DD149075A5 - METHOD FOR PRODUCING MICROPOROESIC BODIES INCLUDING ONE OR MORE ACTIVE AGENTS - Google Patents

Abstract

A novel process is described for the formation of microporous bodies which include active agents, such as enzymes, in their interior. The process consists of mixing a polymer solution with the contemplated active agent, dissolved or dispersed, in a liquid phase miscible with the solvent soluble in the dissolution of the polymer. The final form of the microporous bodies can be imparted by extruding, extruding, dropping and otherwise.

Description

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AP C08J / 219

Process for producing microporous bodies Field of application of the invention

The invention relates to a process for the preparation of microporous bodies which include one or more active agents and, in particular, those agents which exhibit a biological activity.

Characteristic of the known technical olution s

From ITr-PS 836 482 it is known that it is possible to immobilize enzymes and these containing preparations in the interior of fibrous polymer structures. This process consists, according to said patent, of previously preparing an emulsion of an aqueous solution of the enzyme and a solution of the polymer in a solvent which is immiscible with water, the emulsion then being extruded through a spinneret into a coagulating bath to form a filament which includes in its interior the enzymatic solution originally present in the emulsion.

By doing so, one obtains biological catalysts which have high activity, but their practical utility is often limited by the shape given to them by the above process and by the comparatively complicated production.

Object of the invention

The object of the invention is to provide an improved process for the production of microporous bodies, which include active ingredients of any nature and in particular

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In particular, those exhibiting a biological activity present in any arbitrary form, so that their practical application, i. their use in the commercial performance of reactions in which such agents are used is in no way limited.

Explanation of the essence of the invention

It is an object of the present invention to provide a method of production in which microporous bodies including one or more active agents can be obtained in any desired form.

According to the invention, this process comprises mixing an organic solution of a polymer matrix with the active agent in question or a solution or dispersion thereof in a compatible medium which is miscible with the organic solvent for the polymer matrix, and a subsequent shaping step.

By using the method of the present invention, for example, there are obtained biological catalysts having a high efficiency, the utility of which is in no way limited by practical arrangements to be made, due to the various forms in which they can be obtained.

In particular, the invention provides a process for the preparation of microporous bodies which include one or more active agents selected from among those listed below, this process comprising the steps of:. ·

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a) the preliminary formation of a solution of a polymeric matrix in an organic solvent,

b) if necessary, the formation of a dispersion or a solution of the respective active agent in a medium that is compatible with both, as well as miscible with the organic solvent for the polymer matrix,

c) optionally adding to the active agent or to the product obtained in stage b) an additive or additive whose nature and function are explained in more detail in the course of the following Beschx-eibung,

d) mixing the solution of the polymer matrix with the active agent as such or with a solution of the agent or a dispersion corresponding to the preceding stage b), optionally containing the addition of stage c),

e) feeding the mixture coming from the above step d) to the shaping treatment.

The active agents which can be included in the method of the invention are biological compositions, such as enzymes or enzyme cells or, enzyme-containing or enzyme-like cells, antigens, antibodies, antisera, hormones, coenzymes which are suitably bound to macromolecular matrices or also Substances which have other types of activities or which develop special behavior, such as sequestering agents, colorants or dyes with a carefully selected molecular weight and, in general, all such sub-

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In contrast, in the contemplated case, they can be recovered for re-use or re-activation before reuse, as the case may be. It should also be added that it is also possible, according to the procedure described above, to re-entrap bodies already containing the active agents according to the method of the invention or otherwise included. It is also possible to include other substances derived from a physical or chemical combination of the active agents with suitable substrates. In this way it becomes possible, for example, to achieve high protection of the active agent. The active agent may also be included as such, or after the addition of suitable inert fillers. The practice of the present invention has been found to be particularly effective in the preparation of biological catalysts, as well as due to the above-noted ability to obtain such catalysts in a variety of forms. It has thus become possible to make fibers, fibrids, cylindrical bodies of various sizes, ellipsoids, spherical bodies, powders of many grain sizes, even more, since the desired shape is a function of the sequence of steps followed in the final molding treatment.

