Scientific relevance. The immunoglobulin A (IgA) impurity content in parenteral human immunoglobulins should be determined in accordance with the State Pharmacopoeia of the Russian Federation by kinetic nephelometry, radial immunodiffusion, or enzyme immunoassay (ELISA) with a reference standard. The International Standard (IS) for the content of IgA is certified using gravimetry and radial immunodiffusion. However, neither of the existing standards for the content of IgA in human immunoglobulins is currently certified using all three compendial methods. This prevents analysts from comparing test results obtained by different methods and may lead to an underestimation of the IgA content in human immunoglobulins.Aim. This study aimed to determine the procedure for the development, certification, and use of a pharmacopoeial reference standard (RS) for the content of IgA in human immunoglobulins.Materials and methods. The authors studied candidate RSs for the IgA content derived from human plasma for fractionation. The IgA content determination involved kinetic nephelometry, radial immunodiffusion, and ELISA, as well as commercial test kits and the IS. The authors quantified the IgA impurity in samples of commercial human immunoglobulins from various manufacturers. The data analysis involved descriptive statistics and variance analysis using Microsoft Excel and Statistica 10.Results. The authors established a pharmacopoeial standard with a certified IgA content of 1.98 mg/mL (expanded uncertainty, 0.44 mg/mL; coverage coefficient, k=2; confidence level, 95%) for IgA impurity quantification in human immunoglobulins by radial immunodiffusion and ELISA and that of 1.31–2.64 mg/mL (expanded uncertainty, 0.67 mg/mL; coverage ratio, k=3; confidence level, 99%) for intralaboratory quality control of IgA impurity quantification by kinetic nephelometry, radial immunodiffusion, and ELISA.Conclusions. The pharmacopoeial standard developed in the study has been included in the register of standards of the State Pharmacopoeia of the Russian Federation as the Reference Standard for the Content of Immunoglobulin Class A (IgA) (Registry No. 3.1.00454). The pharmacopoeial standard is intended for the standardisation of analytical methods for the-determination of the IgA impurity content in parenteral human immunoglobulins.

The competitive enzyme-linked immunosorbent assay (ELISA) used for the quantification of specific antibodies, anti-D (Rho) IgGs, in human anti-D (Rho) immunoglobulin preparations relies upon the competition between anti-D antibodies in the preparation and anti-D monoclonal antibodies (mAbs) for immunochemical binding to D-antigen epitopes of the immunosorbent, human erythrocytes of the ccDEE phenotype immobilised on a solid support. The immunosorbent is prepared in-house under laboratory conditions. The certification of the International Standard (IS) for anti-D immunoglobulin included attempts to use the CCDee phenotype, which is more common (16.01%) than the ccDEE phenotype (2.16%). It is possible to use the CCDee phenotype, as long as the user adheres to the acceptance criteria for the results, developed during method validation. The aim of this study was to develop the acceptance criteria for the results of anti-D IgG quantification in human anti-D immunoglobulin preparations by the ELISA method that would allow using CCDee erythrocytes to prepare the immunosorbent should there be no erythrocytes of the ccDEE phenotype available. Materials and methods: the study used human anti-D immunoglobulin preparations; anti-D IgG–spiked samples; the IS for anti-D immunoglobulin; the reference standard (RS) for anti-D IgG–free immunoglobulin; anti-D mAbs; and erythrocytes of the CCDee, ccDEE, and ccddee phenotypes. The authors quantified anti-D IgG antibodies by the competitive ELISA. The statistical analysis used parametric and nonpar ametric tests. Results: the study demonstrated the possibility of using CCDee erythrocytes for competitive immunochemical binding with anti-D mAbs and anti-D IgG of various human anti-D immunoglobulin preparations. The suitability of the analytical procedure with the CCDee phenotype was confirmed by validation parameters: trueness, intermediate precision, linearity, selectivity, specificity, and robustness of the method and comparability of the results obtained when using the CCDee and ccDEE phenotypes. The authors developed the acceptance criteria: the relative values of anti-D IgG content in the test sample should range within ±20% of the nominal value; the coefficient of determination (R2) should be at least 0.9; the relative standard deviation (RSD, %) of three absorbance values for each concentration should not exceed 20%. Conclusions: these acceptance criteria guarantee the reliability of assay results of anti-D IgG quantification by the ELISA, which allows them to be used by specialists of control and analytical laboratories to assess the quality of human anti-D immunoglobulin preparations.

