RU2671537C2 - Hyaluronidase preparation and method of its preparation - Google Patents

Abstract

FIELD: pharmaceutical industry.SUBSTANCE: invention relates to pharmaceutical industry, namely to process for obtaining of a hyaluronidase preparation. Method for obtaining the hyaluronidase preparation includes grinding the testes of cattle, extraction with acetic acid with constant stirring, centrifuging the extract, concentrating the resulting centrifugate, diafiltration, Sterilizing filtration, ampouling and freeze-drying under certain conditions. Preparation of hyaluronidase.EFFECT: above described method allows to increase the yield and activity of the final product.4 cl, 5 dwg, 7 ex, 1 tbl

Description

Field of Invention

The invention relates to the pharmaceutical industry and can be used in medicine to obtain a drug having hyaluronidase activity.

State of the art

Lidase is an enzyme derived from the seminal glands of cattle. The active component of the drug is the enzyme hyaluronidase. This enzyme activates the process of splitting the main element of the intermediate substance of the connective tissue - hyaluronate (glycosaminoglycan, whose metabolites provide the strength of the connective tissue). Hyaluronidase has the following positive properties: reduces the viscosity of connective tissue; enhances the permeability of tissues and blood vessels; facilitates the movement of interstitial fluids; reduces predisposition to edema; promotes healing of scars; stimulates the healing of hematomas; increases joint mobility; reduces the severity of contractures and prevents their development.

Lidase is used for various purposes: for cicatricial lesions subcutaneously (under scar-changed tissues) or intramuscularly (near the lesion site), for traumatic lesions of the nerve plexuses and peripheral nerves, in ophthalmic practice, for the treatment of pulmonary tuberculosis (prescribed in complex therapy to increase the concentration antibacterial drugs in the lesions), externally, in the form of dressings soaked in a solution of the drug, with electrophoresis to increase efficiency.

Methods of obtaining this drug are known from the prior art.

A known method of producing lidase according to patent SU 566875 (publ. 07/30/1977), including extraction with acetic acid from the testes of cattle, separating the extract, followed by drying of the target product, the resulting extract is concentrated and the ballast proteins are separated, then the extract is treated with alcohol and the obtained the precipitate is washed repeatedly with acetone and then with sulfuric ether. The disadvantages of this method: the use in the production of fire hazardous and environmentally hazardous substances, as well as the low yield of the final product (1.3% based on the weight of the feedstock) with a relatively low specific activity (5.12 cu / mg).

A slightly different method for producing lidase is described in RU 1448443 (publ. 08/15/1994). According to this method, raw materials are treated with acetic acid, centrifugation, precipitation with acetone, dissolution of the precipitate in water, followed by filtration and drying of the target product, using testis cake after separation of a substance with hyaluronidase activity, or a mixture of testes and cake, is treated with acetic acid at pH 3.5-4.0 for 12-24 hours at a temperature of 10-15 ° C with the addition of 0.5 g / l of zinc chloride, and the target product is obtained by precipitation with three volumes of acetone at a temperature of from -5 to -10 ° C . This method involves a complex, lengthy and multi-stage process, including precipitation at sufficiently low temperatures by a fire hazardous and volatile reagent (acetone), which leads to an increase in production hazard and a decrease in environmental safety.

Also known is a method of producing lidase according to patent SU 111012 (publ. 01.01.1957), including autolysis of testicular tissue of sexually mature cattle in the presence of acetic acid at pH 4.6, purification of the obtained lysate in a known manner with acetone, followed by centrifugation and dissolution in distilled water, s further sterilization and filtration. The final product is lyophilized and ampouled. This method also involves the use of a flammable reagent - acetone, which greatly complicates the production process, makes it flammable and reduces environmental safety.

Obtaining lidase is also known from the patent SU 827068 (publ. 07.05.1981). The method includes treating the testes of cattle in the presence of acetic acid, precipitation with acetone, dissolving the precipitate in water, followed by filtration and freeze-drying, while extraction is carried out with 0.1N acetic acid at a temperature of 5-10 ° C, before precipitation, reprecipitation is carried out at 0 -1 ° C, and after filtration, dialysis at 0-10 ° C for 12-16 hours. This method does not allow to obtain a high yield of product (maximum - up to 1.2 g / kg of feedstock). The specific activity of the obtained preparation was 2.55 cu / mg. This method is also environmentally impractical and not safe, because involves the use of acetone, volatile and toxic substances.

