RU2339401C2 - Preparation of heterologous antirabic immunoglobulin for intravenous both intramuscular introduction and method for its obtaining - Google Patents

Abstract

FIELD: medicine, pharmacology.

SUBSTANCE: invention concerns biotechnology, namely manufacture of medical biological preparations. It is developed a virus-safe method of obtaining of a heterological antirabic immunoglobulin from a high-avid heterological antirabic Serum using a capril-alcohol method with peptic treatment which allows obtaining a high cleaning heterological antirabic immunoglobulin for intravenous and intramuscular administration. Thus as initial raw materials use a high-avid antirabic Serum obtained from immunization of animals-producers by the cleared virus of furiousness, grown up on culture of cells Vero or PSH. The content of γ- globulene fraction in a preparation makes not less than 90%, molecular weight distribution: units no more than 1.0%, diabodies and monomers less than 10.0%, F(ab)₂ - fragments not less than 84.0%, Fab - and Fc - fragments less than 5.0%, low anticomplementary activity, specific activity not less than 150 ME/ml. An osmolarity of a ready preparation is within 250-350 mosm/kg.

EFFECT: obtaining of high cleaning heterological antirabic immunoglobulin, stable at storage and transportation.

10 cl, 3 ex, 3 tbl

Description

The invention relates to biotechnology, namely to the production of medical biological preparations, and can be used to obtain highly purified heterologous anti-rabies immunoglobulin for intramuscular and intravenous administration.

Rabies is still a significant medical and economic problem. This is due to the fact that there is a constant threat of human infection with rabies virus due to the existence of natural foci of the disease and the spread of this infection among wild and domestic animals [1, 2, 3]. According to the WHO, in the world on average 500,000 cases of active-passive treatment and 35,000-50000 deaths from rabies are recorded every year on average [4, 5, 6], with 90% of deaths occurring in developing countries [7].

The clinical picture of rabies is characterized by severe damage to the nervous system and, without specific treatment, leads to 100% death. In this disease, it is effective to use an anti-rabies vaccine for the prevention and for the treatment of anti-rabies serum or anti-rabies immunoglobulin of hetero- or homologous origin. The WHO rabies committee recommended the use of antibodies in all cases of hydrophobic infection [8].

The use of heterologous rabies serum for the prevention of post-exposure to rabies can lead to complications. Its administration is often accompanied by allergic reactions in the form of serum sickness and is associated with a risk of anaphylaxis.

Human rabies immunoglobulin (ChAIG) is well tolerated and has rare cases of allergic reactions, but it is not available to most consumers. The high cost and low production volume associated with the difficulties of immunizing volunteers limits the demand for Chaig drugs in low-income developing countries. In addition, the method of obtaining a homologous anti-rabies immunoglobulin cannot completely exclude the risk of adverse reactions and transmission to the recipient of various pathogens of infectious diseases, therefore this drug cannot be considered absolutely safe [9].

Abroad, the question of obtaining monoclonal antibodies to rabies virus is under study, the use of which should help to reduce the development of post-exposure anaphylactic shock. The disadvantages of the new generation of drugs are: lack of practical basis for clinical use; narrow specificity of action; the possibility of mutation of the antigenic composition of rabies virus; the need to purify monoclonal antibodies to an extremely high level, complex control methods, which leads to the high cost of the drug.

More cost-effective (approximately five times than ChAIG) is the prevention and treatment of rabies with purified equine rabies immunoglobulin (LAIG). The latest generation of purified LAIG is much safer than horse rabies serum, it can be considered an acceptable and safe alternative to chaig. True, over the past twenty years, all world producers (Behring, Sclavo, Bema) have refused to produce LAIG because of the pressure exerted on them by animal rights defenders [10]. In developing countries where rabies is particularly prevalent, national pharmaceutical companies are involved in LAIG production. Their preparations are poorly purified from ballast proteins and have a high reactogenicity [11]. Low quality and low production volume do not satisfy national needs and do not solve the problem of LAIG deficit.

M.A. Selimov et al. Proposed receiving LAIG by immunization of production horses with a fixed rabies virus grown on a primary cell culture of Syrian hamster kidneys (PSC) or newborn guinea pigs [12].

