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Scientific relevance. The clinical effects and the expiration of patents for original (reference) biotechnological medicines based on monoclonal antibodies (mAbs) stimulated the development of biosimilar mAbs. The quality profile of a biosimilar mAb should correspond to the quality of the reference medicinal product. When demonstrating biosimilarity and determining the activity of medicines as part of batch quality control, analysts should study the biological properties of mAbs using suitable reference standards. The lack of international standards (ISs) makes mAb manufacturers use in-house reference standards. There is a risk of obtaining non-uniform quality and efficacy data because of the use of in-house reference standards, the heterogeneity and structural complexity of mAbs, and the relationship between the biological activity and efficacy of mAbs.Aim. This study aimed to analyse the relevance of and need for ISs for the biological activity of biotherapeutic mAbs and to define the role of reference medicinal products and ISs in assessing biosimilarity and testing medicines throughout their lifecycle.Discussion. This review covers the issues arising from the lack of ISs for assessing the biological activity of mAbs and the role and significance of reference products and ISs for biosimilars. The authors describe the specifics of studying the biological properties of mAbs and summarise the data on the need to develop and use ISs for the standardisation of biological tests. This review presents the results of studies on the first ISs established by the World Health Organisation to assess the biological activity of mAbs; these results suggest the need to standardise mAbs using ISs to ensure the quality, safety, and efficacy of mAb therapy.Conclusions. The use of ISs for mAbs plays a key role in harmonising biological activity assessments. Publicly available ISs serve as primary standards for the calibration of secondary reference materials. Moreover, ISs are required for the harmonisation of activity evaluation (in IU) between laboratories and for the consistency of the activity of various medicinal products from different manufacturers that share the same INN. The use of ISs by mAb manufacturers will contribute to ensuring the quality of mAbs and clinical monitoring of the effectiveness of their use.

One of priority issues of the present-day healthcare system is development of new vaccines and improvement of existing ones due to decreasing immunocompetence of the population, emergence of new infections and reemergence of old ones which were previously thought to be under control. Adjuvants have proven to be integral and important components of modern vaccines, as they enhance immune response to the vaccine antigen. However, despite a lot of effort put into their development, only a small number of adjuvants are currently used in clinical practice. The aim of the study was to systematise literature data on the adjuvants’ mechanisms of action, their specific structure, composition, and stimulation effects that mediate their immunoadjuvant properties. The paper summarises data on adjuvants used as components in licensed vaccines, describes their characteristics, analyses molecular mechanisms of their action in order to establish correlation between their structure and activity, which is important for the development of more efficacious and safe adjuvants. The paper cites advanced developments aimed at enhancing stimulation effects of existing adjuvants. It concludes by stating that the key research area aimed at improving vaccination efficacy is the study of mechanisms that contribute to the development of effective protection against infectious agents, as well as analysis of how to use adjuvants to stimulate the body’s defensive mechanisms, primarily by impacting the innate immunity.

Assessment of specific activity of Russian and foreign-made filgrastim products by biological in vitro methods is performed using different types of dyes. It is important to choose one cell staining dye in order to align the procedure of filgrastim specific activity assessment using cell culture. The aim of this study was to perform comparative assessment of tetrazolium and resazurin dyes in tests determining filgrastim ability to activate proliferation of sensitive cells. Materials and methods: NFS-60 (mouse myelogenous leukemia) cell line, 2nd International Standard for Granulocyte Colony Stimulating Factor (IS), as well as МТТ, MTS, WST-1, and alamarBlue dyes were used in the study. Proliferative activity of cells was assessed in vitro. The level of cell proliferation was assessed by fluorescence or absorbance intensity. Origin Pro 9.1. and Microsoft Excel applications were used for statistical processing of the obtained results. Results: the paper compares characteristics of the most widely used dyes. It describes the procedure for choosing optimal test conditions for some of the studied dyes. The authors analysed the potential of some factors, such as duration of cell suspension incubation with IS and with a dye, composition of the lysis buffer (for MTT staining), and different readout modes, to influence the final results. Despite the fact that all the studied dyes gave reproducible dose–response curves under the given test conditions, 50% effective concentrations showed no statistically significant differences in tests with only three dyes: МТТ, MTS, and alamarBlue (р > 0.05). Conclusions: better reproducibility of results was obtained in tests using МТТ and alamarBlue. The test procedure using alamarBlue is easier to perform and less time-consuming, it does not include the cell lysis stage and does not require additional reagents, therefore this dye may be recommended for harmonisation of the test procedure to be elaborated for the Russian Pharmacopoeia.

