Method for obtaining composition for determining content of antialphastaphylolysin

Abstract

FIELD: medicine.SUBSTANCE: invention relates to industrial transfusion medicine, pharmaceutical biotechnology and standardization, namely to a method for producing a composition for the quantitative determination of antialpha-staphylolysin in donor blood plasma and human immunoglobulin preparations. Method for obtaining a composition for determination contents antialpha-staphylolysin in donor plasma containing antibodies to staphylococcal alpha-toxin or human immunoglobulin medicinal products, characterized by the fact that human immunospecific plasma containing antialpha-staphylolysin at a concentration of at least 3 IU / ml is selected, from which a fraction is isolated using the ethanol-cold method γ – globulins with an antialpha-staphylolysin concentration of at least 28 IU / ml, the resulting solution is stabilized with trehalose to a concentration of 40 g / l and bovine serum albumin to a concentration of 2 g / l, the pH is adjusted to a value in the range from 4.8 to 5.3 using 1 M sodium hydroxide solution or 1 M hydrochloric acid solution, sterilely dosed into vials in a volume of 1.5 to 3.0 ml and lyophilized to a residual moisture content of no more than 1%, quality indicators are determined, including a dissolution time of no more than 15 minutes, a protein content from 50 g / l to 110 g / l, a content of IgG dimers and monomers of at least 95%, polymers and IgG aggregates of no more than 5%, the certified value of specific activity and its uncertainty are established by the method of neutralizing the hemolytic properties of staphylococcal alpha-toxin.EFFECT: increased accuracy of quantitative determination of antialpha-staphylolysin in donor plasma and human immunoglobulin preparations.1 cl, 5 tbl

Description

[0001] The invention relates to the field of industrial transfusion science, pharmaceutical biotechnology and standardization and can be used to obtain a standard for the quantitative determination of antialphastaphylolysin in immunospecific donor plasma and human immunoglobulin medicinal products.

[0002] Biological medicinal product standards must meet a number of requirements, one of which is similarity in composition and properties to the standardized material. Furthermore, international reports from the World Health Organization (WHO) recommend releasing biological medicinal product standards in lyophilized form, as freeze-drying significantly improves the preservation of antibodies during production and use (WHO Technical Report Series No. 932, 2006, Annex 2, WHO Meating Report, 2005). In accordance with domestic requirements, the certified value of the primary quality indicator and its statistical uncertainty must be established for the standardized reference material.

[0003] Immune-specific plasma and preparations of normal and anti-staphylococcal human immunoglobulins are used for the comprehensive treatment of staphylococcal infections. The primary quality indicator of these medicinal products is their specific activity—the content of anti-alpha-staphylolysin.

[0004] Specific activity is determined using a pharmacopoeial method based on the antibody neutralization reaction of the hemolytic properties of staphylococcal alpha-toxin. The method involves the use of a standard sample (SS) of antialpha-staphylolysin. The pharmacopoeial SS of antialpha-staphylolysin FSO 3.1.00342 (SS) produced in the Russian Federation is horse blood serum stabilized with glycerol, with a specific activity of 24 ± 1.5 IU / ml. The shelf life of the SS is 18 months when stored at a temperature of (6 ± 2) °C. This analogue is a biomaterial of non-human origin and, therefore, does not meet one of the requirements for SS – similarity in composition and properties to standardized medicinal products. In addition, the release of the SS in the form of a solution reduces its stability and effectiveness.The claimed method for obtaining the composition, unlike its analogue, involves obtaining a reference sample for determining antialphastaphylolysin from human blood plasma in the form of a lyophilisate.

[0005] International CO is used abroad – The 3 rdThe International Standard for Staphylococcus Alpha Antitoxin (NIBSC code: STA) is obtained from equine serum and lyophilized. Each ampoule contains 220 IU of antibodies to staphylococcal alpha-toxin. The disadvantages of this analogue include its failure to meet one of the key requirements for a standardized reference material—similarity in composition and properties to the standardized objects. The statistical uncertainty of the certified value of specific activity is not specified, which does not meet Russian requirements for standardized reference materials. Furthermore, there are logistical difficulties in delivering this international standard to the Russian Federation. Unlike this analogue, the method for producing this composition involves the use of human plasma as raw material, determination of the certified value of anti-alpha-staphylolysin content, and establishment of its statistical uncertainty.

