RU2144379C1 - Antiviral preparation and method of preparing immunoglobulin for prophylaxis and treatment of patient with viral diseases - Google Patents

Abstract

FIELD: medicine, clinical immunology. SUBSTANCE: invention relates to development and use of the new immunological preparation against viral hepatitis B. Method involves immunization of donors checked for the absence of hepatitis B markers (HBsAg and anti-HBs) with recombinant yeast vaccine against hepatitis B by urgent schedule on background of effect of immunomodulating agents. Active protein fraction is isolated by fractionation with ethyl alcohol, purified and concentrated on ultrafiltrating membranes at retention threshold of substances 100 kDa by molecular mass. Before sterilizing filtration small-dispersed impurities are removed by microfiltration. Obtained preparation is the fraction isolated from donor plasma by proposed method. Preparation has anti-HBs as an active component (titer is 50 IU/ml, not less), proteins - 10-16-% and glycine (2.25 ± 0.75-%) as stabilizing agent and 0.9-% sodium chloride solution. The preparation containing immunoglobulin against hepatitis B is developed first. EFFECT: novel antiviral preparation, improved method of preparing. 4 cl, 1 tbl, 3 ex

Description

 The invention relates to medicine, namely to clinical immunology, and for the development and use of a new immunoglobulin preparation against viral hepatitis B.

 The known preparation of anti-hepatitis B immunoglobulin (IgG HBs), obtained from plasma of patients with hepatitis B, containing a high titer of anti-HBs in the complete absence of HBsAg (Sorinson S.N. Viral hepatitis - St. Petersburg: TEZA 1998, p. 111).

 However, there is evidence that in the absence of HBsAg, HBV DNA continues to be detected in the blood of some patients, which characterizes their potential epidemiological danger (Elghouzzi M.N., Courouce A.M., Magnius LO, et al. Transmission of hepatitis B virus by HBV-negative blood transfusion. - Lancet, 1995, V. 346, N 8980, H. 964). In Russia, in accordance with the instructions, people who have had hepatitis B are suspended from donation indefinitely ("Instructions for Donor Plasmapheresis" (approved by the Ministry of Health of the Russian Federation. May 29, 1995). There is a known method for producing an immunoglobulin preparation for the prevention and treatment of bacterial and viral diseases. Distilled water is added to precipitate B / III Cohn fraction /, chloroform is added to precipitate B extract, and after 2 hours sodium chloride is added, centrifuged until a clear centrifugate is obtained.

 The precipitate is removed. A 50% PEG / m.m solution is added to the centrifugate to isolate the immunoglobulin fraction. 6 thousand /. The precipitate of immunoglobulins is separated by decantation and centrifugation, washed with distilled water, acidified to pH 5, dissolved in a cooled 0.9% sodium chloride solution to a protein content of 5-8%, pH 7.2. After dissolution of the precipitate, the drug is centrifuged to remove insoluble impurities. As a stabilizer, glucose up to 2% is used (RU 2084229, 1997, A 61 K 35/14).

There is also a method of producing specific immunoglobulins from serum (plasma) of donors immunized with the appropriate antigens and tested for the absence of hepatitis B virus surface antigen (HBsAg), antibodies to human immunodeficiency virus (HIV), antibodies to hepatitis C virus, by the method of ethyl alcohol fractionation at a temperature below 0 ^o C (Regulation for the production of immunoglobulin from the blood of immunized donors N 28-97, approved 14.02.97).

 Known antiviral drug, including human interferon and human immunoglobulin in a mass ratio of 1: 600-300000, respectively. As a human interferon, the preparation contains recombinant human alpha, beta, or gamma interferon, and a mixture of IgA, IgM and IgG as human immunoglobulins.

 The preparation further comprises pharmaceutically acceptable target additives (RU 2073522, 1997, A 61 K 38/21).

 However, methods for producing anti-hepatitis B immunoglobulin from the plasma of immunized donors are not known. In addition, there is evidence that, during alcohol fractionation, aggregated proteins irreversibly form, the amount of which increases during the lyophilization process and which can enter the final preparation. The presence of aggregated molecules can cause intolerance reactions with intravenous administration, adverse reactions with intramuscular administration, which significantly worsens the preventive value of immunoglobulins.

