SE410737B - PEARLY, IN THE WATER SWELLABLE COPOLYMERISAT AS CARRIER FOR BIOLOGICAL ACTIVE SUBSTANCES, WHICH COPOLYMERISAT CONTAINS EPOXY GROUPS - Google Patents

Description

7310177-6 10 15 20 25 '30 35 40 or by hydrolysis. Carriers based on polysaccharides, such as cellulose, starch, dextran, agarose and its derivatives can be degraded enzymatically, which can be done by microbiological attack or the presence of hydrolytic enzymes. The "bleeding" of biologically active substances often observed in these carriers can be attributed both to such degradation and to incomplete binding. When binding biologically active substances on the surface of inorganic or synthetic materials with a defined, compact shape, such as glass beads or synthetic fibers, a special activation is required. The bonding ability is -very low due to the relatively small available area.

According to a method by Nilsson and Mosbach, enzymes embedded in plastics in the form of beads, which as a filling in a column allow a high flow rate, are accessible by allowing unsaturated monomers to be polymerized in an enzyme-containing aqueous solution during curing. The resulting aqueous polymer beads contain the enzyme without chemical bonding. In this process, only dilute enzyme solutions can be used, binding only about 3% of active protein.

Even with concentrated enzyme solutions, no higher coating of the support with active material is obtained, as this is damaged by the radical polymerization reaction. From the finished product, enzymes can constantly bleed out during use through swelling and decompression. On the other hand, if the bridging or hardening is so strong that the enzyme is constantly entrapped, high molecular weight substrates can no longer migrate into the beads and react with the enzyme. The same applies to all processes in which biologically active substances are embedded in membranes, foils or other semipermeable layers.

DT-OS 1 908 290 describes crosslinked, highly swellable copolymers of acrylamide and maleic anhydride for fixing protein, which are prepared in a complicated multi-step process. These copolymers, like other anhydride group-containing carriers, have the disadvantage that the binding-active anhydride groups are easily hydrolyzed in water and that free carboxyl groups are formed upon binding of proteins or The carrier thereby acquires a strong anionic character, which is a disadvantage when using the carrier-bound 'protein, since anionic substrates are rejected from the anionic carrier matrix, while cationic substrates are retained in the surface with little permanent ionic bonds. In several cases, the interaction between the substrate and the protein molecules bound within the carrier is made more difficult. Bead polymers which chemically bind biologically active substances from an aqueous solution are known from German Offenlegungsschrift 2,062,818. They are prepared by dissolving a solution of maleic anhydride, vinyl ethers and divinyl ethers in a strongly polar solvent, emulsifying and polymerizing the solution. in a hydrocarbon to droplets. This results in large-mesh space-branched, swollen polymer beads, which are initially relatively hydrophobic. Only by hydrolysis of the anhydride groups to carboxyl groups does their hydrophilic properties increase and thus also swellability with water. To the extent that the hydrolysis and swelling on contact with an aqueous phase proceeds from the outside and inwards, a biologically active substance dissolved in the aqueous phase can also diffuse into the beads and bind through the anhydride groups remaining after extensive hydrolysis.

The hydrophobic nature of the starting materials has an aggravating effect on the bead polymerization process. The monomers are soluble even in the hydrocarbon phase and polymerize there to a considerable extent during the formation of a finely divided precipitate polymer, which can only be separated with difficulty from the real polymer beads.

The precipitating polymer eliminates some of the advantages inherent in the pearl form of the enzyme carrier. This is because it makes filterability more difficult, does not allow complete separation from a substrate solution and reduces the flow rate in column reactors.

A significant disadvantage of the polymers lies in the lack of hydrophilic property in the initial stage. The diffusion and binding of the biologically active substances must always be preceded by hydrolysis and hydrophilization. The anhydride groups are mostly hydrolyzed completely before biologically active molecules, “especially high molecular weight substances, can penetrate into the swollen area. The binding capacity of these enzyme carriers is therefore relatively limited, as is the specific activity, ie. the proportion of the chemically bound material that has retained its biological activity. The object of the invention is to provide improved enzyme carriers. They must be in defined pearl form and not together with finely divided precipitated polymers, not be enzymatically degradable and covalently bind biologically active substances in high yield and while maintaining their biological activity. The resulting enzyme products must be easily and completely separable from substrate solutions and allow a high throughput through a column filled with them. The prerequisite for this is a large free volume between individual particles in a loose mass of such.

