CH644387A5 - PROCESS FOR THE PREPARATION OF MICROPOROUS BODIES INGLOBANTS ONE OR MORE ACTIVE AGENTS. - Google Patents

Description

The present invention relates to a process for the preparation of microporous bodies incorporating one or more active agents particularly those having an activity in the biological field, a process which includes a mixing of an organic solution of a polymeric matrix with the active agent which you are interested, or its solution or dispersion in a. a medium compatible with it, and miscible with the organic solvent of the polymeric matrix, and a subsequent forming stage.

It is known from the Italian patent n. 836 482 that it is possible to immobilize enzymes or their preparations within filamentous polymeric structures. The process is carried out, according to said patent, by preliminary forming an emulsion between an aqueous solution of the enzyme and an urn solution of the polymer dissolved in a solvent immiscible with water and subsequently extruding the same through a chain in a clot bath for are a filament that encloses the enzymatic solution initially present in the emulsion.

In this way biological catalysts of high activity are obtained, but of practical use often limited by the form to which the aforesaid process forces them and by the relative complexities of the preparation itself.

The present invention, as mentioned, refers to an improved process for the preparation of microporous bodies incorporating active agents of any nature, those active in the biological field in particular, and in any form, so that their practical use in industrial construction, that is, of the reactions involving said agents, it is in no way limited: for example, highly efficient biological catalysts are obtained and whose use is absolutely not limited by practical arrangements to which they are forced, precisely because of the diversity of forms under which can be obtained. In detail, the present invention offers a process for the preparation of microporous bodies incorporating one or more active agents, selected from those listed most having, a process which comprises the following stages:

a) formation of a preliminary solution of a polymeric matrix in an organic solvent b) when necessary, formation of a dispersion or solution of the active agent which is involved in a medium compatible with it and miscible with the organic solvent of the polymeric matrix c ) possible addition to the active agent or to what obtained in point b) of an additive whose nature and function we will specify in the following description d) mixing of the solution of the polymer matrix with the active agent, as such, or with a solution thereof or dispersion according to point b), whether or not containing the additive referred to in point c)

e) sending the mixture obtained in the previous stage to the forming treatment.

The active agents that can be incorporated according to the method described in the present application are biological compositions such as enzymes or enzymatic cells, antigens, antibodies, antisera, hormones, coenzymes suitably linked to macromolecular matrices, or also substances active in other fields or exhibiting particular functions, as sequestering agents, dyes of suitable molecular weight and, in general, all those substances for which disposable use is envisaged and which, in this case, can be recovered at the end of their use and possibly reactivated for subsequent uses; to add that, according to the previously described methodology, it is also possible to incorporate bodies already incorporating the above agents

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obtained according to the present technology or with other methods or also substances deriving from the union, chemical or physical, of active agents with suitable substrates: in this way, for example, high protection of the active agent can be obtained. The active agents can be incorporated as such or with the addition of suitable inert substances. The method has proved particularly effective for the preparation of biological catalysts also, as mentioned above, for the plurality of forms under which said catalysts can be obtained. It is thus possible to obtain fibers, fibrids, cylindrical bodies of various sizes, ellipsoids, spheroidal bodies, powders of various granulometry, the desired shape depending on the process followed in the final forming treatment.

This can be accomplished by coagulating the mixture consisting of the solution of the polymeric matrix and the active agent or of its dispersion or solution in a non-solvent medium of the polymeric matrix.

Such coagulation can take place by dripping the mixture into the medium, and thus, for example, spherical or spheroidal shaped bodies are obtained, or by making the mixture flow directly inside the medium, thus obtaining fibers or similar forms.

Another forming process consists in the dry extrusion, with evaporation of the solvent, of the mixture defined several times: a continuous wire is thus obtained which can be cut to obtain cylindrical or similar bodies, with sections of various shapes.

A further forming process consists in expelling the above mixture from a pressurized environment, with or without a propellant, thus obtaining the formation of fibrids or powders of various granulometry. The polymeric matrices that can be used according to the present process are, among others: cellulosic polymers, esterified and etherified cellulosic polymers, polyamides, polymers or copolymers of acrylonitrile, butadiene and isoprene, acrylates or copolymers of vinyl-dene chloride, styrene, vinyl butyrate, y-methyl-glutamate and the like.

Cellulose esters have proved particularly suitable for the purposes of this method.

