Introduction. A reliable assessment of the quality and safety of medical products (MP) by biological indicators is directly related to the use of culture media (CM) that meet the established requirements. However, when conducting trial, it is necessary to take into account the peculiarities of culture media due to the presence in their composition of components of biological origin. In particular, a high degree of variability and unstable stability of raw materials, as well as possible mistakes in producing and storage of media, create the prerequisites for deviations. The use of industrial culture media also does not guarantee their suitability for the appropriate test. Therefore to obtain reliable results, the compliance of each batch CM with the specified criteria be confirmed must be confirmed by standard methods using the full range of meaningful indicators. The current lack of a state standard in the Russian Federation regulating the requirements for the use of culture media when confirming the quality of medical products makes it much more difficult to assess the suitability, comparability and reliability of the analysis results, both themselves CM and drugs. Text. The article discusses the draft General Pharmacopoeia Article (GPM) "Culture media", which presents the general requirements for of culture media when used in pharmacopoeia analysis. The standard is intended for specialists involved in the development, improvement and application of methods and / or techniques necessary for confirming the properties of medical products for the purpose of registration, entry into civil circulation, as well as in their production. The document was drawn up taking into account modern domestic and international approaches of good pharmaceutical practices to the assessment of the drugs that involve the use of culture media. The project identifies the key factors that most affect the quality of culture media. Based on the principle of continuity, compiled and described in detail a set of standardized control methods, including tests on physical-chemical and biological indicators. For exploring of specific properties, there are requirements for test cultures have been established. Governs special requirements for culture media used to obtain biological medical products are regulated. The procedure for storage and disposal of culture media unsuitable for use is described. Conclusion. The introduction of the GPM "Culture media" into the practice of domestic pharmacopoeic analysis will increase the reliability of the results of the evaluation of the effectiveness and safety of drugs, and as a result will lead to a reduction in production risks and risks of harm to health patients during in pharmacotherapy and immunoprophylaxis. The proposed approaches can also be used in other branches of science and industry.

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.

In accordance with the requirements of the State Pharmacopoeia of the Russian Federation, 14th edition, Clostridium novyi 198 test strain is used to determine growth promoting properties of thioglycollate medium when testing sterility of immunobiological medicinal products. The preservation of C. novyi 198 as spores requires a special liquid medium containing insoluble calcium chloride casein (CChC). At present, there is no reference standard available on the Russian market for the quality control of CChC used as a component of the media for preservation of C. novyi 198. The aim of the study was to develop and certify the first batch of calcium chloride casein industry reference standard (IRS) for the quality control of CChC batches and liquid media containing CChC which is used to preserve C. novyi 198 as spores. Materials and methods: thermally denatured CChC which was chemically synthesized from industrial CChC was used as candidate material. The quality control was performed using the test procedures described in procedural guidelines MUK 4.2.2316-08. C. novyi 198 spores were obtained as described in the «Sterility testing» section of MUK 4.2.2316-08 (preparation, inoculation and incubation in the medium containing calcium chloride casein IRS). Results: a list of quality parameters was developed for calcium chloride casein IRS: «Spore formation» (certifiable parameter), «Appearance», «Loss on drying», «Clarity of medium». The first batch of the candidate calcium chloride casein IRS was certified. The value of the «Spore formation» certifiable parameter was found to be (32.6 ± 7.3) %, the IRS shelf life was 5 years when stored in a dry place protected from light at a temperature of 15 to 25 °C. A documentation package for the IRS 4228-444-2018 was prepared and approved. The liquid medium containing CChC reference material (0.3 g/10 mL) allows for preservation of the biological properties of the C. novyi 198 working culture for at least 12 months. Conclusions: the certification results demonstrate that the calcium chloride casein IRS meets the applicable requirements and can be used as reference material to control the quality of new CChC batches and to assess the suitability of liquid medium containing CChC for preservation purposes. The use of the calcium chloride casein IRS makes it possible to standardise and greatly simplify the procedure for preserving the stability of C. novyi 198, to increase the efficiency and reliability of immunobiologicals quality control in terms of Sterility, and to reduce costs when using a freeze-dried strain.

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.).