Spherical parts with a highly uniform size meet this condition, it has hitherto not been possible to produce enzyme carrier substances in the form of a uniform bead polymer.

According to the invention, an improved enzyme carrier has been obtained by means of a pearlescent, crosslinked, water-swellable polymer which has the characteristics stated in the following claim 1.

Due to the proportion of water-soluble monomers, the monomer mixture used for the bead polymerization as a whole is more hydrophilic and sharper delimited from the surrounding hydrophobic medium than is the case in the preparation of bead polymer according to German Offenlegungsschrift 2,062,818. accompanying precipitating polymer. Due to their hydrophilic nature, the bead polymers according to the invention swell rapidly and strongly in water, whereby dissolved, biologically active substances dissolved in the swelling water simultaneously penetrate and bind covalently. The advantageous properties of the products according to the invention are clear from the following comparison with products according to the German Offenlegungsschrift 2,062,818. (butanediol- (butanediol1- (acrylamide / 9lYCidY1 'divinyl ether / divinyl ether] acrylate, 421 mOl) maleic acid-ethylvinyl ether / anhydride maleic acid- 1: 2 mol) anhydride 1: 1: pl mol) Reaction ratio (dry support material: 1 trypsin) 2: 1 N: 1 Degree of swelling (0.05 mo1.phosphate buffer pH 7.5) 3 30 11 Protein content of the reaction product 0.7U 1 1.9% 8% Yield bound enzyme (used 2) % 2% 3 enzyme) 1.5% 30% Mean) activity (mU / mg resin) 1.9 1.5 11.2 Stability of the enzymatic activity in repeated use no no yes 1) 1 mÜ corresponds to the cleavage of 1 x 10 -5 μmol peptide bonds per minute as an average at a sale action time of 60 minutes measured by automatic titration. 2) The values were obtained after multiple washes without achieving a constant final activity. The values for covalently bound protein should be even lower. The reactive groups of the monomers mentioned under a) are those groups which at temperatures of 0-40 ° C in aqueous solution react with primary amino groups and hydroxyl groups to form a covalent bond to the oxygen or hydrogen atom of the hydroxyl and amino groups, respectively. Since water is always present in considerable excess relative to the hydroxyl and amino groups, groups which react spontaneously with water, such as, for example, isocyanate groups, are less suitable. Preferably these are activated carboxyl groups known from peptide chemistry, or N- or O- -alkylating agents such as alkyl halide or epoxide groups.

Examples of activated carboxyl groups used in peptide chemistry for the formation of peptide bonds are carboxylic acid chloride, carboxylic anhydride and carboxylic acid groups and the phenyl esters and carbonic acid esters of hydroxylamine derivatives of the general formula R Examples of group (a) are glycidyl esters of α, β-unsaturated carboxylic acids, especially glycidyl acrylate and methacrylate.

When combined with enzymes, enzyme substrates, inhibitors, hormones, antibiotics, antibodies, antigens and peptides, the epoxy groups in aqueous solution at temperatures of 0-40 ° C form covalent bonds to 0 or N atoms. If the biologically active substances have an egg white character, these in each case contain a primary end amino group and in many cases additional OH or NH groups in the amino acid ester of nitine, citrulline, arginine, lysine, serine, threonine or tyrosine. Non-egg white biologically active substances can only be bound if they contain at least one primary amino or hydroxyl group, such as, for example, polysaccharides, nucleic acids, ampicillin, tyramine. The epoxide groups are sensitive to hydrolysis and can, if they do not react with the substances present, turn into hydrophilic hydroxyl groups in the presence of water. The activated groups therefore occur in the carrier material in usually larger numbers than are required for binding of the enzyme protein. 10 15 20 25 30 35 40 'where a covalent bond occurs. 7310177-6 7 However, the epoxide groups react more easily with hydroxyl or amino groups than with water. The electrically neutral character of the matrix is also maintained after binding of the biologically active material.

