RU2181893C2 - Method for producing reference palette for controlling test-systems and vaccines against hepatitis b - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves selecting donor serum samples by applying immunoenzyme analysis and PCR (polymerizno-chain reaction) for detecting hepatitis B virus DNA and anti-HBS antibodies. Diluting solution is prepared on the base of selected sera. HBSAg standard is diluted with the diluting solution. Solution thermodegradation and stabilization test is carried out. EFFECT: improved vaccine quality control; constant virus protein concentration on reference palette within long time period. 5 cl, 1 dwg, 2 tbl

Description

 The invention relates to the field of biotechnology and immunology and can be used in the technology of manufacturing panels of sera containing antigens of the most dangerous viral infections, and in the production of test systems for determining antigens as control positive sera. Certified HBsAg contents in reference panel samples allow these standard materials to be used to control the quality of the hepatitis B vaccine, in which the HBsAg content must be strictly standardized.

Known methods for the preparation and certification of sera containing certain amounts of specific proteins should be used together in the manufacture of sera with a certified level of concentration of HBsAg and require the development of:
- a reliable way to prepare a non-infectious HBV drug;
- physico-chemical method for determining the concentration of HBsAg in human serum;
- selection of the stabilizer composition and freeze-drying mode of the reference panel samples to obtain preparations with a normalized concentration of HBsAg, which remains unchanged for a long time in accordance with the requirements of the WHO Expert Committee on reference materials.

 Maintaining a constant concentration of a specific protein is a complex problem. An additional problem that one has to face when certifying the amount of HBsAg in blood serum is that the optical density (OD) of the same solution changes when using different test systems and even for one test system when using different analysis modes .

For example, a standard sample (CO) of the P. Erlich Institute subtype ay containing 0.9 IU / ml HBsAg has an OD of 1.3 o in the HBsAg, Labsystems test system. E. for incubation mode A and OD = 0.9 p.u. for regime C. The Murex Wellcozyme HBsAg test has a threshold sensitivity of 0.08 ng / ml when incubating samples overnight and a sensitivity of 0.17 ng / ml when incubating for 30 min at 50 ^° C [3]. In addition, in the general case, the OD value of the CO solution is different for different diluting solutions. The solution to the problem of certification of panel sera for HBsAg content is proposed to be carried out in the following way:
- to use a purified and recombinant plasma antigen belonging to different subtypes with known mass content of HBsAg and without infectious danger to create a reference panel;
- use a pool of sera of healthy donors as a diluting solution that do not contain markers of major infections and do not significantly suppress the OD of HBsAg in ELISA relative to the OD of buffered saline containing an inert protein to prevent a hook effect (for example, PBS + 0.2% casein );
- use substances that do not affect the level of the analytical signal as stabilizer components.

 The main criterion for the sensitivity of diagnosticums for serological analyzes is the value of the analytical signal of the control positive serum. In this case, a prerequisite for the operability of test systems is the level of this analytical signal, which should not be lower than the certified value.

 Known methods for producing control sera with a normalized level of IgG antibodies used in serological analyzes [RF patent 2094807 - anti-HIV panel]. However, there are no known methods for the manufacture of sera in which the quantitative content of viral markers is normalized.

 Boston Biomedical Inc. serum panels are known. (USA): Hepatitis In Surface Antigen Sensitivity Panel (PHA 806) [1]. The panel includes serum HBsAg concentrations from 2.5 ng / ml to 0.1 ng / ml and negative sera. However, these panels are not used as standard US reference materials and are intended for scientific use only. In addition, the serums that make up these panels are presented in liquid form and therefore should be stored at low temperatures. As a result, very strict (temperature) requirements for their storage and distribution create serious problems for use in other countries that do not have a reliable cold chain [Instructions for use PHA 806. W. Bridgewater, MA 02379, 1996].

 These serum panels can be used as an analogue for the proposed reference panels.

 The closest technical solution (prototype) is the method for preparing serum panels developed in the Central Laboratory of Dutch Red Cross standards for the preparation of control panels of the Pelichek brand [2].

 These panels represent 8-10 diluted samples of a standard HBsAg preparation in a pool of donor sera that do not contain markers of dangerous viral infections.

The prototype method includes the preparation of standard sera containing HBsAg and having different concentrations of this protein. The main steps in the production of standard serums are:
- collection of reactive sera for the preparation of the standard HBsAg;
- collection of donor sera that do not contain anti-HBs antibodies and markers of viral infections;
- the manufacture of diluting serum, not containing fibrinogen and lipids, from a pool of selected donor serums;
- dilution of the standard HBsAg in diluting serum 4, 8, 16, 32, 64, 128, 256, 1024, 2048 and 8192 times;
- conducting a thermal degradation test to establish the shelf life of the panel.