Разработка критериев иммобилизации эритроцитов человека фенотипов R1R1 и R2R2 на твердой фазе при определении содержания анти-D-антител IgG в препаратах иммуноглобулина человека антирезус Rh o (D) методом иммуноферментного анализа 1 Федеральное государственное бюджетное учреждение «Научный центр экспертизы средств медицинского применения» Министерства здравоохранения Российской Федерации, 127051, г.Москва, Российская Федерация 2 Федеральное медико-биологическое агентство, 123182, г

Ebola outbreak in eastern parts of the Democratic Republic of the Congo in 2018–2020 proved that the virus remains highly hazardous for humans, and the outbreak in West Africa in 2014–2016, which was the largest Ebola outbreak in history, showed that it could be imported to other continents, including Russia. In 1993 the Federal State Budgetary Institution “48th Central Scientific Research Institute” of the Russian Ministry of Defence developed a specific equine immunoglobulin for emergency prophylaxis of Ebola in risk groups. The evaluation and improvement of the product’s properties is an important area in the development of biological defence technologies.The aim of the study was to examine the properties of the equine anti-Ebola immunoglobulin which had been stored for a long time at 2–8 °C.Materials and methods: the authors studied batches of heterologous anti-Ebola immunoglobulin that had been stored for 17–22 years. The properties of the product were evaluated according to the requirements of the State Pharmacopoeia of the Russian Federation, 14th ed. (Ph. Rus. 14 ed.). The specific activity of the product was determined in a plaque reduction neutralisation test using Ebola virus and African green monkey kidney cells (GMK-AH-1(D)). Immunoglobulin molecular parameters were determined by size-exclusion high-performance liquid chromatography using the test methods described in the European Pharmacopoeia 9.6 and Ph. Rus. 14 ed.Results: the storage of anti-Ebola immunoglobulin for 17–22 years at 2–8 °C resulted in a four-fold reduction of the level of virus-neutralising antibodies against Ebola, decrease in the proportion of monomers from 98 to 74–90%, increase in the proportion of dimers and polymers, and formation of immunoglobulin molecules’ fragments. Signs of toxicity for mice were observed in one of the three product batches. Conclusions: the obtained results suggest the need to perform more studies to test the quality of antiEbola immunoglobulin batches that were stored for shorter periods of time in order to assess the stability of their initial characteristics.

Human immunoglobulin and human albumin products can negatively affect patients’ health. The residual content of haemagglutinins, anti-D antibodies, prekallikrein activator, as well as the level of anticomplementary activity, are controlled during both production and testing for compliance with normative documentation. Methods for their quantitative evaluation are based on the effects of erythrocytes hemolysis or agglutination and on the amidolytic product properties of cascade reactions. These methods require the obligatory use of reference materials (RMs). The development of RMs, acting as the carrier of the quantitative characteristics of the human immunoglobulins and human albumin impurities, is fraught with the difficulty of selecting a candidate, substantiating the certification methodology, and determining the relevant characteristics. In this research, we used human immunoglobulin solutions with normalized haemagglutinin content, anti-D antibodies or anti-complementary activity in a different range, whose quality was studied using haemagglutination and chromogenic methods, and the complement fixation test. Methodological approaches to the RM development and certification were justified using system analysis and forensic audit methods. The results demonstrate the necessity of using RMs to assess the stability of analytical work by applying anti-complementary activity determination, haemagglutinin content or anti-Dantibody methods. It is established that RMs allow the quantitative content of haemagglutinins, anti-D antibodies and prekallikrein activator to be established. The value of the CRM characteristics should be approximated to the qualitative composition of these impurities in the analysed products, but in amounts not detectable (to produce negative RM components) or exceeding permissible maxima (to produce positive RM components).