In the aqueous enzyme solution, the carrier material swells to several times the original volume. In this case, not only water but also the biologically active substance dissolved therein diffuses into the interior of the small parts. The swellability of the polymer beads depends on their content of hydrophilic groups. Monomer units with hydrophilic groups form an essential component of poly- They are derived from water-soluble, neutral, As water-soluble meritate. salty, acidic or basic monomers. in accordance with the invention, such monomers apply which, at room temperature, give at least 10% aqueous solutions. Among neutral monomers of this kind, those with an amide group, especially acrylic or methacrylamide or N-vinylpyrrolidone, are particularly important. Monomers containing hydroxyl groups, such as ethylene glycol monoacrylate or monomethacrylate, are slightly less hydrophilic. Monomers with saline groups are extremely hydrophilic and can participate in relatively small amounts in the construction of the copolymers according to the invention.

Monomers of this kind are, for example, vinylsulfonic acid and its salts and the quaternized aminoalkyl esters of d, β-unsaturated carboxylic acids, for example methacryloxyethyl-trimethyl-ammonium chloride. Monomers with carboxyl groups form salts in alkaline and monomers with secondary or tertiary amino groups in acidic, aqueous environment, but these monomers and the monomer units obtained therefrom are hydrophilic even if they are not in the form of salts.

The swellability of the carrier materials in water depends not only on the hydrophilic groups of the monomers summarized in component c) but also on the free hydroxyl groups formed by hydrolysis of epoxy groups. A proportion of up to 50 mol% of the monomers may be water-insoluble and have a less hydrophilic nature. On the other hand, certain proportions of hydrophobic components may be required for an optimal effect of the bound, biologically active substances. Examples of non-water-soluble monomers are acrylic and methacrylic acid esters of aliphatic alcohols, nitriles of these acids, vinyl esters of lower fatty acids, such as vinyl acetate or vinyl propionate, vinylidene chloride and the like. However, the incorporation of such monomers is limited by the polymerization process. The co-use of monomers which are dissolved with preference in the continuous phase, for example consisting of higher aliphatic hydrocarbons, such as, for example, acrylic and methacrylic esters of alcohols with four or therefore several carbon atoms, is not permitted.

A sufficiently hydrophilic property of the copolymer is obtained if a corresponding copolymer produced without curing monomers - at least after the hydrolysis of the activated groups - were soluble in water. By curing by means of such monomers containing two or more radical polymerizable carbon double bonds, such as, for example, glycol dimethacrylate or diacrylate, divinylbenzyl, triallyl cyanurate, methylene-bis-actylamide or methacrylamide and the like, the water solubility is eliminated. The optimal degree of cure, ie. the choice of the amount of curing agent within the limits of 0.2 to 5 mol%, is suitably determined empirically.

The degree of curing can be considered sufficient when the polymer particles in water swell to 5-100-fold volume or more, but as a unit particle retain the nearest spherical shape even when they settle to an aqueous environment. There must be free spaces between the particles, filled only with the aqueous phase. With too little curing, the particles are deformed during the deposition so that no interstices are formed, while with too strong curing no or only a small swelling occurs, for example to less than double the initial volume. A curing with a particularly large-scale space branching can be achieved by allowing the polymerization to take place in the presence of a non-aqueous swelling agent, which is described in more detail below.

Due to the hydrolysis sensitivity of the epoxide groups, the polymerization is carried out in as anhydrous medium as possible. At a water content of, for example, 1% in the monomer phase, no appreciable hydrolysis of the epoxide groups generally occurs, but the water content must, on the other hand, be kept below the limit at which a appreciable proportion of the reactive groups necessary for enzyme formation are hydrolyzed. This limit is differently high depending on the water sensitivity of the reactive groups and usually between 2 and 5% by volume. In essence, it is preferable to work in the complete absence of water.