The disadvantage of this method is the high extraction temperature, which in combination with a high acid concentration leads to a high yield of ballast proteins in the solution and, as a result, a low yield in specific activity of the final product (per mg / protein).

Patent SU 1666534 (with a priority of 05/12/1989) describes a method for producing lidase, which involves the extraction of crushed cattle testes with an aqueous solution of acetic acid at a pH of 4.5-4.7 and a temperature of 2-40 ° C for 2-4 hours subsequent separation of the extract and purification of the target product, characterized in that, in order to simplify the method, increase the yield and specific activity of the drug, the extraction is carried out in an inert gas atmosphere, and the purification is carried out by sorption of the extract with white clay in an amount of 25-50 g per 1 liter of extract within 1-2 hours and d sorption of the desired product with an aqueous acetic acid solution at pH 4.5-4.7 at a weight ratio of the sorbent and the solution was 1: 10-15. Moreover, this method has a fairly low yield compared to the claimed method (only 7.5-9% by weight of the feedstock), and the activity of the resulting lidase is 1020-2050 cu per 100 mg. Additionally, when implementing this method, it is necessary to ensure an inert gas atmosphere and the tightness of the equipment used, which, in turn, leads to additional costs and an increase in the cost of the product.

The closest analogue of the present invention is a method for producing hyaluronidase described in patent RU 2027759 (published on 01/27/1995). The method involves extracting an enzyme from crushed cattle testes with an aqueous solution of acetic acid, separating the extract by centrifugation, purification of the target product on a sorbent and drying, while the extract obtained after centrifugation is concentrated by tangential flow ultrafiltration with a molecular weight retention limit of 50 KDa and precipitation is carried out ballast proteins with polyethylene glycol; purification is carried out by cation exchange chromatography with sorption of the enzyme at pH 4.5-4.9 and elution at the same pH 0.6 MNa Cl. However, this method has its drawbacks. In particular, a relatively low yield of the product (0.078% by weight of the feedstock) and low specific activity of the obtained lidase (140 conventional units / ml). In addition, the use of polyethylene glycol (PEG) for the deposition of ballast proteins can increase the viscosity of the system, difficulties in chromatography and elution, and unbound PEG can remain in the final product, which is extremely undesirable for injection forms.

Thus, it is necessary to develop a method for the preparation of a hyaluronidase preparation that does not involve the use of toxic and / or hazardous substances, which has a high product yield and, if possible, meets the requirements of environmentally friendly production (see Fig. 1).

Description of figures

FIG. 1 illustrates the main stages of the methods for producing a hyaluronidase preparation according to the prior art (left) and the method according to the present invention (right)

FIG. 2 shows the dynamics of the accumulation of total protein depending on the time of extraction;

FIG. 3 shows the dynamics of the accumulation of the enzyme hyaluronidase depending on the time of extraction;

FIG. 4 shows the dynamics of accumulation of the total amount of the active enzyme hyaluronidase depending on the time of extraction.

FIG. 5 is an electrophoretogram of the obtained extract (electrophoresis of samples obtained by concentrating and washing the extract of cattle seed test was performed in PAGE). The frame shows the bands corresponding to the molecular weight of the hyaluronidase enzyme. The numbers in FIG. 5 are indicated:

1 - Bio Rad marker (molecular weights 97.4; 66.2; 45; 31; 21.5; 14.4 kDa)

2 - GSO Lidaza

3 - Cattle Seed Extract

4 - Concentrate obtained on a cassette of 8 kDa

5 - Concentrate obtained after washing on a cassette of 8 kDa

6 - Concentrate obtained on the cartridge 30 kDa

7 - Concentrate obtained after washing on a cassette 30 kDa

8 - Concentrate obtained on the cartridge 50 kDa

9 - Concentrate obtained after washing on a cartridge of 50 kDa

SUMMARY OF THE INVENTION

A method of obtaining a hyaluronidase preparation includes the following steps:

1) grinding of reproductive tissue of cattle (testes);

2) extraction with 0.1 n acetic acid for no more than 2 hours at a temperature of 4-10 ° C at pH 4.3-4.7 with constant stirring;

3) centrifugation of the extract obtained in the previous step;

4) concentration of the obtained centrifugate using ultrafiltration membranes with a pore size that allows particles to pass above 30 to Da;

5) diafiltration through membranes with a similar pore size,

6) sterilizing filtration on membranes with a pore diameter of 0.22 μm;

7) filling the drug into ampoules;

8) freeze-drying.