The known method does not provide for the purification of antigen from bovine serum and cellular detritus. Ballast proteins and other antigenic substances that have a source of origin of the nutrient medium on which the virus grew, being often complete antigens, cause the formation of corresponding non-specific antibodies specific for the antigen during immunization, which enhances the reactogenicity of seropreparations and their sensitizing properties [13]. In addition, in this method for producing rabies immunoglobulin, a method for isolating the γ-globulin fraction is not disclosed.

There is a method of producing rabies serum by immunization of sheep with a homologous brain suspension infected with rabies virus [14].

The disadvantage of this method is the use to obtain anti-rabies serum of an untreated 5% brain suspension, the use of which as a substrate for reproduction of the virus leads to the formation and accumulation in the serum of producers of cytotoxic anti-brain antibodies (cytotoxins), which are one of the causes of reactogenicity of seropreparations and cause adverse reactions when administered to the person [15]. In addition, there is a likelihood of infection of animal producers by prions, which may be contained in the crude antigen.

Known methods of purification and concentration of antitoxic sera Diaferm-3 and Prosdis-2 [16]. Along with the advantages, these methods have a number of significant drawbacks, one of which is low electrophoretic purity, low yield of specific activity, and the possibility of pyrogenization of the drug at the dialysis stage.

A known method of fractionation of proteins of rabies serum according to the variant of the Tomsk NIIVS, developed by N.B.Plakhova, V.G. Mehanikova and A.I. Deeva (1960), in which the gamma-globulin fraction is isolated by alcohol deposition in the cold at pH 6, 4 and dilution of globulins in a triple volume of saline [16].

The disadvantages of this method are the complexity and multi-stage procedure for the isolation of proteins, the narrowness of the intervals of physicochemical constants at which fractionation is performed, the relatively high loss of proteins during production, the significant consumption of alcohol, the need for the entire process at low temperatures, which requires a large amount of refrigeration equipment. In addition, in heterologous immunoglobulins obtained by the method of alcohol fractionation, the sensitizing properties are most pronounced [17].

A known method of producing an immunoglobulin preparation for the prevention and treatment of infectious diseases of humans and animals from blood serum of cattle, including coagulation and removal of ballast proteins and lipids by caprylic acid [18].

The disadvantage of this method is that in the process of obtaining an immunoglobulin preparation does not occur concentration of the titer of specific antibodies. The antibody titer in the final preparation remains at the same level as in the original serum. This method allows to obtain oral immunoglobulin preparations for the prevention and treatment of intestinal infectious diseases of humans and animals, as well as drugs for the treatment and prevention of infectious diseases of cattle for intramuscular and intravenous administration. Known drug is not intended for intramuscular and intravenous administration to humans.

In the Russian Federation and the CIS countries, two LAIG preparations for intramuscular injection of the company “Biolek” (Ukraine) and RosNIPCHI “Microbe” (Russia), obtained by one technology, including the isolation of the γ-globulin fraction by the rivanol-alcohol method, and having the same quality indicators [ 19, 20].

The prototype of the present invention was selected LAIG drug company "Biolek" (Ukraine), which has long been known in the Russian market.

Obtaining a well-known drug rivanol-alcohol fractionation method involves the use of rivanol, which refers to harmful substances and belongs to hazard class III [21]. The maximum permissible concentration of rivanol in the air of the working area is 2 mg / m ³ . Currently, rivanol is almost discontinued in Russia and in almost all countries of the world. The high cost of foreign rivanol makes it inaccessible for use in large-scale production of heterologous anti-rabies immunoglobulin. Currently, rivanol is not used to produce homologous immunoglobulin. In addition, the complexity of the process of fractionation with rivanol, the need for multiple dilutions of plasma to avoid protein denaturation complicate the process of obtaining γ-globulin [22].

The electrophoretic homogeneity incorporated into the Biolek LAIG FSF provides for a low content of γ-globulins - not less than 80% and high α- and β-globulins - not more than 20%, which have high sensitizing activity and can lead to adverse reactions . According to B.L. Cherkassky (1985), the LAIG firm Biolek company causes adverse allergic reactions in 20–40% of vaccinees [23].

