RU2236853C1 - Method for producing immunotropic sterile apyrogenic tolerant preparation based on low-molecular native deoxyribonucleic acid sodium salt - Google Patents

Abstract

FIELD: medicine, pharmaceutical industry.

SUBSTANCE: invention relates to new high-manufacturing technologies allowing to prepare immunotropic sterile high-quality safe medicinal preparations based on deoxyribonucleate sodium prepared from defatted milt of sturgeon and salmon fishes, calf thymus, chicken blood. Invention provides enhancing the therapeutic effectiveness of the preparation and reducing ecological loading in manufacturing the claimed preparation.

EFFECT: improved producing method.

1 tbl, 1 dwg, 5 ex

Description

The invention relates to new technologies for producing biologically active substances from animal raw materials and can be used in biotechnology and the pharmaceutical industry, and the resulting immunotropic sterile pyrogen-free sodium tolerant deoxyribonucleate - in medicine, veterinary medicine, perfumery, cosmetology and research practice.

Modern environmental requirements for biological production impose restrictions on the content of biologically active substances in wastewater, which requires the creation of additional treatment facilities with a higher cost of the process, or the use of technologies based on a closed cycle or recycling.

The production of pharmaceutical substances should be carried out under conditions that require the use of neutral resistant materials in contact with the resulting product. The design and material of the equipment must allow careful processing and be resistant to disinfectant reagents, the process diagram should include sterilizing equipment, as well as other conditions that are not the subject of the proposed method.

The use of typical magnetostrictive ultrasound emitters in the production of low molecular weight DNA is limited to the material of the membrane used. The modern membrane material - stainless steel with a high nickel content - does not have permission for use in the pharmaceutical industry. During operation, the membrane undergoes erosion and metal particles can enter the final product, therefore, with modern requirements for drugs, it is necessary to develop new conditions for ultrasonic treatment.

A number of methods for producing sodium deoxyribonucleate are known. The main disadvantages of most of them is that these methods are usually small-scale, and the receipt of the target product on an industrial scale does not take into account the increased modern requirements for pharmaceutical industries and medical equipment, as well as for drugs.

International rules for the production of pharmaceutical substances and finished dosage forms based on them severely limit the content of ballast and impurity substances in them.

A known method for the allocation of sodium salt of DNA (Patent RU 61 K 35/60, C 07 H 21/04 No. 2017496, 1990), the essence of which is to increase the yield of the target product. However, the obtained sodium salt of DNA contains high impurities of protein (1.5%) and polysaccharides (2%), which causes the danger of pyrogenic reactions in the body when used in drugs and does not meet modern requirements for drugs.

A known method of obtaining a powder of native sodium salt of deoxyribonucleic acid (Patent RU C 07 H 21/04 No. 2041885, 1996). The proposed method is unreasonably complicated by repeated technological operations, which leads to an increase in the duration of the process, additional energy costs, to significant losses of the target product, the yield of which is only 3.9% of the feedstock, i.e. 50% of the DNA content in raw materials, a similar process leads to the release of contaminated wastewater and to obtain a product with reduced biological activity.

Thus, the proposed method is expensive, time consuming and low productivity. In addition, the quality of the resulting product does not meet modern requirements for the substances used in the manufacture of medicines.

A known method of obtaining a sterile solution of the native sodium salt of deoxyribonucleic acid in a solution of sodium chloride (Patent RU C 07 H 21/04 No. 2055837, 1996). The proposed method in the functional diagram of the installation for the production of the target product involves the double use of ultrasonic treatment in stainless steel strippers, which significantly reduces the yield (by mol. Mass) of the final product with a high content of impurities. The final product is characterized by a high content of proteins (0.09 g / l) and other ballast substances, which prevents its use in medical practice.

Closest to the proposed invention are a method for the production of sodium salt of deoxyribonucleic acid from animal raw materials and installation for its implementation (Patent RU C 07 H 21/04 No. 20055724, 1993).

However, modern requirements for the production of pharmaceutical substances dictate more stringent conditions both for the implementation of the technological process (the use of inert materials of equipment, aseptic production conditions, minimization of waste, etc.) and the quality of the target product (high content of the main substance, minimum allowable impurities) for use in the pharmaceutical industry for the production of drugs.

The objective of the present invention is to develop an industrial, highly productive method for the production of a pharmaceutical preparation based on sodium deoxyribonucleate, corresponding to modern, increased requirements for therapeutic efficacy and safety for pharmaceutical preparations, on equipment that meets modern requirements of pharmaceutical production: with minimal losses, optimal consumption of reagents, allowing to reduce the environmental burden from this production environmental impact, taking into account modern requirements for the organization of relevant industries relating to the protection of the product from microbial contamination during the process.

