The urgency of the rabies problem for all mankind and the search for new ways of eradicating the disease entailed the creation of a new global initiative for rabies elimination ‒ «United Against Rabies» which sets a highly ambitious goal of achieving zero rabies human deaths by 2030. The many years of international experience in elimination of street dogs, which account for 99 % of rabies cases, did not produce the desired results, therefore the focus was shifted to mass vaccination of dogs (minimum 70 % of dog population). The rabies problem is complex and global, it requires efforts from all the parties involved as well as hefty investment. The paper presents the results of a continuous long-term analysis of the rabies situation in Russia and across the world, as well as analysis of the current state of vaccination against rabies which plays an important, if not crucial, role in prevention of rabies in humans who got bitten by infected animals. The paper formulates the main currently existing ways of solving the rabies problem, namely: mass vaccination of dogs; improvement of dosing schedules and administration routes of medicines against rabies; analysis of immunity development mechanisms in immunocompromised patients; progressive implementation of vaccination of people who got bitten by infected animals, and alternative administration routes; development of an express method of the neutralising antibody titer determination; raising public awareness about disease hazards.

European competent authorities began to elaborate scientific principles of development of non-innovator biotherapeutic (biosimilar) products in the early noughties, and in 2009 these principles were approved at the WHO International Conference in Seoul gathering participants from countries with a well-developed pharmaceutical industry. The USA adopted the law on biosimilar products in 2012, it was based on the documents and recommendations prepared by the EMA and approved by the WHO. In 2015, the FDA published the new revised versions of the guidelines dealing with biosimilar products. The US regulatory requirements for development and authorisation of biosimilar products are based on a step-by-step comparative assessment of biosimilar and innovator products in terms of their quality, efficacy, and safety in accordance with the WHO/EMA recommendations. At the same time the US regulatory requirements differ from those of other national authorities, including EMA (WHO), when it comes to the design of comparative quality studies (studies of products’ physicochemical and biological properties), the assignment of International Non-Proprietary Names and the interchangeability of biosimilar products.

The manufacturer (developer) has to prepare a specification for each newly developed biomedical cell product (BCP) that has passed the stage of preclinical studies. The specification is included into the registration dossier when applying for marketing authorisation of a BCP. In accordance with the Order of the Ministry of Health of the Russian Federation No. 14n of 19 January 2017 «On approval of the specification format for a biomedical cell product» the specification should contain information about authenticity of the cell line used in the BCP, namely: morphological characteristics, expression of specific markers, expression of specific genes, expression of specific proteins, as well as markers of cell line stability. At present Russia has no practical experience in BCP quality evaluation. The aim of the study was to substantiate methodological approaches to authentication of cell lines used in BCPs as illustrated by quality evaluation of the DF-2 model cell line using test methods that allow for characterisation of the morphological, genetic, immunophenotypic, and cytogenetic profile of the cell line. Materials and methods: the study analysed the DF-2 cell line — human dermal fibroblasts (mesenchymal stem cells) obtained from the Institute of Cytology of the Russian Academy of Sciences (St. Petersburg). The following analytical test methods were used in the study: morphological analysis; flow cytometry for immunophenotyping of the DF-2 model cell line; short tandem repeats for creating an allelic profile of the model cell line; cytogenetic analysis — differential DAPI staining of metaphase chromosomes. Results: the paper summarises methodological approaches to identification testing of medicines containing living human cells (BCP analogues) currently used in international practice, and presents the results of authentication of the model cell line. Conclusions: methods used for BCP identification testing should ensure unambiguous authentication of the cell line according to its specification. The study performed helped to work out the procedure of authentication of a model cell line.

Recombinant tissue plasminogen activator (international nonproprietary name — alteplase) which was developed by «GENERIUM» (Russia) and received a marketing authorisation in Russia is completely analogous to Actilyse® which is used to treat medical conditions accompanied by thrombosis, such as acute myocardial infarction, pulmonary embolism, and ischemic stroke. The aim of the study was to carry out a comprehensive comparison of physico-chemical and biological properties of Revelyse® and the reference product Actilyse® in order to assess their biosimilarity. Materials and Methods: comparative peptide mapping and determination of comparability of chromatographic profiles of tryptic hydrolysates was performed using RP-HPLC and massspectrometry; the molecular weight distribution was determined by mass-spectrometry and polyacrylamide gel electrophoresis (Laemmli method). The purity and homogeneity of products as well as the content of related impurities (oligomers and fragments) were determined using gel filtration; N-glycosylation profile was analysed by hydrophilic HPLC, total sialic acid was quantified by the Svennerholm resorcinol method. Protein binding to fibrin and human fibrinogen was assessed by surface plasmon resonance, and the specific activity was compared by fibrin clot lysis. Results: the research demonstrated a complete overlap of the products’ peptide maps, which indicates the identity of аlteplase amino acid sequences in the two medicines being compared. The authors of the study also determined the molecular weight and the content of the intact single-stranded form of the protein, and quantified post-translational modifications, the content of sialic acids and neutral sugars. The analysis of the N-glycosylation profile revealed insignificant differences in the percentage of multiantenna complex glycans. The specificity of alteplase was evaluated by analysing the formation of protein complexes with natural alteplase ligands – fibrin and plasminogen activator inhibitor-1, but no significant differences were found. The comparison of specific activation of plasminogen fibrinolytic activity was performed based on the results of the assay analysing the fibrin clot lysis rate, and it demonstrated comparability of Revelyse® and Actilyse®. Conclusions: comparative experimental studies have shown no differences in the structure, charge distribution heterogeneity, impurities content, and specific activity of alteplase as a component of Revelyse® and the reference product Actilyse®, which leads to the conclusion that they are similar in terms of physicochemical and biological properties.