The method differs from the production methods described in the prior art in that acetone is not used as an extractant or precipitating agent.

Also, according to the invention, a hyaluronidase preparation prepared according to the described method is provided. The resulting preparation also differs in that it does not contain acetone.

DETAILED DESCRIPTION OF THE INVENTION

The purpose of the invention is the creation of environmentally friendly production, increasing output and increasing the specific activity of the final product while reducing costs.

The problem solved by the present invention is the creation of environmentally friendly production, reducing costs, increasing output and increasing the specific activity of the final product.

The technical result consists in increasing the yield and increasing the specific activity of the final product.

As a result of the study, it was found that it is possible to increase the yield of the drug and its specific activity if the preparation is carried out in a way that includes the steps of:

1) grinding of reproductive tissue of cattle (testes);

2) extraction with 0.1 n acetic acid for no more than 2 hours at a temperature of 4-10 ° C at pH 4.3-4.7 with constant stirring;

3) centrifugation of the extract obtained in the previous step;

4) concentration of the obtained centrifugate using ultrafiltration membranes with a pore size that allows particles to pass above 30 to Da;

5) diafiltration through membranes with a similar pore size,

6) sterilizing filtration on membranes with a pore diameter of 0.22 μm;

7) filling the drug into ampoules;

8) freeze-drying.

At the same time, it is the combination of the optimal extractant (0.1 n acetic acid), time and constant stirring that allow to achieve maximum yield and high specific activity of the obtained hyalurolidase.

In addition, it was found that under these conditions, the extraction process can be significantly reduced in time, up to 2 hours, because it is under these conditions that extraction is most effective, and with extraction over 2 hours, the yield of the target product decreases (see Fig. 2).

A particular embodiment of the present invention includes a method according to which the frozen reproductive tissue of cattle (testes) is crushed, extracted in 1.5-2.0 volumes of 0.1 N. acetic acid for 2 hours at a temperature of 4-10 ° C with constant stirring, the ballast substances are separated in a centrifuge (3000 rpm, 30 min, 4-10 ° C), then clarifying filtration is carried out on membranes with a pore size of 9 μm, then, the resulting solution is fed to ultrafiltration through membranes with a pore size passing particles above 30 kDa, then diafiltration (washing in 10-fold volume) is carried out through membranes with the same pore size, after which the concentrate is sterile filtered, poured into vials (ampoules) based on 1500 ME per bottle, sublimi comfort.

Another implementation option involves a method in which the frozen reproductive tissue of cattle (testes) is crushed, extracted in 2 volumes of 0.1 n acetic acid for 2 hours at a temperature of 4-10 ° C at pH 4.3-4.7 at with constant stirring, concentrate 5 times using ultrafiltration membranes with a pore size that allows particles to pass above 30 kDa, after which the concentrate is sterile filtered, poured into ampoules and sublimated.

The resulting preparation has a finished product yield increased by 2.4-4.5 times compared with the known production methods while maintaining and increasing specific activity up to.

Examples

The invention relates to the pharmaceutical industry and can be used in medicine to obtain a drug having hyaluronidase activity.

Example 1

A known method of producing lidase, according to which the frozen testes of mature bulls are crushed, the crushed mass is defrosted, a solution of 12% acetic acid is added at the rate of 100 ml per 1 kg of raw material. Autolysis is carried out for 18-20 hours at a pH of 4.5-4.6, at a temperature of 39-40 ° C, after which the liquid fraction is separated, sterilized by filtration through a Zeitz filter. The filtered lysate is cooled to a temperature of 1-3 ° C and poured into 4 volumes of acetone with the same temperature, stand 3-5 hours in the cold and the liquid layer is separated. The hyaluronidase precipitate is dissolved in distilled water cooled to 2-3 ° C at the rate of 300 ml per 1 liter of the initial lysate. The solution is transferred to sterile filtration, poured into vials, freeze-dried. The disadvantage of this method is the high extraction temperature, which in combination with a high acid concentration leads to a high yield of ballast proteins in the solution and, as a result, a low yield in specific activity of the final product (per mg / protein).

