INTRODUCTION. The distinctive nature and composition of biological medicinal products present certain challenges for the detection of microbial contamination, which affects the reliability of sterility control results. The main challenges associated with testing biologicals include changes in the appearance and physical properties of culture media or samples, an increased risk of contamination or false-positive results in direct inoculation tests, filtration issues, etc. When drafting product specification files and analytical method validation reports in dossiers for biologicals, manufacturers need adequate, reproducible, and pharmacopoeia-based sterility testing procedures for specific medicinal products, including live and inactivated, viral and bacterial, single- and multi-component vaccines, bacteriophages and interferons, sera and immunoglobulins. These challenges can be addressed through providing recommendations for developing analytical procedures based on compendial methods of direct inoculation and membrane filtration.AIM. This study aimed to offer key recommendations for the development of sterility testing procedures for biologicals based on pharmacopoeial methods.DISCUSSION. This article presents a retrospective analysis of the results of reviewing regulatory documentation for biological medicinal products submitted to the testing centre of the Scientific Centre for Expert Evaluation of Medicinal Products in 2021–2023. Having considered major issues with the analytical procedures described in the Sterility section of regulatory submissions, the authors offer the following recommendations for addressing these issues. The authors advise against using low-pH Sabouraud culture media to test blood products by direct inoculation. Biologicals containing mercury preservatives should be tested using a thioglycollate medium at two temperature settings. When testing various biologicals, analysts should refrain from plugging vials with cotton gauze. Instead, they should pour culture media into glass vials with rubber stoppers and crimps. Biologicals in large primary packaging (≥10 mL) should be tested by membrane filtration. Membrane filtration tests for biologicals should use solvents and rinsing agents selected in validation studies. It is also recommended to set the maximum volume of a biological medicinal product that can be filtered through a membrane filter. A reduction in the filtration speed is recommended to minimise foaming when testing blood products.CONCLUSION. The recommendations presented in this article can guide the development of sterility testing procedures for inclusion in the relevant section of regulatory submissions for biologicals. Manufacturers, control laboratories, and regulatory authorities may implement these recommendations to harmonise their approach to sterility testing of biologicals.

Preventive vaccination against SARS-CoV-2 infection is currently receiving close attention in the Russian Federation. Improving public confidence in immunisation with new vaccines largely depends on a guarantee of the absence of side effects caused by contamination. A high risk of contamination is inherent to biological products, including coronavirus prevention vaccines, due to their properties and the nature of raw materials used. This risk adds to the need for using effective contaminant detection approaches.The aim of the study was to evaluate the possibility to improve sterility testing of preventive vaccines against SARS-CoV-2 infection.This article presents an analysis of the procedures proposed by pharmaceutical developers for sterility testing of ten Russian vaccines approved in the country for COVID-19 prevention. The authors considered specific characteristics of these vaccines, including their physical and chemical properties, the presence of antimicrobial components, and other critical factors affecting the correctness of the experimental setup. The results suggest that it is possible to improve sterility testing. According to the authors, the main directions for its improvement are the proposal to develop an alternative procedure based on compendial method 2 (OFS.1.2.4.0003.15, Ph. Rus. XIV), as well as the use of a universal culture medium. If used for refining the established procedures and developing new ones, the authors’ recommendations will improve the reliability and applicability of sterility testing during both manufacturing and pre-approval regulatory assessment of updated coronavirus vaccines for subsequent release to the market. The proposed approaches can be applied to testing other medicinal products for sterility.

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.

Objective of the study - analysis of standard indicators and methods, utilized for determination of presence of contaminating microorganisms in live bacterial vaccines. Materials and methods. We used the data from the State Pharmacopeia of the USSR, 9th-11th editions; State Pharmacopeia of the Russian Federation, 12th-14th editions; as well as regulatory documentation/manufacturer’s pharmacopoeial monographs for 9 items of live vaccines. Results and discussion. Taking into account the specificity of live vaccines, the process of their manufacturing and quality control must target elimination of the possibility of contamination with microorganisms that differ from production strains. It is established that currently there is no unified terminology for determining the indicator in the Russian Federation, as well as clear-cut criteria for interpretation of test results for quality assessment of live bacterial vaccines when testing sterility/contamination with foreign bacteria and fungi. In compliance with the requirements of the current RF Pharmacopeia editions, detection of contamination in live vaccines for parenteral administration should be carried out using various methods and assessment criteria (General Pharmacopeia Monograph (GPM) “Sterility” and GPM “Microbiological purity”). Performed investigations have revealed the necessity to enhance the regulatory framework in regard to detection of contamination with foreign bacteria and fungi. It is advised to use unified nomination of the indicator, specifically, “Absence of foreign bacteria and fungi” in the normative documents. Given are the recommendations on improvement of methods and requirements to the assessment of live bacterial vaccine contamination. Developed proposals on harmonization of the quality assessment requirements of vaccines containing other live microorganisms can be used for drawing up corresponding normative-regulatory documents (GPM, Pharmacopeia monograph, regulatory documentation et al.).

