Antiviral drug and a method of producing immunoglobulin for the prevention and treatment of viral diseases

 

(57) Abstract:

Usage: refers to medicine, namely, clinical immunology, and the development and use of a new immunoglobulin drug against hepatitis C. the invention consists in the fact that donors are tested for the absence of markers of hepatitis b (HBsAg and anti-Bs), subjected to immunization with recombinant yeast vaccine against hepatitis b emergency scheme against the background of immunomodulators. The active protein fraction allocate alcohol fractionation, purify and concentrate on ultrafiltration membranes with a threshold detention substances on the molecular mass of 100 KD, fine impurities before sterilizing filtration removed by microfiltration. The product obtained according to the invention, is a fraction, isolated from plasma of donors proposed method. The preparation contains as active principle anti-HBs with a titer of at least 50 IU/ml, protein - 10-16% and as a stabilizer glycine of 2.25+0.75% and 0,9% solution of sodium chloride. First developed product containing the immunoglobulin against hepatitis C. 2 S. and 2 C.p. f-crystals, 1 table.

The invention relates to medicine, namely to clinical hepatitis B.

Known for the preparation of immunoglobulin against hepatitis B (HBs IgG) derived from plasma recover from hepatitis B, containing a high titer of anti-HBs in the complete absence of HBsAg (Sorenson S. N. Viral hepatitis - SPb.: TESA 1998, S. 111).

However, there is evidence that in the absence of HBsAg in the blood of some patients continues to detect HBV-DNA that characterizes their potential epidemiological risk (Elghouzzi M. N., Courouce, A. M., Magnius L. O., et al. Transmission of hepatitis virus In by HBV-negative blood transfusion. - Lancet, 1995, V. 346, N 8980, H. 964). In Russia in accordance with the guidance of a person with a history of hepatitis B, by donation suspended indefinitely ("instruction on conducting donor plasmapheresis" (approved by the MOH RPF. , 29.05.95 year). A method of obtaining immunoglobulin preparation for the prevention and treatment of bacterial and viral diseases. To the precipitate B /III fraction of Koh/ add distilled water. To extract sediment B add chloroform and 2 h add sodium chloride, centrifuged to obtain a clear centrifugate.

The precipitate is removed. To centrifugate to allocate fractions of immunoglobulins add 50% solution of PEG /m m 6 thousand/. The precipitate immunoglobulins separated by decantation and centrifugual the sodium to the protein content of 5-8%, pH of 7.2. After dissolving the precipitate drug centrifuged to remove insoluble impurities. As a stabilizer use glucose to 2% (EN 2084229, 1997, A 61 K 35/14).

Also known is a method of obtaining specific immunoglobulins from serum (plasma) donors immunized with the corresponding antigens and tested for the absence of the surface antigen of hepatitis B virus (HBsAg), antibodies to human immunodeficiency virus (HIV), antibodies to hepatitis C virus, fractionation of the ethyl alcohol at a temperature below 0oC (Regulation of production of immunoglobulins from the blood of immunized donors N 28-97, approved 14.02.97).

Known drug antiviral activities involving human interferon and immunoglobulin at a mass ratio of 1: 600-300000. As human interferon drug contains recombinant alpha-, beta - or gamma-interferon is human, and as human immunoglobulins mixture lgA, lgM and lgG.

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

However, methods of obtaining immunoglobulin against hepatitis B from plasma immunities aggregated proteins, the number of which increases during lyophilization and which could be included in the final product. The presence of aggregated molecules can cause intolerance reactions when administered intravenously, adverse reactions with intramuscular injection, which significantly impairs the preventive value of immunoglobulins.

The objective of the invention is to develop a method of obtaining a new effective drug - immunoglobulin against hepatitis B that does not cause any adverse reactions when introduced into the body by immunization of donors recombinant yeast vaccine against hepatitis B and due to the high degree of purification of the target product.

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

The invention consists in the following.

For immunization of donors using recombinant yeast hepatitis b vaccine production JSC SPC "Chembiotech Ltd., representing the surface antigen of hepatitis B virus (HBsAg), selected from the producer strain Saccharomyces cerevisiae, adsorbed on aluminium-hydroxide (RU 2088664 fir is erenik in the absence of markers of hepatitis B (HBsAg and anti-HBs), subjected to immunization with recombinant yeast vaccine against hepatitis B emergency scheme against the background of immunomodulators, active protein fraction allocate alcohol fractionation, then further purified and concentrated by ultrafiltration membranes with a threshold detention substances on the molecular mass of 100 KD, and fine impurities before sterilizing filtration removed by microfiltration.

