RU2696480C1 - Method of producing a gamma-globulin semi-product with high content of antiviral antibodies from slaughter blood of vaccinated cows - Google Patents

Abstract

FIELD: biology.

SUBSTANCE: invention relates to biological industry, in particular to a method of producing a gamma-globulin semi-product from slaughter blood of cattle. Method of producing a gamma-globulin semi-product from slaughter blood of cattle by chemical reagents, wherein the raw material used is slaughter blood in the place of its preparation and separation in the meat-packing plant conditions, and plasma samples as they are manufactured are placed in saturated 100 % of saturation or 4.05 M solution of ammonium sulphate with 0.75 % didecyldimethylammonium chloride, in ratio solution: plasma 1:1.5 by volume, to concentration of 40 % of saturation of ammonium sulphate and 0.3 % of didecyldimethylammonium chloride, formed suspension is centrifuged so that a gamma-globulin fraction precipitate is obtained, dry ammonium sulphate powder is added to the formed supernatant while stirring to obtain a final concentration of ammonium sulphate of 75 % of saturation, and pH is adjusted to 4.6, suspension is mixed and centrifuged, precipitating the albumin fraction, remaining after sedimentation of albumin fraction supernatant is used for resuspension of gamma-globulin residue, final concentration of ammonium sulphate in the gamma-globulin suspension is not less than 60 % of saturation and not less than 0.27 % of didecyldimethylammonium chloride.

EFFECT: said method enables to obtain a gamma-globulin semi-product which is stored for 180 days at room temperature while preserving its properties.

3 cl, 1 dwg, 7 tbl, 1 ex

Description

The invention relates to the biological industry, to the procurement and processing of blood for the production of semi-finished products in non-standard conditions without observing the sterility of the instruments and the proper cleanliness of the production facilities. The prepared semi-finished products do not contain contaminating microflora and active forms of natural proteases. For short-term storage of semi-finished products and their transportation, refrigeration equipment, including the "cold chain", is not used. At the same time, the activity of antibodies from the IgG class is preserved as part of the gamma-globulin semi-finished product at all observation periods for 180 days.

The safety of human blood plasma preparations obeys the well-known algorithm for forming a reactor bookmark. First, the reactor bed is formed from sterile blood donations of the donor contingent, which under sterile conditions are subjected to centrifugation in order to obtain sterile plasma samples intended for fractionation. Viral safety is monitored additionally and in advance using enzyme-linked immunosorbent assays and based on PCR diagnostics. The lack of reactogenicity of the final forms of biological products is achieved due to standard production conditions based on the implementation of all stages of the technological process in rooms with an appropriate level of purity [1. WHO Recommendation for the production, control and regulation of human plasma for fractionation [Electronic resource]. Fifty-sixth Report. 2007. Available from: http://www.who.int/biologicals/expert_committee/Full%20Text%20TRS941.pdf; 2. Guidance on plasma-derived medicinal products. European Medicines Agency. EMA / CHMP / BWP / 706271/2010 .; 3. Directive 2002/98 / EC of the European Parliament and of the Council of 01/27/2003 setting standards of quality and safety for the collection, testing, processing, storage and distribution of human blood and blood components and amending. [Electronic resource]. Available from: https://ec.europa.eu/health//sites/health/files/files/eudralex/vol-1/dir_2002_98/dir_2002_98_en.pdf; 4. GOST P 53420-2009. Donor blood and its components. General requirements for quality assurance in the collection, processing, storage and use of donated blood and its components].

Based on the foregoing, standard schemes of technological processes for the production of medical plasma biological products exclude fractionation of potentially pyrogenic raw materials (blood plasma). In particular, applied research of an international scale is not focused on the development of methods, techniques, stages, designed to inactivate microflora in colloidal solutions, complex in composition of individually distinctive proteins. Known technological processes, in principle, are not able to initiate an improvement in plasma production during conveyor production of slaughter blood in non-standard (unacceptable) conditions without observing the sterility requirements of raw materials and the proper cleanliness of the respective premises. In a meat processing plant at the place of slaughter blood procurement, the plasma production process will always be technologically unacceptable, since it provides microbial contamination of the raw material procurement and the associated pyrogenicity of the finished plasma biological preparations.

The standard technological process for the production of medical plasma biological products includes stages [4. GOST R 53420-2009. Donor blood and its components. General requirements for quality assurance in the collection, processing, storage and use of donated blood and its components]:

- short-term storage of raw materials at low temperatures. It is known that the low-temperature regime of plasma storage provides bactericidal and bacteriostatic conditions and inhibition of natural proteases in frozen plasma samples;

- use of the “cold chain” to ensure the transportation of plasma in a frozen state from the place of its production to the place of its fractionation in order to ensure the industrial production of pharmaceutical plasma biological products.

The physical chemistry and biochemistry of protein, as well as the applied focus on the development of achievements in this field of knowledge, require strict observance of the low-temperature regime, which does not allow repeated freezing and thawing of samples intended for industrial fractionation and production of plasma biological products. [five. Chard T. "Radioimmunological methods." M .: Mir, 1981, p. 48]. In stationary conditions, compliance with this regimen is amenable to proper monitoring. In the context of transportation of raw materials, there are risks of a purely technical nature.

Another problem that is characteristic of the international bioindustry concerns the infectious safety of medicines, the production of which is based on the fractionation of natural biological resources. [6. Zubkova N.V. Biotechnological aspects of effective and safe processing of donor plasma: problems and prospects // Biological products. - 2014. - No. 1. - S. 4-10 .; 7. Burnouf T. Modern plasma fractionation. Transfus Med. Rev. 2007; 21 (2): 101-17.]. In accordance with the requirements of the GMP standard and its domestic counterpart [8. GOST R 52249-2009. The rules for the production and quality control of medicines] increasing the infectious safety of medical biological products is achieved at the stage of procurement of raw materials by improving the optimal combination of screening tests. Moreover, in the medical industry, the optimization of a combination of screening tests is carried out mainly due to the expansion of diagnostic procedures in relation to human viral infections. For example, individual donor blood donations are tested for the absence of HIV1, HIV2, hepatitis B and C. Foreign diagnostic tests additionally monitor the absence of T-cell human leukemia viruses, as well as herpes viruses and cytomegaloviruses. In particular, for these purposes, enzyme-linked immunosorbent kits for detecting antibodies and viral proteins in human serum are commercially available. A similar goal is achieved through the release of diagnostic kits designed on the basis of PCR. An important factor in ensuring the infectious safety of the reactor bookmark is also the principle of quarantine of human blood donor blood plasma, which provides a repeated diagnostic study of the same donor during its subsequent blood supply. [9. Order of the Ministry of Health of the Russian Federation of May 7, 2003, No. 193 “On the implementation of the method of quarantining freshly frozen plasma in the practice of the blood service in the Russian Federation”; 10. Guide to the preparation, use and quality assurance of blood components. Recommendation No.R (95) 15 / 9th edition. - Council of Europe. - 2003.].