The formation of cured, spherical parts of the support material with a narrow particle size distribution is achieved by a special bead polymerization process in which a strongly non-polar organic medium, which is not a good solvent for the monomers to be polymerized, is used as a continuous phase. Aliphatic hydrocarbons are particularly suitable as such, mainly those with 8 and more carbon atoms in the molecule.

A clear polarity difference between the monomer phase and the continuous phase is a prerequisite for a phase precipitation to occur at all and the monomer mixture at the beginning of the polymerization is present in discrete droplets. Only under this condition do spherical polymer particles of uniform size occur. The less polar the participating monomers are, the greater their tendency to dissolve, at least in part, in the non-polar medium. This tendency can be counteracted by diluting the monomer phase with good solvents for the monomers which are immiscible with the non-polar organic medium, for example formamide, dimethylformamide or dimethylsulfoxide, in amounts of, for example, 20 to 200% by weight based on the amount of monomer. . This measure also contributes to a large-mesh space branch and a good swellability of the polymers. A limited solubility of the monomers in the continuous organic phase is not disturbing as the polymerization of this monomer moiety in the continuous phase is excluded. To the extent that the monomers in the droplets react through the polymerization, a re-diffusion of the dissolved monomers into the droplets can then occur. Therefore, polymerization initiators which dissolve to a small extent in the organic medium are preferably used.

The monomer mixture is distributed in the organic medium by vigorous stirring into small drops. In order to maintain the state of distribution of the monomer phase - which may possibly contain solvents - brought about by the stirring during the entire polymerization process, a dispersant which is soluble in the non-polar organic phase is also used. For this purpose, random copolymers or block or graft polymers are used, which are composed of constituents of different polarity. Statistical copolymers 7310177-6 10 15 20 25 30 35 40 10 of a predominant proportion, for example 60-95 base mole percent of a non-polar, long chain alkyl radical containing monomer, such as acrylic or methacrylic acid esters of alkyl alkyl alcohols having at least 4 carbon atoms in the alkyl radical, vinyl esters with at least 4 carbon atoms containing fatty acids, etc., and to the rest of the polar, water-soluble monomers, such as methacryloylcholine chloride, dimethylaminethyl methacrylate, its salts with inorganic or organic acids, acrylic or methacrylic acid or acrylic or methacrylamide, good dispersion effect. However, it is also possible to use graft or block polymers prepared according to known methods, in which monomers of the two mentioned types are not statistically distributed but differ from each other in different molecular blocks or in the base chain and the side chain of a graft polymer. The dispersants are already able, in an amount of 0.02%, based on the weight of the continuous phase, to maintain the suspension state until the end of the polymerization. The larger the amount of dispersant selected and the more vigorous stirring, the smaller parts are formed. A particle size of 0.1 to 1 mm is sought in the unsoiled state.

The ratio between the monomer phase - with or without the addition of a solvent - and the continuous phase may be between 1: 1 and 1: 10, but the ratio between 1: 1, 5 and 1: 4 is preferred. The smaller the density difference between the two phases, the greater the stability of the distribution state.

The density of the continuous phase can be adjusted by adding halogenated hydrocarbons, such as perchlorethylene, trichlorethylene, chloroform or carbon tetrachloride, to the mostly larger and during the course of the polymerization process further increasing the density of the monomer phase.

The polymerization is initiated with a radical-forming initiator which is readily soluble in the monomer phase and in the continuous phase as difficult as possible, for example azobis-cyanovaleric acid. With this initiator, the polymerization occurs at temperatures of 50 to 80 ° C, which means the disadvantage that the solubility of the monomers in the continuous phase at these temperatures becomes inappropriately high, so that there is a risk of a precipitation polymerization. It is therefore preferable to trigger the polymerization at a lower temperature, for example 15 to 30 ° C, by means of a redox initiator system. A suitable one is benzoyl peroxide / dimethylaniline. With good stirring and heat dissipation, the polymerization can be completed in 1 to 3 hours. The polymerization beads are separated by filtration of the continuous phase and, if appropriate, purification of adhesive residues of the liquid phase is carried out by washing with an anhydrous solvent. If the beads have been prepared in the presence of formamide, dimethylformamide or dimethylsulfoxide, these solvents are generally not removed from those with these swollen beads, but are allowed to react in the resulting moist state with the aqueous enzyme solution. It is t.o.m. it is advantageous to swell the beads before the reaction with such solvents, when they are not used in the polymerization.