The review is devoted to specific aspects of the development of post-vaccination immunity following immunisation with different types of antiviral vaccines, as well as to ways of increasing immunogenicity of vaccines and effectiveness of preventive vaccination. Vaccines containing highly purified and recombinant antigens obtained using modern technologies have lower reactogenicity and a higher safety profile, but are less immunogenic compared to live vaccines. Effective vaccines have not been developed for many viral infections yet. Therefore, it is critical to search for ways to enhance immunogenic properties of vaccines in order to increase the efficiency of vaccination, and to develop new vaccine formulations that provide reliable protection of the body against infection. The aim of the paper was to analyse specific aspects of immune response development following immunisation with antiviral vaccines, and approaches to increasing their immunogenicity using adjuvants. It reviews different types of antiviral vaccines, as well as specific aspects of immune response development depending on the nature of a specific antigen. The paper substantiates the use of adjuvants to enhance and regulate the induced immune response. It analyses mechanisms that determine the stimulating effect of adjuvants and summarises data on the adjuvants used in the licensed vaccines for human use. The authors highlight the need for further research to increase the efficiency of vaccination and suggest that one of potential solutions is the use of adjuvants based on recombinant human cytokines.

Biotechnological products, like all other medicinal products, have to comply with efficacy, safety and quality requirements. Quality evaluation of medicines includes assessment of test methods used to control medicinal product quality (described in product specification files provided by the manufacturer), laboratory testing of samples using these methods, as well as assessment of the registration dossier materials, including materials on test method validation included into the product specification files. One of the most important quality parameters of biotechnological products is biological activity, i.e. specific ability of a product to induce a desired biological effect. The article presents the results of a detailed analysis of methods used for determination of biological (specific) activity that are described in product specification files of various biotechnological products. The aim of the study was to demonstrate the importance of proper presentation of methods used for assessment of biological (specific) activity of biotechnological products and familiarise specialists engaged in elaboration of product specification files with the principles of presenting data in the «Biological (specific) safety» section. The analysis of documentation helped summarise the most common mistakes and omissions, formulate general recommendations concerning the description of methods, develop a general structure of the «Biological (specific) safety» section with detailed guidance on what to include in each of the subsections. Rationalisation of information presented in this part of the product specification files will help reduce the number of expert body’s requests for additional information/documents and will help ensure that laboratory testing is performed at a high professional level and within a prescribed period of time.

The review looks into various aspects of assessing specific activity of biotechnological products, which is one of their key quality parameters. Approaches to the analysis of this parameter and the choice of test procedures are governed by the nature and characteristics of a medicinal product. Test procedures should be adequate and have sufficient sensitivity and specificity. Specific activity of the products in question can be assessed by biological methods both in vivo using laboratory animals, which demonstrate the most adequate response to the tested product, and in vitro using sensitive cell lines. Assessment of specific biological activity helps to characterize the product’s pharmacological action and systematically examine the mechanisms of therapeutic effects in clinical practice. Therefore, specific activity of biotechnological products should be assessed using methods appropriate for the proposed mechanism of action. Many biotechnological products, such as cytokine system products, mAbs, fusion proteins and some others call for individual methods for assessment of their specific activity. Biotechnological products are successfully used in the treatment of autoimmune, infectious, oncological, and allergic diseases.

The paper presents the data on safety and efficacy assessment of adjuvants and vaccines with adjuvants at the stages of development and preclinical studies based on international recommendations. The presence of an adjuvant in vaccine content ensures higher expressing and prolonged specific immunity. The origin and nature of adjuvants currently used for various vaccines are different. Adjuvants can be classified by origin, mechanism of action, physical and chemical properties. The finished vaccine may contain one or more adjuvants which can be intended for one antigen or a number of antigens in vaccine content. Antigen-adjuvant combination is a key part in the production of a vaccine with an adjuvant. Due to the diversity of adjuvant nature and their physical and chemical properties and mechanisms of action, as well as to antigens in adjuvant vaccines content, it is rather complicated to plan and to conduct preclinical studies. The present article describes various aspects of preclinical studies of adjuvants and vaccines with adjuvants based on the analysis of current guidelines and requirements.