The means, in which the active agents must be dissolved or dispersed when it is not possible to start from them - as said, are chosen, as mentioned, from those compatible with the active agent itself or miscible with the solvent of the polymer matrix. They can be chosen from the following classes of compounds, the preferred ones being within the parentheses: water,

alcohols (methanol, ethanol, propanol, butanol, ter-bu-tanol, ethylene glycol, glycerin),

ketones (acetone, methylethylketone),

ethers (dioxane, terahydrofuran, ethoxyethanol),

esters (ethyl and methyl acetate, propyl, ethyl formate etc.),

acids (aectic, propionic, formic acid),

pyridine, acetonitrile, cyclohexane, dimethylformamide. In turn, the solvents of the polymeric matrix can be chosen from a wide range of compounds in relation to the nature of the polymeric matrix.

For example the following compounds can be listed: acetone, methylisobutylketone, cyclohexanone, methyl acetate, methylcellosolve acetate, methylcellosolve, ethyl lactate, methylene chloride, propylene chloride, tetrachloroethane, nitromethane, chlorophenol, meta-cresol, acetic acid, formic acid, dimethylformamide, dimethylsulphoxide, alcohols, dioxane, hydrocarbons such as benzene, toluene, ketones, esters, oiridine, chloroform, mixtures of ethanol - H20, ethanol - ZC14, isopropanol - methylethylketone.

The above mentioned additives have the function of aggregating or crosslinking the active agent in the mixing phase of this with the polymeric matrix.

Among the additives that can be used, the following can be particularly mentioned:

a) polymers of different molecular weight insoluble in the solvent of the polymeric matrix: for example polyamines such as polyethylenimines, cationic and anionic polyacrylamides, polyamino acids such as polysilines, sulphonated polystyrene, polycarboxylic acids, polyvinyl alcohols, polyvinylpyrro-lidone,

b) polyfunctional reagents such as aliphatic aldehydes, isocyanates, thioisocyanates etc.

All the operational details will be evident from the reading of the following examples, which have the sole purpose of illustrating the invention without limiting its purposes.

Example 1

Spheres of ß-galactosidase obtained by englobing cells of Sac-charomyces lactis.

A 10% by weight solution of cellulose acetate (Fluka) in acetone (Carlo Erba) is prepared. At 375 gr. 154 gr. of this polymeric solution are added under stirring. of cellular paste (dry weight = 35.8 gr.) coming from two liters of fermentation of Saccharomyces lactis. A cellular dispersion is obtained which, introduced into a steel container, is extruded through a 0.4 mm diameter capillary by simple nitrogen pressure. The thin extruded wire, after a fall of about 20 cm, turns into a succession of drops which, falling into a water bath, coagulate in the form of spheres.

800 g are collected. of wet spheres which dried in a current of air weigh a total of 73 gr. One gram of these spheres are incubated at 25 ° C with 200 ml of an anhydrous lactose solution (4.75%) containing MgSO 4. 7H20 2 mM, E.D.T.A. 1 mM, in phosphate buffer 0.1 M pH = 7. After two hours of reaction, 80% of the lactose is transformed into glucose and galactose as shown by the glucose analysis performed with the glucose test (Boehrin-ger). The hydrolysis of lactose with the same spheres was repeated 20 consecutive times without observing any loss of activity.

Example 2

Penicillin-acylase spheres obtained by incorporating E. Coli cells after treatment with poly-ethylenimine.

E. Coli cells (dry weight = 20 gr.) Containing 1,106 units are suspended in 875 gr. of water and treated for 10 'under stirring with 25 gr. of a 3.3% solution of poly-ethylenimine (Polysciences). Cell aggregates are formed which easily sediment.

The cellular paste is then resuspended in a small volume of water (total weight = 127 gr.) And vigorously mixed with 157 gr. of a 10% solution of cellulose acetate (Fluka) in acetone (Carlo Erba). With the technique described in example no. 1 prepare 35 gr. of spheres (dry weight). A part of these spheres (5 gr.) Is incubated at 37 ° C in 400 ml of a 6% solution of penicillin G (Squibb) in 0.02 M phosphate buffer pH = 8. The initial activity is 60 000 units (micromoles / hour of hydrolyzed penicillin G) and the total hydrolysis is completed in about 4 hours. The same spheres, used repeatedly for 20 consecutive hydrolysis, retain 60% of the initial activity.

Example 3

Penicillin-acylase spheres obtained by incorporating E. Coli cells after treatment with poly-ethylenimine and glutaral-deide.

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E. Coli cells (dry weight = 20 gr.) Containing 1,106 units are suspended in 400 gr. of water and treated under stirring for 10 'with 25 gr. of a 3.3% polyethylene imine solution (Polysciences, Inc.) and 5 gr. of a 25% solution of glutaraldehyde (Merck).

The cells collected by sedimentation are vigorously mixed with 157 gr. of a 10% solution of cellulose acetate (Fluka) in acetone (Carlo Erba).

