RU2455014C1 - Method for producing lyophilised preparation of laky blood - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, namely a method for producing a lyophilised preparation of laky blood. The method for producing a lyophilised preparation of laky blood involving including the erythrocyte lysis of defibrinated blood, the introduction of a sodium chloride solution, the centrifugal elimination of ballast particles and the cooling in a swinging bucket rotor centrifuge, the filter sterilisation through membrane or ceramic filters, the preparation cooling in ampoules that is followed by the frost penetration (cryotrapping) and the lyophilisation under certain conditions.

EFFECT: method enables producing the preparation with long-term storage stability.

3 cl, 2 dwg, 3 tbl

Description

The invention relates to biotechnology for the manufacture of a lyophilized hemolysed blood preparation. The drug is used as a plague microbe growth stimulator on artificial nutrient media in sanitary and clinical microbiology, epidemiological monitoring, and scientific research. The invention can be used in a biotechnological scheme for the production of hemolyzed blood, lyophilisate for the preparation of a solution for diagnostic purposes.

The hemolyzed blood preparation is used as a stimulating additive to nutrient media, improves their growth qualities, increases sensitivity during excretion, and increases efficiency during cultivation of Yersinia pestis. Along with this, hemolized blood can be used to enrich liquid and agar artificial nutrient media in the cultivation of other types of microorganisms of the Enterobacteriaceae family. Blood hemolysed active substances are blood proteins, in particular hemoglobin, low molecular weight nitrogen compounds, metabolic products, etc.

The hemolyzed blood preparation is horse or sheep blood, lysed with sterile distilled water in a ratio of 1:10 (one part of blood + nine parts of distilled water) and filtered twice through filters F-140 and SF-140. Hemolized blood is produced in liquid form and has a shelf life of 2 years [1]. Undoubtedly, liquid forms of release are cheap and convenient to use, since they do not require unnecessary manipulations, except opening the ampoule and selecting the drug. However, as a rule, preparations in liquid form are unstable, therefore, their shelf life does not exceed 2 years. The difficulties associated with the duration of storage of liquid preparations made on the basis of animal blood serums are due to the instability of serum proteins, especially globular ones. Protein instability, expressed by the appearance of a precipitate at the bottom of an ampoule or bottle with a liquid preparation, is caused by a spontaneous and continuous process of their irreversible denaturation, which often sharply increases under the influence of numerous denaturing factors (the effect of proteolytic enzymes contained in serums, etc.). Denaturation of proteins leads to the loss of their specific properties. In liquid blood hemolized during storage, the oxidation of hemoglobin occurs, which leads to a change in the color of the drug from saturated red to brown. The formation of a small precipitate at the bottom of the ampoules, although it is allowed by the technical specifications, however, the precipitate is undesirable for the consumer.

In addition, liquid preparations require a certain storage and transportation regime - from 2 to 8 ° C, violation of which, both upward and downward temperature, can lead to a complete loss of activity.

The method of lyophilization can solve the problem of stabilization of the initial properties of biological preparations. Dry preparations obtained by lyophilization do not undergo chemical changes and do not lose their inherent properties during prolonged storage even at positive temperatures, as a result of which the conditions of transportation and storage are simplified, and the shelf life is increased. Lyophilization, as a rule, are subjected to microorganisms and bacteriophages. It is widely used to obtain blood plasma (dry plasma) capable of long-term storage and its individual fractions, immune serums, immunoglobulins, vaccines, antibiotics, hormones, tissue transplants; therefore, various modes of freezing and drying have been worked out for them. The lack of a unified technology of lyophilic drying of biological objects necessitates studies to stabilize each specific drug [3].

Enough data has been accumulated on the stabilization by drying of blood components, blood substitutes [4, 5, 6] and preparations obtained from animal blood, for example, the method for producing hematogenous powder according to the patent [7], which consists in forcing blood clots through a mesh with subsequent sterilization at a temperature of 60 -65 ° C for 24 hours, while the mass of hemoglobin is used to prepare the powder, dried at a temperature of 35-40 ° C for 36-48 hours and crushed subsequently. In the method, the drying is carried out without freezing.

A known method of producing hemoglobin [8], in accordance with which receive the erythrocyte hemolysate by diluting the erythrocyte mass with distilled water and freezing, followed by concentration of the hemolysate by ultrafiltration, sterilized filtration and lyophilization. The proposed method eliminates blood defibrinization and allows to obtain hemoglobin while maintaining its native properties. The described method is characteristic for obtaining preparations containing hemoglobin.