 The advantages of the prototype method include the development of a general scheme for the preparation of the HBsAg standard and the preparation of diluting serum, which is implemented in the manufacture of control sera for serum panels with other markers.

The main disadvantages of the prototype:
- breeding serum is an idealized model (i.e., fibrin, fibrinogen and lipids have been removed from the serum) as a model of native human blood serum;
- in the Pelicheck panel, the concentration of HBsAg was not determined and not quantified, and only the multiple dilution method was used. For another pool of sera, other HBsAg concentrations will respond to the same dilutions;
- serum panel prototype is not stabilized and not lyophilized.

 As follows from the above description, the delivery and storage of prototype panels require strict adherence to low temperature conditions.

 The present invention is the creation of such a method that provides the manufacture of a reference panel of sera with normalized concentrations of HBsAg in the range from 6 ng / ml to 0.25 ng / ml, to assess the quality of the test systems and allows you to save the specific characteristics of these sera for a long time time (at least 2 years).

 The problem is solved in that in a method for producing serum panels for quality control of test systems and vaccines against hepatitis B, which includes the selection of donor serums that do not contain specific antibodies to the main markers of viral infections, the preparation of a dilution solution based on the selected ones, dilution of the HBsAg standard in the dilution solution, conducting a thermal degradation test to establish the shelf life of the reference panel, according to the invention, the selection of diluting serums for the panels is carried out according to the scheme, including their at testing by ELISA and PCR for the presence of HBV DNA (hepatitis B virus) and titration of HBsAg samples; moreover, negative panel sera are formed from native sera with a zero titer, which have a negative result in PCR of HBV DNA, and in ELISA - the OD value is less than critical (in range 0.5-0.8 OD crit). Plasma purified and recombinant antigen belonging to different subtypes of HBsAg containing at least 95% of the main protein is used as HBsAg; plasma and recombinant HBsAg are diluted with a pool of donor serums containing the following stabilizing composition: 15-20 g BSA, 45-55 g sucrose , 7-15 g of KCl, 5-10 g of casein per 1 liter of mixed serum; the panel is formed of 10 sera with an HBsAg concentration of from 6 to 0.25 ng / ml and 6 native donor sera; the prepared whey is dried to a residual moisture content of not more than 1.5 wt.% by lyophilization.

The selection of donor sera is carried out in 2 stages:
a) selection of sera that do not contain antibodies to HIV, hepatitis B and C viruses, syphilis;
b) the selection of sera having a suppression ratio of less than 1.3.

 Donor sera selected after two stages are pooled. The stabilizer is added to the pool in dry form, the components of which also do not have an inhibitory effect on the analytical signal of HBsAg. To prevent bacterial growth, bactericidal components are added to the pool.

 A pool of donor sera prepared in this way is used as a diluting solution for preparing reference panel samples containing HBsAg in the required concentration from 6 ng / ml to 0.25 ng / ml (a further increase in the HBsAg value does not affect the OD value, as can be seen in the drawing )

 Serums reactive to HBsAg are selected according to the results of screening for native blood serum in serological laboratories using test systems certified in the order of the Ministry of Health of the Russian Federation on the 1st list. Reactive sera are processed to remove Dane particles to inactivate the HBsAg solution. In the processes of separation and concentration of proteins, fractions with an HBsAg content higher than 95% are isolated in accordance with electrophoresis data. In the process of concentration of HBsAg, the sera are treated with trypsin, filtered through 0.45 μm and 0.22 μm filters, high-speed centrifugation and concentration. The resulting product, called "purified plasma HBs antigen" is certified for protein content. To establish a relationship between the analytical signal of ELISA and the amount of HBsAg in the solution of diluting serum, it is necessary to use ELISA test systems, which in the range from 6 to 0.25 ng / ml show a direct relationship between the optical density and the amount of HBsAg in the solution.

 Samples of prepared stabilized reference serum reference panels with normalized HBsAg content are analyzed in various test systems of various formats (one- and two-stage, accelerated and with night incubation) of domestic production. If the obtained concentrations of HBsAg differ from the set values, their content is adjusted upward by introducing a plasma and recombinant antigen or downward using a dilution solution. Liquid samples are lyophilized according to a regimen specially developed for this type of preparation and stabilizer composition according to the method described in [5].

 A known method of stabilizing the specific properties of positive serums [4], and in the present invention, to stabilize the level of HBsAg, it is proposed to additionally use a stabilizer comprising 5-10 g of casein.

