RU2137835C1 - Method of preparing water-soluble immobilized enzyme preparation - Google Patents

Abstract

FIELD: biotechnology, biochemistry. SUBSTANCE: proteolytic enzyme protosubtilisin is immobilized on polyethylene oxide (molecular mass of the latter is 1 500-4 000 Da) by irradiation of the brake γ-radiation at the dose 0.9-1.1 Mrad and at the ratio enzyme : carrier = 1:(2-6). Before immobilization the reaction mixture is purified from inert proteins by the salt precipitation with sodium phosphate and calcium chloride followed by addition of polyethylene oxide (molecular mass is 4 000 Da) as a vehicle at amount 50-300 mg/ml and lyophilic drying. Method ensures to obtain the lyophilized immobilized enzyme preparation that retains stability for 2 years at storage at temperature below +20 C. Method provides preparing the medicinal form of the preparation as the stable tablet-like porous mass. Invention can be used in medicine and veterinary science for treatment of different diseases where an enzymatic hydrolysis of proteins of necrotic tissues and suppurative masses is required. EFFECT: improved method of preparing. 3 cl, 3 tbl, 4 ex

Description

 The invention relates to biotechnology, in particular to a method for immobilizing proteolytic enzymes, and may find application in medicine and veterinary medicine in the preparation of preparations of immobilized enzymes.

 Various methods for immobilizing enzymes on carriers are known.

 Known methods of immobilization by adsorption of the enzyme on carriers with a highly developed surface, as well as by incorporating the enzyme into various gel structures (Immobilized Enzymes. Ed. By IV Berezin. T.1, M., Moscow State University Publishing House, 1976, p. 79-140).

 The disadvantage of these methods is the low bond strength of the enzyme with the carrier, as a result of which the enzyme can diffuse from the carrier into the solution during operation.

 Immobilization methods are also known, which consist in covalently binding an enzyme to a preactivated carrier. Activation of the carrier is necessary to create functional groups on it that can interact with enzyme molecules.

 A method is known, for example, for producing an immobilized proteolytic complex, comprising dissolving an aldehyde-containing carrier in a buffer solution and then attaching a proteolytic complex obtained from Aspergillus oryzae KC. As the carrier, xyluronide is used, dissolved in 0.1 M Tris-hydroxymethylaminomethane methanol buffer pH 8.05, and the enzyme is attached at a carrier-enzyme ratio of 1: 1 - 3 (A.S. N 1041567, class C 12 N 11 / 10, publ. 15.09.83. Bull. N34).

The disadvantages of this method is a scarce and expensive carrier - xyluronide, low stability of the target product, the need to store the drug in a refrigerator at 0-4 ^o C.

There is also known a method of obtaining a water-soluble immobilized enzyme preparation (IFP), which includes the following successive stages (and.with. N 730694, class C 08 B 37/02, publ. 30.04.80, Bull. N16):
1. Activation of the carrier - dextran with a pier. mass of 20 kDa, in the reaction of periodic oxidation (oxidation state of the carrier γ = 6-48%).

 2. Preparation of the reaction mixture: oxidized dextran is dissolved in water to a concentration of 50 mg / ml and mixed with a solution of the terrilithin enzyme in 0.1 M borate buffer pH 8.5.

3. Enzyme immobilization: the reaction mixture is stirred for 2 hours at 21 ± 1 ^° C, then a 0.1 M solution of borate buffer containing sodium borohydride is added and stirred for another 40 minutes.

4. Desalting IFP: by dialysis in the cold (4 ± 2 ^o C) against distilled water for 48 hours.

 5. Separation of excess unbound enzyme by gel filtration.

 6. Freeze drying.

 Get the drug with a specific activity of 7.4 PE / mg.

 The disadvantages of this method are the complexity of the process (a long process of activating the media, a significant investment of time, energy and raw materials).

 - low quality of the target product.

Closest to the claimed method, the prototype is a method of obtaining an immobilized proteolytic complex, which consists in the following (a.p. N 1442548, class C 12 N 11/00, publ. 07.12.88, Bull. N45):
1. Protosubtilin G10X, which is a complex of bacterial neutral and alkaline proteases from you. subtilis, dissolved in 0.05 M borate buffer pH 8.4 at 2-4 ^o C for 30 minutes, and the insoluble precipitate was separated by centrifugation at 7000 g for 30 minutes To the obtained supernatant add a 10% aqueous solution of polyethylene oxide per mol. weighing 600 - 2000 Yes, mainly 1500 Yes, in the ratio of the carrier - protosubtilin 1: (7.5-8.5). A mixture of the enzyme with the carrier is irradiated with inhibitory γ - radiation in a dose of 2.3 - 2.7 Mrad. A water-soluble immobilized enzyme preparation with a proteolytic activity of 80-120 PE / ml is obtained. The drug is a clear yellowish liquid, which is stored at 4 - 8 ^o C.

The disadvantages of this method are:
- insufficiently high quality of the target product;
- low stability of the drug during storage (after two weeks of storage at room temperature, the drug loses 80% activity).