Known hemophilic microorganism growth stimulator (SRHM), manufactured according to TU 9385-016-7809326-2007 (Federal State Institution "State Scientific Center for Applied Microbiology and Biotechnology", Obolensk, Moscow Region), used as a component of culture media for the cultivation of certain hemophilic microorganisms: meningococci, gonococci, legionella, corynebacteria, tularemia microbe (with the exception of hemophilic bacillus and pneumococci). Whole blood of cattle can be used as a raw material for producing a stimulant. SRHM obtained by the method of enzymatic hydrolysis of black albumin, followed by drying in open cuvettes. However, this method is not suitable for the preparation of a lyophilized preparation hemolyzed blood, since drying in open cuvettes does not ensure sterility of the preparation, and also involves its further packaging.

There is a known technique of freezing and drying an animal blood product that affects hematopoiesis [9], in which deer blood taken during the period of greatest physiological activity is hemolized with bidistilled water in a ratio of 1: 0.5, subjected to preliminary purification, sterilized by ultrafiltration, followed by freeze drying . Lyophilization in this case allows for standardization of the drug, facilitates the calculation of individual dosages. The resulting mass is used for the preparation of various dosage forms. The process includes the step of freezing the filtrate (at a temperature of -156 ° C), followed by drying the drug in a container in a vacuum chamber for 22-24 hours at a vacuum of 10-3 mm Hg. However, such a deep freeze before sublimation is not practical for obtaining a lyophilized preparation, the blood hemolyzed in ampoules because, firstly, the freezing of the glass of ampoules can cause its destruction, and secondly, the eutectic zone of hemolyzed blood is within -20 ° С - 60 ° С therefore, the maximum temperature for effective freezing is -60 ° C.

According to Henderson L.O. at all. [10], Matsuda Y. [11] lyophilization without the introduction of stabilizing components often leads to a significant or complete loss of the specific activity of some drugs, and the lyophilized material is often “smeared” along the bottom of the vial due to the low content of total solids.

It is known that stabilizers are used at the stages of freezing and drying, for example, stabilizing compositions for preparing lyophilized blood serum preparations, including carbohydrates, for example, sucrose and glucose [4] or a mixture of glycine and mannitol [12]. This allows you to reduce the drop in biological activity at the stage of lyophilization and to form a bulk tablet.

However, the described stabilizers make it difficult to achieve a predetermined moisture content of the preparations (1-3%) in the process of their lyophilization, require special drying modes, which greatly complicates the process of obtaining the final product. In addition, such stabilizers do not provide long-term storage of dry preparations at positive temperatures.

Known stabilizing composition for the production of lyophilized serums, including peptide and / or a mixture of peptide and carbohydrate components, in which ethylene diamine tetraacetate (EDTA) is additionally introduced [13]. This composition is designed specifically to reduce the loss of specific activity of positive control sera (during storage) used in test systems to determine specific antibodies to viruses and microorganisms, and ensures signal preservation in ELISA.

It is known to use as a stabilizer a medium consisting of human serum albumin, sodium chloride, N-acetyl tryptophanate, sodium caprylate, sodium azide and a phosphate buffer solution with a pH of 8.0-10.0 [14], and a medium including bovine serum albumin , EDTA, peptone, sucrose, ascorbic acid, potassium chloride and bacteriostatic [15]. These stabilizers are intended for lyophilization of injection preparations containing IgM antibodies in an amount of about 5 mg / ml, as well as reference sera containing IgM antibodies to viral antigens.

All of the above stabilizers are unsuitable for use in the lyophilization of hemolyzed blood, as they will change the composition of the target drug and may affect its stimulating properties.

A known method of freeze drying a biological product [16], for example, a solution of conjugate of immunoglobulin G with horseradish peroxidase in 0.02 M phosphate buffer, which includes the introduction of a biological product into the solution before drying the protective medium. A filler substance is additionally introduced into the composition of this protective medium, the diluted aqueous solution of which crystallizes upon freezing in a concentration sufficient to form an amorphous-crystalline structure in the volume of the liquid phase of the biological product remaining after water crystallization. As the filler substance, sodium chloride, potassium chloride or glycine is used. This creates the conditions that make it possible to lyophilize materials with a low collapse temperature (-30 ° C and below) at higher temperatures. However, in the described method, in addition to the filler substance, a protective medium (stabilizer) is introduced into the biological product, which cannot be used in the preparation hemolized blood.