 α-Casein has M.M., close to the mass of HBsAg, and therefore the introduction of an inert molecule of a similar size will not create steric difficulties for the specific interaction: Ar + At. The introduction of casein is equivalent to creating an additional protective shell around the desired molecule for hydrophobic regions of the molecule. At the same time, the introduction of a new component into the composition of the protective medium causes a change in the temperature of the phase transformations of the diluting solution, which entails a change in the lyophilization regime.

 The applicability of the reference preparations created according to the present invention — serums with a normalized level of HBsAg — is checked by sending panels to various laboratories and control services of manufacturers of test systems in accordance with their certification program developed by the national control authority of the Russian Federation. L.A. Tarasevich. According to the results presented by participating analytical laboratories, the certified HBsAg concentrations in the samples of the reference panel are finally established.

 Thus, the certification panel serum certification procedure includes three stages, the results of which are independent and allow reliable identification of the amount of HBsAg.

The concentration of HBsAg established during this certification is entered into the passport on the reference panel. The service life of the reference preparations created according to the present invention is determined according to the results of the thermal degradation test at temperatures of 4 ^° C and 37 ^° C in accordance with [6].

New in comparison with the prototype is:
- the use as a HBsAg of a plasma purified and recombinant antigen belonging to different subtypes of HBsAg containing at least 95% of the basic protein;
- the use as a diluting solution of a pool of donor serums that do not contain markers of major viral infections and do not reduce the analytical signal from HBsAg relative to buffered saline;
- the manufacture of serum with a normalized concentration of HBsAg in the range from 6 to 0.25 ng / ml;
- creation of reference panels in a dry, stable form;
- the use of casein as part of a stabilizing medium.

 The proposed method for obtaining a reference panel of sera for monitoring the quality of test systems and vaccines against hepatitis B is not described in the literature, therefore, we can conclude that the technical solution meets the criteria of the invention of "novelty" and "inventive step".

 The drawing shows a titration curve of a working standard in a diluting solution.

Examples of the method
Example 1. Preparation of a reference panel.

 Donor K-serums are obtained with SECs that are certified free of anti-HIV, anti-HCV, HBsAg and anti-syphilis antibodies. In the laboratory, serums are further monitored for the presence of anti-HBs and anti-HBc antibodies. Selected at the 1st stage K-serum (plasma) is filtered and centrifuged. To separate serum (plasma) from flakes and clots, a filtration unit from a flask and funnel is collected. As a filter element, 3 layers of gauze are used, between which a layer of cotton wool is laid. Then the donor K-serum (plasma) is centrifuged at a speed of 1200 rpm for 10 minutes The precipitate is discarded. At the 2nd stage, working solutions of purified plasma HBsAg ayw2 subtype (manufactured by Imbio CJSC, Nizhny Novgorod) and recombinant HBsAg ayw4 subtype (manufactured by Kombiotekh AO) in donor serum and PBS casein are prepared to obtain two working standards with the calculated concentration of 10 ng / ml each.

 The prepared solutions are titrated simultaneously on one microplate in the Vectogep 2 test system in steps of 3. In the direction of the titration curves and the ratio of OD (PBS) / OD (test serum), a conclusion is drawn on the suitability of using the test serum as a component of the dilution solution. In particular, when OD (PBS) / OD (i.e., solution) <1.3, when the titration curves of both antigens and their solutions in PBS-casein are parallel, serum is included in the pool of donor serums for the preparation of a dilution solution.

At the same time, a stabilizer is prepared per 1 liter of mixed serum, sequentially dissolving BSA, sucrose, KCl and EDTA in a pool of donor serums in the following amounts, g:
BSA - 15-20
Sucrose - 45-55
KCl - 7-15
Casein - 5-10
The dilution solution was adjusted to a pH of 6.8-7.1 with 0.1 M NaCl. As a bactericidal additive, merthiolate is introduced in an amount of 0.01% by weight of the solution. The serum pool with stabilizer components added is filtered sequentially through 0.8 μm and 0.45 μm filters.

 On the prepared reconstituting solution, solutions of plasma Ag and recombinant Ag are prepared to obtain standard solutions with a concentration of 10 ng / ml. The obtained working standards are titrated in diluting serum in three repetitions and test systems: Vektogep 1, Vektogep 2 (ZAO VectorBest) and in the test HBsAg IFA (ZAO Vector Meistar). At the same time, a solution of SB HBsAg (GISC or other) with a concentration of 10 ng / ml in PBS-casein is titrated on the tablets.