Clinical trials of tabletted pox vaccine revealed development of tonsillitis as a postvaccinal reaction in some volunteers: ulceronecrotic lesions in the tonsils, lymphadenitis, hyperthermia and asthenia. The main cause of the local inflammatory reactions was activation of the host opportunistic microflora including hemolytic streptococci and Staphylococcus aureus. For the treatment of the infectious complications systemic antimicrobials, such as benzylpenicillin, amoxicillin, ampicillin, cefazolin and fluoroquinolones (ciprofloxacin) in combination with the symptomatic therapy were used. The treatment course of 9 days provided complete elimination of the postvaccinal reactions, the specific antibody generation being not affected.

Представлены материалы по разработке методики определения содержания активатора прекалликреина в лекарственных препаратах иммуноглобулинов и альбумина человека. Установлено, что адекватность определения содержания активатора прекалликреина хромогенным методом зависит от активности используемого реагента прекалликреина, количественного соотношения компонентов реакционной смеси и времени её инкубации, концентрации используемого хромогенного субстрата. Обоснованы критерии приемлемости реагентов прекалликреина коммерческого производства. Представлены результаты разработки основных параметров методики и материалы валидационных исследований, подтверждающие её линейность в диапазоне от 0 до 35 МЕ/мл, правильность и внутрилабораторную прецизионность, соответствующие таковым для биологических методов анализа. Разработанная методика может быть использована для оценки качества лекарственных препаратов иммуноглобулинов и альбумина человека по содержанию активатора прекалликреина.

Method development for determining prekallikrein activator in medicinal preparations of human immunoglobulins and albumin found that the adequacy of chromogenic determination of prekallikrein activator depends on the activity of the used prekallikrein reagent, the quantitative component ratio in the reaction mixture and its incubation time, and the concentration of employed chromogenic reagent. Acceptance criteria for commercial prekallikrein reagents are justified. The main method parameters and validation test results confirming the linearity in the range 0 – 35 IU/mL, accuracy, and intralaboratory precision are presented and correspond to those for biological analytical methods. The elaborated method can be used for quality assessment of prekallikrein activator content in medicinal preparations of human immunoglobulins and albumin.

In Russia in the second half of the XIX century research schools, developing anthrax vaccines for agriculture were formed. Their level was highly competitive with the Western analogues. At the end of the 1930 Soviet military scientists were the first in the world to create the anthrax vaccine for medical use on the basis of the spores of unencapsulated live strains of B. anthracis. In the 1940-1960-ies Russian scientists determined the principles of the development of anthrax vaccines, which allowed to avoid failures of their Western colleagues, when developing the vaccines capable of protecting the population from biological weapons with anthrax spores as the harmful agent. Russian military scientists in the 1990s managed to keep the vaccine strains of anthrax bacteria and restore the technological capability for their manufacture, which helps to protect the population of the Russian Federation from natural anthrax outbreaks and from biological terror. At a time when there is a need in creating immunity in humans against infection caused by inhalation of anthrax spores, the most reasonable decision for the upcoming decades is to use the domestic combined vaccine that combines unencapsulated live strains of Bacillus anthracis and anthrax toxin protective antigen.