It should be noted that the described system for the procurement of human donated blood, intended for the production of medical biological products, is not possible to reproduce in full for the production of biological products for veterinary use. First, in the biological industry, the optimal combination of screening tests is designed in its most general form. The infectious safety of individual blood donations of animal donors is determined independently by the manufacturing enterprise on the basis of regional and Russian anti-epizootic programs. Secondly, despite fundamental works in the field of viral infections of animals [11. Syurin V.N., Samuilenko A.Ya., Soloviev B.V., Fomina N.V. "Viral diseases of animals." - Moscow, VNITIBP, 2001.

928 pp., Ill.] There are no predictive and planned studies of a scientific and practical nature, on the basis of which it would be possible to create diagnostic kits for the primary viral threats inherent, for example, in dairy and beef cattle breeding. Thirdly, the molecular biology of viruses that cause human infectious diseases has been the most studied. It is the molecular biological principle of viral particles that allows you to create technologies for the industrial production of components in order to create the necessary diagnostic kits. With regard to viral agents that infect domestic animals or wild animals, this kind of technology is extremely underdeveloped, primarily due to the lack of fundamental knowledge in the field of molecular virology.

From an engineering and technical point of view, designing a site for the procurement of raw materials in accordance with the principal technologies in force in the medical industry also causes certain problems in the design of similar production facilities intended for the use of slaughter animal blood.

Firstly, it is necessary to organize the production of:

- systems and specials. bags with a volume of more than 10 liters (such as a GEMASIN container (Kurgansyntez OJSC) for intravital and sterile bleeding of animals received at a meat processing plant;

- special centrifuges with cooling, equipped with a rotor with a spider, to ensure sterility centrifugation operations special. packages with individual volumes of blood supply of about 10 liters each;

- special laminars for the design of the workplace to enable the procurement of the obtained sterile plasma in packaged form;

- cooling units for freezing about 500 l of plasma produced during a shift of an average meat processing plant;

- special automobiles within the framework of the requirements of the “cold chain” for the delivery of raw materials from the place of blood collection and plasma production to the manufacturer of veterinary plasma biological products.

Secondly, to concentrate efforts in the field of fundamentally oriented and applied research for:

- introducing certainties into the infectious (viral) safety of animal husbandry as part of the requirements for optimizing screening tests for laboratory diagnostic control;

- deciphering the molecular biological structure of viral agents, potentially capable of inducing viral infections, with different levels of coverage of livestock farms;

- introducing specifics into the mechanism of the formation of viral reassortants, including details of the relationship of the vaccine immunogen with the transformation of viral particles represented by field and vaccine strains.

Let's note the main thing. It is rather difficult to reproduce the engineering design of plasma production at the place of slaughter blood production in a meat processing plant on the basis of well-known technological standards. However, the creation of appropriate conditions in accordance with the requirements of well-known technological standards is also a difficult task for pharmaceutical bio-industry enterprises using the immune donor plasma of animals for the production of biological products for veterinary or medical purposes. In addition, excessive factology has been published, according to which the most modern system for the formation of an infectious-safe raw material blank does not guarantee the viral safety of plasma biological preparations made from a virus-safe reactor bookmark. [12. Garmaeva T.Ts., Kulikov S.M., Mikhailova E.A., Gemdzhyan E.G., Gaponova T.V., Grumbkova L.O. and others. Long-term results of infection with hepatitis B and C viruses in patients with diseases of the blood system. Therapeutic Archive. 2011; 7: 17-26 .; 13. Kulikov S.M., Garmaeva T.Ts., Zingerman B.V., Filatov F.P., Sudarikov A.B., Mikhailova E.A. et al. Viral safety of blood transfusions and methods for its assessment. Hematology and transfusiology. 2008; 4: 3-5 .; 14. Farshid M. Viral safety of plasma-derived products. PDA J. Pharm. Sci. Technol. 2011; 65 (6): 737-53.] In realities and in fact in the organizational and methodological field of activity, it is possible to achieve the same level of danger = safety if the meat processing plant is the starting point for the production of pharmaceutical plasma biological preparations, which allows for the production of plasma at the place of collection of slaughter blood. Note that only at a meat processing plant it is possible to exclude slaughter blood as a raw material supply according to the results of pathological examination. We emphasize once again that the safety of plasma raw materials is subsequently determined by the ability of biological products to transmit the infectious principle as part of “safe” drugs. [15. Panov V.P. Principles for ensuring the viral safety of blood products (review) // Chemical and Pharmaceutical Journal. - 2004 - T. 38. - No. 3. - S. 39-47]

An analysis of published materials and patent search results allows us to conclude that at the present stage of the development of science and technology, it is not possible to objectively guarantee the generalized safety of medicines. At the same time, the safety level of medical biological products is significantly higher compared to their analogues of veterinary use. The last conclusion is based on the standards of existing technologies and the associated requirements for the material and technical base for the production of medical plasma biological preparations.

At the same time, the Russian market of veterinary medicinal products is represented by a wide range of biological products - plasma derivatives of animal donors. The domestic bioindustry produces 20 types of treatment and prophylactic serums and 6 types of targeted immunoglobulin biological products against infectious diseases characteristic of large agricultural and small domestic animals. The listed blood biologics are made on the basis of donor blood donations of animal producers, which are kept in isolation under conditions that allow their health to be monitored through systematic clinical examinations and maintain proper zoohygienic requirements for the content of the donor contingent. Immunological research methods are used in this technology only as an approach to determine the level of antiviral antibodies induced by vaccination of animal producers.