To date, the technology of live plague vaccine production uses a combined method of concentrating microbial cells which consists of three operations with a total duration of 18 hours. The procedure of obtaining concentrate, which is used in the current vaccine production technology, has a number of disadvantages, namely: multiple operations, high energy consumption, long duration, and, as a consequence, low yield of concentrated suspension (0.04 l from 1 l of native culture). The aim of the study was to optimise the procedure of Yersinia pestis ЕV microbial cell concentration using the system for tangential flow microfiltration with the ASF-020 filter support unit. Materials and methods: the vaccine strain used in the study was Yersinia pestis ЕV derived from NIIEG cell line (the strain of the Research Institute of Epidemiology and Hygiene). Submerged cultivation of the native culture was performed using the BIOR-0.25 reactor. The content of live microbial cells was determined by cytorefractometry. Oxidative metabolism was assessed using the chronoamperometric method. Physico-chemical and immunobiological properties of the dry live plague vaccine were assessed according to the monograph FS.3.3.1.0022.15 of the State Pharmacopoeia of the Russian Federation, 14 edition. Results: the equipment’s design features made it possible to carry out membrane filtration of the microbial suspension using the BIOR-0.25 reactor as an intermediate storage unit, thereby excluding three technological stages. The total concentration of microbes in the suspension obtained by the routine and the optimised methods was not less than 120 billion microbial cells/ml. A comparative study of the effect of various hydrodynamic regimes in the working cavities of ASF-009 and ASF020 filter units did not significantly affect the morphometric and physiological properties of microbial cultures. Experimental data helped to determine the process mass balance of membrane filtration. The optimised technology gave 0.17 l yield of the concentrate from 1 l of native culture, and the process duration was reduced to 4 hours. Conclusions: the process of concentrating Y. pestis EV microbial cells during production of plague vaccines was optimised. A comparative study of morphometric and physiological properties of plague microbe cultures that was carried out during their concentration using the optimised technology did not reveal any significant differences as compared to the routine one.

According to the existing international practice, reference materials, which are used to assess specific activity of purified tuberculin products, are meant to be used for several decades and are therefore characterised by high stability. For instance, PPD-S tuberculin produced in the USA in 1944 has been used ever since as the international reference standard of purified tuberculin and is stored as lyophilisate in ampoules (PPDT) containing 5000 IU each. The Russian PPD-L-2 industry reference standard is made from material produced by the Leningrad Research Institute of Vaccines and Sera in 1973. It has been used for many years to control production batches of PPD-L purified tuberculin because of its high stability that is regularly confirmed in large-scale studies using various experimental models in accordance with the requirements of the World Health Organisation. The aim of the study was to evaluate the long-term stability of the substance of industry reference standard of purified tuberculin (PPD-L-2 IRS) by determining its specific activity relative to the international standard of purified tuberculin, and determine the feasibility of using this substance for the preparation of lyophilised reference product samples. Materials and methods: PPD-L-2 IRS specific activity was determined relative to the PPDT international standard in accordance with the procedure specified in the monograph FS.3.3.1.0023.15 of the State Pharmacopoeia of Russian Federation (14 edition) using guinea pigs vaccinated with various BCG substrains or sensitized by virulent mycobacteria («live» or «inactivated») in accordance with the recommendations of the WHO (WHO TRS 45, 1987). Results: the analysis of the obtained data showed that there were 3–4-fold differences in PPD-L-2 IRS relative potency depending on the BCG substrain used for guinea pigs vaccination (animal sensitization model). This effect of the titration model manifested itself when comparing specific activity of tuberculins obtained by various methods of tuberculoprotein precipitation (PPD-L-2 and PPDT), i.e. different in antigen composition. The specific activity of the previously established dose of PPD-L-2 IRS was shown to be equivalent to the international reference standard in animals sensitized with mycobacteria tuberculosis. Conclusions: the results of PPD-L-2 IRS specific activity assessment obtained in this study are consistent with the data obtained during development and certification of this reference material in the 1980s and confirm the long-term stability of the intermediate powder product of the Russian reference standard of purified tuberculin. At the same time, the production of a freeze-dried form of the IRS, in addition to being economically feasible, would rule out some potential errors that are inevitable during annual preparation and control of the reference standard dilutions, and would make it possible to spare the substance which would improve prospects for the future long-term use of PPD-L as an industry reference standard.