To date, the Bacillus Calmette–Guérin (BCG) vaccine has been the only medicinal product for active mass childhood immunisation against tuberculosis in the Russian Federation. Industrial-scale batches of the BCG vaccine are manufactured using a seed-lot system, which provides for producing the vaccine for civil circulation from a single batch of seed material, a lyophilisate of Mycobacterium bovis BCG. National and international documents touch upon the evaluation of BCG vaccine seed material in terms of its quality attributes in small separate sections containing brief descriptions and/or lists of attributes and control methods. It is relevant to bring together the information on receipt, certification, and storage of the inoculum (the seed lot) for production of the Russian BCG vaccine. The aim of the study was a comparative assessment of the main characteristics of and control methods for the inoculum of the Russian vaccine strain, M. bovis BCG-I, set out in the national and international requirements for BCG vaccines. The article summarises literature data on the history of BCG substrains and the variability of their characteristics and presents a brief account of the origin of the Russian BCG-I substrain. It considers the control methods specified in the national and international requirements for the inoculum for the BCG vaccine. The study demonstrated the practical possibility of identifying BCG down to the substrain level with subsequent determination of genetic properties that characterise genomic stability of the substrain. The article presents the results of the comparative analysis of data on stability of lyophilisates of M. bovis BCG-I seed lots (Russia). Particular attention is paid to biological methods for controlling the seed lot (determination of residual virulence, including BCG survival) and the immunobiological method for controlling BCG for the absence of the genes responsible for virulence antigen expression (animal skin tests with Diaskintest®). The authors concluded that the control of stability of genetic and biological properties throughout the entire period of seed lot production and storage makes it possible to obtain BCG vaccines that meet all the regulatory requirements.

Human anti-D immunoglobulin preparations derived from human immune plasma are much needed and highly effective for specific anti-D prevention of perinatal complications and treatment of primary immune thrombocytopenia. The effectiveness of immune suppression is a direct function of the active ingredient dose received with the medicinal product. To improve the accuracy of anti-D antibody quantification, it is recommended to use certified reference materials with values assigned in international units (IUs). The aim of this study was to analyse the main stages in the development of the international standards (ISs) for human anti-D immunoglobulin potency testing and to substantiate the need for a national standard for anti-Rh_o(anti-D) antibody quantification. The article describes the creation of the first and subsequent ISs, the procedure for establishing the IU equivalent for the anti-Rh_o(anti-D) antibody concentration, the characteristics of the raw materials and preparations used, and the anti-Rh_o(anti-D) antibody assay methods applied to certify the ISs. According to the study conclusions, it is necessary to develop and certify a national standard for the content of anti-Rh_o(anti-D) antibodies that will meet the requirements of the corresponding Russian regulations.

Shigellosis (bacterial dysentery) is an acute infectious disease caused by Shigella spp., members of the Enterobacteriaceae family. The disease has the highest mortality rate amongst bacterial enteric infections. A considerable proportion of Shigella infections occur in children under the age of five. In 2017, WHO included Shigella spp. strains into the list of “priority pathogens” that are resistant to most antibiotics and pose a threat to global public health. This provided a stimulus for the development of new antibiotics to treat shigellosis. Apart from the creation of new antimicrobial therapies for Shigella infections, an important role in fighting against shigellosis belongs to the preventative measures set out in WHO’s Immunisation Agenda 2030. These include sanitation, hygiene, consumption of clean water, and vaccination. The development of Shigella vaccines has been a priority of the WHO programme for more than 20 years. The aim of the study was to analyse promising approaches to Shigella vaccine development. According to the analysis of literature, only one vaccine against shigellosis has been approved so far—Shigellvac, the Russian polysaccharide dysentery vaccine against Shigella sonnei. This study covers a number of vaccine candidates (whole-cell, polysaccharide, polysaccharide conjugate, protein antigen-based vaccines, etc.) that are at different stages of clinical trials. The importance of researching combination (multivalent) vaccines against Shigellа spp. and other enteric pathogens is noted. However, the authors consider subunit vaccines based on Ipa proteins, providing broad cross-protection against Shigellа spp., and conjugate polyvalent vaccines for children under 5 the most promising for further development.

The competitive enzyme-linked immunosorbent assay (ELISA) used for the quantification of specific antibodies, anti-D (Rh_o) 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.