In the FSP, the known preparation does not contain molecular parameters. According to the requirements of the European Pharmacopoeia on animal-derived immune sera for human use [24], the control of molecular parameters associated with anaphylactic and anticomplementary activity (AKA) is mandatory in heterologous serum preparations [17]. Also, the European Pharmacopoeia does not correspond to the concentration of protein in the drug, which should not exceed 10%.

The isoelectroelectric point of immunoglobulin lies between pH 5.8–7.3; therefore, liquid immunoglobulin preparations with a pH of 7.0 are unstable and tend to spontaneously increase AKA during storage.

All seropreparations should correspond to the osmolarity of the isotonic solution and be as close as possible to 300 mOsm / kg. The osmolarity of the anti-rabies immunoglobulin from Biolek and RosNIPCHI Mikrob is at the normal range of 600 mOsm / kg. In addition, they should be monitored for the content of all substances added during the preparation process. The glycocol stabilizer used in the known preparation is not determined in the finished preparation. In addition, the drug is not monitored for the presence of antibodies to the substrate on which the rabies virus is grown.

Known methods for producing heterologous anti-rabies immunoglobulin do not allow to obtain a drug with a high degree of purification, suitable for intravenous administration. The quality of a heterologous anti-rabies immunoglobulin depends not only on the fractionation method, but also on the quality of the antigen used to immunize animal producers, i.e. from the substrate on which the rabies virus is obtained, and the degree of purification of the virus from ballast proteins. Thus, the problem of obtaining highly purified heterologous anti-rabies immunoglobulin has not yet been resolved.

The problem to which the invention is directed, is to obtain highly purified heterologous anti-rabies immunoglobulin for intravenous and intramuscular administration.

The technical result of the proposed method is to develop an industrial method for producing a virus-safe heterologous anti-rabies immunoglobulin from the highly specific heterologous anti-rabies serum using the caprilate-alcohol method with pepsin processing and obtaining highly purified heterologous anti-rabies immunoglobulin for intravenous and intramuscular administration, stable during storage and transportation.

The essence of the invention is as follows: as a raw material for producing an anti-rabies immunoglobulin, highly specific heterologous anti-rabies serum obtained from immunization of animal producers (horses, small cattle, pigs) with purified rabies virus grown on a Vero cell culture or PLC in which antibodies Vero cells or PLC less than 0.5 μg / ml [25], which is treated with caprylic acid, pepsin and ethanol. Pepsin treatment is carried out in the presence of maltose 0.5-3.0% and with a chloride content of 0.2-3.0%. Excess sodium chloride and residual caprylic acid, pepsin and ethanol are removed by dialysis and / or ultrafiltration, or ion exchange chromatography, or activated carbon. Glycine is introduced into the finished preparation at a concentration of 0.7-1.5%, the sodium chloride content is adjusted to 0.1-0.45%, maltose to 0.5-3.0%. The combination of glycine, sodium chloride and maltose is selected in such concentrations as to ensure the osmolarity of the drug in the range of 250-350 mOsm / kg. Set the pH to 5.0-7.5, the protein content of 4.5-10.0%. The final preparation of choice is subsequently prepared in liquid or dry form.

The claimed method allows to obtain highly purified heterologous anti-rabies immunoglobulin for intravenous and intramuscular administration, stable during storage and transportation, which is an F (ab) ₂ preparation with a specific activity of at least 150 IU / ml, with a protein concentration in solution of 4.5-10 , 0%, in which the content of the γ-globulin fraction is at least 90.0%, α- and β-globulin is less than 9.0%, albumin is less than 1.0%, having a molecular weight distribution: aggregates of less than 1, 0%, dimers and monomers of less than 10,0%, F (ab) ₂ fragments n less than 84,0%, Fab- and Fc-fragments less than 5.0%, less than 1.0 ACA 50 CH / mg protein, antibodies to Vero cells or PAF - less than 0.5 g / ml.