The task is achieved by the following new technical solutions.

1. The introduction of 2 stages of homogenization:

a) at the first stage: purification of the homogenate by centrifugation in a settling centrifuge with separation and removal of the supernatant containing ballast substances and fats,

b) in the second stage: repeated homogenization of the purified substrate, followed by treatment with a 3% solution of DDC in 45% ethanol (a 2-fold decrease in the concentration of DDC); significant cooling of the reaction mass (up to 15 ° C) and the use of a smaller amount of SCC.

2. a) Ultrasonic treatment of the cooled reaction mass with an oscillation amplitude of 4-10 μm in bathtubs with piezoceramic emitters installed in the bottom, with a uniform distribution over the entire area.

b) The design of the bottom of the ultrasonic bath, which does not have protruding parts and recesses. The material of the bathtub is polished food stainless steel, approved for use in pharmaceutical industries.

3. The introduction of an additional washing of kieselguhr with permeate (from the concentration stage), followed by its return to the concentration stage.

4. The introduction of a sterilizing filtration procedure (pores 0.04 μm of the obtained concentrate for separating microbes and solids from the finished product concentrate before the stage of isolation with ethyl alcohol.

5. Reducing the drying time of the precipitate under aseptic conditions to 4 hours

6. Preparation of sterile solutions and sterile filling in therapeutic doses of drugs.

The technical result achieved by the invention is to develop a method for the industrial production of a drug based on sodium deoxyribonucleate that meets modern requirements of pharmaceutical production with an optimal consumption of reagents and raw materials, with minimal waste, with an increased yield of an immunotropic product of high quality with a reduced negative impact on the environment. .

The method is as follows: animal raw materials (milk of sturgeon or salmon fish, calf thymus, chicken erythrocytes, etc.) are homogenized in a crushed state in a citrate-salt solution, then the homogenate is centrifuged in a settling centrifuge, then the supernatant is decanted and removed, the resulting the substrate is re-homogenized, the homogenate is treated with detergent and concentrated sodium chloride solution at elevated temperature; then the reaction mixture is cooled to 15 ° C, sonicated, in baths with piezoceramic emitters for 10-80 minutes with the usual frequency and current intensity, with an oscillation amplitude of 4-10 μm, the “voiced” mass is mixed with kieselguhr in a ratio of 5 respectively: 1-25: 1 (wt.), The liquid phase is separated by filtration, the filter cake is additionally washed with permeate (from the concentration stage), the total filtrate is concentrated by baromembrane filtration and the concentrate is filtered through a sterilizing filter; sodium deoxyribonucleate is precipitated from the concentrate with ethyl alcohol and the precipitate is dried under aseptic conditions at 20-60 ° C, for 4 hours, while sterile air / nitrogen is continuously fed into the drying chamber.

The yield of sterile pharmaceutical substance sodium deoxyribonucleate in the form of an odorless white amorphous powder, readily soluble in water, amounts to 7% of the loaded raw materials and has the following physicochemical characteristics:

- the content of the basic substance is not less than 105.5%, residual humidity 7-10%;

- the content of ballast substances:

protein not more than 0.5%,

RNA no more than 0.5%,

- polysaccharides not more than 0.1%;

- hypochromic effect of at least 40%.

The substance is used to produce sterile solutions of 0.25% and 1.5% sodium deoxyribonucleate in a 0.1% aqueous solution of sodium chloride.

The production of sodium deoxyribonucleate on an industrial scale is implemented through the proposed installation shown in the drawing.

Below the invention is illustrated by specific examples of its implementation.

Example 1. 1 kg of milk of salmon fish is ground in a meat grinder, homogenized in 2 liters of citrate-salt solution, the homogenate is centrifuged in a settling centrifuge at 2500 rpm, the supernatant is decanted and removed, the precipitate is homogenized again in 2 liters of citrate-salt solution; the homogenate is placed in a reactor with heated citrate-salt solution (7 liters), 1.9 kg of a 3% solution of sodium dodecyl sulfate in 45% ethanol are added and the mass is kept at 60 ° C for 1.5 hours with stirring. Then add 14.1 kg of 5M sodium chloride solution and continue stirring at the same temperature for 1.5 hours. After that, the reaction mass is cooled to a temperature not exceeding 15 ° C. The cooled mass (26 kg) is treated with ultrasound for 80 min, with an oscillation amplitude of 8 μm. The voiced mass is mixed with kieselguhr in a ratio of 8: 1 (wt.), Then the liquid phase is separated on a suction filter, the precipitate is washed with permeate (10 L) and combined with the filtrate. After additional microfiltration, the filtrate was concentrated (by the baromembrane method) on a membrane with a pore size of 0.04 μm. Get 5 kg of concentrate containing 1.1 wt.% Sodium salt of deoxyribonucleate. The target product is precipitated from the concentrate with ethyl alcohol. The resulting precipitate was separated by centrifugation and dried at 20-40 ° C for 4 hours.