The article describes general principles of evidence-based quality assessment research of a recombinant granulocyte colony stimulating factor preparation (G-CSF) under development as well as a confirmation of its similarity to reference preparation (authorized original preparation). Since the quality of biotech preparations is determined by the manufacturing process, when developing a biosimilar one should focus on the manufacturing process details, starting from the selection of the expression system, the composition of excipients, on to the methods of isolation and purification of recombinant protein. Recombinant protein should be characterized in more details, than the quality parameters of the original preparation, included in the specification of the substance or the preparation. Comparative studies include characterization of the active substance and the assessment of the quality of the finished product. The reference preparation in the development of a biosimilar G-CSF

The article provides with the information on certification of an industrial reference standard (IRS) for determination of filgrastim activity. In order to confirm the quality of the IRS candidate, the following tests have been performed: biological activity, description, identification, clarity, colority, sterility, pH, foreign impurities, bacterial endotoxins, as well as the assay of: Filgrastim, Acetate ion, Polysorbate 80, residual Host Cell Proteins, residual Host Cell DNA. The results fully meet the requirements for filgrastim samples. The reference standard is certified for biological activity. The purpose of the IRS for determination of filgrastim activity is the assessment of acceptability of the results of biological activity tests in the quality control of substances and drugs based on filgrastim. Biological activity of the IRS candidate has been assessed by interlaboratory studies as compared against the second international standard NIBSC-09/136. The certified value for «Biological activity» of the IRS for determination of filgrastim activity has been set as 32.2±5.35 million IU/ml. The shelf-life under the storage conditions of 2 to 8oC has been set as not less than 2 years. The results of long-term stability studies the IRS confirm its stability during the monitored period.

One of the most complicated issues of modern medicine is diagnosis, treatment and prevention of rare diseases. In the structure of rare diseases the vast majority accounts for genetically determined disorders. The introduction of genetic testing methods helped to identify a lot of hereditary diseases, that currently reach the number of 8,000 cases and according to estimations the frequency of their detection is about to increase. Medicinal products for diagnosis or treatment of rare diseases form the group of orphan drugs. Biologicals are the most effective medicinal products in the whole group, due to their ability to bind specifically with the «target receptor», which allows to use them as targeted drugs. Another advantage of biologicals, compared with preparations of chemical origin, is their low toxicity. Orphan drug provision is the most severe issue because the treatment of severe forms of rare diseases may require the prescription of an expensive drug. Manufacturing of orphan drugs may be unprofitable for the reason of the low prevalence of rare diseases. Therefore, the availability of orphan drugs for patients depends on the amount of state participation in manufacture stimulation. In various countries regulatory requirements for the recognition of a disease as a rare disease, and marketing authorization procedures for orphan drugs are developed with due regard to the social and economic characteristics of the country. The article presents the analysis of the regulatory requirements for various countries for the detection of rare diseases, and a common approach to marketing authorization procedure for the drugs used in treatment of rare diseases, exemplified by biological orphan preparations.

The article provides the information on issues related to the clinical use of preparations of monoclonal antibodies (MAb), and the prospects for developing preparations based on modified MAbs. MAb preparations based on recombinant proteins show the consistency of physical and chemical properties as well as high specificity. New class of preparations based on MAbs show great potential of targeted therapeutic influence on important pathogenetic mechanisms of disease development. It has proved to be successful against severe chronic diseases such as autoimmune cancer, infectious and allergic diseases, as well as in transplantation for the treatment and prevention of transplant rejection. The development and manufacture of MAb preparations based on a whole immunoglobulin molecule, as well as preparations of modified antibodies with a specific set of functions associated with individual structural elements of the molecule is now possible due to genetic engineering and transgenic animals technologies. Preparations based on modified MAb can exist in

The review deals with the issues related to the special aspects of the development of the first available similar biopharmaceuticals/biological analogues («biosimilars») based on monoclonal antibodies. In June 2013 the Committee for Medicinal Products for Human Use (CHMP) of the European Medicines Agency (EMA) approved for licensing Remsima and Inflectra which are biosimilars of the brand-name product Remicade® (infliximab). The review describes the general principles of the development of the mentioned biosimilars. It highlights the features of the quality assessment studies, including characterization of the physical and chemical properties, specific biological activity, as well as comparative preclinical and clinical trials confirming the similarity of the candidate and the brand-name (reference) product. The review provides with the analysis of the results of comparative studies to assess the clinical relevance of the differences detected at the stage of quality assessment. The data substantiating the possibility of extrapolating the results obtained in clinical trials, against the approved standards for the brand-name product is provided.