An urgent safety concern associated with biological products is contamination with mycoplasmas, which may originate from donor tissues and organs, virus harvests, culture medium components, trypsin, animal blood serum, as well as be transmitted by personnel involved in the manufacture of medicines. Currently, due to an increase in the range of biologicals available, there is a need for more sensitive and specific test methods. In the Russian practice, microbiological (culture-based) testing of finished pharmaceutical products for mycoplasma contamination is performed using complex culture media whose sensitivity depends on the quality of proteins, ingredients, and reagents used. Growth promotion properties of the media are determined according to the State Pharmacopoeia of the Russian Federation, 14th ed., using a single test strain — Mycoplasma arginini G230 (M. arginini G230 industry reference material). The aim of the study was to analyse current Russian and foreign requirements for the quality control of culture media that are used for mycoplasma detection, in order to update and improve the quality control procedure in Russia. It was demonstrated that a compelling advantage of the State Pharmacopoeia of the Russian Federation is the possibility of using a semi-liquid culture medium which does not require special aerobic or anaerobic incubation conditions and allows for quantification of mycoplasma colonies and determination of mycoplasma titre in culture medium while testing its growth promotion properties using reference М. arginini G230 test strain. The analysis revealed some differences in Russian and foreign requirements for quality evaluation of culture media. These differences were taken into account when developing recommendations for improvement of the Russian test procedure, i.e. enlarging the range of test strains used and development of respective reference standards.

Sterility is one of the key parameters of biological safety of immunobiological medicinal products. The article traces the history of the development of sterility test methods for immunobiological medicinal products from as far back as 1961 and up to the current requirements laid down in the State Pharmacopoeia of the Russian Federation, 13th edition. The article provides a detailed analysis of major approaches to the improvement of medicines quality evaluation based on this parameter, namely to the choice of: optimal growth media and methods of their evaluation, sensitive test strains, incubation conditions, the number of test samples (i.e., sample size) required for reliable demonstration of batch sterility; as well as approaches to the development of a test design that would accommodate specific aspects of production and use of immunobiological products. The article dwells upon the longstanding use of the sterility testing scheme developed in the national agency for control of immunobiological products — L.A. Tarasevich State Institute for Standardization and Control of Medicinal Immunobiological Products. The article analyses the current status of harmonisation of requirements for sterility testing of immunobiological products and other groups of medicines with those of the leading world pharmacopoeias, and prospects of using these requirements in the Eurasian Economic Union.

The safety of biological products derived from animal cells is associated with the properties of the cells themselves or their components, as well as with the possible presence of contaminants of microbial and viral origin. The suitability of cell substrate for production of prophylactic preparations is often determined by the features of the production process, which allows to benefit/risk ratio of the product. One of the common contaminants of cell substrates are mycoplasmas, type Mollicutes. As distinguished from other microorganisms they don’t have cell membranes, and parasitize a wide range of animals and plants, on the surface or inside the cells. Some mycoplasma species of are potentially pathogenic to humans, they compete for nutrients with cells in vitro , cause chromosomal aberrations, interfere with normal cell metabolism. The detection of mycoplasma contamination in cell substrates when manufacturing prophylactic preparations is required by regulatory documents.

Reliable evaluation of blood products sterility is a very important and one of the most complex and critical control methods in the context of microbiological safety. The membrane filtration method that uses a closed-circuit system has, for many years, been the main and the most preferable sterility test method for all known medicinal products. However, Russian manufacturers perform sterility testing of heterologous serum products by direct inoculation method only. The present study was aimed at exploring the feasibility of performing sterility testing of heterologous serum products by membrane filtration. It was shown that sample preparation could be modified by dilution of samples to, on average, 1.5-2 times the initial volume with a sterile 0.9 % sodium chloride solution followed by membrane filtration at a geared-up rate which lowers protein sorption by the membrane filter. The study helped to determine the range of protein impact on the membrane when testing serum products according to the State Pharmacopoeia 13th ed. It was shown that various types of filter elements from mixed cellulose esters and Durapore® (PVDF) could be used to test serum products by membrane filtration with a protein impact range of up to 12 g of protein per membrane. The results of heterologous serum products sterility testing by direct inoculation method and by membrane filtration were found to be comparable. Adaptation of the procedure to protein-containing products makes it possible to perform sterility testing by a more reliable and modern method. The authors can recommend the incorporation of the above-cited test procedure into quality standards for products concerned together with the currently used direct inoculation method.