The latter treatment can be carried out by treating the mixture formed from the solution of the polymer matrix and the active ingredient or its dispersion in a medium which is not a solvent for the polymer matrix.

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The coagulation can take place by dropping or dropping the mixture into the medium, and, for example, bodies having a spherical or spheroidal or spheroidal shape or by direct inflow or inflow of the mixture into the interior of the medium are obtained, so that fibers or similar structures can be obtained. _;

An alternative molding procedure is dry extrusion or dry extrusion with evaporation of the solvent of the mixture as defined above. There is thus obtained a continuous filament or a continuous fiber which can serve to obtain cylindrical bodies and the like having cross-sectional shapes of different shapes.

Another molding operation is to eject from a pressurized space, with or without the aid of a propellant, the above mixture so that formation of fibrids or powders of various particle sizes or semolina sizes can be achieved.

The polymer matrices that can be used in the process of the invention include, but are not limited to, cellulosic polymers, esterified or etherified cellulosic polymers, polyamides, acrylonitrile polymers and copolymers, butadiene and isoprene, acrylates and methacrylates, vinyl esters, vinyl chlorides, polymers and copolymers of vinylidene chloride, styrene, vinyl butyrate, y-methyl glutamate and the like.

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The cellulose esters have been found to be particularly suitable for the purposes of the present process.

The media in which the active agents can be dissolved or dispersed, if it is not possible to start from the active agents as such, are, as mentioned above, selected from those which are compatible with the agent in question or those with the solvent for the polymer matrix can be mixed. They can be selected from the following compound classes, with the preferred ones in parentheses:

Water,

Alcohols (methanol, ethanol, propanol, butanol, tert-butanol, ethylene glycol, glycerol),

Ketones (acetone, ethyl ketone),

Ether (dioxane, tetrahydrofuran, ethoxyethanol), esters (ethyl and methyl acetates, propyl acetate, ethyl formate

and other),

Acids (acetic acid, propionic acid and formic acid), pyridine, acetonitrile, cyclohexane, dimethylformamide.

The solvents for the polymer matrix may in turn be selected from a wide range of compounds that are consistent with the nature of the polymer matrix in question.

For example, the following compounds can be mentioned:

Acetone, methyl isobutyl ketone, cyclohexanone, methyl acetate, methyl cellosolve, ethyl lactate, methylene chloride, propylene chloride, tetrachloroethane, nitromethane, chlorophenol, metacrere

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sol, acetic acid, formic acid, dimethylformamide, dimethyl sulfoxide, alcohols, dioxane, hydrocarbons such as benzene, toluene, ketones, esters, pyridine, chloroform, mixtures of ethanol and water, ethanol and CCl ^, isopropanol, methyl ethyl ketone.

The abovementioned additives have the task of aggregating or cross-linking the active agent or the active ingredient at the stage of its mixture with the polymer matrix. <'>

Among the additives that can be used, you can find the following special mention:

a) polymers of different molecular weight, which are not soluble in the solvent for the polymer matrix: for example, polyamines, such as polyethyleneimine, cationic and anionic polyacrylamides, polyamino acids, such as polylisines, sulfonated polystyrene, polycarboxylic acids, polyvinyl alcohols, polyvinylpyrrolidone.

b) polyfunctional eeagents, such as aliphatic aldehydes, isocyanates, thioisocyanates and the like.

Execution is 1

The invention will be explained in more detail below with reference to some examples. The following examples serve to further explain working details; they are not intended to be limiting.

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example 1

Beads of β-glactooxidase obtained by inclusion

of Saccharomyces lactis cells.

A 10% solution (Gevu base) of cellulose acetate (Fluka) in acetone (Carlo Erba) is prepared. 375 g of this polymer solution are added to 154 g of cellular paste (dry weight 35 g, 8 g), never from 2 l of a fermentation of Saccharomyces lactis, while stirring.