Known requirements for the quality of therapeutic and prophylactic serums of directed action and their derivatives of gamma-globulin biologics are evaluated primarily by antibody titers to infectious agents (antigens) used to induce the immunization process in the body of animal donors. It is the titer of antibodies to specific infectious agents that is the basis for providing passive immunoprophylaxis or treatment and prophylactic measures. The stated technological approach ensures the removal of a potential contaminating microflora from the biological product. However, it is not possible to discuss the objective safety of this quality of medicines at the present stage of development of science and technology. First, immunoserum is a colloidal solution of many individually unique biopolymers, which contain biologically active proteins, for example, group substances of the blood, providing reactogenicity of the final product. Secondly, the conditions for the procurement of individual blood donations, the manufacture of whey from the blanks, its bottling and capping require a real embodiment of the GMP standard or its domestic counterpart [8. GOST R 52249-2009. Rules for the production and quality control of drugs], which ensures the pyrogen-free nature of the drug. However, the main problem boils down to the creation of an effective combination of screening tests, allowing not to use potentially hazardous raw materials for the production of targeted therapeutic and prophylactic serums.

In the practice of production of medical biological products, donated blood, immune donor plasma or serum are not classified as medicines, but are considered as a national resource intended for the production of pharmaceutical biological products. The stated limitation is due, first of all, to the impossibility of guaranteeing the infectious safety of native plasma through the use of the most modern clinical and medical history and laboratory diagnostic approaches.

Known methods of plasma disinfection based on physical methods of exposure. For example, ultraviolet irradiation of freshly frozen plasma, providing a virocid effect [16. Evaluation of Viral Inactivation Efficacy of a Continuous Flow Ultraviolet-C Reactor (UVivatec) Bae, Jung Eun, Eun Kyo Jeong [et al]. Kor. J. Microbiol. Biotechnol. 2009, Vol. 37, No. 4, p. 377-382]. Inactivation of bacterial and viral agents on the basis of ultrafast (millisecond) low-temperature (50-55) ° С pasteurization-sterilization (MCT technology) is being worked out [17. N.N. Nosik, D. Dolgopolov, N.G. Kondrashina. Novel method of virus inactivation in plasma with millisecond technology. International Meeting on Emerging Diseases and Surveillance, 2016].

The disadvantage of these methods is the use of human blood plasma samples, which according to the production method should be sterile with relatively low volumes of raw materials, incomparable, for example, with 500-1000 l of slaughter plasma for a shift of a meat factory.

Physicochemical approaches for the decontamination of infectious agents are used. For example, treatment with methylene blue and exposure to visible light. The disadvantage of this method is its binding to stationary factory conditions, focused on the disinfection of small volumes of sterile donor blood plasma, which passed control on the absence of infectious agents that cause intractable human viral infections.

Known chemical methods of inactivation of infectious agents in samples of donated human blood plasma. One of the most effective methods of disinfection is the introduction of iodoacetaldehyde (inactin) into the plasma in the conditions of solvent-detergent incubation of the treated plasma [18. Guidelines on viral inactivation and removal procedures intended to assure the viral safety of human blood plasma products // WHO Technical Report, Series No. 924, 2004, p. 151-219; 15. Panov V.P. Principles for ensuring the viral safety of blood products (review). // Chem.-farm. magazine. 2004, t. 38, No. 3, p. 39-47]. The solvent-detergent plasma treatment itself is one of the most popular methods for the deactivation of viral agents in human donor plasma [19. Peter Hellstern, Bjarte G. Solheim. The Use of Solvent / Detergent Treatment in Pathogen Reduction of Plasma. Transfus Med Hemother, 2011, 38: 65-70].

One of the methods of antiviral treatment of human blood donor plasma is based on the introduction of psoralen followed by exposure to ultraviolet light [20. Wages D., Smith D., Walsh J. et all. Virally inactivated fresh frozen plasma transfusion in normal volunteers // Vox Sang 1998; 74, Suppl 1: p. 1290].

A disadvantage of the known method is the binding of the method to the process of technological fractionation of donor plasma. The dose-effect-exposure of the antiviral treatment is a short-term stage preceding the stage of industrial fractionation of the reactor bookmark.

Summing up the above methods for disinfection of infectious safe (according to the results of clinical and laboratory control) plasma, we note the main drawback of the known scientific and technical solutions. Methods-stages of inactivation of infectious agents in the plasma include their placement on the territory of the factory unit, carrying out the direct process of fractionation of raw materials. The necessary requirements for the concentration of the antiviral compound and its exposure in the plasma are carried out due to the short incubation interval in the composition of the reactor bookmark before the very first stage of the fractionation process. It is the very first fractionation stage that is focused on reducing the concentration of the disinfectant itself and associated technological impurities that provide disinfecting conditions for the incubation of the plasma loaded into the reactor (reactor bookmark). It should also be noted that the known methods are of a short-term nature. Known scientific and technical solutions do not allow discussing the introduction of a disinfectant in blood plasma in order to ensure the proper effect without the negative effect of the bactericidal-virocidal compound on the target proteins that form the active principle of pharmaceutical biological preparations. The reverse effect is repeatedly noted. Known disinfectants have a protein-denaturing effect, which manifests itself including subsequently at the stages of storage of immunoglobulin biological products [21. Chechetkin A.V., Kasyanov A.D., Soldatenkov V.E., Makeev A.B. The effect of pathogen inactivation on the quality and indicators of the biological usefulness of donor blood components // Biomedical Journal. T. 16, ST. 72.S. 779-790; 22. van Beers M.M., Bardor M. Minimizing immunogenicity of biopharmaceuticals by controlling critical quality attributes of proteins // Biotechnol J. 2012, v. 7 (12), p. 1473-1484. doi: 10.1002 / biot.201200065]. A general disadvantage of the listed methods of decontamination of potential infectious agents is the lack of information on bactericidal and bacteriostatic effects associated with targeted antiviral effects. Existing methods for the disinfection of individual or pooled plasma samples suggest their sterility, which is ensured by the engineering design of individual donor blood donations and the manufacture of individual human blood plasma samples intended for the production of medical biological products.