The problems of quality and safety products derived from human blood plasma and hyperimmune animal sera as well as recombinant blood products resolved strict government regulation of their production processes. The risk of implications is minimized by plasma fractionation and purification of a specific drugs from various impurities (immunoglobulin aggregates, protease, plasmin, plasminogen, prekallikrein activator, IgA and IgM etc.). Viral safety is achieved by multi-step manufacturing process that includes at least two independent methods (treatment with solvent/detergent + incubation at low pH or pasteurization, combined with polyethylene glycol processing). It was justified that for today the technological process of the development of plasma preparations and hyperimmune animal sera has reached its limit. Their further development is the most likely to refer to specific improvements. The improvements will relate to increasing the efficiency of manufacturing technologies and methods of clinical use (preparations for subcutaneous administration, combinations of different immunoglobulin preparations, etc.), viral safety, ways to eliminate component, that were previously not considered to be able to influence the outcome of clinical use (soluble molecules CD4, CD8, HLA, thrombin, trace amounts of blood clotting factors VIII, IX, X, XI, XII etc.). At the same time new genetic engineered preparations with well-characterized molecular composition and a high selectivity for target impact are expected to appear on the market because of these unsolved issues. These are recombinant blood factors with altered properties; cocktails of recombinant antibodies and Fab-fragments of IgG, highly affine for toxin epitopes, etc. Therefore, in the upcoming years it is necessary to create in Russia a new system for assessing the quality, efficacy and safety of blood products, taking into account the future course of their development.

Typing of bacterial populations and identification of microorganisms are the overriding priorities in the field of prophylaxis, diagnosis and treatment of bacterial infections. Over the recent two decades rapidly developing approaches to molecular typing of bacterial populations have become an essential tool for the mentioned task. The information encoded in nucleic acids is more complete as compared to other characteristics of an organism. It can be obtained by either direct determination of genome sequences or by using a variety of techniques, that allow to indirectly assess the nucleotide composition in a representative genome locus of a microorganism under study. The article provides an overview of the literature data on direct and indirect methods of determining nucleotide composition of DNA sequences in microorganisms (multiple locus sequence typing, multiple sequeneed sites typing, denaturing gel electrophoresis, high-sensitivity melting curve analysis, DNA macro(micro)arrays).

Developing specific, effective and safe biomedical cell culture products (BCCP) assumes the following standard operating procedures: a) developing cell line with specific cell composition; b) obtaining a sufficient amount of cells of a specific cell line for therapeutic use as a part of BCCP; c) quality control and standardization of the developed cell line. Experts believe mesenchymal stromal cells (MSC) to be the first choice for the purpose of stem cell therapy. The development of a cell line with a specific cellular composition includes five sequential steps: selection of donors, sampling of biological material, cell isolation from donor material, preparation of standardized homogeneous population of cells, characterization of cell line composition of the cell line and its certification. The process of obtaining a sufficient amount of cells of a specific cell line for therapeutic use as a part of BCCP includes the selection of culture medium for cultivation of MSC, in special bioreactors for cultivation and culture control parameters. Quality control and standardization of the developed cell line assumes the elaboration of general requirements for quality parameters, characterizing all cell lines; as well as specific requirements for the assessment of therapeutic preparations in accordance with their proposed indications (for example, for the treatment of diabetes, bone grafting, restoration of blood supply to ischemic myocardium). The article describes typical procedures and identifies possible checkpoints for common technological methods used in BCCP manufacture and their quality assessment. It also outlines the requirements for the content of documents submitted by BCCP developer to an official authority in marketing authorization dossier as well as the expert evaluation procedure.

Представлен анализ современных технологических подходов к обеспечению специфической безопасности препаратов иммуноглобулинов и альбумина человека. Показана перспективность включения в технологию препаратов иммуноглобулинов человека скрининга доноров по групповой и резус-принадлежности, а также этапов хроматографической очистки с целью снижения содержания в них анти-А и анти-В гемагглютининов, анти-D антител. Рассмотрены технологические возможности снижения антикомплементарной активности и тромбогенного потенциала препаратов иммуноглобулинов, а также снижения содержания активатора прекалликреина как в препаратах иммуноглобулинов человека для внутривенного введения, так и в препаратах альбумина человека.