Get 65 g of the sodium salt of deoxyribonucleate, having the following characteristics: mol. weight 480 kD, protein content 0.2%, content (RNA + polysaccharides) 0.2%, humidity 8%, hyperchromic effect 44%. The calculated amount of powder is weighed under aseptic conditions, dissolved in a 0.1% sterile solution of sodium chloride and sterilely dispensed in therapeutic doses.

Analytical passport of the preparation obtained:

Authenticity - corresponds;

Color is colorless;

Transparency - consistent;

Sterility - sterile;

Toxicity - non-toxic;

Pyrogenicity is pyrogen-free;

The sodium content of deoxyribonucleate is 0.015 g / ml;

pH 6.8;

The content of sodium chloride is 0.08%;

Hyperchromic effect 42%;

RNA content 0.2%;

The protein content of 0.2%.

Example 2. 1 kg of milk of sturgeon fish is ground in a meat grinder, homogenized in 2 liters of citrate-saline solution, the homogenate is centrifuged in a settling centrifuge at 2500 rpm, the supernatant is decanted and removed, the precipitate is homogenized again in 2 liters of citrate-saline solution; the homogenate is placed in a reactor with heated citrate-salt solution (7 liters), 1.9 kg of a 3% solution of sodium dodecyl sulfate in 45% ethanol are added and the mass is maintained at 60 ° C for 1.5 hours with stirring. Then add 14.1 kg of 5M sodium chloride solution and continue stirring at the same temperature for 1.5 hours; then the reaction mixture is cooled to a temperature not exceeding 15 ° C, then for 80 minutes it is treated with ultrasound with an oscillation amplitude of 7 μm. The voiced mass is mixed with kieselguhr in a ratio of 8: 1 (wt.), And the liquid phase is separated on a suction filter. The filter cake was washed with permeate (10 kg), the washing liquid was combined with the filtrate.

After additional microfiltration, the filtrate was concentrated (by the baromembrane method) on a membrane with a pore size of 0.04 μm. Get 5 kg of concentrate containing 1.3 wt.% Sodium salt of deoxyribonucleate. Sodium deoxyribonucleate is isolated by precipitation from the concentrate with ethyl alcohol. The resulting precipitate was separated by centrifugation and dried at 20-40 ° C, for 4 hours.

Obtain 66 g of sodium salt of deoxyribonucleate, having the following characteristics: mol. weight 420 kDa, protein content 0.3%, content (RNA + polysaccharides) 0.4%, humidity 7%, hyperchromic effect 44%. The calculated amount of powder (for the preparation of a 1.5% solution) is weighed under aseptic conditions, dissolved in a 0.1% sterile solution of sodium chloride and sterilely poured in therapeutic doses.

Analytical passport of the preparation obtained:

Authenticity - corresponds;

Color is colorless;

Transparency - consistent;

Sterility - sterile;

Toxicity - non-toxic;

Pyrogenicity is pyrogen-free;

The sodium content of deoxyribonucleate is 0.014 g / ml;

pH 7.0;

The content of sodium chloride is 0.12%;

Hyperchromic effect 44%;

RNA content of 0.4%;

The protein content of 0.3%.

Example 3. 1 kg of calf thymus is ground in a meat grinder, homogenized in 2 liters of citrate-saline solution, the homogenate is centrifuged in a settling centrifuge at 2500 rpm, the supernatant is decanted and removed, the precipitate is homogenized again in 2 liters of citrate-saline solution; the homogenate is placed in a reactor with heated citrate-salt solution (7 liters), 1.9 kg of a 3% solution of sodium dodecyl sulfate in 45% ethanol are added and the mass is kept at 60 ° C for 1.5 hours with stirring. Then add 14.1 kg of 5M sodium chloride solution and continue stirring at the same temperature for 1.5 hours. After that, the reaction mixture is cooled to a temperature not exceeding 15 ° C. The cooled mass (26 kg) is treated with ultrasound for 80 min with an oscillation amplitude of 10 μm. The voiced mass is mixed with kieselguhr in a ratio of 8: 1 (wt.), Then the liquid phase is separated on a suction filter, the precipitate is washed with 10 L permeate and combined with the filtrate. After additional microfiltration, the filtrate was concentrated (by the baromembrane method) on a membrane with a pore size of 0.04 μm. Get 5 kg of concentrate containing 1.1 wt.% Sodium salt of deoxyribonucleate. The target product is precipitated from the concentrate with ethyl alcohol. The resulting precipitate was separated by centrifugation and dried at 20-40 ° C for 4 hours.