[0006] The technical task is to develop a method that allows obtaining a composition for assessing the content of antialphastaphylolysin in donor plasma and human immunoglobulin medicinal products, similar in composition and properties to the controlled objects, in the form of a lyophilisate.

[0007] The technical result of the claimed invention is to increase the accuracy of the quantitative determination of antialphastaphylolysin in donor plasma and human immunoglobulin preparations.

[0008] The technical result is achieved by selecting immunospecific plasma from donors with an antialphastaphylolysin content of at least 3 IU / ml, ethanol-cold fractionation of the selected immunospecific plasma, concentrating the γ-globulin fraction to an antialphastaphylolysin content of at least 28 IU / ml, adding stabilizers of 40 g / l trehalose and 2 g / l bovine serum albumin, adjusting the pH to a value in the range from 4.8 to 5.3, sterilizing filtration, dosing into vials in a volume of 1.5 to 3.0 ml, lyophilization to a residual moisture content of no more than 1%, evaluating the quality indicators of the resulting composition (dissolution time of no more than 15 minutes, protein content from 50 g / l to 110 g / l, content of IgG dimers and monomers of at least 95 %), establishing the certified value of the content of antialpha-staphylolysin and its uncertainty by the method of neutralizing the hemolytic properties of staphylococcal alpha-toxin.

[0009] The obtained composition can be used as a reference material for the quantitative determination of antialphastaphylolysin in donor plasma and immunoglobulin preparations, as well as for assessing the accuracy of the results of control of the main indicator of their quality – specific activity.

[0010] The possibility of implementing the claimed invention is disclosed in the following examples.

[0011] Disclosure of the method

[0012] Apheresis plasma from donors containing at least 3.0 IU / ml of anti-alpha-staphylolysin is collected. Each individual unit of immunospecific plasma is tested for the absence of markers of blood-borne infections (surface antigen and nucleic acid of the hepatitis B virus, antibodies to human immunodeficiency virus types 1 and 2 (HIV-1, HIV-2, HIV-1 p24 antigen) and nucleic acid of the human immunodeficiency virus, antibodies to the hepatitis C virus and nucleic acid of the hepatitis C virus, antibodies to the causative agent of syphilis).

[0013] The γ-globulin fraction is isolated from donor plasma using the cold ethanol method. Fibrinogen is precipitated, the β- and γ-globulin precipitate is isolated and purified, and the prothrombin, β-globulin, and lipid fractions are removed.

[0014] Carry out clarifying filtration of the solution containing γ-globulins, diafiltration and concentration to a refractive index value in the range of (1.350 ± 0.002).

[0015] Determine the concentration of anti-alpha-staphylolysin in the resulting solution containing the γ-globulin fraction by neutralizing the hemolytic properties of staphylococcal alpha-toxin. Its value should be at least 28 IU / ml. If the result is less than the specified value, continue concentrating the solution until the specified value is reached.

[0016] Trehalose is added to a solution containing anti-alpha-staphylolysin at a concentration of at least 28 IU / ml, at a rate of 4 g per 100 ml of solution, and bovine serum albumin is added at a rate of 0.2 g per 100 ml of solution. The pH is adjusted to a value in the range of 4.8 to 5.3 by adding 1 M hydrochloric acid or 1 M sodium hydroxide. This results in a solution of the composition.

[0017] The solution is sterilized-filtered and dispensed into vials in volumes ranging from 1.5 ml to 3.0 ml. The vials are immediately capped to the score line, leaving a gap for the escape of water vapor.

[0018] Vials filled with the composition solution are placed in a lyophilization chamber. The composition solution is frozen, reducing the temperature inside the chamber to minus (45 ± 2) °C, and maintained at this temperature for at least 12 hours but no more than 48 hours.

[0019] The composition solution is lyophilized with a gradual increase in temperature from -40°C to 30°C under a vacuum of 12 Pa. Lyophilization lasts 24 hours from the moment the vacuum is applied. Once lyophilization is complete, the vials are tightly sealed with stoppers secured with aluminum caps.