It should be noted that the technological methods used in known sources of scientific, technical and patent information, stabilizing compositions and freezing and drying modes cannot be used in the manufacture of a lyophilized preparation hemolyzed blood, characterized by other stability parameters with respect to temperature, pH, physical stresses and chemical agents, as a result of which it is required to develop both a stabilizing composition and conditions for lyophilization of hemolytic blood for th e preparation of a dry formulation with specific characteristics remaining unchanged for a long time.

Due to the fact that the presence of stabilizing substances in hemolyzed blood can affect its stimulating properties, one of the objectives of the invention is the selection of such a filler substance that would ensure the maintenance of stimulating activity and exclude a qualitative change in the composition of the drug.

The problem to which the invention is directed, is to obtain a lyophilized preparation hemolyzed blood.

The technical result of the invention consists in the development of a technology for the preparation in a lyophilized form of the drug hemolized blood, stably preserving properties during storage with a longer shelf life.

The technical result is achieved by adding distilled water in a ratio of 1: 9 (one part of blood + eight parts of distilled water) to defibrinated blood, followed by the introduction of sodium chloride as a filler in a volume equal to the volume of defibrinated blood to a final concentration of 0 15 mol / l. The obtained semifinished product of hemolized blood was centrifuged at 4000 g in a centrifuge with a bucket rotor with cooling to 4-8 ° C for 30 min and sterilized by filtration through membrane or ceramic filters with a pore size of 0.2 μm. The obtained hemolized blood filtrate is poured into 2 ml into sterile ampoules, cooled to a temperature of - (45 + 5) ° С and frozen. Then the drug is lyophilized for 24 hours to a final material temperature of 25 ° C, while the sublimation stage averages 14 hours, and desorption 10 hours. Cooling can be carried out in fast or slow mode. In fast mode, the drug is cooled to - (45 ± 5) ° C for 3-4 hours, in slow mode - for 12 hours. The total time of hemolyzed blood cryostabilization, including cooling and freezing, for both modes is 18-20 hours.

In the inventive method, the accepted ratio of defibrinated blood and distilled water 1: 9 is due to the need to maintain the blood concentration adopted in the liquid preparation (1:10), and one part of the water is replaced by a solution of sodium chloride.

The use of sodium chloride as a filler substance allows, without practically changing the hemolized blood composition, to obtain a well-formed tablet after freeze drying that dissolves in 2 ml of distilled water for 1-2 minutes. The stimulating activity of the lyophilized preparation at the time of its preparation is 96.4-98.1% of the activity of the initial liquid preparation. The claimed sequence of adding sodium chloride to the drug is determined experimentally and ensures the preservation of its stimulating properties.

The selected freezing and drying modes are determined by the protein content in the preparation — 32.0–35.0 g / l, which was determined spectrophotometrically at two wavelengths — 280 and 495 nm in accordance with FS 42–3874–99 [17], and lack of stabilizer.

The figure 1 presents graphs reflecting the claimed modes of cooling the drug hemolized blood. Two cooling modes were worked out - fast, characterized by the formation of a fine-crystalline structure of water, and slow, characterized by the formation of large crystals. The working cooling temperature - (45 ± 5) ° С with the fast method is achieved within 3-4 hours. In the slow cooling mode, the required temperature - (45 ± 5) ° С is reached within - 12 hours. Upon reaching the operating temperature, the ampoules are frozen in a low-temperature refrigerator for 8-14 hours. The total time of cryostabilization of hemolyzed blood before the start of sublimation is 18-20 hours.

The figure 2 presents a graph of the drying of three series of the drug hemolized blood, reflecting the claimed regime of lyophilization. The initial temperature parameters of the condenser of the sublimation unit are -60 ° С, the initial temperature of the heating plate is -30 ° С, the material temperature is from -42 to -45 ° С, the vacuum depth is 5.3-5.9 Pa. The shelves were heated at a material temperature of -47-50 ° C and a vacuum of at least 0.1-0.2 Pa was reached. The duration of the sublimation process averaged 14 hours (from 13 to 15 hours), desorption - 10 hours (from 9 to 11 hours). The maximum heating temperature of the material at the desorption stage is (26 ± 1) ° С.

The claimed method allows to obtain in a lyophilized form, a hemolysed blood preparation with the following characteristics:

1. Appearance. A hemolized dry blood preparation is an amorphous mass of red-brown color.

2. Solubility. The contents of hemolyzed dry blood ampoules are completely dissolved in 2 ml of distilled water, the dissolution time is 1-2 minutes. After dissolution, a clear liquid of a deep red color without foreign inclusions.

3. pH. From 7.5 to 8.0.