In contrast to the prototype, in the claimed method, in addition to horses, livestock and pigs are used as animal producers. Highly specific serum obtained from the immunization of producers with purified rabies virus grown on a Vero cell culture or on a primary PLC cell culture in which antibodies to the substrate cells is less than 0.5 μg / ml is used as a feedstock. Fractionation is carried out by the caprilate-alcohol method with pepsin treatment, which is carried out in the presence of maltose at a concentration of 0.5-3.0% and with a chloride content of 0.2-3.0%. Excess sodium chloride and the residual content of caprylic acid, pepsin and ethanol are removed by dialysis or ultrafiltration, or ion exchange chromatography, or activated carbon. The final immunoglobulin preparation contains maltose in a concentration of 0.5-3.0%, glycine 0.7-1.5%, sodium chloride 0.1-0.45%, protein 4.5-10.0%, has a pH value 5.0-7.5 and can be obtained in dry form. The combination of excipients is selected in concentrations that ensure the osmolarity of the drug in the range of 250-350 mOsm / kg.

In the claimed preparation of a heterologous anti-rabies immunoglobulin, the normative limit of electrophoretic homogeneity is increased. The specific activity of the drug is increased. In the finished product, the content of molecular parameters, antibodies to the substrate on which the rabies virus, AKA, and the content of all substances added and remaining in it after technological processing are controlled. The residual ethanol content was reduced to 0.1%. The claimed drug is intended for intravenous administration.

The set of distinctive features of the claimed method allows to obtain the following advantages.

Using raw materials of highly specific heterologous rabies serum obtained from immunization of animal producers with purified rabies virus grown on a Vero cell culture or PLC, as well as controlling the initial serum and final preparation for the content of antibodies to the substrate on which the virus was obtained, can significantly reduce reactogenicity of heterologous anti-rabies immunoglobulin. The need for introducing this control is due to the possibility of an anaphylactic reaction in chinchilla rabbits after intravenous administration of a heterologous anti-rabies immunoglobulin containing an increased level of antibody to the substrate [26].

The use of caprylic acid for fractionation involves a more gentle and higher purification from ballast proteins and lipids, inactivation of enveloped viruses into 4-5 logarithms [27], and the possibility of fractionation at room temperature and in non-sterile conditions.

Pepsin treatment in the presence of maltose 0.5-3.0% and with a chloride content of 0.2-3.0% allows one to practically obtain only the F (ab) ₂ preparation, to prevent the formation of aggregates and a decrease in antibody titer due to uncontrolled proteolysis. Hydrolysis of ballast proteins can increase electrophoretic uniformity, reduce the number of aggregates and AKA. Thus, it is possible to significantly reduce the anaphylactic activity of an immunoglobulin preparation due to the maximum cleavage of the most reactogenic part of antibodies, the Fc fragment [28].

Isolation of the γ-globulin fraction by ethanol contributes to a significant concentration of protective anti-rabies (antiviral) antibodies and an increase in the specific activity of the drug.

Treatment of serum with caprylic acid, pepsin and ethanol significantly increases the electrophoretic uniformity and specific activity of the drug, ensures the inactivation and removal of viruses that are possibly present in the plasma that are pathogenic to humans, and thereby guarantee the safety of the claimed drug. The fractionation process is economical; fractionation agents are available.

The use of dialysis, ultrafiltration, ion exchange chromatography, activated carbon allows you to effectively clean the drug from caprylic acid, pepsin, ethanol and excess sodium chloride. In addition, these methods are effective in removing pyrogen.

The combination of stabilizers used ensures the osmolarity of the drug, which meets the standards of the European Pharmacopoeia, at least 240 mOsm / kg (29) and the stability of IVIG drug quality indicators (molecular parameters, AKA, specific activity) during storage and transportation (Tables 2, 3). In addition, with freeze-drying, the use of maltose in a concentration of 0.5-3.0% prevents the formation of aggregated forms of immunoglobulin and contributes to the stability of AKA. Maltose and glycine used as a stabilizer are well tolerated by the intravenous route of administration due to the ability of kidney cells to metabolize these substances.

In the final preparation, the sodium chloride content is reduced to a concentration of 0.1-0.45%, a high content of which causes temperature instability, aggregation, turbidity, lower antibody titers in immunoglobulin preparations [30] and can lead to the development of acidosis or cell dehydration [31] ].