Obtain 70 g of sodium salt of deoxyribonucleate having the following characteristics: characteristic. pier weight 380 kD, protein content 0.4%, content (RNA + polysaccharides) 0.8% humidity 9%, hyperchromic effect 44%. The calculated amount of powder is weighed under aseptic conditions, dissolved in a 0.1% sterile solution of sodium chloride and sterilely poured in therapeutic doses (1.5% solution).

Analytical passport of the preparation obtained:

Authenticity - corresponds;

Color is colorless;

Transparency - consistent;

Sterility - sterile;

Toxicity - non-toxic;

Pyrogenicity is pyrogen-free;

The sodium content of deoxyribonucleate is 0.015 g / ml;

pH 7.0;

The sodium chloride content is 0.09%;

Hyperchromic effect of 45%;

RNA content of 0.3%;

The protein content of 0.4%.

Example 4. 1 kg of chickens blood is homogenized in 2 liters of citrate-saline solution, the homogenate is centrifuged in a settling centrifuge at 2500 rpm, the supernatant is decanted and removed, the pellet is homogenized again in 2 liters of citrate-saline solution; the homogenate is placed in a reactor with heated citrate-salt solution (7 liters), 1.9 kg of a 3% solution of sodium dodecyl sulfate in 45% ethanol are added and the mass is maintained at 60 ° C for 1.5 hours with stirring. Then add 14.1 kg of 5M sodium chloride solution and continue stirring at the same temperature for 1.5 hours; then the reaction mixture is cooled to a temperature not exceeding 15 ° C, then for 80 minutes it is treated with ultrasound with an oscillation amplitude of 4 μm. The voiced mass is mixed with kieselguhr in a ratio of 8: 1 (wt.) And the liquid phase is separated on a suction filter. The filter cake was washed with permeate (10 kg), the washing liquid was combined with the filtrate. After additional microfiltration, the filtrate was concentrated (by the baromembrane method) on a membrane with a pore size of 0.04 μm. Get 5 kg of concentrate containing 1.3 wt.% Sodium salt of deoxyribonucleate. Sodium deoxyribonucleate is isolated by precipitation from the concentrate with ethyl alcohol. The resulting precipitate was separated by centrifugation and dried at 20-40 ° C for 4 hours.

69 g of the sodium salt of deoxyribonucleic acid are obtained having the following characteristics: molar mass 450 kDa, protein content 0.2%, content (RNA + polysaccharides) 0.3%, humidity 8.5%, hyperchromic effect 40%. The calculated amount of sodium deoxyribonucleate powder is weighed under aseptic conditions, dissolved in a 0.1% sterile aqueous solution of sodium chloride and sterilely dispensed in therapeutic doses (1.5% solution).

Analytical passport of the preparation obtained:

Authenticity - corresponds;

Color is colorless;

Transparency - consistent;

Sterility - sterile;

Toxicity - non-toxic;

Pyrogenicity is pyrogen-free;

The sodium content of deoxyribonucleate is 0.016 g / ml;

pH 6.7;

The content of sodium chloride is 0.11%;

Hyperchromic effect 46%;

RNA content of 0.3%;

The protein content of 0.3%.

Example 5. To the stage of concentration, as in example 3.

After additional microfiltration, the filtrate was concentrated (by the baromembrane method) on a membrane with a pore size of 0.04 μm. Get 5 kg of a concentrate containing 1.3 wt.% Sodium salt of deoxyribonucleate. The concentrate is filtered through a 0.2 μm sterilizing membrane and transferred to a crystallizer vessel under aseptic conditions. The target product is isolated by mixing the concentrate with sterile ethyl alcohol. The resulting precipitate is separated by centrifugation and dried at 20-40 ° C for 4 hours under aseptic conditions with continuous supply of sterile dried air into the drying chamber. (Aseptic conditions - a sealed room with controlled air parameters: air exchange rate of at least 20, temperature 21-24 ° C, dust content - not more than 350,000 particles / m ³ (zone C) and not more than 3500 h / m ³ (zone A), the content of microbes is not more than 100 CFU / m ³ (zone C) and less than 1 CFU / m ³ (zone A)).