The preparation of the spheres takes place with the technique described in the previous examples. 40 gr are obtained. of dry spheres of which a part (5 gr.) is used for the control of the activity in the same conditions of the example n. 2. The activity is 50,000 units and hydrolysis is completed in just over 4 hours. Unlike the spheres prepared in the absence of glutaraldehyde, these after 20 consecutive hydrolysis keep the initial activity unchanged.

Example 4

Glucose-isomerase spheres obtained by incorporating cells of Arthrobacter sp. after treatment with polyacrylaxnmide (Pro-defloc A / 1S).

A 10 liters of Arthrobacter sp. containing the enzyme glucose isomerase, 500 gr are added at the end of the fermentation. of a 3% 0 solution of Prodefloc A / 1S. The cell suspension is stirred for 20 'after which the cells settle. The cellular pasta (dry weight = 150 gr.) Collected is resuspended with water up to a weight of 600 gr. and vigorously mixed with 1500 gr. of a 10% solution of cellulose acetate (Fluka) in acetone (Carlo Erba). With the technique described in the previous examples, the spheres are prepared. One gram of these spheres are incubated at 60 ° C in 100 ml of a 50% glucose solution (weight / weight) containing MgSO 4. 7H20 5. IO "3 M, CoCl2 IO" 4 M, Na2S03 0.1 M pH = 7. Using polarimetric measurements, the activity is determined to be 100 international units (fructose micromoles produced / minute) with a yield (carried out activity / incorporated activity) of 56%.

These spheres used continuously for 20 consecutive days in the test conditions for 20 consecutive days do not lose activity.

Example 5

Glucose-isomerase spheres obtained by incorporating the enzyme in solution after treatment with polyethylene imine and glutaraldehyde.

The enzyme solution is prepared by dissolving 2 g. of glucose-isomerase powder (Godo Shusei) in 8 gr. of water.

This solution is added under stirring of 4 g. 3.3% polyethylene imine solution (Polysciences, Inc.) and 4 gr. of a 2.5% solution of glutaraldehyde (Merck). This enzymatic preparation is mixed at room temperature with 100 gr. of a 10% solution of cellulose acetate (Fluka) in acetone (Carlo Erba).

With the technique described in the previous examples, spheres are prepared whose weight after air drying is 12 g. A part of these spheres (2 gr.) Is incubated at 60 ° C with 100 ml of a 50% glucose solution (weight / weight) containing MgSO 4. 7H20 5. IO "3 M, CoCl210 ~ 4 M, NaSOs 0.1 M, pM = 7 for the determination of the activity which turns out to be 500 international units with a yield of 30-40%.

Example 6

Cylindrical bodies of cellulose acetate incorporating the polyethylene imine.

15 g of cellulose acetate (Fluka) are dissolved with 85 gr. of acetone (R. P. Carlo Erba). To the polymeric solution are added slowly and under stirring 15 gr. of a solution in water of polyethyleneimine hydrochloride at 33% (weight / weight) (Polisciences, Inc.) and 5 gr. of a 1% solution of glutaraldeidb (Merck). The mixture is poured into a steel cylinder connected at the top of a nitrogen cylinder and at the bottom of a die immersed in a water bath.

By exerting a nitrogen pressure the mixture flows from the die and by coagulation a continuous thread is obtained which is broken by a cutter in pieces of 1-2 cm. One gram of these cylindrical bodies is contacted for 4 hours with 50 ml of a copper solution at the concentration of 18.3 ppm obtained by dissolving CuS04. 5H20 in. distilled water. With an atomic absorption spectrophotometer (Varian-Techtron 1200) the copper content (1.1 ppm) is measured in the solution treated with the cylinders.

After washing the cylinders with 50 ml of HCl 1 N, 17 ppm of copper are dosed in the water. The cylinders are again in contact for 4 hours with the aforementioned copper solution and the copper content which is 1.2 ppm is dosed again in solution. The procedure described for is repeated

10 times without the cylinders losing the ability to sequester copper.

Example 7

Invertase fibrids obtained by incorporating the enzyme in a solution added with polyvinylpyrrolidone.

A solution (50 gr.) Of invertase (B. D. H.) after adding 10 gr. of polyvinylpyrrolidone K - 30 (Fluka) is vigorously mixed with 333 gr. of a 15% solution of cellulose acetate (Fluka) in acetone (Carlo Erba).