A thus-obtained cell dispersion, which is introduced into a steel container, through a capillary tube with a

Diameter of 0.4 mm by mere press ^ with. Nitrogen extruded or extruded. The thin extruded filament, or thin extruded fiber, after a fall of about 20 cm, is converted into a crown (rosary) of beads that drip into a vesicle bath and coagulate in the form of beads.

About 800 g of moist beads are obtained which, when dried in a stream of air, weigh a total of 73 g. 1 g of these beads is incubated at 25 ⁰ C with 200 ml of a solution of aqueous lactose (4.75 wt .-%), the 2 mWöl MgSO ₄ . After 2 hours of reaction, 80 % of the lactose is converted to glucose and galactose as shown by analysis of glucose after the glucose assay (Boehringer).

The hydrolysis of the lactose with these beads was carried out 20 consecutive times without any loss of activity.

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Example 2 · diminutive of reins of penicillin acylase obtained by inclusion of E. coli cells that have been previously treated with polyethyleneimine. ;

I G

E. coli cells (dry weight 20 g) containing 1 × 10 units were slurried in 875 S water and treated with 25 g of a 3% by weight solution of polyethyleneimine (Polysciences) for 10 minutes with stirring. The result is the formation of cell aggregates that settle easily.

The cell paste is then reslurried in a small volume of water (total weight 127 g) and stirred vigorously with 157 g of a 10% strength by weight solution of cellulose acetate (Fluka) in acetone (Carlo Erba). According to the procedure described above in Example 1, 35 ε beads (dry weight) are obtained. A portion of these beads (5 g) is concentrated at 37 ⁰ C in 400 ml of a solution of penicillin G (Squibb) 6%. The original activity which developed was 60,000 units (micromoles of penicillin G hydroylated in one hour) and total hydrolysis was achieved within about 4 hours. The same beads, when used repeatedly for 20 consecutive times (hydrolysis), retain 60% of their original activity.

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Example 3.

Pellets of penicillin acylase obtained by inclusion of E. coli cells previously treated with polyethyleneimine and glutaraldehyde.

E. coli cells (dry weight 20 g) containing 1 × 10 units were slurried in 400 g of water and stirred for 10 minutes with 25 g of a 3 _f / 3% solution of polyethyleneimine (Polysciences Inc.). and 5 g of a 25% solution of ^ glutaric aldehyde.

The cells obtained on settling were stirred vigorously with 157 g of a 10% solution of cellulose acetate in acetone.

The preparation of the beads was carried out according to the procedure described in the preceding examples. 40 g of dry beads were obtained, of which one part of 5 g was used to study the activity under the same conditions as in Example 2. The activity which unfolded was 50,000 units and the hydrolysis was completed within a period of little more than 4 hours. In contrast to the beads made without glutaraldehyde, the beads of this example retain their activity as compared to the original one.

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Example 4-

Glucose isomerase beads obtained by entrapment of Arthrobacter sp. Cells previously treated with polyacrylamide (Prodefloc A / 1S)

To 10 l of a culture broth of Arthrobacter sp. Cells containing the glucose isomerase enzyme, 500 g of a 0.3% solution of Prodefloc A / 1S were added after the fermentation had ended; the cell slurry was stirred for 20 minutes, whereupon the cells were allowed to settle. The cell paste (dry weight 150 g) was recovered and re-slurried with water until a total weight of 600 g was obtained and mixed vigorously with 1500 g of a 10% solution of cellulose acetate in acetone. Beads were prepared according to the procedure described in the previous examples. 1 g of these beads solution of glucose was added at 60 ⁰ C in 100 ml of a 50% (wt./wt.) Incubated 5 x 10 -3 mole of MgSO ₄ 7H ₂ O χ, 10 "" ⁴ mol CoCl _2, 0.1 mol of ITa ₂ SO ₃ ; pH 7. Polarimetric measurements determined activity in 100 international units (micromoles of fructose prepared in 1 minute) with a yield (unfolded activity / entrapped activity) of 56 % . These beads did not lose their activity when used continuously for 20 consecutive days under the study conditions.

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Example 5

Beads of glucose isomerase obtained by inclusion of the enzyme in solution upon treatment with polyethyleneimine and

Gl u * arald e hyd .