The reaction with the aqueous enzyme solution forms a competitive reaction between the desired covalent bond of the biologically active substance and the undesired hydrolysis of the epoxide groups. The higher the concentration of the biologically active substance in the aqueous solution, the more favorable the desired fixation. The solutions must be as free as possible from inactive sequential proteins or other hydroxyl- or amino-group-containing substances, which can otherwise be bound to the carrier material. Unless a specific pH range must be contained to maintain the activity, the reaction with the biologically active substances is allowed to take place at pH = 5 - 8, preferably at temperatures of 0-30 ° C. The reaction is initiated to the extent that the polymer beads swell in water. After a reaction time of a few hours to a few days with gentle stirring, most of the enzyme has been bound and the excess active groups have been hydrolyzed.

Most of the biologically active substances are bound in the large-mesh "cavities" in the interior of the swollen parts, where the sensitive substances are protected against degradation due to shear forces that can occur during stirring or at high flow rates in column reactors, but are Only extremely high molecular weight substrates are apparently initially attacked mainly by the molecules bound to the polymerization surface and can, with decreasing molecular size, increasingly react with those in the internally bound substances. The molecules should also be the reason for the better maintenance of the enzyme activity during the binding to the polymer, in comparison with enzyme carriers which are constructed in a different way.

These are usually activated as solid particles and bind the enzyme predominantly on the surface, where it can easily be damaged by shear forces. In the binding of proteins to uncured copolymers of maleic anhydride during simultaneous curing, enzyme molecules are probably also bound in the interior of the carrier particles, but these become so strongly entrapped by subsequent curing that a reaction with substrate molecules is no longer possible.

Among the biologically active substances which can be bound to the beaded carriers according to the invention, the enzymes, in particular hydrolase, are of particular importance. Examples of suitable enzymes are mainly proteases, such as fungal and bacterial processes, trypsin, chymotrypsin, pancreatin and amylases, ribonucleases, deoxyribonucleases, etc. The high activity of the bound enzymes in relation to high molecular weight substrates is particularly noticeable.

By means of bound enzyme substrates or inhibitors, enzymes dissolved in aqueous media or enzymes contained in biological materials can be selectively removed or rendered inactive.

By changing the pH or electrolyte content of the unbound medium, the taken up enzymes can be separated from the carrier-bound substrates again and in this way re-insulated. The numerous additional possibilities for influencing biological system reactions to carrier-bound hormones, such as insulin or hypertensin, or antibiotics, such as ampicillin or antibodies, antigens, biogenic amines and various peptides, may be mentioned here. Mixtures of different biologically active substances can also be bound at the same time. Biologically active substances, which in the free state would interfere with each other's action, can be bound separately from each other to carrier substances according to the invention, after which the products obtained are mixed and the mixture is used. The carriers reacting with biologically active substances typically contain 5 to 50, preferably 10 to 30% by weight of bound material, based on the dry matter of carriers and biological material. The products are present in water as gel-like, spherical particles, usually 1 to 5 mm in diameter. Due to their strong swelling, they have a density which differs from the aqueous medium only slightly, so that they can be kept in suspension by only slight stirring. When used as a column or reactor filling, the flow rate of the substrate solution is limited only by the diffusion rate of the substrate.

The loss of activity of the bound, biologically active material during use and storage is clearly less than when storing the unbound substances under the same conditions. As a result, through frequent repeated re-use during use or through long service life during continuous use, a significantly higher utilization of the biological activity is possible than is the case with the use of the unbound substances.