The acidic pH value of 5.0-6.0 increases the stability of the quality characteristics of IVIG, is better tolerated by intravenous administration and allows the preparation to be prepared in liquid form with stable parameters [24]. The liquid form significantly reduces the cost of the drug and is more convenient for the consumer.

The lyophilized form of the drug provides greater stability during storage and transportation, and also increases the shelf life of the target product.

According to the requirements of the European Pharmacopoeia on IVIG [29], the drug is monitored for the content of all substances added to it and remaining in it after technological processing (Table 1).

The control of the content of molecular parameters and AKA, which are indicators of the safety of intravenous immunoglobulins (IVIG), has been introduced. Molecular parameters have a molecular weight distribution: aggregates less than 1.0%, dimers and monomers less than 10.0%, F (ab) ₂ fragments no less than 84.0%, Fab and Fc fragments less than 5.0% AKA less than 1.0 CH50 / mg protein.

The compliance of the drug with the requirements for IVIG and the stability of these indicators during storage and transportation (Tables 1, 2, 3) makes it suitable for intravenous administration. The intravenous route of administration has an advantage in time of action and allows you to get the maximum effect in a short period of time, which is especially important in severe cases of rabies infection, when the therapy is given out of time. The effect of action will occur within 20 minutes after intravenous administration, and not on the second or third day, when the drug is administered intramuscularly.

The specific activity of the claimed drug is 1.5 times higher than the prototype, therefore, the load of a foreign protein and, accordingly, the reactogenicity of the drug are reduced.

Purified heterologous anti-rabies immunoglobulin obtained from the blood of pigs and small cattle may be less reactogenic due to the fact that the human body is not sensitized to their protein and due to the specific features of their serum. So V.V. Pushkarev (1970) showed that the most pronounced anaphylactogenic properties of native sera were found in horse sera, then in a decreasing sequence - in pigs, rams, bulls, goat serums were the least anaphylactogenic.

The preparation of heterologous rabies immunoglobulin was controlled by the following methods.

Electrophoretic homogeneity by electrophoresis on films of cellulose acetate (FS 42-3874-99, p.20). Molecular parameters by gel filtration (FS 42-3874-99, p. 28). Anti-complementary activity - 1 mg of immunoglobulin protein should not consume more than 1 unit. (SN50) complement (MUK 3.3.2.1063-010). Specific activity in the in vivo protective protection test on white outbred mice weighing (10 ± 1) g with fixed rabies virus strain CVS versus 100-1000 LD ₅₀ relative to the industry standard specimen of specific activity of anti-rabies gammaglobulin (OSO 42-28-200-92). The content of antibodies to Vero cells and PLC by the method of missile immunoelectrophoresis, the sensitivity of the method is 0.5 μg / ml. Sodium chloride according to FS 42-3874-99, p. 59. Determination of glycine according to MUK 4.1 / 4.2.588-96, p.114. The maltose content was determined colorimetrically (with Feling's reagent) and qualitatively (by chromatography on paper). Determination of caprylic acid by ion-exclusion chromatography [32]. Residual ethanol according to MUK 4.1 / 4.2.588-96, p.107. Proteolytic activity was determined in terms of 1 μg tyrosine [33]. The osmolarity was viewed on an MT-4 milliosmometer. A study of the stability of the drug was carried out in tests for mixing and accelerated aging. The mixing test, which is an analogue of the conditions of drug transportation, was carried out by stirring for 2 hours at a frequency of 80 horizontal oscillations per minute at a temperature of 20 ° C. The accelerated aging test was carried out by keeping the drug in a thermostat at a temperature of 37 ° C for 4 weeks and is an analog of storage conditions for 2 years [29].