 The objective of the invention is to develop a method for producing a new effective drug - anti-hepatitis B immunoglobulin, which does not cause any adverse reactions when it is introduced into the body through immunization of donors with a recombinant hepatitis B yeast vaccine and due to the high degree of purification of the target product.

 The problem is solved by the proposed method for producing immunoglobulins (IgG) and using the developed drug for the prevention of viral diseases.

 The invention consists in the following.

 For immunization of donors, a recombinant yeast vaccine against hepatitis B produced by AOZT NPK Kombioteh Ltd. is used, which is a surface antigen of hepatitis B virus (HBsAg), isolated from a producer strain Saccharomyces cerevisiae, adsorbed on aluminum hydroxide (RU 2088664, Combiotech company Combiotech Combiotech State Register of Medicines under N 94/266/1 and 96/144/53/7).

 Donors tested for the absence of hepatitis B markers (HBsAg and anti-HBs) are immunized with a recombinant hepatitis B yeast vaccine according to an emergency schedule against immunomodulators, the active protein fraction is isolated by alcohol fractionation, then it is further purified and concentrated on ultrafiltration membranes with a threshold for retention of substances a molecular weight of 100 kD, and finely dispersed impurities are removed by microfiltration before sterilizing filtration.

 A comparative analysis of the essential features of the proposed method and the closest analogue indicates that they have common features: immunization of donors with a specific antigen, production of immune plasma by plasmapheresis, isolation of the active fraction by alcohol fractionation.

Distinctive features in the proposed method are:
- screening of donors before immunization for the presence of anti-HBs,
- immunization with a recombinant yeast vaccine against hepatitis B according to the emergency schedule against the background of the introduction of immunomodulators,
- purification and concentration of immunoglobulin (KOH-III fraction) by ultrafiltration on membranes with a threshold for retention of substances by molecular weight of 100 kD, followed by removal of fine impurities by microfiltration.

A causal relationship between the totality of the essential features of the claimed invention and the achieved result is as follows:
Detection of anti-HBs in the absence of HBsAg allows us to exclude people who have previously had hepatitis B from the number of donors, and thereby reduce the potential risk of infection of patients with the use of blood and preparations prepared from it.

 The developed scheme of donor immunization with a recombinant hepatitis B vaccine against the background of immunomodulators provides a pool of plasmas used to prepare immunoglobulin with an anti-HBs level of at least 2000 mIU / ml.

 The use of ultrafiltration for purification and concentration of immunoglobulin allows you to get a drug with a maximum content of monomer fraction in the complete absence of polymers (table. 1).

 Thus, the proposed method allows to obtain anti-hepatitis B immunoglobulin from the plasma of immunized donors with high levels of anti-HBs in a short time with improved molecular parameters.

The method is as follows:
To obtain the proposed drug, a contingent of donors of predominantly young age (20-40 years) is selected according to the "Instructions for the medical examination of blood, plasma, blood cells" approved by the Ministry of Health of the Russian Federation M., 05/29/95.

 Donors tested for the absence of hepatitis B virus surface antigen (HBsAg), antibodies to human immunodeficiency virus (HIV), antibodies to hepatitis C virus, are additionally tested for the presence of antibodies to hepatitis B virus (anti-HBs).

 Donors that do not contain hepatitis B markers in the blood are actively immunized with a recombinant yeast vaccine against hepatitis B against the background of an immunomodulator (lactobacterin or thymogen).

 Recombinant yeast hepatitis B vaccine is injected intramuscularly into the deltoid muscle three times, while against the background of the use of immunomodulators, a pool of plasmas with a high titer of specific antibodies is obtained.

