LU82166A1 - PROCESS FOR THE PREPARATION OF MICROPOROUS BODIES CONTAINING ONE OR MORE ACTIVE AGENTS - Google Patents

Description

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The present invention relates to a process for the preparation of microporous bodies containing one or more active agents, and more particularly those of agents having biological activity.

5 This process comprises mixing the organic solution of a polymer matrix with the active agent concerned, or else with a solution or a dispersion of this agent in a medium compatible with it and miscible with the organic solvent used for the matrix. polymer, then a shaping operation.

. According to the specification of Italian Patent No. 836,482, it is known that enzymes and preparations containing them can be immobilized inside filamentous polymer structures. This process is carried out according to this patent 15 by first preparing an emulsion of an aqueous solution of the enzyme and of a solution of the polymer dissolved in a solvent immiscible with water, said emulsion then being extruded through a die in a coagulation bath to provide a filament which contains therein the solution of enzyme which was initially present in the emulsion.

In doing so, biological catalysts are obtained which have a high activity but their practical implementation is often limited by the form which is given to them by the process mentioned above and by the relative complexity of the preparation.

The present invention, as indicated above, relates to an improved process for the preparation of microporous bodies containing active agents of all kinds and more particularly those which exhibit biological activity, these bodies having any shape of their choice, so that their practical use, that is to say their use in the industrial preparation of the reactions in which these agents are involved, is not prevented by any means: thus obtained for example biological catalysts having a high yield and whose l 'exploitation is absolutely not limited by the practical provisions which they must undergo, precisely because of the different forms *] under which they can be obtained.

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For more detail, the present invention provides a process for the preparation of microporous bodies containing one or more active agents, chosen from those mentioned below, said process comprising the following operations: a) formation of a preliminary solution of a polymer matrix in an organic solvent, b) if necessary, formation of a dispersion or of a solution of the active agent concerned in a medium which is both compatible with the agent and miscible with the organic solvent for the polymer matrix, c) optional addition to the active agent or to what has been obtained in operation b) of an additive whose nature! and the function will be explained further below] 15 of this memo, d) mixing the solution of the polymer matrix with the active agent as it is or with a solution of said agent,! or a dispersion according to stage b) above, containing or j, not containing the additive of stage c), !: 20 e) sending of the mixture originating from stage d) above to the shaping treatment .

i j j Active agents which can be enclosed according to; the method disclosed in the present application are biological compositions such as enzymes or enzyme cells, antigens, antibodies, antisera,! = hormones, co-enzymes, appropriately linked | to macromolecular matrices, or also substances • with other types of activity or which have! particular behaviors such as chelators, | 30 dyes with a clearly determined molecular weight, | and in general all those substances which are not intended to be recovered but which, in the case in question, can on the contrary be recovered to be reused or ji to be reactivated with a view to being reused depending on the case..

It should be added that, according to the method described above, we! can also enclose several bodies containing the active agents at once according to the process of the present 1) Invention or in another way. It is also possible to also enclose other substances originating from the physical or chemical union of active agents with suitable substrates. In doing so, it becomes possible, for example, to obtain great protection for the active agent. The active agent can also be enclosed as it is, or after having added to it suitable inert charges.

The process of the present invention has proved to be particularly effective for the preparation of biological catalysts, also because of the possibility, underlined 1 ° above, of obtaining these catalysts in a large number of forms. It was thus possible to obtain fibers, fibrids, cylindrical bodies of various dimensions, ellipsoids, spheroidal bodies, powders of very different particle sizes, insofar as the desired shape depends on the successive operations followed in the final treatment. of shaping.

This latter treatment can be carried out by bringing the mixture, formed by the solution of the polymer matrix and of the active agent or of the dispersion thereof, into a medium which is not a solvent for the polymer matrix,

Coagulation can take place by abruptly immersing the mixture in the medium and one obtains for example bodies having the spherical or spheroidal shape, or else by making the mixture flow directly inside the medium in order to obtain fibers or analogous structures. Another shaping process is dry extrusion with evaporation of the solvent from the mixture defined above, thus a continuous filament is obtained which can be cut to obtain cylindrical bodies and the like 30 with cross-sectional shapes of various contours. . Another shaping method consists in ejecting the above-mentioned mixture from a pressure vessel, with or without the aid of a propellant, so that the formation of fibrids or powders of various particle sizes can be thus performed.