4. Mass loss on drying. No more than 2%.

5. The content of amino nitrogen 360 ± 5 mg / l.

6. Sterility. The drug must be sterile.

7. Specific activity. With the addition of 1% (v / v) blood hemolyzed to culture media, more than 30 colonies from 100 seeded microbial cells of the vaccine strain Y. pestis EV of the NIIEG line should grow.

The implementation of the invention is confirmed by the following example.

The blood of a horse or a sheep is taken sterile from the jugular vein into a defibrator (a bottle with glass beads). After the end of the bloodletting procedure, the bottle is shuttled (shaken) for 30-40 minutes. After the formation of the fibrin clot, the defibrinated blood is separated by filtering it into a sterile bottle through a sterile cotton-gauze filter. Sterile distilled water is added to defibrinated blood in a ratio of 1: 9 (one part of blood + eight parts of distilled water), followed by mechanical stirring for 15-20 minutes at a temperature of 18-20 ° C and an exposure of 18-20 hours at a temperature of 2-4 ° C. Then, a solution of sodium chloride in an amount equal to the volume of defibrinated blood is added to the obtained hemolyzed blood with constant stirring. The concentration of sodium chloride in the added solution should be such that in a final dilution a concentration of 0.15 mol / L is obtained. For example, to prepare 10 liters of hemolyzed blood, you need to take 1 liter of defibrinated blood, add 8 liters of distilled water and 1 liter of a 1.5 M sodium chloride solution (based on 90 g per 1000 ml of water).

It has been experimentally proved that sodium chloride as a filler substance allows, without practically changing the hemolized blood composition, to obtain a well-formed tablet after freeze drying that dissolves in 2 ml of distilled water within 1-2 minutes. The stimulating activity of the lyophilized preparation at the time of its preparation is 96.4-98.1% of the activity of the initial liquid preparation. The claimed sequence of adding sodium chloride was determined experimentally.

The obtained semifinished product of hemolized blood is centrifuged at 4000 g in a centrifuge with a bucket-rotor while cooling to 4-8 ° C for 30 min in order to free from suspended particles formed after the destruction of red blood cells or passed through a cotton-gauze filter at the previous stage, and sterilized through membrane filters with a pore size of 0.2 μm.

Hemolyzed blood filtrate is poured into sterile ampoules ШП-5 НС-1А (ОСТ 64-2-485-85) in 2 ml and subjected to lyophilization. Freezing is carried out in a low-temperature refrigerator in the mode of fast or slow cooling (Figure 1).

Lyophilization is carried out in sublimation chamber devices of the type KC-30, LZ-9, etc. The duration of the lyophilization regime is (25 ± 1) hours (Figure 2). The initial temperature parameters of the condenser of the sublimation unit are - (60 ± 2) ° C, the temperature of the heating shelves is - (42 ± 2) ° C, the temperature of the material is - (50 ± 1) ° C, the vacuum depth is 5.3-5, 9 Pa. Heated shelves are turned on after 1-3 hours when the vacuum reaches at least 0.1-0.2 Pa and the material temperature is from -47 to -50 ° С ° С. The heating rate of the material at the sublimation stage is 0.93 ° C / min, at the desorption stage - 0.083 ° C / min. The duration of the sublimation stage is (14 ± 1) hours, desorption - (10 ± 1) hours. The final temperature of the material is (26 ± 1) ° С.

Sealing of the ampoules after lyophilization is carried out in an atmosphere of dried purified air at normal atmospheric pressure.

The minimum shelf life of the resulting preparation is 3 years (observation period) and can be increased with the accumulation of experimental data, while the shelf life of a liquid preparation hemolyzed blood is 2 years [18]. The lyophilized preparation hemolyzed blood obtained by the claimed method, during storage, stably preserves the physicochemical and biological properties (stability indicators of the lyophilized blood preparation hemolized are shown in Table 1), while the liquid preparation shows a change in pH values, as well as a slight decrease in the degree of stimulation , which amounted to 4.6 and 8.2% for series 15 and series 20, respectively (stability indicators of a hemolyzed liquid blood preparation are presented in table 2). In accordance with the regulatory documentation, the hemolyzed liquid blood preparation should be stored and transported at a temperature of 0 to 8 ° C in accordance with SP 3.3.2.1248-03. Transportation at a temperature up to 20 ° C is allowed no more than 5 days. Freezing is not allowed [18]. A study of the stability of liquid and lyophilized blood products hemolized under conditions of elevated temperature (37 ° C) (accelerated aging test) confirmed the stability of the dry forms, the change in the stimulating activity of which was in the range of 1.9 - 8.7%, while the same the parameter of liquid preparations varied from 31.8 to 38.8%. The change in pH values was 0.03-0.06 and 1.0-1.3 for dry and liquid preparations, respectively. A comparative assessment of the stability of liquid and lyophilized blood products hemolized during storage at elevated temperatures (37 ° C) is presented in table 3.