Maroteaux—Lamy syndrome (mucopolysaccharidosis type VI) is an orphan genetic disease caused by mutations in the arylsulfatase B gene (ARSB), which encodes the lysosomal enzyme arylsulfatase B (ASB). The relevance of the study lies in the need of a Russian recombinant ASB product for patients with the disease in the Russian Federation. Previously, the authors have developed producer lines coexpressing the target ASB enzyme with an auxiliary formylglycine-generating enzyme (FGE), based on Chinese hamster ovary (CHO) cells. Further development of the recombinant ASB preparation places priority on increasing the enzyme yield. The aim of this study was to increase the productivity of producer clones by optimising the culture process and adding calcium chloride and copper sulfate to the culture medium. Materials and methods: a suspension-adapted CHO cell line was used. Monoclonal cell lines were developed using Cell Metric and ClonePix FL systems. The concentration of ASB in the culture liquid was determined using the enzyme-linked immunosorbent assay (ELISA). The authors analysed batch culture and/or fed-batch culture in media supplemented with various concentrations of copper sulfate and calcium chloride. Results: the combined addition of copper sulfate and calcium chloride at concentrations of 300 μM during batch culture of producer clones coexpressing ASB and FGE increases viability and specific productivity of the cells up to 4.58±1.62 pg/ (cell×day). The cultivation of the lead producer clone coexpressing ASB and FGE under fed-batch conditions for 12 days and the addition of copper sulfate to the growth medium at the concentration of 300 μM allow for increasing the yield of the active lysosomal enzyme, arylsulfatase B, to 420 mg/L. Conclusions: the cultivation of producer clones coexpressing ASB and FGE under fed-batch conditions with copper sulfate added to the medium significantly improves cell line growth properties and the ASB yield. This approach to the selection of culture conditions for producer cell lines can be applied to other enzymes of the sulfatase family.

New effective wound healing agents are a priority for modern clinical pharmacology. A promising approach would be to develop medicinal products that promote angiogenesis, which is a critical step in wound healing. The aim of the study was to evaluate the wound healing effect of a medicinal product based on recombinant human angiogenin in gel form in various experimental models. Materials and methods: white outbred male rats were used as experimental ani mals. The study compared healing effects of a regenerating product containing recombinant human angiogenin (0.0025%) in gel form and a reference product in full-thickness excision, incision, and burn wound models. The healing effect of the test product in treating chronic wounds was assessed in a model of alloxan-induced diabetes mellitus. The anti-inflammatory effect of the test product containing recombinant human angiogenin was compared with that of another reference product in a model of adjuvant-induced arthritis. Results: according to the study, the test product based on recombinant human angiogenin exerts higher wound healing effect in treating excision, incision, and burn wounds than the reference product (Solcoseryl gel). Being applied, the test product intensifies tissue repair in chronic wounds in the model of alloxan-induced diabetes. The dissociation of necrotic tissues and the progression towards epithelialisation at wound edges are more rapid. The anti-inflammatory effect of the test product based on recombinant human angiogenin is comparable with that of the reference product (Diclofenac gel). Conclusions: the test product based on recombinant human angiogenin in gel form was found to have pronounced wound healing and anti-inflammatory effects comparable with those of reference products.

The use of sulfated polysaccharides (fucoidans) as active pharmaceutical ingredients or adjuvants poses the challenge of obtaining structurally characterised and homogeneous samples or their oligomeric fractions maintaining high biological activity. The authors obtained a highly purified enzymatic hydrolysate of fucoidan from the brown alga Fucus evanescens and compared its biological activity with that of a native sample. The aim of the study was to compare, in vitro and in vivo, the effects of depolymerised fucoidan from the brown alga F. evanescens and native fucoidan on the effector functions of innate and adaptive immunity cells loaded with ovalbumin (OVA). Materials and methods: the effects of the fucoidan samples (depolymerised and native) on the expression of the main immunophenotypic markers by innate and adaptive immunity cells (neutrophils, monocytes, natural killers, and lymphocytes) were studied in vitro using flow cytometry. The levels of serum OVA-specific antibodies (IgG, IgG1, IgG2а) and cytokines (IFN-γ, IL-2, IL-10, IL-12) were studied in vivo using BALB/c mice immunised with OVA. The statistical analysis of the data obtained was performed using the Statistica 10 software package. Results: in vitro, both fucoidan samples altered the expression of the main immunophenotypic markers by innate and adaptive immunity cells, indicating their activation. In vivo, mice treated with the fucoidan samples demonstrated an increase in the levels of OVA-specific antibodies (IgG, IgG1 and IgG2a) and in the production of cytokines (IFN-γ, IL-2, IL-10). Conclusions: the effects of enzymatically depolymerised fucoidan on functional activity of innate and adaptive immunity cells are comparable to those of native fucoidan. The findings indicate the possibility of using enzymatic hydrolysis products of fucoidan as adjuvants for a wide range of prophylactic and therapeutic vaccines.