The polymer matrices which can be used according to the process of the present invention are inter alia: V cellulosic polymers, cellulose polymers III · I '

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esterified or etherified, polyamides, polymers and copolymers of acrylonitrile, butadlene and isoprene, jj ‘of acrylates and methacrylates, vinyl esters, vinyl chlorides, polymers and copolymers of chloride j; 5 of vinylidene, styrene, vinyl butyrate, gamma-methyl 1 glutamate and the like.

Cellulosic esters have been found to be particularly suitable for the purposes of the present invention.

; 10 The media in which the active agents are | j 4 dissolved or dispersed, whenever it is not possible | j to start from the active agents themselves, are chosen as

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j-j suggests above, among those which are compatible with the agent concerned or which can be mixed with the solvent of jj of the polymer matrix. They can be chosen from the following ij classes of compounds, the preferred substances being jj in parentheses:

Ij water, alcohols (methanol, ethanol, propanol,; butanol, tert.butanol, ethylene glycol, glycerol), 20 ketones (acetone, methyl ethyl ketone), ethers (dioxane, tetrahydrofuran, ethoethanol), esters, (acetates ethyl and methyl, propyl acetate, ethyl formlate and others), acids (acetic, proplonic and formic acids), pyridine, acetonitrile, cyclohexane, dimethylformamide.

: For their part, the solvents for the polymer matrix can be chosen from a wide range of compounds according to the nature of the polymer matrix concerned.

"50 By way of example, the following compounds may be mentioned: I Acetone, methylisobutyleetone, cyclohexanone,!. Methyl acetate, methylcellosolve acetate, ethyl lactate, methylene chloride, propylene chloride, I 35 tetrachloroethane, nitromethane, chlorophenol, metacresol, acetic, formic, dimethylformamide, dlmethylsulfoxyide, alcohols, dioxane, hydrocarbons such as benzene, toluene, ketones, esters, pyridine, chloroform, mixtures of ethanol / 5 and water, ethanol and CClit, isopropanol and methyl ethyl ketone.

The role of the additive products mentioned above is to agglomerate or crosslink the active agent in the operation of mixing the latter with the polymer matrix.

Among the additive products which can be used, the following may be mentioned in particular: a) polymers of different molecular weights, insoluble in the solvent of the polymer matrix: for example polyamines such as polyethyleneimine, cationic and anionic polyacrylamides, polyamino acids such as poly-lysines, sulfonated polystyrene, polycarboxylic acids, polyvinyl alcohols, polyvinylpyrrolidone.

B) Polyfunctional reagents such as aliphatic aldehydes, isocyanates, thio-isocyanates and the like.

The present invention is illustrated by the following descriptive and nonlimiting examples showing all the operational details, EXAMPLE 1 - Beta-galacto-oxidase spherules obtained by occlusion of Saccharomyces lactlfs cells

A 10 # solution (by weight) of cellulose acetate (Fluka) in acetone (Carlo Erba) is prepared. 254 g of cell paste (dry weight 35.8 g) from 2 liters of fermentation of Saoharomyces lactis are added to 375 grams of this polymer solution.

A dispersion of cells is thus obtained which is introduced into a steel container, and which is extruded through a capillary tube having a diameter of 0.4 mm, operating purely and simply under nitrogen pressure. The thin extruded filament, after a fall of about 20 cm, is converted into a chain of pearls which, falling in a water bath, are coagulated in the form of spherules.

About 800 grams of wet spherules are thus collected which are then dried in a stream of air and which weigh a total of 73 g. 1 gram of these spherules is incubated at 25 ° C. with 200 ml of an anhydrous lactose solution \ (4.75% by weight) containing 2 millimoles of MgS0 ,,. 7Hp0, one -ητΤ IL · a «6 millimole of EDTA , in 0.1 M phosphate buffer, pH 7 ·

After a reaction of two hours, 80 # of the lactose are transformed into glucose and galactose as shown by the glucose analysis carried out with the Boheringer test.