An important aspect concerns the volume of disinfection of plasma (serum) of blood. In standard schemes of existing technologies at the enterprises of the medical industry, individual plasma samples are represented by small volumes of about 100-200 ml, and the corresponding reactors do not exceed volumes of 50-200 liters. Thus, the previously described methods for the physical and physico-chemical inactivation of the reactor bookmark are technically adapted to the relatively small volumes of disinfection of raw materials, for example, not more than 50 liters, forming a series. The prepared application material is focused on volumes of 500-1000 liters of plasma production under the conditions of an existing meat processing plant of medium power. There is currently no scaling of technical means designed to provide well-known technical solutions based on physical or physicochemical methods of virocidic-bactericidal effects intended for processing individual samples of 5 liters of slaughter blood plasma or pooled samples of slaughter blood plasma of cattle. Known technical solutions based on the use of physical or physico-chemical methods are not focused on the disinfection of individual samples of slaughter plasma with a volume of 5 liters or their pulled volumes. To do this, it is necessary to solve the technical problem regarding the scaling of the necessary equipment, focused on irradiation, either with visible light or ultraviolet, or gamma radiation [23. Maclean M., Anderson J.G., MacGregor S.J., White T., Atreya C.D. A New Proof of Concept in Bacterial Reduction: Antimicrobial Action of Violet-Blue Light (405 nm) in Ex Vivo Stored Plasma // J. Blood Transfus. V. 2016. Article ID 2920514. 11 p .; 6. Zubkova NB Biotechnological aspects of effective and safe processing of donor plasma: problems and prospects // Biologics 2014; (1): 4-10]. However, this kind of approach will in itself be ineffective, since it requires the organization in the conditions of a meat factory of a factory unit focused on harvesting slaughter blood plasma with a minimum level of microbial contamination.

Existing methods for storing and transporting human blood plasma in low-temperature freezing conditions (-20 ° C and below) allow the use of raw materials for 7 years. Due to the rapid freezing of plasma to a temperature (-35 ° C or -40 ° C), it is possible to increase the shelf life without losing the functional properties of proteins that are significant for the pharmaceutical bio-industry [24. Zhiburt E.B. "Transfusiology: a textbook." St. Petersburg: Peter, 2002, 736 p.]. The possibility of a relatively long shelf life of immunosorts at a temperature of (-5) ° C to (-10) ° C without loss of antibody activity was shown if it was frozen using liquid nitrogen (-196) ° C [5. Chard T. "Radioimmunological methods." M .: Mir, 1981, p. 48].

The disadvantage of low-temperature storage and transportation of plasma (serum) blood is their energy intensity. At the same time, the low-temperature regime of storage and transportation of plasma or blood serum should be maintained strictly up to the stage of industrial fractionation of raw materials [5. Chard T. "Radioimmunological methods." M .: Mir, 1981, p. 48]. For technical reasons, thawing and freezing of plasma samples is not allowed, since such violations have a negative effect on the conformational nativeness and physiological activity of the target proteins.

Known methods of inactivation of contaminating microflora in the composition of the plasma (serum) of blood, prepared in inappropriate conditions.

Fractionation of contaminated microflora of plasma with ethanol allows inactivation of microorganisms already at the very first stages of the technological process [25. Collection "Blood products. Guidance materials on control and production ”edited by SP Burenkov M .: Publishing House of the Ministry of Health of the USSR, 1976, 384 p .; 26. Rusanov V.N., Skobelev L.I. "Fractionation of plasma proteins in the production of blood products." - M .: Medicine, 1983, p. 75-111].

, Cristina Fuentes,

[et al]. Food Control, 2015, Volume 56, p. 77-85], которые используются как реагенты (материалы) в процессе фракционирования плазмы [29. Minipool Caprylic Acid Fractionation of Plasma Using Disposable Equipment: A Practical Method to Enhance Immunoglobulin Supply in Developing Countries. Magdy El-Ekiaby, Mariangela Vargas, Makram Sayed [et al] PLOS Neglected Tropical Diseases, 2015].A similar bactericidal and bacteriostatic effect is possessed by rivanol, caprylic acid [27. Zhurina NA, Shatskaia T.L., Katushkina NV The virological safety and bacterial sterility of a method for fractionating blood plasma proteins with rivanol // Zh. Mikrobiol. Epidemiol. Immunobiol. 1993, 1: p. 45-48 ,; 28. Bactericidal activity of caprylic acid entrapped in mesoporous silica nanoparticles

, Cristina Fuentes,

[et al]. Food Control, 2015, Volume 56, p. 77-85], which are used as reagents (materials) in the process of fractionation of plasma [29. Minipool Caprylic Acid Fractionation of Plasma Using Disposable Equipment: A Practical Method to Enhance Immunoglobulin Supply in Developing Countries. Magdy El-Ekiaby, Mariangela Vargas, Makram Sayed [et al] PLOS Neglected Tropical Diseases, 2015].

A disadvantage of the known methods is the procurement of raw materials in unacceptable conditions, ensuring its contamination with microflora. The freezing of such raw materials should be carried out quickly in order to ensure the inactivation of contaminating microflora. However, bacterial-contaminated plasma contains pyrogens. Removing bacterial pyrogens requires additional stages of purification of the target proteins [6. Zubkova N.B. Biotechnological aspects of effective and safe processing of donor plasma: problems and prospects // Biological products. - 2014, No. 1, p. 4-10 ,; 30. Israfilov A.G. et al. Experience in obtaining pyrogen-free drugs // Abstracts of the IV Russian National Congress “Man and Medicine”, Moscow April 8-12, 1997: collection “Man and Medicine”. Russian nat. Congr. (4; 1997; Moscow). - M .: "PHARMEDINFO", 1997. - p. 54; 31. Patent RU 2192279. A method of obtaining an immunoglobulin preparation. Publ. November 10, 2002 Bull. No. 31].

The closest technical solution to ensure the inactivation of microflora in the blood plasma (serum) is the sequential introduction of phenol to its final concentration (0.7 ± 0.2)% and then dry powder of ammonium sulfate up to 50% of saturation (2.03 M ) in a phenolic sulfate suspension [32. Patent RU No. 23338375. A method for storing plasma or blood serum to obtain immunoglobulin and albumin biological products. Publ. November 20, 2008 Bull. No. 32] (prototype). The above process in a meat factory is carried out within 5 days. Therefore, the problem of the pyrogenicity of the raw material procurement is not solved using the prototype scientific and technical approach. In addition, the proper bactericidal and bacteriostatic conditions are realized initially by adding phenol in the form of a 40% solution in glycerol. It is known that such concentrated phenol solutions have a local protein-denaturing effect. The presence in the composition of phenolized plasma (serum) of blood proteins with elements of denaturation changes will affect the technological parameters of the isolation and purification of the active principle. In particular, the changes discussed concern hydrophobic proteins, which, in particular, include cattle IgG.