The article presents prospects for improving the evaluation of live tularemia vaccine (hereinafter - tularemia vaccine) quality in terms of the following parameters: «Identification», «Specific activity (the number of living microbial cells)» and the «Absence of extraneous microorganisms and fungi». The authors investigated the possibility of improving evaluation of tularemia vaccine quality as regards the «Identification» parameter by using a commercial diagnostic test kit - immunochromatographic test system for express detection and identification of tularemia agent («F. tularensis ICA test system»). In order to optimize and improve the evaluation of tularemia vaccine quality as regards «Specific activity (the number of living microbial cells)» parameter it is recommended to use a ready-to-use growth medium for cultivation and isolation of tularemia microbe - as an additional culture medium for determination of the number of living microbial cells. It is proposed to improve the methodology of tularemia vaccine quality evaluation as regards the «Absence of foreign microorganisms and fungi» parameter by eliminating the stage of subculturing material in thioglycollate medium for 5-7 days, which is reasonable from economic and practical points of view and also reduces the risk of false positive results.

Reliable evaluation of blood products sterility is a very important and one of the most complex and critical control methods in the context of microbiological safety. The membrane filtration method that uses a closed-circuit system has, for many years, been the main and the most preferable sterility test method for all known medicinal products. However, Russian manufacturers perform sterility testing of heterologous serum products by direct inoculation method only. The present study was aimed at exploring the feasibility of performing sterility testing of heterologous serum products by membrane filtration. It was shown that sample preparation could be modified by dilution of samples to, on average, 1.5-2 times the initial volume with a sterile 0.9 % sodium chloride solution followed by membrane filtration at a geared-up rate which lowers protein sorption by the membrane filter. The study helped to determine the range of protein impact on the membrane when testing serum products according to the State Pharmacopoeia 13th ed. It was shown that various types of filter elements from mixed cellulose esters and Durapore ® (PVDF) could be used to test serum products by membrane filtration with a protein impact range of up to 12 g of protein per membrane. The results of heterologous serum products sterility testing by direct inoculation method and by membrane filtration were found to be comparable. Adaptation of the procedure to protein-containing products makes it possible to perform sterility testing by a more reliable and modern method. The authors can recommend the incorporation of the above-cited test procedure into quality standards for products concerned together with the currently used direct inoculation method.

The article presents prospects for improving the evaluation of live tularemia vaccine (hereinafter - tularemia vaccine) quality in terms of the following parameters: «Identification», «Specific activity (the number of living microbial cells)» and the «Absence of extraneous microorganisms and fungi». The authors investigated the possibility of improving evaluation of tularemia vaccine quality as regards the «Identification» parameter by using a commercial diagnostic test kit - immunochromatographic test system for express detection and identification of tularemia agent (« F. tularensis ICA test system»). In order to optimize and improve the evaluation of tularemia vaccine quality as regards «Specific activity (the number of living microbial cells)» parameter it is recommended to use a ready-to-use growth medium for cultivation and isolation of tularemia microbe - as an additional culture medium for determination of the number of living microbial cells. It is proposed to improve the methodology of tularemia vaccine quality evaluation as regards the «Absence of foreign microorganisms and fungi» parameter by eliminating the stage of subculturing material in thioglycollate medium for 5-7 days, which is reasonable from economic and practical points of view and also reduces the risk of false positive results.

A variant of Vero cell line has been obtained, adapted to the Russian Eagle's medium with 8% fetal calf serum. The variant is characterized by intensive cell proliferation, possesses a different content of chromosomes, contains no oncogens and contaminants, specifically, Mycoplasma. The strain was characterized and certified in accordance with the WHO requirements. Inoculation (110 ampules) and working (100 ampules) stocks at the levels of the 170th and 178th passages were placed for storage in liquid nitrogen. Vero (B) cell line is sensitive to herpes simplex virus types 1 and 2, CMV, hepatitis A virus, recombinant variolovaccinia strain expressing HbS antigen, etc. The line retains its biological properties from passage 178 to passage 200 and is recommended for control and preparation of various biomedical agents, including antiviral vaccines.

A high-sensitive, easy and rapid proximate method has been developed to reveal mycoplasmas in the monolayer cell cultures using home fluorescent antibiotic olivomycin. This method has been used to screen many cell lines with its high effectiveness being shown. As based on this method the following control methods are worked out: indication of mycoplasmas in animal blood sera, used for the cultivation of cell lines detection of contaminant microorganisms of the cell cultures (fungi and bacteria) and human ureaplasmas.