A comparative analysis of the essential features of the proposed method and the nearest analogue shows that the General characteristics they are: immunization of donor specific antigen, obtaining immune plasma by the method of plasmapheresis, the selection of the active fraction alcohol fractionation.

Distinctive features of the proposed method are:

- donor screening before immunization in the presence of anti-HBs,

- immunization with recombinant yeast vaccine against hepatitis B emergency scheme against the background of the introduction of immunomodulators,

- purification and concentration of immunoglobulin (fraction KOH-III) ultrafiltration on membranes with a threshold detention substances on the molecular mass of 100 KD, with the subsequent removal of micro dispersed impurities microfiltration and achieve the result is the following:

The detection of anti-HBs in the absence of HBsAg allows to exclude from the number of donors persons who previously had hepatitis B, and thereby reduce the potential risk of infection of the patients using blood and cooked it in drugs.

The scheme immunization of donors recombinant vaccine against hepatitis B in the background immunomodulators provides obtaining the pool of plasmas used for the preparation of immunoglobulin, with the level of anti-HBs is not lower than 2000 mIU/ml

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

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

The method is as follows:

To obtain the proposed drug pick up the number of donors, most of whom are young (20-40 years) according to the "Instructions for medical examination of donors of blood, plasma, blood cells", approved by RF Ministry of health M, 2 human immunodeficiency virus (HIV), antibodies to hepatitis C virus, additional testing for antibodies to hepatitis B (anti-HBs).

Donors that do not contain blood markers of hepatitis B, active subjected to immunization with recombinant yeast vaccine against hepatitis B in the background of the immunomodulator (Lactobacterin or timogen).

Recombinant yeast vaccine against hepatitis B is administered intramuscularly in the deltoid muscle three times, on the background of immunomodulators get a pool of plasmas with high titers of specific antibodies.

Stage alcohol fractionation immune plasma is conducted by a standard method. Then the precipitate KOH-III is dissolved in sterile 0,9% solution of sodium chloride to a concentration of 3-5% for protein. The solution of purified immunoglobulin in membranes with a pore diameter of 3 μm and was subjected to purification and concentration by ultrafiltration. The cleaning solution of immunoglobulin and concentration carried out by ultrafiltration installation, consisting of a peristaltic pump and membrane units with a limit of detention substances on the molecular mass of 100 KD. At the beginning of the lead concentration fraction KOH-III up to 1/5 of the original volume, then spend diafiltration constant volume% solution of sodium chloride. Diafiltration spend up to complete disappearance of nitrogenous substances in the permeate (optical density of less than 0.1 at a wavelength of 280 nm on spectrophotometer readings). After diafiltration spend the final concentration of protein in the preparation of 10-16%, adjusting the pH to (7 0,4), added as a stabilizer glycine concentration (2,250,75)% and before sterilizing filter free from suspended impurities by filtration on membranes with a pore size of 3 μm.

Immunoglobulin against hepatitis B contains not less than 50 IU/ml of anti-HBs, 10-16% protein.

The method is illustrated by the following specific example.

Example

To obtain the proposed drug pick up the number of donors, most of whom are young (20-40 years) according to the "Instructions for medical examination of donors of blood, plasma, blood cells", approved by RF Ministry of health M., 29.05.95.

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

Donors that do not contain blood markers of hepatitis B, the asset is h or timogen).

Schematic diagram of immunization

Before vaccination donors accept within 10 days Lactobacterin 5 doses daily or timogen 50-100 µg intranasal within 10 days.

Recombinant yeast vaccine against hepatitis B is administered intramuscularly in the deltoid muscle triple (single dose of 20 mcg HBsAg) for emergency circuits: 0-7-21 day or 0-1-2 months.

In the process of immunization donors are under systematic medical, clinical-morphological, biochemical, immunological control. On day 14 after the last immunization control antibody titer in the serum of immunized donors.