Biological preparations differ substantially from low molecular weight chemicals in molecular weight, multidimensional structure of an active substance, presence of post-translational modifications, composition of impurities and excipients, drug manufacturing process, etc. These specific features of biological preparations also influence their pharmacokinetic properties. The assessment of pharmacokinetic properties is essential not only for the confirmation of the efficacy of a biological preparation, but it is also key aspect of demonstrating the similarity/analogy of a biosimilar preparation under development and the reference (original) preparation. High molecular weight biological preparations when administered subcutaneously and intramusculary initially migrate from the injection site into lymphatic vessels and then to blood. Pharmacokinetic properties of drugs with Fc region of immunoglobulin, are affected by their ability to interact with Fcy and FcRn receptors. Almost all biological preparations possess immunogenicity, the appearance of antibodies to a drug causes changes not only in pharmacodynamic but also in pharmacokinetic properties of biological products. The assessment of pharmacokinetic properties is a complicated and time-consuming process, for example, the study of drug distribution may in some cases require the use of a radioactive label.

Preparations of recombinant human erythropoietin (rhEPO) are included in the list of vital and essential drugs for medical use. When evaluating the quality of EPO preparations during the manufacturing process, under the state marketing authorization and certification procedures, in order to confirm their quality in terms of assay (specific activity), identification, dimer and high-molecular related substances content, sialic acids, it is required to use erythropoietin reference standard. The present article describes various stages of the development of erythropoietin reference standards. It provides the comparative description of the existing methods for evaluating the quality of erythropoietin preparations using reference standards. The necessity of the development and validation of national erythropoietin reference standard is justified.

Rationale. The task of import substitution in health care and the development of normative legal acts in the framework of the Eurasian Economic Community raises the questions of harmonization of national requirements with international ones, including the standardization of drugs. Thereby, the assessment of the quality of domestic recombinant human erythropoietin in accordance with European Pharmacopoeia requirements is very actual. The main indicator of drug quality is specific activity of drug (rhepo). In Russian Federation it is determined by the influence of the drug on erythropoiesis stimulation. This effect of erythropoietin is measured by counting the number of reticulocytes using the flow cytometry and light microscopy. The latter method of calculation is not included in European Pharmacopoeia. Adequate assessment of this indicator is required for the development of national requirements for quality control of drugs rhepo harmonized with European Pharmacopoeia. Purpose of the work. The study aimed the comparative evaluation of the accuracy of the specific erythropoietin activity determination method using the flow cytometry and light microscopy data to provide the harmonization of the draft General monograph with European Pharmacopoeia. Research methods. The erythropoietin specific activity was determined in vivo in normocythaemic mice using the flow cytometry in accordance with the European Pharmacopoeia and light microscopy. We have compared the effects of two independent dilution series of erythropoietin European Pharmacopoeia standard sample. Results. The results of specific erythropoietin activity determination obtained by two different methods of measurement differed in the offset from the reference value (certified value). In flow cytometry data, the offset varied between 1-8%, the results of light microscopy showed higher variability ranging from 4 to 31%. The estimation of intermediate precision showed twice lower dispersion of specific erythropoietin activity measurements obtained by flow cytometry in comparison with light immersion microscopy data. Conclusion. Specific erythropoietin drug activity determination with flow cytometry is characterized by high accuracy and meets the European Pharmacopoeia requirements. We have considered the possibility of light microscopy usage for the same task in the absence of a flow cytometer. The usage of light microscopy requires increasing its accuracy in order to harmonize the method with European Pharmacopoeia requirements.

Analysis of the publications devoted to the structure, functions, mechanism of action of erythropoietin is given in the article. Erythropoietin preparations derived from recombinant DNA technology are a mixture of isoforms with different biological activity, which determine the biological properties pharmacological activity, pharmacokinetics, efficacy and safety of medicinal product. Erythropoietin preparations derived by using recombinant DNA technology are a mixture of isoforms with different biological activity, that determine the biological properties, pharmacological activity, pharmacokinetics, safety and therapeutic efficacy of the drug. However, at production of erythropoietin, its pharmacological activity is controlled only by the ability to stimulate erythropoiesis, despite the multiplicity of different directions of action of drugs erythropoietin. The drug is dispensed and applied on this indicator. The international reference standard, a European reference biologic drug or calibrated by him enterprise standard samples are used by manufacturers to assess the quality of erythropoietin in the Russian Federation. The urgency of developing domestic standard samples for the evaluation of biological activity and physico-chemical characteristics of erythropoietin preparations produced by recombinant DNA technology.