 The stage of alcohol fractionation of the immune plasma is carried out by the standard method. Then the precipitate KOH-III is dissolved in a sterile pyrogen-free 0.9% sodium chloride solution to a concentration of 3-5% protein. The immunoglobulin solution is purified on membranes with a pore diameter of 3 μm and subjected to purification and concentration by ultrafiltration. In this case, the purification of the immunoglobulin solution and concentration is carried out on an ultrafiltration unit consisting of a peristaltic pump and membrane devices with a substance retention limit of 100 kD in molecular weight. At the beginning, the KOH-III fraction is concentrated to 1/5 of the initial volume, then diafiltration is carried out in a constant volume, for which, with the unit in operation, a diafiltration solution is added to the concentrate — sterile pyrogen-free 0.9% sodium chloride solution. Diafiltration is carried out until the complete disappearance of nitrogenous substances in the permeate (optical density less than 0.1 at a wavelength of 280 nm according to the readings of a spectrophotometer). After diafiltration, the final concentration is carried out until the protein content in the preparation is 10-16%, the pH is adjusted to (7 ± 0.4), glycine is added as a stabilizer at a concentration of (2.25 ± 0.75)% and, before sterilization filtration, it is freed from fine particles impurities by microfiltration on membranes with a pore size of 3 microns.

 Anti-hepatitis B immunoglobulin contains at least 50 IU / ml anti-HBs, 10-16% protein.

 The method is illustrated by the following specific example.

Example
To obtain the proposed drug, a contingent of donors of predominantly young age (20-40 years) is selected according to the "Instructions for the medical examination of blood, plasma, blood cells" approved by the Ministry of Health of the Russian Federation M., 05/29/95.

 Donors tested for the absence of hepatitis B virus surface antigen (HBsAg), antibodies to human immunodeficiency virus (HIV), antibodies to hepatitis C virus, are additionally tested for the presence of antibodies to hepatitis B virus (anti-HBs).

 Donors that do not contain hepatitis B markers in the blood are actively immunized with a recombinant yeast vaccine against hepatitis B against the background of immunomodulators (lactobacterin or thymogen).

Immunization concept
Before vaccination begins, donors take 5 doses of lactobacterin for 10 days daily or thymogen 50-100 μg intranasally for 10 days.

 Recombinant yeast hepatitis B vaccine is administered intramuscularly into the deltoid muscle three times (single dose of 20 μg HBsAg) according to emergency regimens: 0-7-21 days or 0-1-2 months.

 In the process of immunization, donors are under systematic medical, clinical, morphological, biochemical, immunological control. On the 14th day after the last immunization, the titer of antibodies in the blood serum of the immunized donors is monitored.

Donors containing at least 100 mIU / ml anti-HBs in serum are subjected to systematic plasmapheresis twice a month. The anti-hepatitis plasma obtained by this method is accumulated and stored in sterile plastic bags at a temperature of from -20 ^° C to -30 ^° C. Immunoglobulin against hepatitis B is isolated from this anti-hepatitis plasma by fractionating the plasma with ethyl alcohol at low temperatures.

80.0 l of anti-hepatitis plasma are loaded into the reactor and cooled to 0 ^° C. with continuous stirring by feeding a cooling brine into the jacket of the reactor. Then, ethanol pre-cooled to -20 ^° C is added to the reactor to a final plasma concentration of 8.0%, gradually lowering the temperature of the mixture in the reactor during deposition from 0 ^° C to -3 ^° C. The mixture is kept under stirring for one hour at temperature -3 ^o C. The resulting precipitate, consisting of fibrinogen, erythrocyte stroma, insoluble blood proteins, is separated by centrifugation on a high-speed centrifuge SGO - 150 at 15000 rpm.

The centrifuge is placed in a reactor and the globulins are separated from albumin. To do this, add 50% alcohol, pre-cooled to -20 ^o C, with continuous stirring for 4-5 hours, gradually reducing the temperature of the mixture from -3 ^o C to -10 ^o C, to a final concentration of alcohol in the mixture 25% the pH should be 7.0-7.4. If the pH deviates from the indicated values, a correction is carried out using 1 mol / L sodium bicarbonate solution. Then the alcohol-protein mixture is fed to the SGO-150 centrifuge to separate the globulin precipitate from the albumin solution. The total weight of the globulin sediment is 3.6 kg.

In the next stage, beta-globulin and immunoglobulin are separated. For this, the globulin precipitate is dissolved in distilled water cooled to 0 ^° C at the rate of 25 l per 1 kg of sediment. Sodium chloride is added to the resulting solution at the rate of 0.812 g per 1 liter of water. The solution is adjusted to pH (5.0 ± 0.1) by adding 1 mol / L acetate buffer at the rate of 110-120 ml per 1 kg of precipitate and stirred for at least 1 hour. After that, cooled 50% alcohol is added to the solution to the final concentration of 17%. Precipitation is carried out for 4-5 hours with a gradual decrease in temperature in the reactor from 0 ^o C to -5 ^o C. After this, the precipitate of beta-globulin and lipoproteins is separated from the immunoglobulin solution by centrifugation on a SGO-150 centrifuge.