As an example of the simplification of the process, which is possible by carrier-bound enzymes, the isolation of nucleic acids from biological material can be mentioned. Proteins in particular are difficult to separate. They have hitherto had to be disposed of by cumbersome phenol extraction procedures. Although they can also be degraded with soluble proteolytic enzymes, these enzymes are proteins themselves, which in turn must also be removed by phenol extraction. With a carrier-bound protease, the proteins can be easily and completely degraded. After filtering off the carrier-bound enzyme, in addition to the nucleic acids, the substrate solution no longer contains any high-molecular constituents. The nucleic acids themselves are usually divided according to known methods into ribonucleic acid and deoxythibonucleic acid. Even then, the residual disposal of the other components caused considerable work. By means of carrier-bound ribonuclease, residues of ribonucleic acid can now be removed from the deoxyribonucleic acid fraction. Conversely, ribonucleic acid can be recovered from deoxyribonucleic acid if treated with carrier-bound deoxyribonuclease.

Preparation of beaded carrier polymers Example 1 Into a round bottom flask (500 ml) are introduced 87 g of n-heptane, 55 g of perchlorethylene and 0.1 g of emulsifier (statistical copolymer of n-butyl methacrylate / methacryloylcholine chloride 95/5). After cooling to -111 ° C, the following solution is added:

Claims (6)

17.5 g formamide 12 g acrylamide 3 g glycidyl acrylate 0.375 g ethylene glycol dimethacrylate The monomer phase is distributed with stirring in the organic The reaction is started by adding Fine beads were obtained. phase and degassed. 0.5 g of dimethylaniline in two portions. Reaction of the bead polymer with biologically active substances Example 2 Pancreatic trypsin inhibitor is reacted with a bead polymer of Example 1 at pH 9.5 (aqueous bicarbonate buffer) for 30 hours and then washed five times with the aqueous saline solution and three times with 1 % acetic acid and 0.2 M phosphate buffer, pH 7.5. Protein content of trypsin inhibitor beads: 8.5 (based on dry weight). In particular, trypsin can be bound to the inhibitor beads and desorbed in the acidic pH range without loss of activity. Example 3 500 mg of tyramine are reacted in 25 ml of bicarbonate buffer (pH 9) with 1000 mg of the bead polymer prepared in Example 1 at room temperature for 12 hours. After removal of the unbound tyramine by washing with 5% acetic acid, tyramine beads are obtained, which, based on the dry weight, contain 20% tyramine. The tyramine content is determined ultraviolet spectrometrically after alkaline hydrolysis in 0.5 N NaOH * via 100 ° C. In an analogous manner, the antibiotic ampicillin binds to the same bead type. Beaded, crosslinked, water-swellable copolymer for use in biochemical reactions, for example for the preparation of carrier-bound, biologically active substances and carrier-bound enzymes, characterized in that it is composed of a) at least one radically polymerizable compound having an epoxide group, b) a crosslinking comonomer having at least two radically polymerizable carbon double bonds, c) one or more radically polymerizable water-soluble compounds, and d) optionally additional, water-soluble radical polymerizable compounds, wherein the monomers of the groups a ), b) and c) together make up 50 to 100 mole percent and the monomer b) makes up 0.2-5 mole percent, based on the total monomer mixture. Copolymer according to Claim 1, characterized in that it contains units of a glycidyl ester of a U, B-unsaturated carboxylic acid, preferably glycidyl acrylate or methacrylate. 3. A copolymer according to claim 1 or 2, characterized in that it contains units of acrylic or methacrylamide or vinyl pyrrolidone. 4. A copolymer according to claim 1, 2 or 3, characterized in that the individual particles swell in water to 5-100 times the original volume. 5. A copolymer according to claims 1-4, characterized in that it is swollen by 20-200%, based on the weight of the polymer, with formamide, dimethylformamide or dimethylsulfoxide. Use of copolymers according to any one of claims 1 to 5 for the preparation of carrier-bound, biologically active substances. PROMISED PUBLICATIONS: Great Britain 1 132 645 'Germany 1 908 290 (CO8f 15/02), 2 D62 818 (CO8f 27/08) US 3,414 S50 (260-86.7)