CS203207B1 - Agents for evolving cells from tissues or from cultivating vessels surface in the form of powder granulate or mouldings and method of producing them - Google Patents

Description

The invention relates to a process for the production of cell release preparations from tissues or from the surface of culture vessels in the form of powder, granules or moldings (most often tablets or briquettes) prepared from powdered homogenized or granulated mixtures of proteolytic enzymes, chelating agents and excipients mainly consisting of inorganic salts.

For the preparation of cell suspensions! various proteolytic enzymes such as e.g. trypsin, pancreatin, pronase, elastase, chymotrypsin and others. Similar release of cells from the surface of the culture vessels is accomplished by the above enzymes. In addition to solutions of the above-mentioned enzymes, chelating agents such as ethylenediaminotetraacetic acid, citric acid and others are used to release tissues and release cells from culture vessels. Enzymes or chelating agents are usually dissolved in isotonic sodium chloride solution, which is suitably buffered to a certain pH where the enzyme activity is at maximum. To date, these formulations have been produced for use in in vitro cell and tissue culture in the form of sterile solutions or lyophilizates for immediate use (catalog of Difco, USA r.

1976, Gibco, USA r. 1976, Flow, England r. 1977).

In aqueous solutions, trypsin, as well as other proteolytic enzymes, is unstable and subject to proteolytic self-degradation. During storage, enzymatic activity decreases and products become non-standard. When stored at 0.25% trypsin in buffered saline at pH 4 ° C, trypsin is usable for a maximum of 2-3 weeks, and when stored at -20 ° C, trypsin is stable for 6 months (Von A. Mayr, PA Bachmann, B. Bibrack, G. Wittmann, Virologische Arbeitsmethoden, VEB Gustav Fischer, Verlag Jena 1974).

The present invention eliminates the disadvantages that, to increase the stability of cell release preparations and tissue culture surfaces, enzymes, chelating agents and adjuvants predominantly from inorganic salts are homogenized, formulated into granules or formed on suitable compression machines (under the conditions described below) ) in the form of moldings, spherical, cylindrical, lens or other suitable form.

Accordingly, the present invention is directed to cell release formulations of tissue or surface of culture vessels in the form of powder, granules or moldings, in particular of tab203207 aircraft and briquettes, containing 1-40% by weight of proteolytic enzymes, in particular trypsin, chymotrypsin, pancreatin, pronase, collagenases and other or chelating agents, in particular the disodium salts of ethylenediaminotetraacetic acid, individually or in mixtures. Further, the preparations contain 60-99% by weight of excipients such as:

sodium chloride, potassium chloride, glucose, sodium phosphate, potassium phosphate, sodium phosphate, potassium phosphate, sodium phosphate, sodium carbonate, tris-hydroxymethylaminomethane, calcium chloride, magnesium chloride, polyethylene glycol, polyvinylpyrrolidone, methylcellulose, phenol worm, phenol worm

N-2-hydroxyethylpiperazine-N'-2-thiine sulfonic acid, N-tris-hydroxymethylmethylglycine, 2- (N-morpholino) -ethanesulfonic acid, piperazine-N, N'-bis (2-ethanesulfonic acid) acid,

N, N-bis-1- (2-hydroxyethyl) glycine,

N- (tris- (hydroxymethyl) methyl-2-aminoethanesulfonic acid),

2-bis- (hydroxyethyl) -imino-2-hydroxymethyl) -1-3-propanediol, hydroxyethyl piperazine-N'-3-propane sulfonic acid and others.

Enzymes, chelating agents and excipients consisting predominantly of inorganic salts are preferably homogenized in a fluidization apparatus or in a planetary mixer. An aqueous or alcoholic solution of one or more excipients and the total content of chelating agent enzymes or a mixture thereof are added to the fluidized bed composed of excipients. The mixture is gas dried, preferably air to 100 ° C, or is first granulated and then dried. In another case, the excipients are mixed in a planetary mixer and moistened with a solution of ethanol, propanol, acetone, or a mixture thereof in which the oncotic pressure modifying agents are in particular polyethylene glycol, polyvinylpyrrolidone as well as pH indicating agents such as phenol red. Preferably, the humidified mixture is gas-dried to 100 ° C. After drying, an enzyme or a chelating agent or mixture thereof is added to the granulate and, after homogenization, the granulate is compressed into compacts, preferably into tablets or briquettes, at a pressure of 40 to 200 MPa.

The powdered resulting homogenate or granulate, whether or not the tablets were weighed or filled into suitable vials, was sterilized by gamma irradiation at a dose of 2.5 Mrad. The powdered homogenate or granulate or tablets of the cell-release preparations can also be sterilized by filtration through a Milipor filter after being dissolved in a given volume of redistilled water.