The enzyme solution was prepared by dissolving 2 g of glucose isomerase powder in 8 g of water. To this solution is added 4 g of a 3% solution of polyethylenimine and 4 g of a 2.5% solution of glutaraldehyde with stirring. This enzyme preparation is mixed with 10% of a 10% solution of cellulose acetate in acetone at room temperature. According to the procedure described in the preceding examples, beads are prepared whose weight when dried in air is 12 g. A portion of these beads (2 g) solution of glucose is at 60 ⁰ C with 100 ml of a 50% (wt./wt.) Incubated 5 x 10 "* ^ mol MgSO ^. 7H ₂ O, 10"^"4 Mol CoCIp, 0.1 mol Na ₂ SO 4, pH 7, to determine the activity, which is 500 international units boi in a yield of 30 to 40 % .

Example 6

Cylindrical body of cellulose acetate, the polyethylene imine

to include as sequestering agent \

15 g of cellulose acetate are dissolved with 85 g of acetone, pure reagent. To the polymer solution is added slowly and with stirring 15 g of a 33 % (w / w) aqueous solution of polyethyleneimine hydrochloride and 5 g of a 1% solution of glutaraldehyde. The mixture is introduced into a steel cylinder, which is connected at its upper end with a nitrogen cylinder and at the bottom with a spinneret, the

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in, dipping a water bath. By applying nitrogen pressure, the mixture exits the spinneret and coagulates to obtain a continuous filament. This is crushed by a cutter into samples of 1 to 2 cm in length. 1 g of this cylindrical body is treated for 4 hours with a Eupfer-II-J.-ösang; at a concentration of 18.3 parts per million (ppm), which was obtained by dissolving CuSO 4 .5H ₂ O in distilled water. An atomic absorption spectrophotometer (Varian-Techtron 1200) is used to measure the copper content (1.1 ppm) of the solution treated with the cylinders. Washing the cylinders with 50 ml of In-HCl gives 17 ppm of copper. "

The cylinders are again contacted with the above copper solution for 4 hours, and the copper content in the solution is measured again to be 1.2 ppm. This sequence is repeated ten times and it can be seen that the cylinders do not lose their copper sequesterability.

Example 7

Invertase fibrids obtained by including the enzyme in

a solution to which polyvinylpyrrolidone has been added

50 g of a solution of invertase (B.D.H.) are stirred vigorously with 333 g of a 15% strength solution of cellulose acetate in acetone after addition of 10 g of polyvinylpyrrolidone. The preparation is placed in an autoclave and extruded through nitrogen (about -50 bar, 50 atm.) Through a 500 / μm (micron) nozzle, and

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Winnt a dry powder of fibrid type whose particle size in the range of 160 to 1600, to (microns).

The activity dea included in the fibrids Invertaseenzyms was measured on a solution of 20% sucrose in a 0.1M pH 4 at 25 ⁰ C phosphate buffer. The activity expressed as mg of inverted sucrose per minute and per gram of fibrid is in the range of 8 to 50, depending on the fibrid size.

Example 8

Fibers of hydroxypyridimine hydrolase and N-carbamoyl-D-amino acid hydrolase obtained by inclusion of Agrobacterium radio-bacter cells to which polyethyleneimine has been added '___

Agrobacterium radio-bacter cells (dry weight 4 g) were slurried in 100 g of water and treated with stirring with 2 g of a 3% solution of polyethyleneimine. After 10 minutes, the stirring is stopped and the cells which have settled are collected and slurried in 25 g of distiller. The slurry is vigorously stirred with 20 g of a 20% solution of cellulose acetate in acetone. The preparation is then introduced into a steel cylinder, which is connected at its upper end with a nitrogen bottle and at the bottom of a monofilament spinneret (diameter 1 mm). By a metering pump, the preparation is spun through the spinneret in the form of a continuous filament which is allowed to dry in air by evaporation of the acetone during a 4-ra fall. The entire thread (dry weight 8 g) is dried at 40 ^° C. under a nitrogen blanket in a

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0.1 M, pH 8 phosphate buffer containing 4 g of DL-phenylhydantoin, for the determination of the activity of the two enzymes. After 20 hours of reaction, complete conversion of the hydantoin to D (-) - phenylglycine is demonstrated by analysis on an amino acid analyzer. The same thread loses 30 % of its initial activity when used 10 times during equal number of successive hydrolyses.

