Scientific relevance. To date, multiple approaches to genome editing have been developed based on different genome-editing systems (GESs) and genome modifications that result in single- or double-strand DNA breaks, either in vivo or ex vivo, followed by homologous recombination or non-homologous end joining to restore the sequence. However, the use of GESs is associated with a number of potential risks arising from the complex biology of such medicinal products and the fundamental role of their target, i.e. the DNA molecule.

Aim. This study analysed the most relevant trends and risks associated with medicinal products based on genome editing, the ways taken to overcome these risks, and the research methods used to identify and control the development of undesirable effects.

According to the literature, the adverse effects of GESs may arise both from the methods used to deliver GES components into the cell and from the functional activity of the GES itself, which includes insufficient on-target or undesirable off-target effects. This review indicates the main risks associated with the use of GESs. Preferable strategies to mitigate the risks of using GESs include repairing DNA breaks by homologous recombination, selecting GESs and related endonucleases that have greater specificity and restriction accuracy, increasing guide RNA specificity (for CRISPR/Cas), correcting the activity of the system regulating the cell cycle and apoptosis in a controlled manner, regulating the duration of expression and persistence of GES components in cells, etc.

Conclusions. The requirement to include quality, efficacy, and safety data when submitting registration dossiers for advanced therapy medicinal products prompts the discussion of the main risks associated with such products.

Scientific relevance. Botulinum toxin preparations are a good example of using a deadly toxin as a unique therapeutic agent. However, there are many unresolved issues related to biotechnology, biological activity, interchangeability, and standardisation of botulinum toxin preparations.

Aim. To review current opportunities for improving therapeutic botulinum toxin preparations.

Discussion. This review covers botulinum toxin type A preparations and unresolved issues related to them. In the absence of international non-proprietary names recommended by the World Health Organisation or by the Board of the Eurasian Economic Commission, domestic and imported botulinum toxin type A preparations approved in Russia have only similarity-based grouping names. In addition, manufacturers name botulinum toxin preparations at their discretion. Therefore, classifying these preparations under a common nomenclature is essential for clear identification, adequate selection, and correct prescription. Several studies have shown significant variability across botulinum toxin type A preparations. Due to the identified differences in qualitative and quantitative characteristics, botulinum toxin type A preparations cannot be considered similar, which raises the issue of their interchangeability and bioequivalence. To resolve this issue, a unified classification and naming system for botulinum toxin preparations should be established and documented in regulatory standards. According to the literature, manufacturers of botulinum toxin preparations use in-house reference standards. Hence, the same activity unit resulting from toxicity and efficacy studies may express a different protein load for each botulinum toxin preparation. Keeping that in mind, the authors discuss the development of a single international potency standard for existing and pipeline botulinum toxin type A preparations.

Conclusions. The article describes novel pharmaceutical compositions containing botulinum toxin, including those in late development. Summarised data from clinical studies on the safety, efficacy, and cost-effectiveness of botulinum toxin type A preparations can guide prescribing decisions.

Scientific relevance. Intranasal vaccination may dramatically change current approaches to mass immunisation against various infections, shifting the focus from inducing systemic humoral and cellular immune responses to developing mucosal immunity and protective barriers on the mucous membranes, which are entry points for pathogens. Therefore, the safety of switching from intramuscular to intranasal vaccination should be evaluated.

Aim. This study aimed to compare the safety of intranasal and intramuscular vaccination using the intermediate results of the phase III VCI-COV-III clinical trial in healthy volunteers.

Materials and methods. The evaluation of the safety profile was based on the adverse events and adverse drug reactions (ADRs) documented by investigators in the interim report on the randomised, double-blind clinical trial of the Salnavac intranasal vaccine (InV) and the Gam-COVID-Vac intramuscular vaccine (ImV) against coronavirus infection. The report covered 42 days of observation in 137 healthy volunteers.

Results. ADRs were reported for 17/68 (25%) and 30/69 (43.5%) volunteers in the InV group and the ImV group, respectively (p=0.036). The ADRs reported for the InV group were systemic and local, short-term, mostly mild, and generally consistent in incidence and nature with the ADRs typically observed with other nasal products.