In accordance with the requirements of the State Pharmacopoeia of the Russian Federation, 14th edition, Clostridium novyi 198 test strain is used to determine growth promoting properties of thioglycollate medium when testing sterility of immunobiological medicinal products. The preservation of C. novyi 198 as spores requires a special liquid medium containing insoluble calcium chloride casein (CChC). At present, there is no reference standard available on the Russian market for the quality control of CChC used as a component of the media for preservation of C. novyi 198. The aim of the study was to develop and certify the first batch of calcium chloride casein industry reference standard (IRS) for the quality control of CChC batches and liquid media containing CChC which is used to preserve C. novyi 198 as spores. Materials and methods: thermally denatured CChC which was chemically synthesized from industrial CChC was used as candidate material. The quality control was performed using the test procedures described in procedural guidelines MUK 4.2.2316-08. C. novyi 198 spores were obtained as described in the «Sterility testing» section of MUK 4.2.2316-08 (preparation, inoculation and incubation in the medium containing calcium chloride casein IRS). Results: a list of quality parameters was developed for calcium chloride casein IRS: «Spore formation» (certifiable parameter), «Appearance», «Loss on drying», «Clarity of medium». The first batch of the candidate calcium chloride casein IRS was certified. The value of the «Spore formation» certifiable parameter was found to be (32.6 ± 7.3) %, the IRS shelf life was 5 years when stored in a dry place protected from light at a temperature of 15 to 25 °C. A documentation package for the IRS 4228-444-2018 was prepared and approved. The liquid medium containing CChC reference material (0.3 g/10 mL) allows for preservation of the biological properties of the C. novyi 198 working culture for at least 12 months. Conclusions: the certification results demonstrate that the calcium chloride casein IRS meets the applicable requirements and can be used as reference material to control the quality of new CChC batches and to assess the suitability of liquid medium containing CChC for preservation purposes. The use of the calcium chloride casein IRS makes it possible to standardise and greatly simplify the procedure for preserving the stability of C. novyi 198, to increase the efficiency and reliability of immunobiologicals quality control in terms of Sterility, and to reduce costs when using a freeze-dried strain.

Culture media with predetermined specific characteristics play a key role in ensuring safety, quality and efficacy of medicinal products, especially biologicals. It is necessary to formulate clear criteria for culture media suitability, because they are used not only in manufacture, but also in quality control of medicinal products. The expansion of the range of complex high-technology medicinal products containing biologically derived substances as active ingredients, whose quality and properties are tested by biological methods, calls for more stringent requirements for the quality of culture media. The aim of the study was to analyse requirements for the culture media used in the production of medicinal products — in order to identify approaches to the development of a national standard. Currently, culture media are not included into the State Register of Medicinal Products, and there are no standards that could be used by manufacturers to confirm that a culture medium complies with specific safety, efficacy and quality requirements. The paper summarises the results of the analysis of the State Pharmacopoeia of the Russian Federation, 14th ed., and nine leading world pharmacopeias’ requirements for culture media used for various purposes (manufacture, auxiliary purposes, and quality control). Most countries have adopted requirements mainly for those culture media that are used in purity tests. The analysis revealed differences both in the lists of requirements (test strains, incubation and storage conditions, preparation for use, suitability assessment), and the approaches to the establishment and presentation of requirements. The paper discusses the main challenges and risks associated with the quality control of culture media in accordance with the requirements of the State Pharmacopoeia of the Russian Federation, 14th ed. It outlines current national and international ways of improving the quality of culture media used in the production of medicinal products, and substantiates the need to develop an appropriate national standard. The paper suggests approaches to the development and standardisation of requirements for the quality of various types of media used in the production and control of medicines. These approaches could be used in the preparation of the general chapter Culture media.

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.

In accordance with the requirements of the State Pharmacopoeia of the Russian Federation, 14th edition, Clostridium novyi 198 test strain is used to determine growth promoting properties of thioglycollate medium when testing sterility of immunobiological medicinal products. The preservation of C. novyi 198 as spores requires a special liquid medium containing insoluble calcium chloride casein (CChC). At present, there is no reference standard available on the Russian market for the quality control of CChC used as a component of the media for preservation of C. novyi 198.The aim of the study was to develop and certify the first batch of calcium chloride casein industry reference standard (IRS) for the quality control of CChC batches and liquid media containing CChC which is used to preserve C. novyi 198 as spores.Materials and methods: thermally denatured CChC which was chemically synthesized from industrial CChC was used as candidate material. The quality control was performed using the test procedures described in procedural guidelines MUK 4.2.2316-08. C. novyi 198 spores were obtained as described in the «Sterility testing» section of MUK 4.2.2316-08 (preparation, inoculation and incubation in the medium containing calcium chloride casein IRS).Results: a list of quality parameters was developed for calcium chloride casein IRS: «Spore formation» (certifiable parameter), «Appearance», «Loss on drying», «Clarity of medium». The first batch of the candidate calcium chloride casein IRS was certified. The value of the «Spore formation» certifiable parameter was found to be (32.6 ± 7.3) %, the IRS shelf life was 5 years when stored in a dry place protected from light at a temperature of 15 to 25 °C. A documentation package for the IRS 4228-444-2018 was prepared and approved. The liquid medium containing CChC reference material (0.3 g/10 mL) allows for preservation of the biological properties of the C. novyi 198 working culture for at least 12 months.Conclusions: the certification results demonstrate that the calcium chloride casein IRS meets the applicable requirements and can be used as reference material to control the quality of new CChC batches and to assess the suitability of liquid medium containing CChC for preservation purposes. The use of the calcium chloride casein IRS makes it possible to standardise and greatly simplify the procedure for preserving the stability of C. novyi 198, to increase the efficiency and reliability of immunobiologicals quality control in terms of Sterility, and to reduce costs when using a freeze-dried strain.