The objective of the invention is to create a method of inactivation of contaminating microflora without the use of phenol, which currently refers to ecotoxicants and the classification of harmful substances [33. GOST 12.1.007-76. Harmful substances. Classification and general safety requirements] refers to hazard class 4 (highly hazardous substance). For this purpose, a concentrated (saturated) solution of ammonium sulfate (4.05 M) containing 0.75% didecyldimethylammonium chloride (HOUR D) (hereinafter referred to as the bactericidal fractionation solution) is prepared in a 250 liter reactor. Plasma samples are loaded into the bactericidal fractionating solution with constant stirring as the slaughter blood is separated. The loading of plasma samples into the prepared reactor is carried out to reduce the concentration of ammonium sulfate to 40% of saturation (1.62 M). At the same time, the concentration of the disinfectant decreases from 0.75% to 0.3%. After the last addition of a serving of plasma with constant stirring and at a concentration of 40% of saturation (1.62 M) of ammonium sulfate and 0.3% of didecyldimethylammonium chloride, the formed suspension is additionally mixed for another (60 ± 10) min. This is followed by a centrifugation step to separate the precipitate of gamma globulins. Powder of dry ammonium sulfate is added to the supernatant to a concentration of 75% of saturation (3.04 M), and the pH of the incubation medium is reduced to (4.6 ± 0.1). After separation of the albumin precipitate by centrifugation, a postalbumin centrifuge is used to suspend the gamma globulin precipitate.

The problem is solved by using a specially made solution having bactericidal and bacteriostatic (see Table 1 of the Appendix (Other materials)) and protein-fractionating properties. For incubation of samples of slaughter plasma (serum) of blood, a concentrated (saturated) solution of ammonium sulfate (4.05 M) with 0.75% HOUR D (didecyldimethylammonium chloride) is initially used. Plasma samples with a volume of (5 ± 1) L enter the initial (concentrated) solution as blood is separated with an interval of 20-30 minutes. The process of loading the reactor in a meat factory continues, as a rule, (10 ± 5) hours. The reactor loading is stopped if the concentration of ammonium sulfate due to dilution with individual plasma samples decreased from 100% (4.05 M) to 40% (1.62 M) of saturation. This is followed by the stage of separation of the precipitate of gamma globulins by centrifugation, and additional ammonium sulfate is added to the albumin centrifugate to a final concentration of 75% of saturation (3.04 M) and the pH is reduced to (4.6 ± 0.1). The sulfate suspension of the albumin-transferrin fraction is stirred for an additional (60 ± 10) min. and then aggregated proteins are precipitated by centrifugation. The supernatant solution (postalbumin centrifuge) is used to prepare a solution designed to suspend the precipitate of gamma globulins.

The described method has been tested in the laboratory version for the manufacture and storage of gamma-globulin semi-finished products of plasma (serum) of blood of cattle with a volume of 150 ml and 1000 ml, and in an industrial version with a volume of (150 ± 50) liters of blood plasma of cattle.

From the analysis of literature data it follows that with the help of saturated solutions of ammonium sulfate or its dry powder, colloidal solutions, complex in the composition of individual proteins, are fractionated on a laboratory or industrial scale. The fractionating effect is based on the mechanisms of protein aggregation under the influence of ammonium sulfate. Ammonium sulfate in the composition of the colloidal solution provides the destruction of structured water, focused on the screening of hydrophobic and neutrally charged sites on the surface of protein globules. In the absence of a hydration shell, it is precisely at these hydrophobic-neutral sites that aggregation of individually distinctive proteins occurs, which in the process of coprecipitation are represented by a rather wide spectrum of protein impurities [34. Scopes R.K. Protein purification methods. M.: Mir, 1985, 358 p.]. From the mechanism of protein aggregation under the influence of ammonium sulfate follows the technique of its introduction into colloidal solutions of complex biomolecules. [35. J.M. Curling. Methods of plasma protein fractionation. Academic press, 1980, 601 p.] In particular, ammonium sulfate must be introduced on the principle of a gradual increase in its concentration in plasma or blood serum. To do this, use a saturated solution of ammonium sulfate or its dry powder (prototype), which is introduced into the incubation medium gradually or by jet-drop method or in small portions with constant stirring (prototype).

In the proposed method, the reverse order is implemented, i.e. to a saturated solution (100% of saturation (4.05 M)) of ammonium sulfate at pH 7.0, plasma of cattle is fractionally introduced as the separation of slaughter blood. Under such conditions, the maximum destruction of structured water is ensured and, as a consequence of this, the maximum possible level of protein aggregation over hydrophobic-neutral parts of the surface of protein globules is ensured. From the results shown in Table 2 of the Appendix (Other materials), it follows that the theoretically estimated effect of excessive deposition is not pronounced.

From an analysis of the disinfection technique using known disinfectants, it follows that their bactericidal concentrations cannot be used to inactivate microflora that contaminate blood plasma [15. Panov V.P. Principles for ensuring the viral safety of blood products (review) // Chemical and Pharmaceutical Journal. - 2004 - T. 38. - No. 3. - S. 39-47.]. Disinfectants have a pronounced protein-denaturing effect, which, in particular, was expressed in the separation of the plasma and the formation of a hardly soluble or insoluble precipitate.

In the known method (prototype), phenol (0.7 ± 0.2)% and ammonium sulfate 50% of saturation (2.03 M) are used as a disinfectant. However, phenol is an ecotoxicant [36. Food Toxicology - real or imaginary problem? / Eds. G.G. Gibson, R. Walker. - L .: Taylor & Francis, 1985.]. The tendency of phenol to intercalate is also known, therefore, the removal of phenol from the composition of intermediates and the finished medicinal product presents certain difficulties of a scientific and methodological nature.

In the proposed method, instead of phenol, moderately toxic (hazard class 3) is used [33. GOST 12.1.007-76. Harmful substances. Classification and general safety requirements] didecyldimethylammonium chloride compound without the above negative properties. In medical and hygienic practice, this disinfectant is used to sterilize medical instruments, other products and objects requiring periodic disinfection.

In the known method (prototype) uses a concentration of ammonium sulfate 50% of saturation (2.03 M) in the composition of the phenol-sulfate suspension of blood plasma. However, the storage of blood plasma in the form of phenol-sulfate suspensions requires maintaining a strictly defined temperature, since the level of hydrophobic aggregation of proteins and their deposition under the influence of ammonium sulfate depends on the temperature of the incubation medium.