Obtain 66 g of sterile sodium salt of deoxyribonucleate, having the following characteristics: mol. weight 450 kDa, protein content 0.4%, RNA and polysaccharide content 1.2%, humidity 9%, hyperchromic effect 44%. The calculated amount of powder is weighed under aseptic conditions, dissolved in a 0.1% sterile solution of sodium chloride and sterilely poured in therapeutic doses (0.5% solution).

Analytical passport of the preparation obtained:

Authenticity - corresponds;

Transparency - consistent;

Sterility - sterile;

The sodium content of deoxyribonucleate is 0.245-0.255%;

pH 6.8;

The sodium chloride content is 0.08%.

Thus, the claimed method for the production of a sterile pharmaceutical substance of sodium deoxyribonucleate allows to obtain a product of higher purification quality (reduction in the content of ballast protein of not more than 0.5%, RNA + polysaccharides to 2.0%, moisture to 10%) without compromising the overall physicochemical properties, reduce the consumption of expensive and environmentally unsafe reagents (sodium dodecyl sulfate by half, kieselguhr by 20%), increase the manufacturability of equipment and facilitate its maintenance (bathtubs with piezoceramic emit they don’t emit toxic impurities, last longer, warm less, wash better and disinfect), get a product that meets modern high requirements for pharmaceutical substances and preparations (using sterilizing equipment and premises with aseptic conditions excludes the possibility of microbial contamination of the finished product and increases the shelf life of the substance and the duration of the drug). The proposed installation allows you to implement the claimed method of production of an immunotropic sterile preparation.

The drawing shows a functional diagram of the proposed installation.

A plant for the production of a sterile pyrogen-free sodium deoxyribonucleate preparation from animal feedstock includes a cutting table with weights 1, an electric meat grinder 2, a microparticle grinder 3, a centrifuge 4, a reactor for boiling the reaction mass 5, a cooling reactor 6, an ultrasonic treatment device 7, a mixer for mixing the reaction mass with kieselguhr 8, a device for separating the liquid phase 9, a membrane device for clarifying microfiltration 10, a membrane device for concentrating liquid basics 11, a membrane device for sterilizing filtration 12, a reactor for precipitating the target product with ethyl alcohol 13, equipment for separating and drying the precipitate, a centrifuge 14, a Schott funnel 15, an oven 16, a container for 0.1% sodium chloride solution, sterile containers solutions 22, 23, 24 calculated concentrations with dispensers dispensing.

In addition, the installation contains measuring tanks 17 (6 pieces in the diagram) for measuring liquids, mixers for preparing solutions and suspensions at room temperature 18, reactors for preparing solutions and mixtures at a temperature different from room 19, and containers for collecting intermediate products and production waste 20.

Hardware and technological improvement in the production of sodium deoxyribonucleate can increase the production line with a design capacity of 10 kg / year to 20 kg / year.

The controlled molecular weight, preservation of the secondary structure and a high degree of purification guarantee high biological activity, high immunotropy and prolonged stability of the drug.

The table shows comparative data of the substances of the prototype and the invention. From the table it follows that the quality of the drug according to the invention exceeds the prototype.

Claims (1)

A method of manufacturing an immunotropic pyrogen-free tolerant preparation based on sodium salt of low molecular weight deoxyribonucleic acid, which consists in the fact that skim milk of sturgeon fish, salmon fish, calf thymus, blood of chickens are homogenized in a citrate-saline solution, the homogenate is centrifuged in a sedimentation centrifuge / min. the supernatant is removed, the substrate is re-homogenized, then the purified homogenate is incubated in the presence of 3% detergent, during continuous stirring at a higher at a temperature, a concentrated solution of sodium chloride is added to the reaction mass, the reaction mass is cooled to a temperature below room temperature (15 ° C), sonicated, in baths with piezoceramic emitters with an oscillation amplitude of 4-10 microns; then a sorbent, the liquid phase is separated, the sorbent is eluted with permeate, the eluate combined with the liquid phase is microfiltered, concentrated, the sodium precipitate of deoxyribonucleate is isolated with alcohol, centrifuged, separated from the liquid phase, dried for 4 hours under aseptic conditions at 40 ° С, amorphous white a powder with a molecular weight of 270-500 kDa, a hyperchromic effect of at least 40%, a protein content of up to 0.5%, a total content of polysaccharides and RNA of not more than 2%, is dissolved in a 0.1% aqueous solution of sodium chloride in predetermined concentrations and sterile spill ayut at therapeutic doses, with the content of Na-DNA for the 1.5% concentration of 0,14-0,16 g / ml; pH 6.2-7.2, hyperchromism 40-46%, protein content 0.2-0.5%, sodium chloride content 0.08-012%.