[0020] Table 1 presents 3 examples of obtaining a composition by the claimed method.

[0021] Determination of quality indicators of the resulting composition

[0022] A visual assessment of the appearance of the resulting composition is carried out: the composition should be an amorphous white mass in the form of a tablet, tightly adhering to the walls of the bottle.

[0023] Determine residual moisture by drying an accurately weighed portion of the test sample in a Binder VDL 53 vacuum drying oven to a constant weight in accordance with OFS.1.5.3.0007.15. Its value should not exceed 1%.

[0024] Record the dissolution time using a stopwatch SOPpr-2a3-000 while reconstituting the lyophilisate to the initial volume with purified water in accordance with OFS.1.4.2.0019. Dissolution time is no more than 15 minutes.

[0025] Determine the protein content relative to the enterprise standard sample, calibrated according to FSO 3.1.00340, using the colorimetric method with a biuret reagent in accordance with OFS OFS.1.2.3.0012.15. Protein content - from 50 g / l to 110 g / l.

[0026] The content of anti-alpha-staphylolysin is determined by the method of neutralization of the hemolytic properties of staphylococcal alpha-toxin in accordance with OFS.1.8.2.0008.15. The concentration of anti-alpha-staphylolysin is not less than 20 IU / ml.

[0027] Molecular parameters were analyzed by high-performance liquid chromatography using an Agilent 1200 system and a BioSep-SEC-s4000 column (Phenomenex, USA) under the following conditions: mobile phase – 0.1 M phosphate buffer solution, elution rate – 0.5 ml / min, wavelength – 280 nm. Chromatograms were processed using the ChemStation software supplied with the equipment. The relative content of the dimer and monomer fraction was at least 95%, and that of polymers and aggregates was no more than 5%.

[0028] Three series of the composition were obtained using the claimed method, and quality indicators were determined that correspond to the established criteria (Table 2).

[0029] Establishing the value of the certified characteristic of the composition

[0030] The resulting composition is certified for its anti-alpha-staphylolysin content. Three series of certification tests are performed on different days with the participation of two analysts. Five samples of the composition are tested in each test, but no less than 10% of the batch volume. Anti-alpha-staphylolysin content is tested using a neutralization test for the hemolytic properties of staphylococcal alpha-toxin.

[0031] On the day of the test, rabbit erythrocytes from defibrinated or preserved blood are washed three times with a five-fold volume of physiological saline (PS) and centrifuged at 1500 rpm for 15 minutes. A 15% suspension is prepared from the sediment of the washed erythrocytes using PS. To standardize the method, the concentration of erythrocytes in the prepared suspension is established by determining the optical density of the hemolyzed erythrocyte solution. For this, 2.0 ml of purified water are added to 0.5 ml of the 15% suspension of rabbit erythrocytes, mixed in a circular motion, and then the resulting solution is diluted 1:6 (5 ml of purified water are added to 1 ml of the resulting hemolyzed solution). Next, the optical density of the diluted hemolyzed solution is measured at a wavelength of 400 nm in a cuvette with a layer thickness of 1 mm. Purified water serves as a control. The optical density of the prepared solution should be equal to 0.53 ± 0.01.If this indicator is higher than 0.54, FR (V.) is added to the prepared 15% suspension of erythrocytes. доб ) in the volume calculated using the formula:

[0032] V доб = V нач ⋅ A 400 / 0.53 – V нач ,

[0033] where Vinit is the initial volume of 15% erythrocyte suspension;

[0034] A 400 – the value of the optical density of the hemolyzed solution.

[0035] If the optical density value is below 0.52, add washed red blood cells in a volume of (V эр.доб ), calculated using the formula

[0036] V эр.доб = V эр.исх ⋅ 0.53 / A 400 – V эр.исх ,

[0037] where Vэр.исх is the volume of washed erythrocytes used to prepare 1.5% suspension, ml;

[0038] A 400 – the value of the optical density of the hemolyzed solution.

[0039] After adding the FR or washed red blood cells, the optical density of the prepared solution is determined again. The prepared 15% red blood cell suspension with the determined optical density value is suitable for analysis within a day.