5 The hydrolysis of lactose with these spherules is repeated 20 times in succession without any loss of activity being detected.

EXAMPLE 2 Penlcllllne acylase spherules obtained by occlusion of E. coll cells treated beforehand with polyethylene lne

E.coll cells (dry weight 20 g) containing 1.10 ^ units in 875 g of water are suspended and treated for 10 minutes, with stirring, with 25 g of one! 3.3 # solution by weight of polyethyleneimine (Polysciences).

We notice the formation of cell aggregates which deform easily.

; The cell paste is then diluted again in a small volume of water (total weight 127 μ) and stirred vigorously with 157 g of a 10% by weight solution of cellulose acetate (Pluka) in acetone (Carlo Erba).

: · With the process described in Example 1 above, 35 g of spherules (dry weight) are prepared. We put some of these | spherules (5g) incubated at 37 ° C in 400 ml of a solution | tion of penicillin G ("Squibb") concentrated to 6 # in an I 25 0.02 molar phosphate buffer of pH 8. Initial activity I. which is present reaches 60,000 units (micromoles of * penicillin G hydrolyzed in one hour) and hydrolysis is | completed in approximately 4 hours. The same spherules dd which have been used 20 times in succession (hydrolysis) retain S -30 another 60 # of their initial activity.

1 EXAMPLE 3 - Penlcllllne-acylase spherules obtained by 1 occlusion of E. coll cells treated beforehand with polyethylene-ml and glutarlic aldehyde.

S 35 E. coll! J cells (dry weight 20 g) containing 1.10 ^ units are suspended in 400 g of water and I! they are treated, with stirring, for 10 minutes, with 25 g of a 3.3 # solution of polyethylene lmine (Polyscien- ces 7, Inc.) and 5 g of a 25 # solution of glutaric aldehyde (Merck).

The cells collected by decantation are vigorously agitated with 157% of a 10 # solution of cellulose acetate (Fluka) in acetone (Carlo Erba).

The preparation of these spherules is carried out according to the method described in the previous examples. 40 g of dry spherules are obtained, part of which, 5 g, is used to control the activity under the same conditions as in Example 2 above. The activity which is manifested is 50,000 units and the hydrolysis is completed in a little more than 4 hours. Unlike the spherules prepared without glutaric aldehyde, the spherules of the present example retain their activity Unchanged compared to the initial activity.

EXAMPLE 4 Glucose isomerase spherules obtained by occlusion of Arthrobacter sp. previously treated with polyacrylamide ("Prodefloc A / 1S").

At 10 liters of a cell culture broth of Arthrobacter sp. containing the glucose-isomerase enzyme, 500 g of a 0.3 # solution of "Prodefloc A / 1S" are added, once the fermentation is complete. The cell suspension is stirred for 20 minutes, after which the cells decan. tent. The cell paste (dry weight 150 g) is collected and resuspended with water until a total weight of 600 g is obtained and the mixture is mixed vigorously with 1500 g of a solution. 10 # of cellulose acetate (Fluka) in acetone (Carlo Erba). The spherules are prepared with the method described in the previous examples. 1 gram of these spherules is incubated at 60 ° C in 100 ml of a 50% by weight solution of glucose containing 5.10- ^ mole of MgSO 4 HgO, 10 ~ ^ mole of CoCl 2, 0.1 mole of Na ^ O ^, at pH 7 · Pa ** of the polarimetric measurements, the activity is determined in 100 international units (micromoles of fructose 35 produced in one minute) with a yield of 56 # (activity manlestested / actlvlté enclosed). These spherules used continuously for 20 days in a row under the test conditions do not lose their activity.

f- h I ’8 i EXEMPTA 5 - Glucose-isomerase spherules obtained by occlusion of the enzyme in solution after treatment

Iwith polyethylene-lne and glutaric aldehyde.