 The titration curves are processed and the average titration curve is determined for each working standard and the exact initial concentration of HBsAg in the working standards (see drawing). The average titration curve determines the dilution coefficients of the working standards for obtaining samples of the HBsAg reference panel with concentrations: 6 ng / ml, 2.0 ng / ml, 1.0 ng / ml, 0.5 ng / ml and 0.25 ng / ml

The reference panel serum of each number is obtained by mixing the HBsAg reference solution with the dilution solution in the ratio given by the dilution coefficient. Mixing for each number is carried out in a container with a volume of 500 cm ³ mounted on a magnetic stirrer for 5-7 minutes. The determination of the OD of samples of the reference panel of all numbers is carried out in ELISA test systems of the same type that were used in the titration of standard solutions.

 When assessing the correctness of dilution, the OD values obtained by analyzing the prepared samples with the OD of the sera shown in the drawing are compared. Diluted samples suitable for the preparation of a reference panel according to the test results are transferred to a lyophilization operation. For sera whose optical density does not correspond to the average titration curve, the concentration is adjusted using the initial HBsAg standard solution or dilution solution. Thus, 10 panel samples having different HBsAg contents are prepared: 5 samples based on purified plasma Ag and 5 samples based on recombinant Ag with equal concentrations of 6 ng / ml, 2.5 ng / ml, 1 ng / ml, 0.5 ng / ml and 0.25 ng / ml.

 Negative reference panel sera are prepared from donor sera that do not contain anti-HIV, anti-HCV and anti-syphilis antibodies, as well as HBsAg. In total, 6 samples are prepared that do not contain HBsAg, but have increased OD values in ELISA — from 0.5 to 0.8 OD crit.

 All panel serum samples were monitored for the absence of HBV DNA by PCR in the system (Vector Best CJSC).

Serum panels are poured in a 0.7 ml laminar cabinet into 5 ml vials, closed with rubber stoppers and placed in metal cassettes for freezing. Serum freezing is carried out at a temperature of minus (60 +/- 5) ^o С, keeping at this temperature for at least 20 hours. The temperature during the freezing process is maintained automatically and controlled by the apparatchik.

The frozen preparation in vials is transferred to the sublimation chamber of the TG-50 freeze dryer, the shelves of which are cooled to minus 35 ^o С. The material temperature at the freeze-drying stage is kept below its glass transition temperature (-35 ^o С). The average heating rate of the material at the stage of drying is 4 ^o C / min, the aging time at 27 ^o C is 10 hours.

 Lyophilized dried samples of the reference panel have a residual water content of about 1.0-1.5 wt.%. The determination is carried out according to the method [7]. Vials with the drug are sealed under vacuum. The vacuum in the chamber is controlled by a vacuum gauge. The total average drying time is 36 hours. The control of the specific activity of lyophilized sera with normalized levels of HBsAg is carried out in test systems of domestic production. The results of the analysis are shown in table 1.

Example 2. Preparation of the reference panel HBsAg
The selection of donor sera, their filtration and pooling are carried out in the same ways as in example 1. When stabilizing the pool, the same amount of substances is added to the dilution solution, except that casein is added 3 g per 1 liter of solution. The remaining operations are carried out, as in example 1.

Example 3 Preparation of a HBsAg Reference Panel
The selection of donor sera, their filtration and pooling are carried out in the same ways as in example 1. When stabilizing the pool, the same amount of substances is added to the dilution solution, except that casein is added 10 g per 1 liter of solution. The remaining operations are carried out, as in example 1.

Example 4. Preparation of the reference panel HBsAg
The selection of donor sera, their filtration and pooling is carried out in the same ways as in example 1. When stabilizing the pool, the same amount of substance is added to the dilution solution, except that casein is added 15 g per 1 liter of solution. The remaining operations are carried out, as in example 1.

The dilution solutions prepared in Examples 1-4 were used to prepare positive HBsAg panel samples. The thermal stability of samples prepared with different amounts of casein is estimated by the results of the thermal degradation test, carried out at 37 ^o C for 2 weeks. The results are presented in table 2 in comparison with the serum panels stored in the refrigerator at 4 ^o C.

 Serum panels after exposure to a thermostat were supplied in the Vectogep-2 test system and in the HBsAg ELISA system. As follows from the results of table. 2, the optimal amount of casein is from 5 g to 10 g per 1 liter of dilution solution. With a lower concentration of casein, the OD of positive panel samples in ELISA decreases after 2 weeks. For samples prepared with casein added more than 10 g / l, there was no improvement in performance compared to samples of 5 g / l and 10 g / l. The increased casein content leads to an increase in the viscosity of the dilution solution, which makes it difficult to conduct ELISA.