[0040] Next, the hemolytic limit of the staphylococcal toxin is determined. Toxin dilutions are prepared based on the Lh value specified in the product data sheet. Carefully, without touching the walls, add the test toxin to 7 test tubes: the middle tube in a volume equal to the Lh value specified in the data sheet, and the remaining tubes in corresponding volumes, spaced 0.01 ml apart from the middle tube. Then, the FS is added to each tube to bring the volume to 1.0 ml.

[0041] Pharmacopoeial standard sample of antialphastaphylolysin content FSO 3.1.00342 is diluted to a concentration of 1 IU of antialphastaphylolysin in 1.0 ml. To do this, add 0.6 ml of FSO to 13.8 ml of the SS. Then, add 1.0 ml of the prepared FSO dilution to each tube containing the toxin dilutions. Add 2.0 ml of the SS to the control tube (control for the absence of spontaneous lysis of rabbit erythrocytes). The tubes containing the antigen (staphylococcal toxin) and antibody (antialphastaphylolysin) mixture are gently shaken and incubated at 18 to 22°C for 15 minutes. Then, 0.1 ml of a freshly prepared 15% erythrocyte suspension with the established optical density is added to each test tube in the series (test and control). The tubes are gently shaken again and incubated in a thermostat at 37.0 ± 0.5°C for 1 hour.The results are assessed visually based on the degree of hemolysis: (++++) – complete hemolysis of erythrocytes; (+++) – almost complete hemolysis of erythrocytes; (++) – partial hemolysis (50% hemolysis of erythrocytes); (+) – traces of hemolysis of erythrocytes; ( – ) – no hemolysis of erythrocytes.

[0042] There should be no hemolysis of erythrocytes in the control tube.

[0043] The amount of toxin contained in the first test tube, in which almost complete hemolysis (+++) of erythrocytes occurred, is taken as the limit of hemolytic action under the experimental conditions.

[0044] Next, the anti-alpha-staphylolysin content in the certified samples of the composition is determined. Staphylococcal toxin with an established Lh value is diluted with a standard solution to a Lh / 5 content in 1.0 ml (toxin working solution) immediately before analysis. The amount of initial toxin and the volume of staphylococcal toxin working solution required for the analysis are calculated based on the established Lh value of the toxin and the number of test tubes required for the analysis.

[0045] Before conducting certification tests, the resulting composition is reconstituted to its original volume with purified water. To determine the concentration of anti-alpha-staphylolysin in the test samples, dilutions are prepared based on the estimated anti-alpha-staphylolysin content (in IU / ml) according to the scheme (Table 3).

[0046] Each experiment is accompanied by a control procedure to confirm compliance with the test conditions. This control procedure involves assessing the accuracy of the staphylococcal toxin dose used in the experiment and, if necessary, adjusting the experimental results. A control series is prepared in accordance with Table 4, using a dilution of the FS containing 1 IU / ml of anti-alpha-staphylolysin.

[0047] Add 0.5 ml of the required dilutions of the certified samples of the composition, prepared in accordance with Table 3 (test series), and 0.5 ml of the prepared dilutions of the anti-alpha-staphylolysin FSO (control series) to clean test tubes. Then, add 0.5 ml of the prepared dilution of staphylococcal toxin (toxin working solution) to each test tube. The test tubes are gently shaken in a circular motion and incubated at a temperature of 18 to 22 °C for 15 minutes. Then, add 0.05 ml of a freshly prepared 15% suspension of rabbit erythrocytes with the established optical density to each test tube of the test and control series. The tubes are again gently shaken in a circular motion and incubated at 37.0 ± 0.5°C for 1 hour. After incubation, the reaction results are recorded based on the severity of hemolysis. Recording of the results begins with a visual assessment of the degree of hemolysis.For this purpose, the last tube showing the onset of hemolysis (+), all tubes with 50% (partial) hemolysis (++), and the first tube with almost complete hemolysis (+++) are selected from the control and experimental series. To stop hemolysis, the selected tubes are placed in the refrigerator for 10 minutes at a temperature of (5 ± 3) °C. After cooling, the tubes are centrifuged for 5 minutes at 1500 rpm. The supernatant from each tube is quickly and carefully collected into clean, dry tubes and the optical density is determined at a wavelength of 400 nm, using cuvettes with a layer thickness of 1 mm. The reference solution is FR. The optical density corresponding to 50% hemolysis of erythrocytes should be equal to 0.35 ± 0.05.