To ensure the quality of immunobiologicals, it is required to quantify the thiomersal preservative present in a number of them. The authors have previously developed an analytical procedure for thiomersal quantification in non-adsorbed immunobiological medicinal products, which is based on cold vapor atomic absorption spectrometry (CV AAS). The aim of the study was to analyse the possibility of using the CV AAS procedure for thiomersal content determination in adsorbed immunobiologicals and evaluate the comparability of thiomersal quantification results obtained by colourimetry and CV AAS. Materials and methods: the study used the national reference standard of mercury ions content and the pharmacopoeial reference standard of thiomersal content in adsorbed medicinal products (PhRS 3.1.00427), as well as samples of immunobiologicals by different manufacturers: a DTP vaccine, anatoxins, hepatitis B and influenza vaccines, and combined vaccines. The study involved CV AAS and the colourimetric reaction between mercury and dithizone. Results: the specificity of the CV AAS procedure is demonstrated by the coefficient of variation (3.95%) and the coefficient of correlation between the test sample volume and thiomersal content (0.9956). The regression analysis and the Fisher’s test value of 0.16 indicate the absence of bias. The trueness of the method is satisfactory, as the percent recovery differs from the total spiked amount by less than 10%. For the sensitivity of the CV AAS procedure, its quantification and detection limits are 6.9×10^-3 μg/ mL and 2.3×10^-3  μg/ mL, respectively. The Fisher’s test value obtained in the comparability assessment of the results of thiomersal quantification by colourimetry and CV AAS (1.29) is lower than the conventional tabulated one (3.96). Conclusions: according to the study, it is possible to use the CV AAS procedure for thiomersal quantification in adsorbed immunobiologicals. The established detection limit allows evaluating residual amounts of thiomersal in in-process intermediates during the production of preservative-free immunobilogical dosage forms. The comparability assessment of the results of thiomersal quantification by colourimetry and CV AAS, carried out using oneway ANOVA and Fisher’s test, showed the possibility of using PhRS 3.1.00427 to control the consistency of operation when reproducing the CV AAS procedure.

The State Pharmacopoeia of the Russian Federation (Ph.Rus.) requires a preparatory evaluation of culture medium suitability using the reference standard (RS) of the Mycoplasma arginini G230 test strain in order to ensure reliable results when testing medicines for mycoplasmas using a microbiological (culture) method. The RS retains the stability of physico-chemical properties and the certified characteristic value (titre) for one year if stored under the specified temperature conditions (–20 ºC to –30 ºC). Deviations from this range and transportation in ambient conditions can alter the properties of the RS and consequently result in biologicals substandard in terms of mycoplasmas. Experts from the Reference Materials Committee of the International Organisation for Standardisation (ISO), the State System for Ensuring the Uniformity of Measurements, and the WHO Expert Committee on Biological Standardisation emphasise the need to study the stability of RSs not only under the specified long-term storage conditions, but also with short-term deviations from such conditions. The aim of the study was to analyse the stability of the M. arginini G230 test strain RS under the specified long-term storage conditions and with short-term exposure to elevated temperatures. Materials and methods: the study used the RS of M. arginini G230 by the Scientific Centre for Expert Evaluation of Medicinal Products and culture media and solutions required per the Ph.Rus. general chapter on mycoplasma tests (OFS.1.7.2.0031.15). The stability of RS’s characteristics, including the certified value (titre), was determined using Ph.Rus. methods. The experiment involved two testing regimes with short-term exposure to elevated temperatures. Control samples were stored at –20±2 °С. Statistical data processing was carried out by the serial dilution method using McCrady’s table with the calculation of the arithmetic mean of the most probable number (MPN) for the limiting dilution (titre). Results: as demonstrated in the study under the specified conditions, the RS retains its characteristics for a period exceeding its shelf life (for up to 16 months). The main characteristics of the RS remain stable after 30-day exposure to elevated temperatures (25±2 ºC and 37±2 ºC). The certified value (titre) of the RS decreases after exposure to 37±2 ºC for 10 and 30 days. Conclusions: the study proved the possibility of storing and transporting the RS at a temperature lower than 25±2 ºC for up to 30 days with no subsequent changes to the quality within the 1-year shelf life. The conditions of 37±2 ºC cannot be used for the purpose.