15 The enzyme solution is prepared by dissolving 2g of powder / glucose-isomerase (Godo Shusel) in 8 g of water, To this solution and, while stirring, 4 g of a 3.3 $ solution are added polyethylene-lmne (Polysciences, Inc.) and 4 g of a 2.5 $ solution of glutaric aldehyde (Merck), day 10 This enzyme preparation is mixed with 100 g of a 1 ”day solution. $ 10 of cellulose acetate (Fluka) in acetone (Carlo Erba) at room temperature. With the process described in the previous examples, spherules are prepared whose weight after air drying is 12 g. We put one! 15 part of these spherules (2 g) in incubation at 60 ° C with 100ml 1 of a 50 $ solution by weight of glucose containing 5.10 ^; mole of MgSO ^ .YP ^ O, 10-i | mole of CoClg, 0.1 lmole of Na 2 SO 4, pH 7, to determine the activity which is 500 international i i units with a yield of 30 to 40 $.

| 20 EXAMPLE 6 - Cylindrical bodies of cellulose acetate | containing polyethylene-lmlne as a chelator | 15 g of cellulose acetate (Fluka) are dissolved in 85 g of acetone (pure reagent by Carlo Erba). 15 g of a 30% by weight aqueous hydrochloride solution are added slowly to the polymer solution while stirring. of polyethylene-lmlne (Polysciences, Inc.) and 5 g of a j „| $ 1 solution of glutaraldehyde (Merck). The mixture is transferred into a steel cylinder connected at the top to a nitrogen bottle and at the bottom with a die immersed in | 30 a water bath. By compressing with nitrogen, the mixture leaves the die and coagulates, so that a continuous filament is obtained; this is cut with an appropriate device into samples 1 to 2 cm long. One gram of these cylindrical bodies is brought into contact for 4 hours; 35 with a cupric solution having a concentration of 18.3 parts per million (ppm) obtained by dissolving CuSO ^ .SH ^ O in distilled water. With an atomic absorption spectrophotometer ("Varian-Techtron 1200"), the copper content 9 (1.1 ppm) in the solution treated with the cylinders is measured. After washing the cylinders with 50 ml of normal HCl, I7 ppm of copper is found.

The cylinders are brought back into contact for 4 hours with the copper solution indicated above and the copper content in the solution is measured once more and found to be 1.2 ppm. This series of operations is repeated 10 times and it is seen that the cylinders retain their ability to sequester copper.

- · 10 EXAMPLE 7 - Invertase flbrides obtained by enclosing the enzyme in a solution to which polyvinylpyrrolldone has been added.

A solution (50 g) of invertase (BDH), after addition of 10 g of polyvinylpyrrolldone (K30, Fluka), is stirred vigorously with 333 g of a 15 # solution of cellulose acetate (Fluka) in the acetone (Carlo Erba). The preparation introduced into an autoclave is extruded under nitrogen pressure (50 bars) through a 500 micron nozzle and a dry powder of the fibrid type is collected, the particle size of which is between 160 and 1600 micrometers. The activity of the invertase enzyme enclosed in the fibrids was measured on a 20 # sucrose solution in 0.1M phosphate buffer, pH 4, at 25 ° C. The activity, expressed in milligrams of invert sucrose per minute 25 and per gram (g) of layers of fibrids varies from 8 to 50 depending on the size of the fibrids.

EXAMPLE 8 Hydroxyplrlmldlne hydrolase and N-carbamoyl-D-amlno-aclde-hydrolase fibers obtained by occlusion of Agrobacterlum ethylene-mlne cells;

Agrobactérlum radiobacter cells (dry weight 4g) are put in suspension in 100 g of water and treated, with stirring, with 2 g of a 3 # solution of polyethyleneimine (Polysclences, Inc .). After 10 mi-35 minutes, the stirring is stopped and the cells which have settled are collected and suspended in 25 g of water. The suspension is stirred vigorously with 20 g of a

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j 20 # solution of cellulose acetate (Fluka) in acetone -f- / C.