Donors containing serum at least 100 mIU/ml of anti-HBs, subjected to systematic plasmapheresis twice a month. Obtained in this method protivogerpetical plasma accumulate and stored in sterile plastic bags at a temperature of from -20oC to -30oC. From this protivogerpetical plasma secrete immunoglobulin against hepatitis B by fractionating plasma ethanol at low temperatures.

80,0 l protivogerpetical plasma loaded into the reactor and cooled to 0oC with continuous stirring p the>C ethanol to a final plasma concentration of 8.0%, gradually lowering the temperature of the mixture in the reactor during deposition from 0oC to -3oC. Maintain the mixture under stirring for one hour at a temperature of -3oC. the Resulting precipitate, consisting of fibrinogen, stroma of erythrocytes, insoluble proteins in the blood is separated by centrifugation at high speed centrifuge CDF - 150 at 15000 rpm

Centrifugal placed in a reactor and produce Department globulins from albumin. For this reactor type 50% alcohol, pre-cooled to -20oC, under continuous stirring for 4-5 hours, gradually reducing the temperature of the mixture from -3oC to -10oC, to a final concentration of alcohol in a mixture of 25%. the pH should be between 7.0 to 7.4. When the deviation of the pH from these values make the correction using 1 mol/l solution of sodium bicarbonate. Then serves spirit-protein mixture to a centrifuge CDF-150 for the globulin precipitate from a solution of albumin. The total weight of sediment globulin - 3,6 kg

At the next stage, to make a separation of beta-globulin and immunoglobulin. For this globulin precipitate is dissolved in cooled to 0oC distillirovanna is. In the solution set pH (5,0 0,1) added 1 mol/l acetate buffer based 110-120 ml per 1 kg of sediment and stirred for at least 1 h then to the cooled solution add 50% ethanol to a final concentration of 17%. The deposition of lead 4-5 hours while gradually reducing the temperature in the reactor is from 0oC to -5oC. After that make the sludge separation of beta-globulin and lipoproteins from the immunoglobulin solution by centrifugation in a centrifuge CDF-150.

At the next stage, conduct sedimentation immunoglobulin. For this to centrifugate add cooled to 5 mol/l solution of sodium chloride at the rate of 10 ml per 1 l of centrifugate. Set pH (6,9 0,1) by adding 1 mol/l solution of sodium bicarbonate. To the cooled solution add ethanol for 4-5 h, gradually lowering the temperature from -5oC to -10oC to a final concentration of alcohol in a mixture of 25%. Mixture at a temperature of -10oC is stirred for 1 h then separating the precipitated immunoglobulin by centrifugation, collect the precipitate of immunoglobulin and weighed. The weight of sediment - 1.7 kg (fraction KOH-III) or of 21.2 g of purified immunoglobulin from 1 l of plasma.

The precipitate (KOH-III) dissolved in sterile 0.9% saline Filtracia on membranes with a pore diameter of 3 μm. The filtrate (volume 18 l) collected in the tank is stainless steel with two pipes connected with prepared to work ultrafiltration plant, consisting of a peristaltic pump and membrane apparatus with hollow fibres brand TLU -100. Under pressure (0,06 0,02) MPa spend the concentration of the material to 1/5 of the original volume, and then diafiltration at constant volume, which when operating setting is added to the concentrate diafiltrate solution - sterile 0,9% solution of sodium chloride. Diafiltration spend up to complete disappearance of nitrogenous substances in the permeate (optical density of less than 0.1 at a wavelength of 280 nm reading of the spectrophotometer). Then spend the final concentration to a volume of 3.2 l (the total protein content of 11.2 %). The permeate during the entire cleaning process is discharged into the sewer.

In the product set pH (7,0 0,4), added as a stabilizer glycine concentration (2,250,75)% and before sterilizing filter free from suspended impurities by filtration on membranes with a pore size of 3 μm.

The protein content determined by the method of Lowry, immunospecificity activity (titer anti-HBs) is determined by enzyme-linked immunosorbent anzmi human immunoglobulins (IgG) establish a method of zone electrophoresis in polyacrylamide gel.

