Stabilizing composition for obtaining lyophilised preparations based on conjugate of antiimmunoglobulin g and horseradish peroxidase


(57) Abstract:

The invention relates to the field of biotechnology and immunology, and can be used to obtain stable forms of immunobiological preparations, sokhranyayuschikh its own specific characteristics in a long time. A stabilizing composition includes a protein hydrolysate, sucrose, phosphate-saline buffer, bacteriostatic, bovine serum albumin, EDTA (Trilon B), and potassium chloride in the following ratio, wt.% : bovine serum albumin 0,1 - 3,0; sucrose 1,0 - 10,0; hydrolyzed protein 1,0 - 5,0; potassium chloride 2,0 - 8,0; EDTA (Trilon B) 0,03 - 0,2; bacteriostatic 0,01 - 0,02; phosphate-saline buffer else. As hydrolyzed protein used peptone or gelatin, and as bacteriostatic - thimerosal. The stabilizer provides the conservation of the biochemical properties of the drug conjugate anti-IgG and horseradish peroxidase in the process of freeze drying, storage and rehydration at room temperature. 1 C. p. F.-ly, 3 tables.

The invention relates to the field of biotechnology and immunology, and can be used to obtain stable forms of immunobiological preparations, preserving their shirouma compositions of conjugates on the basis of protein A and antivitamin immunoglobulins [1, 2, 3]. The compositions of the stabilizers include carbohydrates in the form of sucrose or lactose, amino acids and BSA.

The disadvantage of these stabilizing compounds is that they increase the stability of drugs in no more than 60% and thus does not give the option to save the drugs for 1 year. Using glucose or lactose as the carbohydrate component of the stabilizer to obtain conjugates specific activity of the preparations is reduced already at the first stage of freezing - thawing on 35-40%.

The closest technical solution (prototype) is a stabilizing composition to obtain a lyophilized preparations on the basis of protein A and horseradish peroxidase (KG BA:HRP), including sucrose (1,3 - 6%, peptone 4 - 12% and phosphate-saline buffer (FSB) else [4].

The disadvantages of this regulator is that it retains well the drug KG BA:HRP, but not fully protects the conjugate of antiimmunoglobulin G and horseradish peroxidase (KG anti - IgG:HRP) freeze at the stage of preparation, and also does not ensure the preservation of specific recognition of immunoglobulins after prolonged storage of the dry drug at positive temperatures.

The problem is solved in that the stabilizing composition to obtain lyophilised preparations based on conjugate of antiimmunoglobulin G and horseradish peroxidase, including hydrolyzed protein, sucrose, phosphate-saline buffer and bacteriostatic, according to the invention, further comprises bovine serum albumin, EDTA (Trilon B), and potassium chloride in the following ratio, wt.%:

Bovine serum albumin - 0,1-3,0

Sucrose - 1,0-10,0

Hydrolyzed protein - 1,0-5,0

Potassium chloride - 2,0-8,0

EDTA (Trilon B) - 0,03-0,2

The bacteriostatic - 0,01-0,02

Phosphate-saline buffer - Rest

And as hydrolyzed protein used peptone or gelatin, and as bacteriostatic - thimerosal.

A comparison of the proposed stabilizing composition with the known analogues shows that the distinctive features of the prototype signs are new, previously unknown property, namely the complete preservation of the activity of the drug KG anti-IgG: HRP at the stage of lyophilization and the increase compared to the prototype and other analogues of the term of his wounds at positive temperature is an economic decision criterion of "inventive step".

Such preservation activity of the conjugate, probably caused by the following reasons. First, the hydrolysate in contrast to the native protein contains a short peptides, which are much lighter and easier to enter into interaction with both hydrophilic and hydrophobic groups on the surface of the conjugate unlike bulky polymer source of protein molecules. Secondly, the introduction of EDTA in the form of its soluble salts of the trylon B is known for its chelating properties, protects gamenow group of horseradish peroxidase from the damaging interactions with ions of heavy metals. In these interactions active metal ions are replaced by passive to the exchange of potassium ions with the introduction of KCl salt.

Example 1. Technology of preparation of a stabilizing composition to obtain a freeze-dried preparations.

First prepare the hydrolyzed protein by high-temperature splitting of a 10% aqueous solution of peptone according to GOST 13805-76 [5] or gelatin according to GOST 11293-78 [6] (pH = 7.0) at 135oC for 3.5 hours Hydrolyzed peptone and gelatin characterized by amino acid composition, viscosity, and fractional composition characterized by gel filtration and cryoscopy.

Com is the so called 40oC under stirring until complete disappearance of the precipitate. The entire weight of the solution add the required amount of the trylon B. as bacteriostatic use of thimerosal in an amount of 0.01%.

The resulting mixture was filtered through a glass filter, and then through the filter Millipor" with a pore size of 0.45 µm.

The filtrate is cooled in snow bath and add to it with stirring, a solution of the drug KG anti-IgG: HRP, reaching breeding in 500 to 1000 times depending on the titer of the conjugate is determined in direct response by ELISA with standard immunoglobulins.