In the proposed method, instead of preparing and storing the plasma in the form of a phenol-sulfate suspension, it is fractionated using ammonium sulfate in the presence of a didecyldimethylammonium chloride disinfectant and resuspended by a gamma-globulin precipitate with a postalbumin centrifuge. Table 2 of the Appendix (Other materials) shows that, depending on the concentration of ammonium sulfate in the initial solution, it is possible to form gamma globulin fractions with different completeness of IgG deposition. For example, upon dilution of the initial (concentrated) solution of ammonium sulfate (100% of saturation (4.05 M)) with plasma samples to a concentration of 27% (1.09 M), 33% (1.34 M) and 40% (1, 62 M) from saturation, it is possible to form non-chromatographic fractions with the highest yield of IgG or albumin in the form of precipitation. In the proposed method, focused on the maximum deposition of antibodies from the class of IgG specific for the infectious rhinotracheitis virus of cattle, a final concentration of ammonium sulfate of 40% of saturation (1.62 M) in the blood plasma of cattle was used. The selected concentration ensures almost complete precipitation of IgG and antibodies from the IgG class as part of the gamma-globulin fraction and leaves proteins for the production of albumin biological products in the supernatant (see Tables 2, 3 and 4 of the Appendix (Other materials)).

In the proposed method, isolated gamma globulin samples are suspended in a postalbumin centrifuge - a solution made after total coprecipitation of proteins from the albumin fraction.

It was established that gamma-globulin semi-finished products made in this way are stored for 180 days at room temperature (20-25) ° C. The most significant properties of proteins remain unchanged in the composition of the gamma-globulin semi-finished product (see Table 5 of the Appendix (Other materials)). The functional activity of antibodies from the IgG class to the cattle infectious rhinotracheitis virus does not change (see Table 5 of the Appendix (Other materials)). Semi-finished products of gamma globulin remain sterile, natural proteases are inhibited by the action of ammonium sulfate in the sediment and the postalbumin centrifuge.

At the present stage of the development of science and technology, the use of sulfate solutions with the addition of quaternary ammonium salts and, in particular, didecyldimethylammonium chloride for incubation of individual and pooled plasma or serum samples is not known as the blood is separated at the place of its preparation in conditions and rooms that do not meet sterility requirements raw materials and proper cleanliness of production facilities. Due to high concentrations of ammonium sulfate, the activity of plasma or serum proteases is almost immediately inhibited (see Table 6 of the Appendix (Other materials)). The optimal concentration of ammonium sulfate in the proposed method is 40% of saturation (1.62 M), which ensures the most complete deposition of IgG and antiviral antibodies from the IgG class, as well as protease inhibition. The Application Figure (Other materials) shows the protein spectrum of fractions obtained from cattle plasma under incubation conditions in a solution of ammonium sulfide with didecyldimethylammonium chloride at initial concentrations of 100% of saturation (4.05 M) and 0.75%, respectively. Moreover, the presence of a relatively high content of albumin impurities in the gamma-globulin fraction at a final concentration of ammonium sulfate of 40% of saturation (1.62 M) (4 track) compared with similar samples manufactured at concentrations of 27% (1.09 M) ( 6 track) and 33% (1.34 M) (5 track) (see Appendix Figure (Other materials)). The precipitating concentration of ammonium sulfate 50% of saturation (2.03 M) as well as 40% of saturation (1.62 M) causes the most complete IgG precipitation in the gamma globulin fraction (see Table 2 of the Appendix (Other materials)). However, albumin impurities in the composition of gamma globulins manufactured at a concentration of 50% of saturation (2.03 M) of ammonium sulfate exceed the coprecipitation effect at a concentration of precipitant 40% of saturation (1.62 M) in the blood plasma (serum). As an example, the protein spectrum of albumin fractions is given, which allows one to judge IgG impurities at concentrations of ammonium sulfate 27% (1.09 M) (3 track), 33% (1.34 M) (2 track) and 40% (1, 62 M) (1 track) from saturation (see Appendix Figure (Other materials)). Moreover, only with a precipitating concentration of ammonium sulfate 40 (1.62 M) and 50% (2.03 M) from saturation, the presence of IgG antibodies specific for the cattle infectious rhinotracheitis virus is not detected in albumin precipitates (Table 7 of the Appendix ( Other materials)).

An example of the implementation of the proposed method Stage 1. Preparation of a bactericidal fractionating solution. The solution is a mixture of ammonium sulfate (hereinafter - CA) and didecyldimethylammonium chloride (hereinafter - HOUR D) at final concentrations of 4.05 M (100% of saturation) and 0.75%, respectively.

For laboratory preparation, 300 ml of distilled water with a temperature of 25-30 ° C are collected in a 500 ml volumetric flask, 3.75 ml of HOUR D is added with stirring. If initially HOUR D is an aqueous or alcoholic solution, the added volume must be recalculated relative to the current substances HOUR D. Stir 5 minutes. Then 266.5 g of dry CA powder is weighed and added to the HOUR D solution with stirring. Next, the level in the volumetric flask is adjusted to 500 ml with distilled water and stirring is continued until ammonium sulfate is completely dissolved. With a decrease in the level of the solution, it is brought to 500 ml with distilled water. Additionally mixed for 30 minutes.

In an industrial version, 50 liters of distilled water with a temperature of 25-30 ° C are poured into a graduated container. 0.75 L of didecyldimethylammonium chloride is added to the vessel with stirring with an anchor stirrer at a speed of 60-70 rpm. After the addition of HOUR D, 53.3 kg of ammonium sulfate are weighed and, while stirring, by the above method, a portion of a portion of ammonium sulfate is added. Next, the level is adjusted with distilled water to 100 l and stirring is continued until complete dissolution of ammonium sulfate. With a decrease in the level of the solution, it is brought to 100 l with distilled water.

Stage 2. Bringing the pH of the bactericidal fractionating solution to neutral values. In a beaker with a volume of 25 ml, 1 ml of solution was taken and 19 ml of distilled water was diluted. The pH values of the diluted solution are measured on a Professional Meter PP-20, Sartorius type ionomer. Then titrate a solution of 1M NaHCO ₃ to a pH of 7.0-7.5. The volume of 1M NaHCO _{3 used} for titration is multiplied by the ratio of the initial volume and the volume taken for titration. The resulting volume of 1M NaHCO ₃ with stirring by the above method is added to the original bactericidal fractionation solution. Stirred for 10 minutes and control the pH in the above way.

Stage 3. Adding plasma to the bactericidal fractionation solution. The plasma or blood serum of cattle is added to the solution in portions or at the same time (jet) with stirring on a magnetic stirrer, preventing foaming of the suspension. The number of portion additions and the duration of addition are not limited. In both cases, plasma (serum) is added in a solution-plasma ratio of 1: 1.5 (by volume).