Conclusions. According to the preliminary data obtained in the clinical trial, the InV demonstrates a high level of safety. Its safety profile is comparable with those of other intranasal and intramuscular vaccines for the prevention of coronavirus infection.

Scientific relevance. Innate immune activation in the early phases of COVID-19 infection and subsequent interferon induction may help control viral replication and protect cells not yet infected with SARS-CoV-2. Thus, immunostimulants that induce interferon (IFN), including double-stranded RNA-based agents, are a promising means of post-exposure prophylaxis and treatment of COVID-19 at early stages.

Aim. The study evaluated the in vitro antiviral activity of a double-stranded RNA sodium salt-based medicinal product against SARS-CoV-2.

Materials and methods. The authors analysed the double-stranded RNA sodium salt-based medicinal product RADAMIN^®VIRO using Vero cells and the Delta variant of SARS-CoV-2 (B.1.617). The virus titre was calculated as the tissue cytopathic dose that caused 50% cell death. The authors measured the content of IFN-α and IFN-γ in the culture fluid by enzyme immunoassay and assessed the viral load by real-time polymerase chain reaction (using the cycle threshold value) and by titration (using Vero cells).

Results. The studied double-stranded RNA sodium salt-based medicinal product at a concentration of 250 or 500 μg/mL induced IFN-α and IFN-γ expression by Vero cells, thus increasing their resistance to SARS-CoV-2. The authors evaluated the antiviral activity of the medicinal product based on the virus titre, viral load, and cell monolayer damage. The antiviral activity became clear 24 h after treatment, which confirmed the ability of the medicinal product to inhibit the replication of the SARS-CoV-2 virus in vitro as early as the first day after infection.

Conclusions. The double-stranded RNA sodium salt-based medicinal product induced IFN-α and IFN-γ synthesis in Vero cells, increasing their resistance to SARS-CoV-2 infection in vitro. These results demonstrate the immunomodulatory and antiviral potential of the medicinal product.

Scientific relevance. Anti-hepatitis B virus surface-antigen (HBsAg) immunoglobulins are used to prevent hepatitis B in adults and children after exposure and to treat mild to moderate acute viral hepatitis B. The clinical effectiveness of human immunoglobulin preparations is determined by their potency, which is assessed by the content of antibodies to hepatitis B virus surface antigen (anti-HBs antibodies). Currently, this assessment involves using immunoassay techniques, such as enzyme-linked immunosorbent assay (ELISA).

Aim. This study examined several mathematical methods for analysing the experimental data obtained in ELISA-based anti-HBs antibody potency assays of human immunoglobulin preparations.

Materials and methods. This study used the international standard for human anti-HBs immunoglobulin, two immunoglobulin preparations, and an ELISA test kit for the detection and quantification of anti-HBs antibodies in serum or plasma samples.

Results. Using sandwich ELISA, the authors ascertained that the measured anti-HBs antibody concentration depended on the choice of calibration curve calculation method (i.e. manual analysis, parallel-line analysis using PARALINE software, linear regression, and 4-parameter logistic regression). The measured anti-HBs antibody concentrations varied by ± 19 IU/mL. According to the study results, an incorrectly selected method of data analysis can lead to an erroneous calculation of the analyte potency (concentration) in the test sample.

Conclusions. The study demonstrated the need for improved mathematical methods for the evaluation of experimental data used to determine the anti-HBs antibody concentration in human immunoglobulin preparations. It is essential to switch from manual to automated calculation (for example, using a 4-parameter logistic model), taking into account the requirements for bioanalytical methods and the capabilities of the available equipment.

Scientific relevance. The quality of medicinal products, particularly vaccines, is contingent on the stability of the manufacturing process at all stages, which can be evaluated using Shewhart charts for data obtained by monitoring the quality attributes of interest.

Aim. This study evaluated the stability of the quality and manufacturing processes of the BCG and BCG-M tuberculosis vaccines using Shewhart charts.