Case Studies

Example 1. As a feedstock for fractionation, 100 l of rabies serum obtained from immunization of horses with purified rabies virus Vnukovo-32 strain grown on a Vero cell culture in which the specific activity is 75 IU / ml, antibodies to Vero cells are less than 0, are used. 5 kg / ml. The serum is diluted to a protein content of 2%, a pH of 4.2 is established, caprylic acid is added at a rate of 20 g / l at a temperature of (22 ± 2) ° C and constant stirring for 1 h. After which the precipitate is removed and maltose is introduced into the centrifugate at a concentration of 3.0% and sodium chloride 0.2%, establish a pH of 3.2-3.3 and add pepsin at a concentration of 1-3 g / l. Hydrolysis is carried out for 1 h at a temperature of (22 ± 2) ° C. The enzyme is removed with activated carbon 5-10 g / l at a pH of 6.0-6.1. Then, at a pH of 7.0 and a temperature of minus 5 ° C, ethanol is added to a concentration of 25% in solution. The resulting pellet was dialyzed against 0.45% sodium chloride. The residual content of caprylic acid, pepsin and ethanol is removed by repeated diafiltration in an ultrafiltration unit using distilled water. After that, the maltose content is adjusted to a concentration of 2.0%, chlorides to 0.1%, glycine is introduced at a concentration of 1.5%, the protein content in the solution is adjusted to 4.5-5.0%, the pH value is 5.0-5, 5 and subjected to sterilizing filtration. Obtain liquid heterologous anti-rabies immunoglobulin for intravenous administration with electrophoretic uniformity: γ-globulins 96.6%, α- and β-globulins 3.2%, albumin 0.2%; having a molecular weight distribution: aggregates of 0.0%, dimers and monomers - 5.2%, F (ab) ₂ fragments 92.5%, Fab and Fc fragments - 2.3%, in which AKA 0, 1 CH50 / mg, specific activity 440 IU / ml, no antibodies to Vero cells, osmolarity of the finished preparation 350 mOsm / kg.

Example 2. For fractionation, 100 l of rabies serum obtained from immunization of pigs with purified rabies virus Vnukovo-32 strain grown on a PLC cell culture in which the specific activity is 80 IU / ml, antibodies to PX cells less than 0.5 kg / ml are used. The serum is treated with caprylic acid as described in Example 1. Then hydrolysis is carried out in the presence of maltose 0.5% and chloride content 3.0%. Residual pepsin, cleavage products and lipoproteins are removed using a DEAE-Sepharose anion exchanger, which is added 10 g / L. Treatment with ethanol is carried out as described in example 1. The resulting precipitate is dialyzed and ultrafiltered. As a stabilizer, glycine is introduced at a concentration of 0.7%, the content of maltose is adjusted to 0.5% and chlorides to 0.45%. Set the protein content of 5.0-5.5%, the pH value of 6.5-7.0. The drug is subjected to sterilizing filtration and freeze-drying. Get dry heterologous anti-rabies immunoglobulin for intravenous administration with electrophoretic homogeneity: γ-globulins 95.8%, α- and β-globulins 4.0%, albumin 0.2%; AKA 0.1 CH50 / mg and the following molecular parameters: aggregates 0.0%, dimers and monomers 6.0%, F (ab) ₂ fragments no less than 90.8%, Fab and Fc fragments 3.2 %, in which the specific activity is 520 IU / ml, antibodies to Vero cells are absent, the osmolarity of the drug is 295 mOsm / kg.

Example 3. The raw material is the serum obtained from immunization of a sheep with purified rabies virus Vnukovo-32 strain grown on a Vero cell culture in which the specific activity of 78 IU / ml, antibodies to Vero cells are absent. The serum is treated with caprylic acid as described in Example 1. Pepsin treatment is carried out with a content of: maltose 1.5% and chlorides 1.0%. Removal of pepsin and low molecular weight cleavage products from the immunoglobulin solution is carried out by dialysis, for which the solution is poured into a dialysis tube and dialyzed against water at pH 4.0. Then it is treated with ethanol as described in Example 1. The resulting γ-globulin precipitate is dialyzed against 0.45% sodium chloride, then concentrated by ultrafiltration. As a stabilizer, glycine is used in a concentration of 1.0%, the maltose content is adjusted to 3.0%, sodium chloride to 0.3%, the protein content is 9.5-10.0%, and the pH value is 6.5-7.0. The final preparation is subjected to sterilizing filtration. Get liquid heterologous anti-rabies immunoglobulin with electrophoretic uniformity: γ-globulins 97.3%, α- and β-globulins 2.4%, albumin 0.3%, AKA 0.2 CH50 / mg and molecular parameters: aggregates 0.0% , dimers and monomers 8.1%, F (ab) ₂ fragments not less than 88.1%, Fab and Fc fragments 3.8%, in which specific activity is 505 IU / ml, antibodies to Vero cells are absent , osmolarity of the finished product 350 mOsm / kg