In the next stage, the precipitate of immunoglobulin is isolated. For this, a cooled 5 mol / L sodium chloride solution is added to the centrifugate at the rate of 10 ml per 1 liter of centrifugate. Set the pH (6.9 ± 0.1) by adding 1 mol / L sodium bicarbonate solution. Chilled alcohol is added to the solution over 4-5 hours, gradually lowering the temperature from -5 ^° C to -10 ^° C to a final alcohol concentration of 25% in the mixture. The mixture is stirred at -10 ^° C for 1 hour. After this, the immunoglobulin precipitate is separated by centrifugation, the immunoglobulin precipitate is collected and weighed. The weight of the precipitate is 1.7 kg (KOH-III fraction) or 21.2 g of purified immunoglobulin with 1 l of plasma.

 The precipitate (KOH-III) is dissolved in a sterile 0.9% sodium chloride solution to a final protein concentration of 2-3%. The resulting solution is freed from fine impurities by microfiltration on membranes with a pore diameter of 3 μm. The filtrate (volume 18 l) is collected in a stainless steel tank with two nozzles connected to an ultrafiltration unit prepared for operation, consisting of a peristaltic pump and a membrane unit with hollow fibers VPU-100. At a pressure of (0.06 ± 0.02) MPa, a material concentration of up to 1/5 of the initial volume is carried out, and then diafiltration in a constant volume, for which, when the installation is in operation, a diafiltration solution is added to the concentrate - sterile pyrogen-free 0.9% sodium solution chloride. Diafiltration is carried out until the complete disappearance of nitrogenous substances in the permeate (optical density less than 0.1 at a wavelength of 280 nm according to the spectrophotometer). Then, the final concentration is carried out to a volume of 3.2 l (total protein content of 11.2%). Permeate is discharged into the sewer during the entire cleaning process.

 The pH is adjusted in the preparation (7.0 ± 0.4), glycine is added as a stabilizer at a concentration of (2.25 ± 0.75)%, and before sterilizing filtration it is freed from fine impurities by microfiltration on membranes with a pore size of 3 μm.

 Protein content is determined by the Lowry method, immunospecific activity (anti-HBs titer) is determined by enzyme-linked immunosorbent assay using Abbott kits (USA) to determine anti-HBs.

 The content of immunoglobulins (IgG) in a human plasma protein is determined by zone polyacrylamide gel electrophoresis.

 The proposed method provides for the production of immunoglobulin (IgG) against hepatitis B with a protein content of 10-16%, an anti-HBs titer of at least 50 IU / ml.

 The isolated fraction is the active component of the anti-hepatitis B drug developed on its basis. The preparation contains the following components: immunoglobulin IgG fraction obtained by the proposed method 10-16 wt. %; glycine in the amount of 1.5-3.0 wt.%, 0.9% sodium chloride solution with a pH of 6.6-7.4 to 1 or 2 ml as a stabilizer.

 The preparation may additionally contain other active components, such as, for example, immunomodulators, immunoglobulins against concomitant infections. Get the drug by the traditional method of mixing all its components. If necessary, the solution of the drug is subjected to lyophilization by any known method.

Example 1
Immunoglobulin fraction lgG, with the content of anti-HBs 50 IU / ml - 10 wt. %
Glycine - 1.5 wt.%
0.9% sodium chloride solution with a pH of 6.6 - Up to 2 ml
Example 2
IgG immunoglobulin fraction with anti-HBs content of 100 IU / ml - 16 wt.%
Glycine - 3.0 wt.%
0.9% sodium chloride solution with a pH of 7.4 - Up to 1 ml
Example 3
The immunoglobulin fraction of IgG, with an anti-HBs content of 100 IU / ml - 15 wt. %
Glycine - 2.0 wt.%
Immunomodulator - - 0.0005 wt.%
0.9% sodium chloride solution with a pH of 7.0 - Up to 1 ml
The drug is a clear or slightly opalescent liquid, colorless or with a faint yellow color. During storage, the appearance of a slight precipitate, disappearing when shaken at a temperature of the drug (20 ± 2) ^o C.