Materials and methods. This study focused on samples of the BCG tuberculosis vaccine and the BCG-M tuberculosis vaccine, a less reactogenic alternative for primary immunisation. Both vaccines were released to the market in 2019–2022. The quality of samples was assessed for stability based on their potency and total bacterial count, which are the key parameters for immunogenicity evaluation. These quality parameters were compared using test results submitted by the manufacturer and obtained at the testing centre. The authors plotted individuals charts (X-charts) and moving range charts (R-charts) in accordance with national standards GOST R 50779.42-99 and GOST R ISO 7870-2-2015.

Results. The quality of the BCG and BCG-M vaccines remained stable during the entire follow-up period (2019–2022). For some periods, the retrospective analysis of R- and X-charts revealed characteristic trends meeting special cause criteria. The Pearson correlation coefficient (r) between the data submitted by the manufacturer and the data obtained at the testing centre ranged from 0.2 to 0.8.

Conclusions. The Shewhart charts demonstrated that the quality parameters of the BCG and BCG-M tuberculosis vaccines tested in 2019–2022 were stable. These vaccines had stable manufacturing processes, as shown by the R- and X-charts. However, the warning signs indicated that additional measures should be taken to standardise the manufacturing processes. The findings suggest that Shewhart charts may be recommended for monitoring the production and quality of tuberculosis vaccines.

Scientific relevance. Currently, two types of methods are used to evaluate the potency of diphtheria and tetanus toxoids: the gold standard, which involves administering toxins to immunised animals, and serological methods, which involve quantifying protective antibodies in the serum of immunised animals. International validation studies of serological methods for assessing the potency of diphtheria and tetanus toxoids have resulted in revisions to the relevant chapters of the WHO Manual for Quality Control of Diphtheria, Tetanus and Pertussis Vaccines, as well as the European, Japanese, and several other pharmacopoeias. Consequently, some diphtheria and tetanus vaccine manufacturers have substituted the potency evaluation methods that require administering toxins to animals with alternative serological methods.

Aim. The study aimed to assess the suitability, reproducibility, and feasibility of an alternative enzyme immunoassay method (ELISA) for assessing the potency of diphtheria and tetanus components of diphtheria, tetanus, and whole-cell pertussis (DTwP) vaccines and to determine the possibility of implementing this method in Russia using standards and reagents manufactured in the country.

Materials and methods. The study used combined vaccines for diphtheria prophylaxis, diphtheria toxin, and reference vaccines. The potency of diphtheria and tetanus toxoids was determined in guinea pigs and mice by the pharmacopoeial lethal challenge method and an alternative ELISA method.

Results. ELISA and lethal challenge methods demonstrated comparable results of potency determination: 230 IU/mL vs 264 IU/mL (diphtheria toxoid), 188 IU/mL vs 160 IU/mL (tetanus toxoid), respectively. As opposed to the lethal challenge, the serological method allows testing the efficacy of several antigens in the same animals. The study showed the possibility of using purified diphtheria and tetanus toxoids manufactured in Russia as coating antigens. The authors obtained comparable antibody titres for each animal, using plates coated with international standards and Russian-made diphtheria and tetanus toxoids.

Conclusions. The authors demonstrated the possibility of using ELISA to determine the potency of diphtheria and tetanus toxoids in DTwP vaccines. Moreover, the study demonstrated the suitability of Russian purified diphtheria and tetanus toxoids as coating antigens. Researchers should continue working on enzyme immunoassay validation with a view to harmonising national and international methods for assessing the potency of diphtheria and tetanus toxoid vaccines, as these efforts will not only facilitate the registration of foreign vaccines in Russia but also accelerate the approval of Russian vaccines in other countries.

Scientific relevance. The content of Bordetella pertussis lipo-oligosaccharide (LOS) and the residual levels of active pertussis toxin (PT) are generally accepted to be the primary factors that determine the reactogenicity of whole-cell pertussis vaccines. To improve the quality of whole-cell pertussis vaccines, it is both relevant and necessary to study the relationship between the toxicity of B. pertussis bacterial cell components and the main quality parameters of these vaccines, including their potency and specific toxicity, as termed in the WHO recommendations and the European Pharmacopoeia.

Aim. This study aimed to analyse the effects of B. pertussis LOS and residual active PT on the specific toxicity and potency of adsorbed diphtheria, tetanus, and whole-cell pertussis (DTwP) vaccines.