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Table 1 Characterization of a heterologous rabies immunoglobulin obtained by the claimed method and prototype at the time of receipt No. p / p Indicators The inventive method M ± SD (n = 8) Prototype M ± SD (n = 8) R one Electr. homogeneous.,% gamma globulin 96.51 ± 1.89 87.23 ± 1.59 P <0.05 α-, β-globulin 3.26 ± 0.68 12.63 ± 1.65 P <0.05 Albumen 0.23 ± 0.16 0.14 ± 0.13 P> 0.05 2 Molecular options, % Aggregates 0.04 ± 0.11 0.04 ± 0.05 P> 0.05 Dimers and monomers 5.21 ± 0.85 11.99 ± 1.16 P <0.05 (Fab) ₂ fragments 92.47 ± 1.72 70.86 ± 2.34 P <0.05 Fragments (Fab + Fc) 2.28 ± 0.87 17.11 ± 0.73 P <0.05 3 AKA, CH50 / mg protein 0.11 ± 0.06 0.16 ± 0.17 P> 0.05 four Specific Activity, IU / ml 439.38 ± 17.82 290.00 ± 24.93 P <0.05 5 The specific activity, IU / mg 11.80 ± 0.94 7.6 ± 0.97 P <0.05 6 Caprylic acid,% 0.03 ± 0.01 - - 7 Ethanol,% 0.01 ± 0.04 0.10 ± 0.13 P <0.05 8 Sodium chloride, % 0.30 ± 0.11 0.90 ± 0.30 P <0.05 9 Glycine% 1.00 ± 0.27 2.13 ± 0.10 P <0.05 10 Maltose,% 1.75 ± 0.79 - - eleven Osmolarity, mOsm / kg 300 ± 30.24 525 ± 75.59 P <0.05 12 Proteolytic activity, μg tyrosine / ml (mm) 0.13 ± 0.07 5.80 ± 0.50 P <0.05 Note: A - aggregates, D - dimers, M - monomers.
AKA is an anti-complementary activity of immunoglobulin.

table 2 Characterization of liquid forms of heterologous anti-rabies immunoglobulin preparations obtained by the claimed method and prototype in an accelerated aging test (heating at 37 ° C for 28 days) No. p / p Indicators The inventive method, M ± SD (n = 8) Prototype, M ± SD (n = 8) P _1,3 R _2,4 before processing (1) after processing (2) P _1,2 before processing (3) after processing (4) P _3.4 one Molecular Parameters,% BUT 0.04 ± 0.11 0.26 ± 0.18 P> 0.05 0.04 ± 0.05 1.94 ± 0.38 P <0.05 P> 0.05 P <0.05 D + M 5.21 ± 0.85 5.80 ± 1.29 P> 0.05 11.99 ± 1.16 11.11 ± 0.81 P> 0.05 P <0.05 P <0.05 (Fab) ₂ 92.47 ± 1.72 89.93 ± 1.25 P> 0.05 70.86 ± 2.34 66.65 ± 1.78 P <0.05 P <0.05 P <0.05 Fab + fc 2.28 ± 0.87 4.01 ± 0.56 P> 0.05 17.11 ± 0.73 20.30 ± 0.78 P <0.05 P <0.05 P <0.05 2 AKA, CH50 / mg protein 0.11 ± 0.06 0.19 ± 0.09 P> 0.05 0.16 ± 0.17 1.00 ± 0.18 P <0.05 P> 0.05 P <0.05 3 Specific Activity, IU / ml 439.38 ± 17.82 405.63 ± 23.06 P> 0.05 290.00 ± 24.93 248.75 ± 21.67 P <0.05 P <0.05 P <0.05 Note: A - aggregates, D - dimers, M - monomers, (Fab) ₂ - (Fab) ₂ fragments, Fab + Fc fragments,
AKA is an anti-complementary activity of immunoglobulin.