Genotyping efficiency depends on resolution of methods which provided by the target sequence of genome, efficiency of enzymes, primers and conditions of DNA restriction and PCR amplification. The most differential tools are methods involving the high evolutionary markers. They are methods MST, MLVA, DGE, HRM against to the method MLST based on the slow evolutionary marker. Methods PFGE, AFLP, DNA chips exploit the preliminary information of genome and have the better resolution than ribotyping and RFLP-PCR limited by certain loci. Monitoring of local outbreaks may be carried out using a rapidly evolving markers (methods RAPD-PCR, MST, or MLVA, DGE, HRM). The most suitable methods for prolongated epidemiological or population researches are MLST, PFGE, ribotyping and pyrosequencing analysis. Polymicrobial samples are investigated with DNA specific methods PCR-RFLP, MLVA, DGE, HRM, MLST, MST, DNA chips. The metagenomic analysis is the most informative to identify the species of bacterium from the polymicrobial sample. It is essential to use the conservative DNA sequences, for example 16S rRNA. The modern genotyping assays provide the informative and technological platform for phylogenetic studies of bacterial strains even through the several generations. The important results are figures of genetic distances between strains involving in complex of epidemiological characteristics: virulence, antibiotic resistance and the others. The computing tools for integration the phenotyping and sequencing data have developed. Whole-genome sequencing is the optimal method for bacterial genotyping. But there are several crucial restrictions of this method. It is still rather expensive and needs to operate with high quality DNA.

Documentation for standardization of the method for determining hemagglutinins (HAs) in human immunoglobulin preparations used in Russia is presented. The proposed general pharmacopoeial monograph "Determination of anti-A and anti-B hemagglutinins in medicinal preparations of human immunoglobulin" allows quality control of human immunoglobulin preparations for HAcontent using indirect hemagglutination under standard conditions. The positive and negative components of the domestic standard sample (SS) of HAs contain 1:32 anti-A and 1:16 anti-B, respectively; less than 1:2 anti-A and -B; and 1:64 components of the HA content limit. Introduction of the SS for contents of anti-A and anti-B HAs into pharmacopoeial practice would ensure that results obtained in various ranges are accurate and enable the quantitative contents of the corresponding HAs in human immunoglobulin preparations to be determined.

Pharmacopoeial analytical methods involving reference standards (RS) are used for manufacture and quality control of medicinal products, including biologicals, these RS should now to be called pharmacopoeal. The introduction of the mentioned term reflects special drug characteristics, which are regulated not by the State Union Standards but by the Russian State Pharmacopoeia in terms of quality control. Drug and RS special characteristics require the establishment of legal and methodological framework. The recommendations stated in ISO REMCO are general do not fully cover the special aspects of the certification of reference standards for each specific area. The documents of the Federal Agency for Technical Regulating and Metrology (Rosstandart) on reference standards can not be used for biological medicinal products due to their specificity as the test methods do not allow to separate systematic and random components of uncertainty of test results, as required by Rosstandart. The regulatory framework for biological RS should be developed on the basis of WHO and ICH Guidelines. The classification of drug reference standards and the list of priority documents required for the elaboration of normative and procedural framework regulating their development, certification, approval and use is considered in the article. The development of the documents for the mentioned system is exemplified by a new pattern for an RS certificate.

Modern technological approaches to ensuring the specific safety of human immunoglobulin and albumin preparations are analyzed. Introduction into human immunoglobulin preparation technology of donor screening for group- and rhesus-affiliations and chromatographic purification steps to reduce the contents of anti-A and anti-B hemagglutinins and anti-D antibodies are shown to be promising. Technological capabilities for reducing the anticomplementary activity and thrombogenic potential of immunoglobulin preparations and decreasing the contents of prekallikrein activator in human i.v. immunoglobulin and albumin preparations are discussed.