 It is known that aqueous solutions of immobilized enzyme preparations (IFP) are unstable during storage, so the task of obtaining highly stable dosage forms of IPP is relevant.

 An object of the invention is to increase the stability of the target product.

 The problem is achieved by the proposed method, which consists in the following.

The reaction mixture is prepared by dissolving a proteolytic enzyme (PF), mainly GZH protosubtilin, in a Na-phosphate buffer solution (pH 7.2 - 8.2) of polyethylene oxide (PEO) with a mol. mass of 1500 - 4000 Yes, with the ratio of enzyme: carrier 1: (2-6). The resulting mixture was purified by salt precipitation of ballast proteins and removing the latter by filtration. The filtrate is subjected to irradiation with bremsstrahlung γ-radiation in a dose of 0.9 - 1.1 Mrad. To the obtained proteolytic enzyme immobilized on PEO, PEO with mol. weighing 4000 Da in an amount of 50-300 mg / ml, the solution is subjected to sterilizing filtration, Packed in bottles with a capacity of 10 cm ³ of 1.2 and 5 ml and freeze-dried.

The result is a preparation with an activity of 460 PE in a vial (commercial name "Imosimase amorphous"), which is a tablet-like porous mass, white to white with a cream tint, readily soluble in water. The shelf life of the drug at temperatures up to 20 ^o C is 2 years.

 Obtained by the claimed method, IFP can be used in any field of medicine and veterinary medicine for the treatment of various diseases where enzymatic hydrolysis of proteins of necrotic tissues and purulent masses is necessary.

The determining significant differences of the proposed method from the prototype are:
1. Covalent binding of the enzyme to the carrier is carried out at an experimentally selected, optimal ratio of enzyme: carrier equal to 1: (2-6), which improves the quality and stability of the target product, since when the ratio of the enzyme-carrier less than 1: 2 is not achieved stable stabilization, and with a ratio of more than 1: 6, a partial precipitation of the enzymatic protein by the carrier occurs.

происходит коагуляция и осаждение балластных белков, чему способствует выпадающий в осадок фосфат кальция. Данная стадия позволяет простым способом (без использования центрифугирования) обеспечить освобождение от балласта, что способствует повышению качества целевого продукта и снижению дозы тормозного γ-излучения в 2-3 раза по сравнению с прототипом (с 2,3 - 2,7 до 0,9 - 1,1 Мрад).2. Before immobilization, the enzyme preparation is purified from ballast proteins by successive addition of sodium phosphate and calcium chloride to the reaction mixture. As a result of the reaction

coagulation and precipitation of ballast proteins occurs, which is facilitated by precipitated calcium phosphate. This stage allows a simple method (without the use of centrifugation) to ensure release from ballast, which improves the quality of the target product and reduces the dose of bremsstrahlung γ-radiation by 2-3 times compared with the prototype (from 2.3 - 2.7 to 0.9 - 1.1 Mrad).

 Before lyophilization, the target additive (filler) is introduced into the IFP, which is used as a PEO with a molar mass of 4000 Da in an amount of 50 - 300 mg / ml, which helps protect the IFP from loss of activity during freeze drying, as well as increase the stability of the drug during storage. Unexpectedly, it turned out that this particular synthetic polymer - PEO with mol. weighing 4000 Da in a concentration of 50 to 300 mg / ml is the optimal filler for the preparation of the IPF dosage form, since it simultaneously serves as both a stabilizer and a structurant, which makes it possible to obtain a tablet-like porous dense mass of the preparation after lyophilization. The use of PEO-4000 as a filler is also caused by its availability (produced by the domestic industry), permission to use in medicine and reasonable price.

 Table 1 presents data on the effect of PEO-4000 additives on the quality of the target product (tablet formation).

 From table 1 it can be seen that the addition of PEO-4000 to IPF in an amount of 50-300 mg / ml, mainly 50-100 mg / ml, ensures the formation of a tablet-like dense mass of the drug.

In the table. 2 and 3 presents data on the storage of the lyophilized dried preparation "Imozimaza" at room temperature (18 - 20 ^o C) in comparison with the storage of the liquid form of the drug obtained by the method prototype.

From the table. 2 and 3 it is seen that after 28 days of storage at a temperature of 20 - 25 ^o C, the IFP obtained by the prototype method loses 50 - 78% of the initial proteolytic activity, and after 12 months of storage the loss of activity is more than 95%. The IFP obtained by the claimed method can be stored at room temperature without loss of activity for 12 months.

 The invention is illustrated by the following examples of specific performance.