The purpose of the invention is the preparation of high-quality dry preparations for the release of cells from tissues and culture vessels in the form of moldings (tablets, briquettes or moldings of another suitable shape) or homogenized powders and granules. perfect homogenization of ingredients, long-term stability, good solubility, solid standard form and weight in the case of moldings and simplified handling during preparation and transport The obtained compositions retain all the properties for release of cells from animal tissues or culture vessel surfaces.

The advantage of product preparation is the standard of use resulting from the increased stability of dry enzymatic preparations in the form of homogenised powders, granules or moldings (tablets, briquettes). The tablets may be aqueous-weighted to dissolve in a defined volume of water. The economic advantage of cell release formulations or tissue culture containers in the form of granules or tablets is the long-term shelf life and reduced cooling space requirements, as well as reduced shipping and packaging costs.

The possibility of producing large batches of products prepared by the proposed method makes it possible to improve the standard of the enzyme preparations. Dry tissue molding can be carried out either on conventional tabletting machines (briquetting machines) of eccentric or rotary or other kind, under normal atmospheric conditions at a suitable compression pressure, or provided with a special coating of punches teflon, silicone or other suitable mass] after moisture treatment starting material and at relative atmospheric humidity of the environment of 10 - 50 ° / o, depending on the type of pressed mixture of preparations for releasing cells from tissues or culture vessels.

Example 1

400 g of sodium chloride p. a., 10 g of potassium chloride p. a., 144.5 g of medium sodium phosphate p. a. and 5 g of magnesium chloride with 6 H2O p. a. A solution of trypsin 12.5 g, chymotrypsin 12.5 g, potassium potassium phosphate p is successively injected into the fluidized bed. a., 10 g, 8 g polyethylene glycol m. in. 6000 in 50 ml of redistilled water. The resulting granulate is dried at a temperature of up to 100 ° C. The dried granulate can be dispensed in vials of 0.6 g and irradiated with gamma rays to a total dose of 2.5 Mrad. After irradiation, the sterile granulate is dissolved in 50 ml of sterile redistilled water and used to release cells from <5 0 3 2 0 7 culture vessels, or the dried granulate can compress tablets weighing 0.6 g at 98 MPa. Tablets can be sterilized by gamma irradiation with a total dose of 2.5 Mrad. One sterile 0.6 g tablet is dissolved in 50 ml of sterile redistilled water before use.

Example 2

340 g of sodium chloride p. a., 20 g of potassium chloride p. a., 54.75 g glucose, 110 g sodium bicarbonate p. a. and moistened with the composition solution; 49 g 95% ethanol, 0.25 g phenol red and 10 g polyethylene glycol of m. in. 6000. The resulting granulate is dried at a temperature of up to 150 ° C. The dry granulate is mixed with 12.5 g of trypsin and 12.5 g of chymotrypsin with a particle size of 0.125 mm. Tablets weighing 0.56 g at 40 MPa are compressed from the dried mixture. The tablets are sterilized by gamma irradiation with a total dose of 2.5 Mrad. One sterile tablet is dissolved in 50 ml of <RTIgt; pg </RTI> before use. UCalkůJ V V-. ίί% j in y.

Example 3

100 g of tris - hydroxyinethylaminomethane are mixed in a planetary mixer. HO), with 25 grams of tris-hydroxymethylaminomethane and

377.5 g sodium chloride p. a. and moistened with a mixture of 50 g of 96% ethanol, 0.25 g of phenol red and 7.25 g of polyvinylpyrrolidone. The resulting granulate is dried at a temperature of up to 139 ° C. After drying, 12.5 g of trypsin having a particle size of 0.100 mm are added to the granulate. After homogenization! Tablets weighing 0.55 g and diameter 12 mm are compressed at a pressure of 60 MPa. One tablet is dissolved in 50 ml of redistilled water before use and the solution is sterilized by ultrafiltration through a Milipor 0.22 µm filter before use. Of course, gamma radiation can also be used for sterilization as in the previous example.

Example 4

400 g of sodium chloride p. a., 10 g of potassium chloride p. a., 144.5 g of medium sodium phosphate with 12 H2O p. a. and 5 g of magnesium chloride with 6 H2O p. a. 12.5 g of tissue culture trypsin, 10 g of potassium phosphate acid p are dissolved in 50 ml of redistilled water. a. and 8 g of polyethylene glycol of m. in. 6000. The solution is filtered through a S 3 glass sintered filter. This solution is then gradually injected into the fluidized bed. The resulting granulate is dried to 100 ° C. The dried granulate is pressed into briquettes weighing 2.4 g at 200 MPa. The tablets were irradiated with gamma rays with a total dose of 2.5 Mrad. One sterile tablet is dissolved in 200 ml of sterile redistilled water before use.