Claims (8)

Erfindungsansprach 1, Process for the preparation of microporous bodies, which include one or more active agents, characterized in that an organic solution of a polymer matrix with the respective active agent or
a solution or a dispersion in a medium compatible therewith and miscible with the organic solvent for the polymer matrix, and then carrying out a shaping step or a shaping process. 2. A process for the preparation of microporous bodies, which include one or more active agents, according to item 1, characterized in that in particular the following steps are carried out: a) Forming a first solution of a polymer matrix-in
an organic solvent, b) possible formation of a dispersion or solution of the
respective active agent in a compatible therewith and with the organic solvent for the
Polymer matrix miscible medium, c) possible addition of an additive to the active agents or to the product obtained under b), which is selected from: 1, polymers of different molecular weight, not in the solvent for the polymer matrix
are soluble, - 17 - 56 982 18 2. polyfunctional reagents selected from aliphatic aldehydes, isocyanates and thioisocyanates, d) mixing the solution of the polymer matrix with the active ingredient as such and with the product obtained under b), with or without the addition of stage c) *, e) immediately converting the mixture obtained in the previous stage to the shaping treatment, A method for producing microporous bodies which include one or more active agents according to item 2, characterized by carrying out stage e) by coagulating the mixture of stage d) in a medium which is not a solvent for the polymer matrix. 4 ·. Process for the preparation of microporous bodies, which include one or more active agents, according to item 3>, characterized in that the coagulation is carried out by dripping or dropping the mixture into the medium. 5. A process for the preparation of microporous bodies which include one or more active agents according to item 3 »characterized in that the coagulation is carried out by directly flowing the mixture into the medium« - 18 - 56 982 18 6. A process for the preparation of microporous bodies, which include one or more active agents, according to item 2, characterized by carrying out stage e) by extruding bzvj, extruding under dry conditions the mixture of stage d). Process for the preparation of microporous bodies including one or more active agents according to item 2, characterized in that one carries out stage e) by ejecting the mixture of stage d) from a pressurized environment. 8. A method for the degradation of microporous bodies which include one or more active agents according to item 2, characterized in that the active agents are selected from enzymes, enzyme cells, antigens, antibodies, antisera, hormones, coenzymes, bound to macromolecular matrices, Sequestering agents and dyes. 9. A process for producing microporous bodies which include one or more active agents according to item 2, characterized in that the polymer matrix is selected from cellulose polymers, esterified and etherified alkyl polymers, polyamides, polymers or copolymers of acrylonitrile, butadiene, isoprene, Aeryates, methacrylates, vinyl esters, vinyl chlorides, vinylidene chloride, styrene, vinyl butyrate, T'-methylglutamate. - 19 -. 56 982 18 A process for the preparation of microporous bodies which include one or more active agents according to item 2, characterized in that the solvent is selected from the polymer matrix under acetone, methyl isobutyl ketone, cyclohexanone, methyl acetate, ethylcellosolve acetate, methyl cellosolve, ethyl lactate, methylene chloride, Propylene chloride, tetrachloroethane, nitromethane, chlorophenol, m-cresol, acetic acid, formic acid, dimethylformamide, dimethyl sulfoxide, alcohols, dioxane, hydrocarbons, ketones, esters, pyridine, chloroform, mixtures of ethanol and water, ethanol and CCl., Isopropanol and methyl ethyl ketone. 1.1. Process for the preparation of microporous bodies, including one or more active agents, according to item 2, characterized in that the medium of stage b) is selected from among non-alcohols, ketones, ethylhexenes, esters, acids, pyridine, Acetonitrile, cyclohexane, dimethylformamide.