[0048] The optical density of the supernatant fluid in the control tubes is determined in a similar manner. Under optimal experimental conditions, 50% erythrocyte hemolysis and an optical density value of 0.35 ± 0.05 are recorded in a tube containing 0.1 IU of anti-alpha-staphylolysin. In this case, the test tube in which 50% erythrocyte hemolysis is recorded contains 0.1 IU of anti-alpha-staphylolysin in a volume of 0.5 ml, which corresponds to a content of 0.2 IU in 1 ml of solution. To calculate the anti-alpha-staphylolysin content in 1.0 ml of undiluted certified sample of the composition, 0.2 IU is multiplied by the dilution value in a given tube. For example, the first tube, in which the supernatant has an optical density of 0.35 ± 0.05, contains a sample at a dilution of 1:100; therefore, 1 ml of undiluted sample contains 0.2 IU⋅100 = 20 IU.

[0049] Due to the influence of various factors, a discrepancy may occur between the toxin dose used in the experiment and the calculated value. In such cases, when calculating the specific activity of the test samples of the composition, a conversion factor (K) is used, determined for each specific analysis using the formula

[0050] K = a / 0.1,

[0051] where a is the concentration (number of IU) of antialphastaphylolysin in the first test tube of the control series, in which hemolysis of 50% of erythrocytes was recorded, giving an optical density of the supernatant fluid of 0.35 ± 0.05.

[0052] The obtained results of determining the values ​​of the certified characteristic of the composition are presented in Table 5.

[0053] It has been established that the certified value of the content of antialphastaphylolysin in the resulting composition after its rehydration to the original volume with purified water is 24.0 IU / ml (the uncertainty of the certified value is from 22.0 IU / ml to 24.2 IU / ml).

[0054] Thus, the possibility of establishing the certified value of the specific activity of the obtained composition and its uncertainty for the quantitative determination of antialphastaphylolysin by the method of neutralizing the hemolytic properties of staphylococcal alpha-toxin has been demonstrated.

[0055] Use of a composition for the quantitative determination of antialphastaphylolysin

[0056] The resulting composition is used to determine the quality indicator of immunospecific plasma or human immunoglobulin preparations – the content of antialpha-staphylolysin (specific activity) by neutralizing the hemolytic properties of staphylococcal alpha-toxin.

[0057] The composition is diluted to a concentration of 1 IU of anti-alpha-staphylolysin per 1.0 ml. To do this, 0.6 ml of the composition, reconstituted to the original volume with purified water, is added to 13.8 ml of 0.9% sodium chloride solution. The resulting solution is used to determine the hemolytic activity limit of staphylococcal toxin and to prepare a control series instead of the FSO.

[0058] The control series is used to confirm compliance with the test conditions. It allows for an assessment of the accuracy of the staphylococcal toxin dose used in the test and, if necessary, correction of the test results using the composition. The control series is prepared in accordance with Table 4. When calculating the specific activity of the test samples, a conversion factor (K) is used, determined for each specific analysis using the formula

[0059] K = a / 0.1,

[0060] where a is the concentration (number of IU) in the first test tube of the control series, in which hemolysis of 50% of erythrocytes was recorded, giving an optical density of the supernatant fluid of 0.35 ± 0.05.

[0061] Thus, the possibility of using the obtained composition as a standard sample for the quantitative determination of antialphastaphylolysin by the method of neutralizing the hemolytic properties of staphylococcal alpha-toxin is demonstrated.

[0062] Using Composition to Assess Correctness

[0063] The resulting composition is used to evaluate the accuracy of the method for determining the anti-alpha-staphylolysin content (specific activity) of immunospecific plasma or human normal and anti-staphylococcal immunoglobulin preparations in the neutralization test for the hemolytic properties of staphylococcal alpha-toxin. For this purpose, the composition is tested as a test sample, and its dilutions are prepared in accordance with Table 3. The determined anti-alpha-staphylolysin content in the composition must be within the uncertainty range of the certified value. Otherwise, the analysis is repeated.