In the laboratory version, the volumes of bactericidal fractionating solution from 100 to 666 ml and from 150 to 1000 ml of plasma (serum) were tested. An example of a laboratory option for adding plasma. In a beaker of at least 0.5 liter volume, pour 100 ml of a bactericidal fractionating solution and add 150 ml of blood plasma (serum) by the above methods. After the addition of blood plasma (serum) is completed, stirring is continued for 1 hour at a temperature of 18-25 ° C. Initial concentrations of substances are diluted with blood plasma (serum) to the final ones: 40% of saturation (1.62 M) in SA and 0.3% in HOUR D.

In an industrial embodiment, 100 l of a bactericidal fractionating solution is poured into a 250 liter reactor. Then, 150 l of blood plasma (serum) are introduced into the solution with stirring with an anchor stirrer at a speed of 60-70 rpm. After the addition is complete, an additional 1 hour is stirred.

Stage 4. Precipitation of the gamma globulin fraction. The resulting suspension is centrifuged in a centrifuge type PC-6 with a gravitational field of 4200 g for 30 minutes. In the industrial version, flow-through centrifuges of the OTR-1 type are used at a flow rate of 60 l / h and a gravitational field strength of 10000-15000 g. The precipitate is a gamma globulin fraction. 50-80% of the total plasma protein passes into it.

Stage 5. Precipitation of the albumin fraction. Dry ammonium sulfate powder was added to the resulting supernatant with stirring at the rate of 241 g per 1 liter of supernatant, thus obtaining a final concentration of ammonium sulfate 75% of saturation (3.04 M). Then the pH is adjusted to (4.6 ± 1) with 1 M hydrochloric acid and the value is checked by the above methods. After adjusting the pH, the suspension was stirred for 1 hour.

The suspension is centrifuged in laboratory conditions using a PC-6 centrifuge with a gravitational field of 4200 g for 30 minutes. In the industrial version, flow-through centrifuges of the OTR-1 type are used at a flow rate of 60 l / h and a gravitational field strength of 10000-15000 g. As a result, the remaining proteins become precipitated.

Stage 6. Resuspension of the precipitate of gamma globulins. The supernatant remaining after precipitation of the albumin fraction (postalbumin centrifuge) is used to resuspend the gamma-globulin precipitate. For this, a postalbumin centrifuge is added to the precipitate of gamma globulins in the ratio: 2 liters of supernatant to 1 kg of crude precipitate. Then mix for 30 minutes as described above, avoiding foaming, to form a homogeneous suspension. The final concentration of ammonium sulfate in the gamma-globulin suspension is not less than 60% of saturation (2.43 M), and HOUR D is not less than 0.27%.

Information used in preparing a patent application

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ATTACHMENT

Table 1. The effect of solutions of phenol, didecyldimethylammonium chloride and ammonium sulfate on the level of inactivation of the contaminating microflora inherent in samples of cattle blood plasma produced in meat processing plants

- среднеарифметическое значение КОЕ; σ - стандартное отклонение; Р - уровень значимости различий; n=5. Уровень бактериальной контаминации определяли по методу, рекомендованному Государственной фармакопеей, изд. XII, ч. 1 ОФС 42-0067-07.Note: HOUR D - didecyldimethylammonium chloride; CA - ammonium sulfate; CFU - colony forming units. K (control) - the number of colony forming units fixed on the surface of meat and peptone agar during the cultivation of microorganisms without making the studied disinfectants and ammonium sulfate; O (experiment) - the number of colony forming units fixed on the surface of meat and peptone agar during the cultivation of microorganisms in the presence of the studied disinfectants and ammonium sulfate;

- arithmetic mean value of CFU; σ is the standard deviation; P is the level of significance of differences; n = 5. The level of bacterial contamination was determined according to the method recommended by the State Pharmacopoeia, ed. XII, part 1 OFS 42-0067-07.

To standardize the preparation of a culture of bacteria used plasma, prepared in a meat factory, which was thermostated for 3 days at 37 ° C. The incubated plasma was seeded on the surface of the meat-peptone agar and thermostated for 2 days. Colonies were washed from the surface with sterile water, the optical density of the bacterial suspension was measured on an SF-46 spectrophotometer at a wavelength of 540 nm and a 1 cm thick quartz cuvette. In parallel, the suspension was seeded with 10 ⁷ dilution on the surface of meat peptone agar to determine CFU in the volume of suspension according to the method described above. Comparing the optical density of the bacterial suspension and the CFU in it, the value of the CFU per 1 unit of optical density was obtained. The obtained value was used for standardized preparation of a control bacterial culture with a known amount of CFU.

Table 2. The distribution of protein in fractions depending on the final concentration of ammonium sulfate.

- среднеарифметическое значение; σ - стандартное отклонение; Р - уровень значимости различий; n=5; К (контроль) - добавление насыщенного раствора сульфата аммония в плазму до достижения требуемой конечной концентрации; О (опыт) -разбавление насыщенного раствора сульфата аммония плазмой до требуемых конечных концентраций согласно предлагаемому способу.Note:

- arithmetic mean value; σ is the standard deviation; P is the level of significance of differences; n is 5; To (control) - the addition of a saturated solution of ammonium sulfate in plasma to achieve the desired final concentration; About (experience) -dilution of a saturated solution of ammonium sulfate with plasma to the desired final concentration according to the proposed method.

Table 3. Determination of the completeness of IgG deposition and the level of inactivation of the contaminating microflora of the gamma globulin fraction when plasma samples are diluted with a saturated solution of ammonium sulfate with 0.75% didecyldimethylammonium chloride to 40% of saturation and didecyldimethylammonium chloride to 0.3%

- среднеарифметическое значение; σ - стандартное отклонение.Note:

- arithmetic mean value; σ is the standard deviation.

The level of microbial contamination was determined by seeding 0.1 ml of samples on the surface of meat-peptone agar, followed by incubation for 2 days.

¹ - The protein distribution is determined on the basis of the total amount of protein in the fractions determined by the biuret method in accordance with the Methodological Guidelines of MUK 4.1 / 4.2.588-96 "Methods of control of medical immunobiological preparations administered to people", p. 46.

² - The level of bacterial contamination is determined by the method recommended by the State Pharmacopoeia, ed. XII, part 1 OFS 42-0067-07.

