims 1 to 8 or Claim 19, wherein the biologically active substance is pharmaceutically useful, in admixture or conjunction with a pharmaceutically suitable carrier.

21. Affinity chromatography, whenever the carrier-bound affinity substance is a carrier-bound biologically active substance as claimed in any one of Claims 1 to 8 or Claim 19.