; ίο j (Carlo Erba). The preparation is then introduced into a steel cylinder connected at the top to a nitrogen bottle and at the bottom to a monofilament die (diameter 1 mm). Using a measured flow pump, the preparation is extruded through the die in the form of a continuous filament which is allowed to air dry by evaporation of the acetone during a descent of 4 meters. The complete yarn (dry weight 8g) is || incubated at 40 ° C. under a nitrogen atmosphere in a 0.1 μM phosphate buffer, pH 8, containing 4 g of DL-phenylhy-: J 10 dantoin to measure the activity of the two enzymes. After 20 to 11 hours of reaction, the complete transformation of the hydantoin to D (-) phenyl-glycine is obtained as shown in ana-i! lysis done with an analyzer for amino acids. The same yarn, when used 10 times to make a large number | 15 of successive hydrolysis loses $ 30 from its initial activity.

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Claims (11)

11 1. Process for the preparation of microporous bodies containing one or more active agents, characterized in that it comprises the operations of mixing an organic solution of a polymeric material with the active agent concerned or with a solution or a dispersion of this agent in a medium compatible with it and miscible with the organic solvent of the polymer matrix, then the execution of an operation. of shaping. 2. Process for the preparation of microporous bodies containing one or more active agents according to claim 1, characterized in that it comprises in particular the following operations: a) formation of a preliminary solution of a polymer matrix in an organic solvent, b) optional formation of a dispersion or solution of the active agent concerned in a medium compatible with it and miscible with the organic solvent of the polymer matrix, c) optional addition to the active agents or to what has been obtained in b) of an additive product chosen from 1. polymers of different molecular weight insoluble in the solvent of the polymer matrix, 2. polyfunctional reagents chosen from aliphatic aldehydes, isocyanates and thlo -isocyanates, “25 d) mixing of the solution of the polymer matrix with 1 active agent as it is, and with what was obtained in b) with, or without, the product of stage c), e) immediate flight of the mixture e obtained in the previous stage towards the shaping treatment. 3. Method for the preparation of microporous bodies containing one or more active agents according to claim 2, characterized in that stage e) is carried out by coagulating the mixture of operation d) in a medium which is not a solvent for the polymer matrix. 4. Process for the preparation of microporous bodies containing one or more active agents according to claim 3, characterized in that the coagulation takes place by i immersing the mixture in the medium. : ' 5. Process for the preparation of microporous bodies i containing one or more active agents according to claim! ‘· H 3, characterized in that coagulation takes place by running the mixture directly into the medium. 6. Process for the preparation of microporous bodies i containing one or more active agents according to claim j 2, characterized in that operation e) is carried out by • i 1 dry extrusion of the mixture of the operation d). 7. Method for the preparation of microporous bodies j containing one or more active agents according to claim I 1 2, characterized in that operation e) is carried out in j,: | ejecting the mixture from operation d) from a pressurized enclosure. 8. Process for the preparation of microporous bodies! containing one or more active agents according to claim! 2, characterized in that the active agents are chosen j- from enzymes, enzymatic cells, antigens, i ‘antibodies, antisera, hormones, co-enzymes, linked || macromolecular matrices, chelators and dyes. i 9. Process for the preparation of microporous bodies; containing one or more active agents according to claim 2, characterized in that the polymer matrix is chosen from cellulosic polymers, esterified and etherified cellulose polymers, polyamides, polymers or copolymers of acrylonitrile, butadiene, isoprene, acrylates, methacrylates, vinyl esters, vinyl chlorides, vinylidene chloride, styrene, vinyl butyrate, gamma-methylglutamate. 10. Process for the preparation of microporous bodies containing one or more active agents according to claim 2, characterized in that the solvent of the polymer matrix i is chosen from acetone, methyl isobutyl ketone, cyclohejanone, l acetate, methylcellosolve acetate, methylcellosolve, ethyl lactate, methylene chloride, propylene chloride, tetrachloroethane, nitromethane, chlorophenol, metacresol, acetic acid, for-r> j mique, dimethylformamide, dimethyl sulfoxide, alcohols,% - 13 dioxane, hydrocarbons, ketones, esters, * pyridine, chloroform, mixtures of ethanol and water, ethanol and CCl ^, Isopropanol and methyl ethyl ketone. 11, A process for the preparation of mieroporeqx ^ bodies containing one or more active agents according to claim 2, characterized in that the medium for operation b) is chosen from alcohols, ketones, ethers, esters, acids, pyridine, acetonitrile, cyclohexane, dimethylformamide. 1 *