Example 2

There is a method of producing lidase, including grinding testes of sexually mature bulls, extraction in 1.5-2.0 volumes of 0.1 N. acetic acid for 4 hours at a temperature of 4-10 ° C, filtering, precipitation of the raw lidase with 99% acetone based on 4 volumes of acetone per 1 volume of extract at a temperature not exceeding 0 ° C, stirring and settling for at least 4 hours at a temperature not exceeding 5 ° С, sediment separation, dissolution of the precipitate in water at the rate of 200-250 ml per 1 kg of feedstock, filtration, lidase reprecipitation with 99% acetone at the rate of 6 volumes of acetone per 1 filtrate volume at a temperature not higher 0 ° С, settling for 4 hours at a temperature not exceeding 5 ° С, washing with 99% acetone m at a temperature not higher than 0 ° C, drying, filling into sterile vials and freeze drying the final product. The resulting product has a specific activity of 0.14 cu / mg.

Example 3

Frozen reproductive tissue of cattle (testes) was crushed, extracted in 1.5-2.0 volumes of 0.1 N. acetic acid for 2 hours at a temperature of 4-10 ° C, the ballast substances were separated in a centrifuge (3000 rpm, 30 min, 4-10 ° C), then clarification filtration was performed on membranes with a pore size of 9 μm, then the obtained the solution was submitted to ultrafiltration through membranes with a pore size that allowed particles to pass above 30 kDa, then diafiltration (washing in 10-fold volume) was performed through membranes with the same pore size, after which the concentrate was sterile filtered, poured into vials (ampoules) at a rate of 1500 ME on the bottle, and subjected to sublimation su shke.

The yield was at least 3.42 g / kg of feedstock.

The specific activity of the obtained lidase was not less than 7.39 cu / mg.

Example 4

Studies were conducted comparing the effect of extraction time on protein yield, specific and total enzyme activity. Raw materials were extracted for 1, 2 and 4 hours. The results are shown in table 1 and in FIG. 2.

According to the results of the study, it can be concluded that the duration of the extraction does not significantly affect the protein yield, specific and total activity.

Example 5

Specific activity was measured by a method known in the literature and being standard for measurements of this kind.

Example 6

To determine the molecular composition of the proteins in the resulting lidase preparation, polyacrylamide gel electrophoresis was performed in the presence of Laemmli sodium dodecyl sulfate (SDS PAGE). The data obtained during ultrafiltration on cassettes with different pore sizes (8, 30 and 50 kDa) are presented in FIG. 5.

As can be seen from the results of the analysis, the obtained extract contains the enzyme hyaluronidase.

Example 7

Obtained by method 3 ampoules with the preparation of hyaluronidase are packaged in boxes of 10 ampoules or vials, put instructions for use and use if necessary for the intended purpose.

Claims (12)

1. A method of obtaining a hyaluronidase preparation, comprising the following stages: 1) grinding of testes of cattle; 2) extraction of 0.1 N. acetic acid with a ratio of raw materials to extractant 1: (1.5-2) for no more than 2 hours at a temperature of 4-10 ° C at pH 4.3-4.7 with constant stirring; 3) centrifugation of the extract obtained in the previous step; 4) concentration of the obtained centrifugate using ultrafiltration membranes with a pore size that allows particles to pass above 30 kDa; 5) diafiltration through membranes with a similar pore size, 6) sterilizing filtration on membranes with a pore diameter of 0.22 μm; 7) filling the drug into ampoules; 8) freeze-drying. 2. The method according to p. 1, characterized in that they do not use acetone as an extractant or precipitating agent. 3. The preparation of hyaluronidase obtained according to the method according to p. 1. 4. The hyaluronidase preparation according to claim 3, characterized in that it does not contain acetone.

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