The active principle of the drug is immunoglobulins, which have the activity of antibodies to surface HBsAg of hepatitis B virus. The drug is used to prevent viral hepatitis B disease, as well as in the following cases:
1. After accidental infections (during injections, dental procedures, blood transfusions, etc.) of people who have not previously been vaccinated against viral hepatitis B, or people whose vaccination is not completed, or in the case when the level of HBs antibodies below protective (less than 10 IU / l) or unknown.

 2. For persons or patients whose vital activity requires constant preventive measures due to the risk of infection with the hepatitis B virus, or for those who cannot be vaccinated or do not lead to the formation of post-vaccination immunity (for example, the staff of the hemodialysis department who is monitoring for patients with a positive result for the HBs antigen or for sexual partners - chronic carriers of the HBs antigen).

An example of the use of the drug:
The drug is administered intramuscularly in the upper outer quadrant of the gluteal muscle. Prior to the injection, the ampoules with the preparation are kept for 2 hours at room temperature (20 ± 2) ^o C. The opening of the ampoules and the administration procedure are carried out with strict adherence to aseptic and antiseptic rules. The dose of the drug and the frequency of its administration depend on the indications for use:
1. After accidental infections, the drug is administered in an amount of 8 ME per 1 kg of body weight within 24 hours after infection. Preferably, within 7 days after passive immunization, a single dose of the recombinant hepatitis B vaccine is necessary and subsequently additional doses of the vaccine are administered in accordance with the instructions for use of the recombinant yeast liquid hepatitis B vaccine.

 2. For risk groups (hemodialysis department staff, sexual partners - chronic carriers of HBs antigen, etc.), as well as in cases where vaccination against hepatitis B is impossible or it does not lead to the formation of a sufficient level of antibodies (below 10 IU / l) - 8 ME per 1 kg of weight once every two months.

 When using large doses (more than 5 ml) of the drug, it is recommended to divide the dose into several injections in different parts of the body. The maximum concentration of antibodies in the blood is reached after 24 hours, the half-life of antibodies from the body is 4-5 weeks.

As a rule, there are no reactions to the introduction of immunoglobulin. In rare cases, local reactions may develop in the form of hyperemia and fever up to 37.5 ^o C during the first days after administration. The drug is released in liquid form in ampoules of 1 or 2 ml. One dose of the drug (1 or 2 ml) contains at least 100 ME HBs antibodies. The drug is stored in a dry and dark room at a temperature of (6 ± 4) ^o C.

Claims (3)

1. Immunoglobulin preparation against viral hepatitis B, which is an immunoglobulin fraction with an anti-HBs content of at least 50 IU / ml, and excipients - glycine and 0.9% sodium chloride solution with a pH of 6.6 - 7.4 at the following ratio of ingredients:
IgG immunoglobulin fraction with an anti-HBs content of at least 50 IU / ml, wt.% - 10 - 16
Glycine, wt.% - 1.5 - 3.0
0.9% sodium chloride solution with a pH of 6.6 - 7.4, ml - Up to 1
2. The immunoglobulin preparation against viral hepatitis B according to claim 1, characterized in that it further comprises an immunomodulator.  3. The immunoglobulin preparation against viral hepatitis B according to claim 1 or 2, characterized in that it is a lyophilized form.  4. A method of obtaining an immunoglobulin preparation against viral hepatitis B, comprising immunizing healthy donors that do not contain any hepatitis B markers in the blood with a recombinant yeast vaccine against hepatitis B against the background of the introduction of an immunomodulator and plasmapheresis with a serum content of at least 100 mIU / ml anti -HBs, after alcohol fractionation, the precipitate is dissolved in a 0.9% sodium chloride solution, the resulting solution is ultrafiltered and a fraction of mol. m to 100 kDa, then its concentration and diafiltration is carried out using a 0.9% sodium chloride solution and the target product is isolated after concentration of the resulting solution by microfiltration on membranes with a pore size of 3 μm.

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