Materials and methods. The authors tested 57 commercial batches of adsorbed DTwP vaccines for compliance with the regulatory standards and product specification files. Vaccine batches that failed specific toxicity tests formed Group 1, and the other batches were designated as Group 2. The potency was tested in F1 CBA/Ca×C57BL/6J hybrid mice with experimentally induced meningoencephalitis that were immunised with DTwP and reference vaccines. The authors assessed the specific toxicity of DTwP vaccines by changes in body weight following intraperitoneal administration. The toxic activity was assessed indirectly by changes in body weight in the first 16–24 h (B. pertussis LOS) and on day 7 (PT) after dosing. The authors used Spearman’s rank correlation coefficient to measure the strength of correlation between the toxic activity of vaccine components and the specific toxicity and potency of the vaccine, which were established using the same vaccine batches.

Results. The authors measured the toxic activity of LOS and residual active PT in the vaccine batches studied. The correlation coefficients between the specific toxicity and potency of the vaccines and the toxic activity of LOS were 0.113 (p>0.05) and 0.049 (p>0.05), respectively. Similarly, the correlation coefficients between the specific toxicity and potency of the vaccines and the toxic activity of PT accounted for 0.595 (p<0.01) and –0.534 (p<0.01), respectively.

Conclusions. The authors studied the toxic activity of B. pertussis LOS and residual active PT in whole-cell pertussis vaccines and found an inverse correlation between the potency of the vaccines and the toxic activity of residual active PT. The study demonstrated that the specific toxicity test for whole-cell pertussis vaccines fails to detect and quantify B. pertussis LOS in the samples. The authors advise to determine the content of LOS in the B. pertussis strains intended for the production of whole-cell pertussis vaccines, which is not yet an accepted practice in the Russian Federation.

Scientific relevance. Lyophilisation is the preferred method at the National Collection of Pathogenic Microorganisms (NCPM) of the Scientific Centre for Expert Evaluation of Medicinal Products of the Ministry of Health of the Russian Federation. Lyophilisation is used to provide for high standards of test-strain deposition, storage, and transportation and to ensure that test strains maintain their properties. Successful lyophilisation requires conducting experiments to establish the key parameters and critical conditions of the process.

Aim. The study aimed to evaluate the effects that the speed and time of freezing, the time of drying, the fill volume of ampoules, and the density of cotton filters have on the quality of NCPM indicator microorganisms lyophilised in a manifold-type apparatus.

Materials and methods. Pseudomonas aeruginosa NCTC 12924, Staphylococcus aureus NCTC 10788, and Salmonella Abony NCTC 6017 were freeze-dried using a manifold-type apparatus (M. S. R. 18, Usifroid). The authors used a low-temperature freezer at –70±2 °C for slow freezing and a mixture of dry ice and alcohol for quick freezing. The statistical analysis was performed using Microsoft Excel and Statistica 10.

Results. The minimum time needed for freezing the samples in a low-temperature freezer at –70±2 °C was 4 hours. Further storage at this temperature for up to 1 month was shown possible without compromising the quality of the final product. The time needed for freezing the samples in a mixture of dry ice and alcohol was under 1 minute. No differences in quality parameters were observed between the lyophilised samples frozen slowly or quickly, except for the cake appearance. Quick freezing resulted in cakes that were non-uniform, crumbled, and pulled away from the ampoule walls, which is considered undesirable. The primary drying stage for ampoules with a fill volume of 0.2 mL took 6–8 hours. The secondary drying stage of 11, 18, 35, and 59 hours resulted in comparable lyophilisate quality: the authors observed no statistically significant differences in viable cell counts (CFU/mL) at the end of lyophilisation and at the end of stress testing. The residual moisture content after 59-hour secondary drying was less than 2%. The cotton filter density had a critical influence on the lyophilisate quality. Therefore, the authors recommend using cotton filters weighing 50 mg or less.

Conclusions. The authors analysed the main stages of the lyophilisation process used for NCPM test strains and considered the effects that the speed and time of freezing, the time of drying, the fill volume of ampoules, and the density of cotton filters have on the quality of the final lyophilised product. The NCPM has implemented the results of this study in its work.