[0064] The disclosure of the method demonstrates the possibility of obtaining a composition in the form of a lyophilisate, the compliance of the obtained composition with established quality indicators, the establishment of certified characteristics of the obtained composition, its use as a standard sample for the quantitative determination of antialphastaphylolysin, as well as for assessing the accuracy of the results of determining the concentration of antialphastaphylolysin in donor plasma and human immunoglobulin preparations.

[0065] Table 1. Controlled parameters during production of the composition

[0066] Parameter name Example 1 Example 2 Example 3 Antialphastaphylolysin content in plasma pool, IU / ml 3,6 4,0 4,0 The value of the refractive index of the immunoglobulin solution 3,352 3,352 3,351 Content of antialphastaphylolysin in immunoglobulin solution, IU / ml 30,0 30,0 30,0 Volume of the resulting immunoglobulin solution, ml 80,0 95,0 86,0 Mass of trehalose, g 3,20 3,80 3,44 Mass of bovine serum albumin, g 0,16 0,19 0,17 pH value of the solution 4,99 5,03 5,05 Dosing volume of the composition when filling into bottles, ml 2,0 2,0 2,0 Freezing temperature of the composition, °C minus 43 minus 45 minus 44 Duration of hardening of the composition, h 18 20 20

[0067] Table 2. Results of determination of quality indicators of the composition for the quantitative determination of antialphastaphylolysin

[0068] Name of the quality indicator Series No. Standard value of the quality indicator 1 2 3 Appearance An amorphous white mass in the form of a tablet, tightly adhering to the walls of the bottle An amorphous white mass in the form of a tablet, tightly adhering to the walls of the bottle Residual moisture, % 0,10 0,10 0,10 No more than 1% Dissolution time, minutes 4,45 4,34 5,01 No more than 15 minutes Protein content, g / l 86,00 86,20 84,60 From 50 g / l to 110 g / l Antialphastaphylolysin content, IU / ml 24 24 24 Not less than 20 IU / ml Molecular parameters, content of di- and monomer fraction, %: 100 100 100 Not less than 95%

[0069] Table 3. Sample dilution scheme for anti-alpha-staphylolysin determination

[0070] Test tube number Estimated antialphastaphylolysin content (IU / ml) Sample dilution The amount introduced into the test tube Test sample, ml Physiological solution, ml 1 2 1:10 1,0 9,0 2 18 1:90 0.5 (from test tube No. 1) 4,0 3 20 1:100 0.5 (from test tube No. 1) 4,5 4 22 1:110 0.5 (from test tube No. 1) 5,0 5 24 1:120 0.5 (from test tube No. 1) 5,5 6 26 1:130 0.5 (from test tube No. 1) 6,0 7 28 1:140 0.5 (from test tube No. 1) 6,5

[0071] Table 4. Scheme of solution dilution to obtain a control series

[0072] Test tube number Concentration of antialphastaphylolysin, IU / 0.5 ml The amount of solution introduced into the test tube standard sample of antialpha-staphylolysin in a dilution of 1 IU / ml Saline solution, ml 1 0,12 1,0 3,17 2 0,11 1,0 3,55 3 0,10 1,0 4,00 4 0,09 1,0 4,56 5 0,08 1,0 5,25

[0073] Table 5. Results of determining the values ​​of the certified characteristic of the composition for the quantitative determination of antialphastaphylolysin

[0074] Sample number, statistical parameter (n = 5) Content of antialphastaphylolysin (IU / ml) in the composition Analyst 1 Analyst 2 in a series of tests… 1 2 3 1 2 3 1 22,0 24,0 24,0 24,0 24,0 24,0 2 24,2 24,0 24,0 24,0 24,0 24,0 3 24,2 24,0 22,0 24,0 24,2 24,0 4 24,0 22,0 24,2 24,2 24,0 22,0 5 24,0 24,0 24,0 24,2 24,2 24,2 median 24,0 24,0 24,0 24,0 24,0 24,0 minimum 22,0 24,0 22,0 24,0 24,0 22,0 maximum 24,2 24,2 24,2 24,2 24,2 24,0 Certified value 24.0 IU / ml Uncertainty of the certified value from 22.0 IU / ml to 24.2 IU / ml