 Example 1

Prepare the reaction mixture. To do this, 30 g of PEO-1500 are dissolved in 300 ml of 0.025 M Na-phosphate buffer (pH 8.2), 7.5 g of protosubtilin GZH (enzyme: carrier ratio 1: 4) are added, the mixture is stirred at a temperature of 18-20 ^o C within 30 minutes. Then carry out the deposition of ballast proteins by coagulation. To do this, 1.3 g of sodium phosphate (Na ₂ HPO ₄ • 12H ₂ O) is added to the mixture until complete dissolution to a final concentration of 0.45% and 1.9 g of calcium chloride (CaCl ₂ • 6H ₂ O) to a final concentration 0.63%. After adding calcium chloride in the reaction mixture, a precipitate of insoluble calcium phosphate precipitates, which adsorbs ballast proteins. The mixture was incubated for 12 hours at a temperature of +4 +8 ^o C for complete precipitation of ballast proteins. Next, the reaction mixture is filtered through paper filters ("white tape"). The filtrate volume is 300 ml. The resulting filtrate is irradiated with bremsstrahlung γ-radiation at a dose of 1 Mrad. In the process of irradiation, the enzyme is immobilized on a polymer carrier. The activity yield is 85%. After irradiation, PEO-4000 in the amount of 15 g per 300 ml of solution (50 mg / ml) is introduced into a solution containing protosubtiline (IPP) immobilized on PEO-1500 and mixed until completely dissolved. Then carry out sterilizing filtration of the resulting mixture through a membrane filter (Millipore) with a pore size of 0.2 microns. The sterile filtrate is poured into sterile vials with a capacity of 10 cm ³ of 1 ml and freeze-dried. After lyophilization, a preparation is obtained containing in one vial 6.4 mg of IPF (protein) with an activity of 300 PE, 100 mg of PEO-1500, 50 mg of PEO-4000.

 The drug dissolves in water for 1 minute, remains stable for 2 years.

 Example 2

Prepare the reaction mixture. To do this, 75 g of PEO-1500 are dissolved in 300 ml of 0.025 M Na-phosphate buffer (pH 8.2), 15 g of protosubtiline are added, the mixture is stirred for 30 minutes and sodium phosphate is added successively until complete dissolution to a final concentration of 0.45% and calcium chloride to a final concentration of 0.63%. The mixture is kept for 12 hours until the ballast proteins are completely precipitated, then the precipitate is filtered off on a paper filter. The obtained purified filtrate (V - 300 ml) is irradiated with bremsstrahlung γ-radiation at a dose of 1.0 Mrad at an electron accelerator ELV-2. The activity yield is 86%. 30 g of PEO-4000 (100 mg / ml) are added to the solution containing IFP, stirred until complete dissolution, sterilized filtration of the resulting mixture is carried out through a membrane filter with a pore size of 0.2 microns. The sterile filtrate is poured into sterile vials with a capacity of 10 cm ³ of 1 ml and freeze-dried. One vial contains 13 mg IPF (protein) with an activity of 660 PE, 250 mg PEO-1500, 100 mg PEO-4000.

The drug remains stable for 2 years when stored at a temperature not exceeding 20 ^o C.

 Example 3

 The method is carried out analogously to example 1, except that the ratio of the enzyme: carrier in the reaction mixture is set to 1: 2. For this, 30 g of PEO-1500 are dissolved in 300 ml of 0.025 M Na-phosphate buffer (pH 8.2), 15 g of protosubtilin are added and the mixture is stirred. To the obtained IPF after immobilization, PEO-4000 is added in an amount of 300 mg / ml. One bottle of the drug contains 17 mg of IPF (protein) with an activity of 720 PE, 100 mg of PEO-1500, 300 mg of PEO-4000.

 Example 4

 The method is carried out analogously to example 1, except that the ratio of the enzyme: carrier in the reaction mixture is set to 1: 6, and PEO-4000 is used as the carrier. For this, 30 g of PEO-4000 are dissolved in 300 ml of 0.025 M Na-phosphate buffer (pH 8.2), 5 g of protosubtilin are added and the mixture is stirred. Before lyophilization, PEO-4000 in an amount of 50 mg / ml is added to the IFP solution.

 One bottle of the drug contains 1.8 mg of IPF (protein) with an activity of 190 PE, 150 mg of PEO-4000.

Using the proposed method allows, in comparison with the prototype:
- improve the quality of the target product;
- increase the stability of the drug IAP ("Imosimase") during storage,
- to provide the possibility of obtaining a stable tablet-like porous mass.

Claims (3)

 1. A method of obtaining a water-soluble immobilized enzyme preparation, comprising preparing a reaction mixture containing protosubtiline with polyethylene oxide, followed by irradiation of the resulting mixture with γ-radiation with a brake, characterized in that the enzyme-carrier ratio is set to 1: (2-6) from the reaction mixture before the irradiation, salt precipitation of ballast proteins is carried out, and the target product is freeze-dried in the presence of polyethylene oxide with a molar mass of 4000 Da in an amount of 50-300 mg / ml.  2. The method according to claim 1, characterized in that polyethylene oxide with a molar mass of 1500-4000 Da is used as a carrier for immobilizing the enzyme.  3. The method according to claim 1, characterized in that the ballast proteins are precipitated by sequentially adding to the reaction mixture first sodium phosphate to a final concentration of 0.45%, and then calcium chloride to a final concentration of 0.63%.

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