The proposed method provides receiving immunoglobulin (IgG) against hepatitis B protein content of 10-16%, the titer of anti-HBs not less than 50 IU/ml

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

The preparation may further contain other active components, such as immunomodulators, immunoglobulins against opportunistic infections. Get the drug to the traditional method of mixing all of its constituent components. If necessary, the solution 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.%

a 0.9% solution of sodium chloride with a pH of 6.6 To 2 ml

Example 2

Immunoglobulin IgG fraction, containing anti-HBs 100 IU/ml - 16 wt.%

Glycine - 3.0 wt.%

a 0.9% solution of sodium chloride with a pH of 7.4 To 1 ml

Example 3

Immunoglobulin Fracci solution of sodium chloride with a pH of 7.0 - To 1 ml

The drug is a transparent or slightly opalescense liquid, colourless or slightly yellow color. In the storage process allows a minor sludge, disappearing with shaking at a temperature of the drug (20 2)oC.

Active principle of the drug are immunoglobulins having the activity of antibodies to the surface of HBsAg of hepatitis B. drug Used for prevention of hepatitis B, as well as in the following cases:

1. After random infections (injections, dental manipulations, blood transfusions, and so on) people that have not been previously vaccinated against hepatitis B, or persons for whom vaccination is not completed, or if the level of HBs-antibody protective (less than 10 IU/l) or unknown.

2. Persons or patients, the activity of which requires permanent preventive measures due to the risk of infection by hepatitis B virus, or those in whom vaccination is not possible or it does not lead to the formation of post-vaccination immunity (for example, staff of the Department of hemodialysis, which is engaged in the surveillance of patients with positive resulte drugs:

The drug is administered intramuscularly in the upper outer quadrant of the gluteal muscle. Prior to the injection of the injectable drug was incubated for 2 h at room temperature (20 2)oC. Opening of the ampoule and the insertion procedure carried out in strict compliance with the rules of asepsis and antisepsis. The dose of the drug and the multiplicity of its introduction depends on indication for use:

1. After random infections the drug is administered in the amount of 8 ME 1 kg weight within 24 h after infection. Preferably within 7 days after passive immunization must enter one dose of recombinant vaccines against hepatitis B and subsequently introduce additional doses of the vaccine in accordance with the instructions in the "Instructions for use of hepatitis B vaccine recombinant yeast liquid.

2. For risk groups (staff of the Department of hemodialysis, sexual partners of chronic carriers of HBsAg and so on), and when the vaccination against hepatitis B is not possible or it does not generate a sufficient level of antibodies (below 10 IU/l) - 8 ME 1 kg 1 every two months.

Ingestion of large doses (more than 5 ml) of the drug is recommended to divide the dose into several injections in different parts of the body. The maximum is t 4-5 weeks.

Reactions to the introduction of immunoglobulin, usually absent. In rare cases, can develop local reactions such as redness and increased temperature up to 37.5oC during the first days after injection. The drug is released in liquid form in ampoules of 1 or 2 ml Single dose (1 or 2 ml) contains at least 100 ME HBs antibodies. The preparation should be stored in a dry and dark place at a temperature of (6 4)oC.

1. Immunoglobulin medicine against hepatitis b, which represents the immunoglobulin fraction with a content of anti-HBs not less than 50 IU/ml, and the excipient is glycine and 0.9% sodium chloride solution with a pH of 6.6 to 7.4 in the following ratio of ingredients:

Immunoglobulin IgG fraction with a content of anti-HBs not less than 50 IU/ml, wt.% - 10 - 16

Glycine, wt.% - 1,5 - 3,0

a 0.9% solution of sodium chloride with a pH of 6.6 to 7.4, ml - Up to 1

2. Immunoglobulin medicine against hepatitis b under item 1, characterized in that it further comprises an immunomodulator.

3. Immunoglobulin medicine against hepatitis b under item 1 or 2, characterized in that it is a lyophilized form.

4. The method of obtaining the immunoglobulin preparation of thee, recombinant yeast vaccine against hepatitis b in the background of the introduction of the immunomodulator and holding plasmapheresis when the content in the serum of at least 100 mIU/ml of anti-HBs after alcohol fractionation residue was dissolved in 0.9% sodium chloride solution, the resulting solution is subjected to ultrafiltration and selected fraction (mol. m to 100 KD, then hold her concentration and diafiltration using 0.9% sodium chloride solution and produce the target product after concentration of the resulting solution by filtration on membranes with a pore size of 3 μm.

 

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