The product is poured in hoods 0.2 ml in vials of 5 ml, closed with rubber stoppers and placed in a metal cassette for freezing. Freezing of the drug in vials carried out at a temperature of counter - 60oC for 10-12 h

Frozen drug in vials are transferred into the freeze chamber lyophilic install TG-50, shelves which chilled to minus 35oC. the Temperature of the preparation at the stage of freeze dehydration support below the glass transition temperature (-30oC). The average heating rate of the material at the final stage is 6oC/min, time vyderzhivala 1,0 - 2.0 wt.%. The determination carried out by the method of [7]. The vials with the product is sealed under vacuum.

The activity of the conjugate control at all stages of preparation of the dry form: when filling, freezing and after lyophilization. As the ELISA test systems use specially selected from one high quality party kits Recombinant anti - HIV 1+2". ELISA is performed in accordance with the Instructions of applying the specified test system.

To substantiate the quantitative content of components in stabilizing the composition prepared as described above options proposed composition (1-6) and structure-prototype (7). The data are given in table. 1.

Example 2. The study of the preservation activity of the conjugate anti-IgG:HRP at the stage of lyophilization.

Comparative studies on the preservation of the activity of the preparation containing the inventive stabilizing composition with different quantitative content of the components (compounds 1-6) or use of the stabilizer composition-prototype (part 7). The data are given in table. 2.

The results show that the most stable during freezing and dehydration are variants of the drug from formulations 1 and 2, one of which sodl. 2, all the compounds ensure the preservation of the activity of the conjugate is at the stage of freeze cooking and within the accuracy of staging ELISA differences statistically insignificant.

Example 3. The study of the activity of the drug containing different options for stabilizing compositions during storage

To verify the effectiveness of the stabilizers when stored at positive temperatures lay in thermostats 30 vials conjugate anti-IgG:HRP, with different ways of compounds stabilizers, and stores these drugs at a temperature of 4 and 37oC. the Results are shown in table. 3. The activity of the conjugate anti-IgG:HRP in storage is determined in % of the dry form of this medication is stored at -20oC ( = 5%).

The results of the analysis table. 3 shows that during storage of the drug in 4oC for 6 months offer a stabilizing composition (N 1 and N 2) has higher characteristics of the conservation of the drug than the prototype. Characteristics are statistically significant during storage of the drug 2 months at 37oC. With increasing storage time of the preparation of the advantages of the proposed stabilizer become even more obvious.

Mark is ralista protein and sucrose, necessary for the formation of the drug in tablet form with uniformly distributed pores. The total protein concentration and sucrose pick up at least 5%. The upper limit concentrations of native proteins is limited by their solubility in the FSB.

The minimum concentration of EDTA is not securely protects the iron ion from the impurity ions in the solution, and the maximum concentration provides an excess of chelating agent which binds to and partially active iron.

Thus, the economic efficiency of application of the invention consists in the following. The stabilizing composition (1 and 2) based on hydrolyzed protein, sucrose and EDTA (table. 2) allows freeze-dried conjugate anti-IgG:HRP without loss of activity. When storing conjugate anti-IgG:HRP at 37oC during 10 months of the inventive stabilizer composition (table. 3) ensures the preservation of the original activity by 80%, and the prototype is at the level of 50% of the initial activity. Thus, compared with the prototype of the proposed stabilizing composition reduces loss of specific activity of the drug in half when storing it for a long time (10 months or more).

Industrial drugs.

Sources of information

1. U.S. patent N 4504579, CL C 12 Q 1/28, C 12 N 9/96, publ. 19/6, (analog).

2. Vorobyev, M. S., Shalamberidze A. G., Kahn A. N. and other Issues of Virology. - 1992, No. 2.

3. Naofumi Hanafusa. - Contributions from the Institute of Low Temperature Science. Ser. B, 1972, N 17, p 1-38 (analog).

4. The patent of Russian Federation N 2046342, CL G 01 N 33/535, C 12 N 9/96, publ. 1995 (prototype).

5. GOST 13805-076. Peptone is an enzymatic. Gosstandart of the USSR, 1976.

6. THE 11293-78. Food gelatin. Gosstandart of the USSR, 1978.

7. MI 123. 39. 002-88. The method of determining the residual moisture of small quantities of material. - 1988. 7 C.

1. Stabilizing composition for obtaining lyophilised preparations based on conjugate of antiimmunoglobulin G and horseradish peroxidase, including hydrolyzed protein, sucrose, phosphate-saline buffer and bacteriostatic, characterized in that it further comprises bovine serum albumin, EDTA (Trilon B), and potassium chloride in the following ratio, wt.%:

Bovine serum albumin - 0,1 - 3,0

Sucrose - 1,0 - 10,0

Hydrolyzed protein - 1,0 - 5,0

Potassium chloride - 2,0 - 8,0

EDTA (Trilon B) - 0,03 - 0,2

The bacteriostatic - 0,01 - 0,02

Phosphate-saline buffer - Rest

2. the e bacteriostatic - thimerosal.


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