Thus, the inventive method allows without the use of stabilizers to obtain the drug hemolyzed blood in lyophilized form, characterized by the stability of storage properties and a longer shelf life compared to a liquid analog.

Literature

1. Industrial regulation No. 01898109-13-07 for the production of hemolyzed blood, solution for diagnostic purposes. Approved February 13, 2007

2. MUK 4.1 / 4.2.588-96. Methodical instructions. Methods of control of medical immunobiological drugs administered to people. Ministry of Health of Russia. Moscow, 1998

3. Belous A.M., Tsvetkova C. D. Scientific basis of the technology of sublimation conservation. - Kiev: Naukova Dumka, 1985 .-- 208 p.

4. Podolsky M.V. Drying blood products and blood substitutes. - M.: Medicine, 1973. - 198 p.

5. Podolsky M.V., Rybolovlev Yu.R. On the lyophilization of human red blood cells // Cryobiology and Medicine. - 1980. - Issue 6. - S. 54-56.

6. Zhvania T., Kamalova M., Chichua Yu., Tolorai G. Lyophilization of platelet mass for long-term storage and use in the clinic // Proceedings of the Research Institute of Hematology and Blood Transfusion. - 1974. - v.14. - Pages. 188-191.

7. RF patent No. 2301068 A method for producing hematogenous powder. - Publ. 06/20/2007. Bull. Number 17.

8. RF patent No. 2080865 A method for producing hemoglobin. - Publ. 06/10/1997.

9. RF patent No. 2017493 A method of obtaining funds that affect hematopoiesis from animal blood. - Publ. 08/15/1994.

10. Henderson L. O., Hazlehurst J.S., Taylor L., Hannon W.H. Preparation of lyophillzed human serum based reference materials with graded levels of apolipoproteins A-I and B. - Clin. Biochem. - 1988. - v.21. - p. 219-223.

11. Matsuda Y. Factors affecting the biological activity of lyophilized myofibrils - protective substances and collapse temperatures, in: Fundamentals and applications of freeze-drying to biological materials, drugs and foodstuffs. - Proceeding of meeting of Commiss. Ci, Tokyo, May 20-22, 1985, Intern. Inst. Refrigeration, 1985, p. 103.

12. Chang B.S., C.S. Randall, Cryobiology, 1992, v. 29, p. 632.

13. RF patent No. 2011202 A stabilizing composition for obtaining lyophilized positive control sera used in test systems for the determination of special antibodies to viruses and microorganisms. - Publ. 04/15/1994.

14. International application (WO) N 90/11091, cl. A61K 39/395, publ. 10/04/1990.

15. RF patent No. 2136313 Stabilizing composition for obtaining reference sera containing IgM antibodies. - Publ. 09/10/1999.

16. RF patent No. 2111426 Method of freeze drying of a biological product. - Publ. 05/20/1998.

17. Physico-chemical, chemical, physical and immunochemical methods for monitoring medical immunobiological preparations. FS 42-3874-99. - Moscow, 1999. - P.71.

18. Instructions for the use of hemolized blood, a solution for diagnostic purposes. Approved 07/04/2002.

Claims (3)

1. A method of obtaining a lyophilized blood preparation hemolized, including hemolysis of erythrocytes of defibrinated blood in a ratio of 1: 8 (one part of blood + eight parts of distilled water), the introduction of a 1.5 M sodium chloride solution in a volume equal to the volume of defibrinated blood (1: 1) purification from ballast particles by centrifugation at 4000 g with cooling to 4-8 ° C for 30 min in a centrifuge with a bucket-rotor, sterilization by filtration through membrane or ceramic filters with a pore size of 0.2 μm, cooling of the drug in ampoules to eratury - (45 + 5) ° C, followed by freezing (kriostabilizatsiey) 18-20 hours and lyophilized for 24 hours to a final temperature of the material of 25 ° C, the sublimation step is on average 14 hours desorption - 10 hours. 2. The method according to claim 1, characterized in that the drug is cooled to - (45 ± 5) ° C for 3-4 hours 3. The method according to claim 1, characterized in that the drug is cooled to - (45 ± 5) ° C for 12 hours

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