Example 5

340 g of sodium chloride p. a., 20 g of potassium chloride p. a., 54.75 g glucose, 100 g sodium bicarbonate ρ. a. and 10.0 g of disodium ethylenediaminotetraacetic acid p. a. and moistened with a solution of 0.25 g phenol red, 10 g sodium bicarbonate in 30 ml redistilled water. The resulting granulate is dried at a temperature of up to 130 ° C. The dried granulate is mixed with 10 g of trypsin and 10 g of chymotrypsin having a particle size of 0.125 mm. The dry powdered homogenized mixture is filled in 0.56 g vials and sterilized by total dose of gamma radiation.

2,5 Mrad.

Claims (9)

1. Preparations for the release of cells from the tissues or surfaces of culture vessels in the form of powder, granules or moldings, in particular tabbet and briquettes, characterized in that they contain 1 to 40% by weight of proteolytic enzymes, in particular trypsin, chymotrypsin, pancreatin, pronase, elastase; collagenase or chelating agent, in particular disodium ethylenediaminotetraacetic acid, singly or in mixtures, and 60 to 99% by weight of excipients consisting of ionic strength modifiers for isotonic solution, in particular sodium chloride, potassium chloride, glucose in an amount of 0.5 to 99% o and o buffering agents, in particular sodium phosphate, sodium phosphate, potassium phosphate, potassium phosphate, sodium carbonate, OF THE INVENTION tris-hydroxymethyl-aminomethane. HCl, tris-hydroxymethylaminomethane, N-2-hydroxyethylpiperazine-N‘-2-ethanesulfonic acid, N-tris-hydroxymethylglycine, 2- (N-morpholino) ethane sulfonic acid, piperazine-N-N‘- (bis-2-ethane sulfonic acid, N, N‘-bis-1- [2-hydroxyethyl) glycine, N-triis (hydroxymethyl) methyl 2-aminomethanesulfonic acid, 2-bis- (hydroxyethyl) -imino-2-hydroxymethyl-1,3-prapanediol, hydroxyethyl piperazine-N-3-propane sulfonic acid in an amount of 1 to 99.5 weight percent. 2. Preparations according to item 1 in the form of granular powder or in the form of moldings, characterized in that they additionally contain, as excipients, oncotic pressure modifiers, in particular polyethylene glycol, polyvinylpyrrolidone, methylcellulose, hydroxyethylcellulose and other soluble cellulose derivatives in water in an amount of 0 01 to 2% by weight. 3. Preparations according to Claims 1 and 2, in the form of powder, granules or moldings, characterized in that they additionally contain, as auxiliary substances, pH-indicating substances, in particular from 0.02 to 0.1% by weight of phenol red. 4. Preparations according to Claims 1 to 3 in powder, granulate or molded form, characterized in that they contain, as auxiliary substances, substances which activate the enzymatic activity of proteolytic enzymes, in particular calcium chloride, magnesium chloride from 1 to 5% by weight. 5. A process for the preparation of cell-release preparations from the tissues or surface of culture vessels according to claim 1, characterized in that 60 to 99 percent by weight of the excipients, based on the weight of the final product, are homogenized preferably In the fluidized bed, an aqueous or alcoholic solution containing at least one excipient as well as a pH-indicating agent and 1 to 40 percent by weight of proteolytic enzymes or chelating agent, based on the weight of the resulting product, at a pH of 3 to 6, wherein the obtained mixture is gas-dried, preferably air-dried, at a temperature of up to 100 ° C, first granulated and then dried. Θ. Process for preparing cell-release preparations from the tissue or surface of culture vessels according to Claims 1 to 4, characterized in that 60 to 99% by weight of excipients are homogenized, granulated and dried, and then 1 to 40% by weight, based on the dry granulate, is added. by weight of the resulting product, proteolytic enzymes or chelating agents or mixtures thereof. 7. A method according to any one of Claims 1 to 4 for preparing cell-release preparations from the tissues or surfaces of culture vessels, characterized in that 1 to 40 percent by weight of proteolytic enzymes or chelating agents and 60 to 99 percent by weight of excipients are homogenized. preferably acetone, ethanol, isopropanol, water or a mixture thereof and subsequently granulated and gas dried, preferably air at a temperature of up to 100 ° C. 8. A process according to any one of Claims 5 to 7, characterized in that the powdered product or granulate is compressed into moldings, preferably into tablets or briquettes, at a pressure of from 40 to 200 MPa. 9. A method according to any one of Claims 5 to 8, characterized in that the powdered product, the granulate or the moldings are sterilized by gamma irradiation, preferably with a total dose of 2.5 Mrad.