³ - The content of the target proteins was determined using densitometry of the electrophoregram of samples of semi-finished products (PAAG concentration of 3-25%, the amount of protein applied - 40 μg / track, Davis-Lamley electrophoresis, native samples under dissociating conditions) in accordance with the Methodological Guidelines of MUK 4.1 / 4.2 .588-96 "Methods of control of medical immunobiological drugs administered to people", p. 2.

Table 4. Testing the optimal concentration of ammonium sulfate 40% of saturation and didecyldimethylammonium chloride 0.3% in relation to the completeness of deposition of antibodies from the class of IgG specific for the virus of infectious rhinotracheitis in cattle

- среднеарифметическое значение; σ - стандартное отклонение; Р - уровень значимости различий; (n=5); ОП 450 нм - оптическая плотность субстратной смеси; К (контроль) - фракции, полученные предлагаемым способом без использования дидецилдиметиламмония хлорида; О (опыт) - фракции, полученные предлагаемым способом с использованием использования дидецилдиметиламмония хлорида.Note:

- arithmetic mean value; σ is the standard deviation; P is the level of significance of differences; (n = 5); OD 450 nm - the optical density of the substrate mixture; To (control) - fractions obtained by the proposed method without the use of didecyldimethylammonium chloride; About (experience) - fractions obtained by the proposed method using didecyldimethylammonium chloride.

Fractions are represented by the corresponding sulfate suspensions. Suspensions were centrifuged at 4200 g for 20 minutes. Sulfate precipitates were dissolved to a protein concentration of 40 mg / ml. Antibody activity was determined by the IRT-SEROTEST test system (Vetbiokhim LLC) according to the attached instructions. The OD of samples of albumin fractions corresponds in magnitude to the OD of the negative control sample of the test system.

Table 5. Comparative characteristics of the semi-finished product of gamma globulins, which has been stored for 180 days at a temperature of (20 ± 5) ° С

- среднеарифметическое значение; σ - стандартное отклонение; Р - уровень значимости различий; (n=5); Контроль - полуфабрикат гамма-глобулинов без внесения дидецилдиметиламмония хлорида в условиях хранения при температуре (20±5)°С; Опыт - полуфабрикат гамма-глобулинов, полученный предложенным способом в условиях хранения при температуре (20±5)°С; КОЕ - количество колониеобразующих единиц, посчитанных на поверхности мясо-пептонного агара после термостатирования в течение 48 часов.Note:

- arithmetic mean value; σ is the standard deviation; P is the level of significance of differences; (n = 5); Control - prefabricated gamma globulins without didecyldimethylammonium chloride in storage at a temperature of (20 ± 5) ° С; Experience - semi-finished gamma globulins obtained by the proposed method under storage at a temperature of (20 ± 5) ° C; CFU - the number of colony forming units calculated on the surface of meat-peptone agar after incubation for 48 hours.

Fractions are represented by the corresponding sulfate suspensions. Suspensions were centrifuged at 4200 g for 20 minutes. Sulfate precipitates were dissolved to a protein concentration of 40 mg / ml. Antibody activity was determined by the IRT-SEROTEST test system (Vetbiokhim LLC) according to the attached instructions.

Table 6. The effect of the used concentrations of ammonium sulfate and didecyldimethylammonium chloride on the proteolytic

trypsin activity in the manufacturing process of semi-finished products.

- среднеарифметическое значение; σ - стандартное отклонение; Р - уровень значимости различий; n=5; ОП₄₀₅ - оптическая плотность при длине волны 405 нм; ОП₄₀₅(Ф+СА+Б4) - оптическая плотность реакционной среды в присутствии фермента, сульфата аммония и дидецилдиметиламмония хлорида; ОП₄₀₅(Ф) - оптическая плотность реакционной среды в оптимальных условиях. Эксперимент проводился согласно методике, изложенной в «Практическая энзимология» / X. Биссвангер; пер. с англ. - М.: Бином. Лаборатория знаний, 2010. - c. 163.Note: CA - ammonium sulfate;

- arithmetic mean value; σ is the standard deviation; P is the level of significance of differences; n is 5; OP ₄₀₅ - optical density at a wavelength of 405 nm; OD ₄₀₅ (F + CA + B4) is the optical density of the reaction medium in the presence of an enzyme, ammonium sulfate and didecyldimethylammonium chloride; OP ₄₀₅ (F) is the optical density of the reaction medium under optimal conditions. The experiment was carried out according to the method described in Practical Enzymology / X. Bisswanger; trans. from English - M .: Binom. Laboratory of Knowledge, 2010. - c. 163.

Table 7. Determination of the activity of specific antibodies against infectious rhinotracheitis virus in cattle in fractions obtained from cattle blood plasma

Note: the activity of specific antibodies was determined using the test system "IRT-SEROTEST" (LLC "Vetbiochem") according to the attached instructions.

Claims (3)

1. The method of obtaining gamma-globulin semi-finished product from slaughter blood of cattle using chemical reagents, characterized in that the raw material is slaughtered blood at the place of its preparation and separation in a meat factory, and plasma samples are placed in saturated 100 as they are manufactured % of saturation or 4.05 M solution of ammonium sulfate with 0.75% didecyldimethylammonium chloride, in the ratio solution: plasma 1: 1.5 by volume, to a concentration of 40% of the saturation of ammonium sulfate and 0.3% didecyldimethylammonium chloride, vol the resulting suspension is centrifuged to obtain a precipitate of the gamma-globulin fraction, dry ammonium sulfate powder is added to the resulting supernatant with stirring, obtaining a final concentration of ammonium sulfate 75% of saturation, and the pH is adjusted to 4.6, the suspension is stirred and centrifuged, precipitating the albumin fraction remaining after precipitation of the albumin fraction, the supernatant is used to resuspend the gamma globulin precipitate; the final concentration of ammonium sulfate in the gamma globulin suspension is not less than e 60% of saturation and not less than 0.27% of didecyldimethylammonium chloride. 2. The method according to p. 1, characterized in that didecyldimethylammonium chloride is used as a disinfectant, which does not have the properties of an ecotoxicant and is approved for use for the disinfection of medical instruments, work surfaces and industrial premises. 3. The method according to p. 1, characterized in that the deposition of IgG from the composition of the pulled plasma samples is carried out during their incubation by diluting individual plasma samples with a saturated solution of 100% of saturation or 4.05 M ammonium sulfate to a concentration of 40% of saturation or 1.62 M.

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