The anodic synthesis of a poly-o-phenylenediamine film at gold, platinum, and ITO electrodes and peculiarities of its doping–undoping are studied by cyclic voltammetry. Absorption spectrum of undoped films is obtained in visible and near-IR light. [ABSTRACT FROM AUTHOR]

Spectroelectrochemical properties of poly-o-phenylenediamine (PPD) and the synthesized composite of PPD and polyaniline—two chemically related polymers containing an amino-substituted benzene ring but having different conduction nature—are studied. The polyaniline synthesis on PPD-modified electrodes involves stages of the reaction initiation, the copolymer formation, and the formation of a polyaniline layer at the copolymer/solution interface. [ABSTRACT FROM AUTHOR]

A voltabsorptometry study of the redox behavior of poly(3,4-ethylenedioxythiophene) films in tetrabutylammonium tetrafluoroborate solutions in acetonitrile shows the polymer to exhibit heterogeneous properties when both reduced and oxidized. The measurements are taken at characteristic wavelengths λ selected on the basis of stationary absorption spectra. The reduced polymer's heterogeneity is explained under the assumption that the polymer contains simultaneously linear (λ = 530 nm) and spiral (λ = 660 nm) structures. The formation of two oxidized polymer states is proved experimentally. Both states presumably result from strong (λ = 880 nm) and weak (λ = 1050 nm) interactions between the anion and the positively charged polymer chain. The oxidized 880-nm state is shown to arise from the 660-nm state, whereas the 1050-nm state forms of the 530-nm state. [ABSTRACT FROM AUTHOR]

The sorption and hydrophilic properties of composite membranes PAN/MF-4SK, obtained by a chemical template synthesis, are studied. The synthesis is performed in static and dynamic conditions for perfluorinated sulfo-cationite membranes MF-4SK in 0.01 M aniline solutions in 0.5 M H2SO4 and 0.01 M FeCl3 in 0.5 M H2SO4. The aniline sorption and polymerization kinetics is studied by conductimetry, radioisotope, and optical methods. The polymerization rate is found to depend on the oxidant concentration and the sequence of the membrane saturation with aniline. An increase in the intensity of the color of a composite film, which depends on the balance between the quinoimine, amine, and radical-cation fragments in the perfluorinated matrix, does not lead to any substantial changes in the moisture content or the ac electroconductivity. Implanting polyaniline chains into the perfluorinated matrix presumably leads to a molecular reorganization of water at the expense of a change in its association at the point where redox fragments of polyaniline converge with side segments of the template matrix. [ABSTRACT FROM AUTHOR]

The redox behavior of polybithiophene in 0.1 M Bu4NBF4 solutions in acetonitrile is studied using cyclic voltammetry, potentiostatic spectroelectrochemical measurements, and differential cyclic voltabsorptometry. Comparing differential cyclic voltabsorptograms, obtained at wavelengths of 420, 475, and 520 nm for the undoped state and 720, 850, and 1050 nm for the doped state, with the cyclic voltammetry data reveals the polymer heterogeneity. The heterogeneity of the undoped polymer is ascribed to the formation of polymer chains of different lengths during the polybithiophene synthesis. The heterogeneity of the doped polymer is attributed to the existence of two types of an oxidized state. Both states presumably form as the result of the occurrence of two parallel processes. The first oxidized state is probably connected with the formation of a strong complex (associate) of positively charged fragments of a polymer chain with the dopant anion, while the second oxidized state is caused by a weak interaction between the dopant and the doped polymer. [ABSTRACT FROM AUTHOR]

A cyclic voltammetry study shows the anodic polymerization of aniline in 2 M HClO4 water–acetonitrile solutions to proceed via an EC: EC mechanism. In the chemical reaction, pernigraniline catalyzes the aniline oxidation, recovering from emeraldin in the second stage of the electrochemical process. [ABSTRACT FROM AUTHOR]