Table 3 Characterization of liquid forms of heterologous anti-rabies immunoglobulin preparations obtained by the claimed method and prototype in a mixing test (80 horizontal oscillations per minute for 2 hours) No. p / p Indicators The inventive method, M ± SD (n = 8) Prototype, M ± SD (n = 8) R _1.3 R _2,4 before processing (1) after processing (2) P _1,2 before processing (3) after processing (4) P _3.4 one Molecular Parameters,% BUT 0.04 ± 0.11 0.23 ± 0.19 P> 0.05 0.04 ± 0.05 1.75 ± 0.25 P <0.05 P> 0.05 P <0.05 D + M 5.21 ± 0.85 5.61 ± 0.74 P> 0.05 11.99 ± 1.16 11.41 ± 0.71 P> 0.05 P <0.05 P <0.05 (Fab) ₂ 92.47 ± 1.72 90.35 ± 1.02 P> 0.05 70.86 ± 2.34 68.01 ± 1.44 P> 0.05 P> 0.05 P <0.05 Fab + fc 2.28 ± 0.87 3.81 ± 0.47 P> 0.05 17.11 ± 0.73 18.83 ± 0.80 P <0.05 P <0.05 P <0.05 2 AKA, CH50 / mg protein 0.11 ± 0.06 0.20 ± 0.15 P> 0.05 0.16 ± 0.17 0.60 ± 0.25 P <0.05 P> 0.05 P <0.05 3 Specific Activity, IU / ml 439.38 ± 17.82 426.50 ± 21.09 P> 0.05 290.00 ± 24.93 276.88 ± 16.01 P <0.05 P <0.05 P <0.05 Note: A - aggregates, D - dimers, M - monomers, (Fab) ₂ - (Fab) ₂ fragments, Fab + Fc fragments.
AKA is an anti-complementary activity of immunoglobulin.

Claims (10)

1. A method of obtaining a highly purified heterologous anti-rabies immunoglobulin, comprising isolating the γ-globulin fraction with ethanol and introducing glycine and sodium chloride into the finished preparation, characterized in that highly specific anti-rabies serum obtained from immunizing animal producers with purified rabies virus grown on Vero cell culture or PLC, fractionation is carried out with caprylic acid with pepsin treatment, which is carried out in the presence of maltose 0.5-3.0% and soda Rye Chlorides 0.2-3.0%, purified by primestnyh substances and auxiliary substances are administered in combination, providing the finished product osmolality within 250-350 mOsm / kg. 2. The method according to claim 1, characterized in that the highly specific anti-rabies serum obtained from immunization of horses, small cattle, and pigs is used for fractionation. 3. The method according to claim 1, characterized in that a highly specific heterologous anti-rabies serum is used for fractionation, in which the antibodies to Vero cells or PLC are less than 0.5 μg / ml. 4. The method according to claim 1, characterized in that the excess content of sodium chloride and the residual content of caprylic acid, pepsin and ethanol are removed by dialysis, and / or ultrafiltration, or ion exchange chromatography, or activated carbon. 5. The method according to claim 1, characterized in that to stabilize the drug use maltose in a concentration of 0.5-3.0%, glycine 0.7-1.5%, sodium chloride 0.1-0.45%. 6. The method according to claim 1, characterized in that the finished product is obtained in liquid or dry form. 7. A heterologous anti-rabies immunoglobulin preparation for intravenous and intramuscular administration, obtained by the method according to claim 1, with a specific activity of at least 150 IU / ml, with a protein concentration in the solution of 4.5-10.0%, in which the content of the γ-globulin fraction is not less than 90.0%, α- and β-globulins less than 9.0%, albumin less than 1.0%, anti-complementary activity less than 1.0 CH50 / mg protein, having a molecular weight distribution: aggregates less than 1.0% dimers and monomers of less than 10,0%, F (ab) ₂ fragments of at least 84,0%, Fab- and Fc-fragments less than 5.0%, antibodies to taphole Vero or PAF - less than 0.5 g / ml. 8. The drug according to claim 7, characterized in that the osmolarity of the drug is in the range of 250-350 mOsm / kg 9. The drug according to claim 7, characterized in that the drug has a pH value of 5.0-7.5. 10. The drug according to claim 7, characterized in that the drug can be in liquid or dry form.