The preparation, introduced into an autoclave, is extruded by means of nitrogen pressure (50 Atm) through a nozzle of 500 | i collecting a dry fibrous type powder whose particle size varies between 160 and 1600 | i. The activity of the invertase enzyme incorporated in the fibrids was measured on a 20% sucrose solution in 0.1 M phosphate buffer pH = 4 at 25 ° C. The activity expressed as mgr of inverted sucrose per minute and per gram of fibril varies between 8 and 50 depending on the size of the fibride.

Example 8

Hydroxypyrimidine hydrolase and N-carbamyl-D-amino-acid hydrolase fibers obtained by incorporating Agrobacte-rium radiobacter cells after addition of polyethylene imine.

Agrobacterium radiobacter cells (dry weight = 4 gr.) Are suspended in 100 gr. of water and treated under stirring with 2 gr. of a 3% solution of polyethylene imine (Polysciences, Inc.), after 10 'the stirring is stopped and the sedimented cells are collected and suspended in 25 gr. of water. This suspension is vigorously mixed with 20 gr. of a 20% solution of cellulose acetate (Fluka) in acetone (Carlo Erba). The preparation is introduced into a steel cylinder connected at the top to a nitrogen cylinder and at: bottom to a single-burr die (0 = 1 mm). Using a metering pump, the preparation is extruded from the die in the form of a continuous thread which dries in the air by evaporation of the acetone during a fall of 4 meters. The whole wire (dry weight = 8 gr.) Is incubated at 40 ° C under a nitrogen head in phosphate buffer 0.1 M pH = 8 containing 4 gr. of D-L-phenylidanfoin 'for dosage in the at-. the two enzymes. After 20 hours of reaction, the complete conversion of redirantoin to D (-4) phenylglycine is obtained. as demonstrated by the analysis performed on the ämininö- \ acid analyzer. The same thread used repeatedly for 10 consecutive hydrolysis loses 30% of the initial activity.

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Claims (11)

644387 1. Process for the preparation of microporous bodies incorporating one or more active agents consisting of a mixing of an organic solution of a polymeric matrix with F active agent in which one is interested or with a solution or dispersion in a medium compatible with it and miscible with the organic solvent of the polymeric matrix is in a subsequent forming step. 2. Process according to claim 1, particularly comprising the following steps: a) formation of a preliminary solution of a polymeric layer in an organic solvent b) possible formation of a dispersion or solution of the active agent which is involved in a medium compatible with it and miscible with the organic solvent of the polymeric matrix c) possible addition to the active agent or to what obtained in b) of an additive selected from i) polymers of different molecular weight insoluble in the solvent of the polymeric matrix ii) polyfunctional reactants selected from the aliphatic aldehydes, isocyanates and thioisocyanates d ) mixing the solution of the polymeric matrix with the active agent as such, and with what obtained in b) with or without the additive referred to in point c) e) immediate sending of the mixture obtained in the previous stage to the forming treatment. 2 Process according to one of claims 1 or 2, characterized by the fact referred to in point d) in a non-solvent medium of the polymeric matrix. Process according to one of claims 1, 2 or 3, characterized in that coagulation occurs by dripping the mixture into the medium. 5. Process according to claim 3, characterized in that coagulation occurs by making the mixture flow directly into the medium. 6. Process according to claim 2, characterized in that step e) is carried out by dry extrusion of the mixture referred to in point d). 7. Process according to claim 2, characterized in that step e) is carried out by expelling the mixture referred to in point d) from an environment under pressure. 8. Process according to claim 2, in which the active agents are selected from enzymes, enzyme cells, antigens, antibodies, antisera, hormones, coenzymes bound to macromolecular matrices, sequestering agents, coloring agents. 9. Process according to claim 2, characterized in that the polymeric matrix is selected from cellulosic polymers, esterified and etherified alkyl polymers, polymers, polymers or copolymers of acrylonitrile, butadiene, isoprene, acrylates, methacrylates, vinyl esters, vinyl chlorides, vinylidene chloride, styrene, vinylbutyrate, y-methylglutam- mato. 10. Process according to claim 2, characterized in that the solvent of the polymeric matrix is selected from acetone, methylisobutylketone, cyclohexanone, methyl acetate, methylcellosolve acetate, methylcellosolve, ethyl lactate, methylene chloride, propylene chloride, tetrachoro-ethane. , nitromethane, chlorophenol, metacresol, acetic acid, formic acid, dimethylformamide, dimethylsulphoxide, alcohols, dioxane, hydrocarbons, ketones, esters, pyridine, chloroform, ethanol mixtures -H20, ethanol -CCI4, isopropanol-methylethyl-ketone. 11. Process according to claim 2, characterized in that the medium referred to in point b) is selected from alcohols, ketones, ethers, esters, acids, pyridine acetonitrile, cyclohexane, dimethylformamide.