From the test results it follows that the concentration of HBsAg in the tested sera decreased significantly when the casein content was less than 5 g / l and practically did not change when the content was from 5 to 15 g / l after storage for 4 weeks at a temperature of 37 ^o C. However, the increase the concentration of casein in the sample is accompanied by a decrease in OD in ELISA, which is undesirable. Therefore, the recommended casein content per liter of stabilized solution is from 5 to 10 g / l.

Based on the results obtained, it is possible to evaluate the shelf life of drugs with a normalized level of HBsAg by a standard method [7]. For storage of drugs at a temperature of 4 ^o With expiration date:
t = 4 weeks * 24 = 96 weeks

 Thus, the estimated shelf life of the serum HBsAg panels meets the requirements of WHO experts on standard biological products.

 The economic efficiency of the proposed method for the manufacture of serums with a certified concentration of HBsAg lies in the fact that the desired protein is diagnosed in the blood of patients with hepatitis B at all stages of infection. It appears in the blood of infected people 2-3 weeks after infection. It is the reduction in the time needed to establish diagnoses for infected patients and the earlier medical care and treatment that make up socially significant and economic efficiency associated with the reduction in the time required for treatment and the period of labor incapacity for people.

 In addition, the introduction of serology calibrators with a known concentration of HBsAg into serology creates the basis for checking the correctness of blood tests for this marker and thereby the legal basis for resolving conflicts between manufacturers and users of HBsAg test systems and between manufacturers and users of hepatitis vaccines In which should contain a certain amount of HBsAg.

 Industrial applicability. The invention can be used in biotechnology, medicine and veterinary medicine in assessing the quality of test systems to identify markers of various viral or bacterial infections.

Sources of information
1. Newsletter of Boston Biomedica, Inc., 1996, Hepatitis B surface antigen sensitivity panel (PHA806).

 2. Pelicheck sensitivity HBsAg panel. Central Laboratory of the Netherlands Red Cross. Amsterdam, 1996.

 3. Janot S., Courouce A.-M., Maniez-Montreuil M. et al. Reevaluation of the sensivity of 19 HBs antigen screening kits. La Revue francaise des Laboratories, fevrier / mars 1996, 282 (translation).

 4. E. A. Gutova, A. N. Kanev, E. Yu. Shalaev. Stabilizing composition for control sera positive for antibodies to viral and microbial antigens. RF patent 2011202, 1993.

 5. A.N. Kanev, A.P. Sadovsky, E.Yu. Shalaev, M.Yu. Rukavishnikov. A method for determining the technological parameters of lyophilization of biological products. RF patent 2010220, 1993.

 6. OST 42-2-72 "Medicines. The procedure for establishing the shelf life." Ministry of Health of the USSR, Moscow, 1972.

 7. "Determination of residual moisture in small quantities of biological products", MI 123.39.002.89, SSC WB "Vector", 1990.

Claims (5)

 1. A method of obtaining reference panels of sera containing HBsAg for quality control of test systems and vaccines against hepatitis B, including the selection of donor serums that do not contain specific antibodies to the main markers of viral infections, the production of a dilution solution based on selected sera, dilution of the HBsAg standard in diluting solution, conducting a thermal degradation test to establish the shelf life of the panel, characterized in that the stabilization of the diluting solution is carried out, and the selection of the diluting serum is carried out by a analysis of donor sera by enzyme immunoassay and polymerase chain reaction for the presence of hepatitis B virus DNA and anti HBs antibodies, non-specific suppression of HBsAg, titer HBsAg samples in a diluted stabilized solution to a concentration of HBsAg from 6 to 0.25 ng / ml, and as diluting serums choose serum with a negative result in these methods and having a value of optical density less than critical (in the range of 0.05-0.08 OD crit).  2. The method according to p. 1, characterized in that as the HBsAg use a plasma purified and recombinant antigen belonging to different subtypes of HBsAg containing at least 95% of the main protein.  3. The method according to p. 1, characterized in that the stabilizer contains 15-20 g BSA, 45-55 g sucrose, 7-15 g KCL, 5-10 g casein per 1 liter of mixed whey.  4. The method according to p. 1, characterized in that for the formation of a standard reference panel 10 sera are selected with a concentration of HBsAg from 6 to 0.25 ng / ml, including 5 samples prepared on the basis of plasma purified antigen, and 5 samples prepared on the basis of recombinant antigen belonging to different subtypes of HBsAg, and 6 native donor sera.  5. The method according to p. 1, characterized in that the prepared whey in the reference panel is freeze-dried to a residual moisture content of not more than 1.5 wt. %

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