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.

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 production of biological medicinal products used for prophylactic, diagnostic and therapeutic purposes includes the use of inherently variable biological processes and materials. In order to ensure the quality of the finished biological product it is necessary to determine the source, nature and fitness for use of the starting materials, including reagents, culture media, buffer solutions, sera, and enzymes. The article summarises literature data on the structure, properties and mode of action of the animal-derived reagent trypsin — proteolytic enzyme widely used in the production of biological medicinal products. The article dwells upon the sources of trypsin, methods of its production, requirements for trypsin products of different purity grades intended for human and veterinary use as well as for use as reagents in the production of vaccines, advanced therapy medicinal products and genetically engineered products. The article describes the role that trypsin plays in the human and animal intestinal digestive enzyme systems, in dissociation of cells in cultures in the process of cell passaging, in the mechanism of proteolitic activation and inactivation of a wide range of viruses, and in the examination of proteins primary structure.

Medicinal products fail sterility testing if visual observation shows the growth of microorganisms that manifests itself as turbidity, sedimentation, flocculation and other changes in the growth medium. A key factor allowing robust determination of changes in the culture that may be suspected of contamination is the quality of growth media used, namely their transparency, and absence of foreign matter detectable by microscopic examination of the growth media smears. The presence of such foreign matter makes it especially difficult to interpret the results of testing of immunobiological products, namely live bacterial vaccines, because they cause turbidity of the media due to their specific composition. The article dwells upon the results of testing (in terms of Transparency and Microbial content) of dehydrated growth media recommended by the State Pharmacopoeia of the Russian Federation, 13th ed., General monograph 1.2.4.0003.15 for sterility testing of immunobiological medicinal products. The study revealed the presence of microorganisms, including pathogenic ones, in the growth media. In view of the fact that certificates of analysis and technical documentation accompanying components of growth media and dehydrated growth media produced by most national and foreign manufacturers do not contain any data on the acceptable levels of microorganisms it is argued that these products have to be tested for microbial content. The study also investigated the ways of improving the quality of commercial dehydrated growth media at the preparation stage.

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.

Comparative trials were carried out new commercial nutrient media made by Russian (Mirogen Research-and-Production Association, Ministry of Health of the Russian Federation; State Research Center for Applied Microbiology and Biotechnology, Obolensk, Serpukhov District, Moscow Region; and I. I. Mechnikov Biomed, Moscow Region; Research Center of Pharmacotherapy, Saint Petersburg) and foreign (NovaMed Ltd, Israel) manufacturers to isolate staphylococci. Their quality was assessed in terms of physicochemical and biological parameters. The test nutrient media provided a good growth of specifically important test staphylococcal strains and inhibited Enterobacteriaceae. The technique of using the traditional inoculum to Petri dish ensures a higher sensitivity and inhibition of nutrient media as compared to that of applying the inoculum to NovaStreak.

The paper gives data on the use of techniques to detect and register Salmonella in the water objects, by applying a new liquid nutrient medium. Experimental and field studies have shown its advantage over the accumulation media widely used in practical healthcare. It has been ascertained that the nutrient medium not only accumulates biomass, but also provides the restoration of the biological properties of uncultivated Salmonella species. The use of the nutrient medium at practical laboratories makes it possible to unify guidelines for the examination of water objects with varying degrees of biological pollution and to obtain the comparable results of analyses.

The comparative studies of biological properties of test-systems NovaStreak (Novamed Ltd, Israel) were made. The sensitivity of these systems allows to detect total bacterial count in food products. To assess microbial cleanliness for other groups of microorganisms (coliforms, Salmonella, fungi) them sensitivity is not enough and in case a negative results during analysis of samples are obtained additional control by means of traditional methods should be made.

In this work the results of the trial of three selective nutrient media for Salmonella accumulation, viz. Leifson's selenite broth produced by the Research and Manufacturing Amalgamation (RMA) "Nutrient Media", tetrarathionate broth (Müller's medium), laboratory-prepared, and newly developed MA-broth produced by the RMA "Nutrient Media", are presented. The results of 6- and 24-hour incubation in these selective media was evaluated. As found in this study, the laboratory-prepared medium was most effective for the accumulation of salmonellae (S. paratyphi, S. typhi, S. gallinarum). With respect to the possible concomitant microflora (Escherichia coli and shigellae), the inhibiting properties of MA-broth were superior to those of Leifson's medium, but inferior to those of Müller's medium.