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.

Short tandem repeat analysis (STR) is a well-established international method of authentication and genetic stability testing of cell lines (CLs). Therefore, the development and introduction of this method into routine practice of cell banks and cell culture collections is a pressing concern. In addition, the expansion of the field of cell-line based biomedical cell products (BСPs) necessitates the implementation of STR as a tool of identification testing during quality control. The State Pharmacopoeia of the Russian Federation does not require mandatory use of STR for cell line identification, while other countries have been using this method for cell line quality control for about a decade. The use of identified CLs in medical practice will ensure the efficacy and safety of BCPs.The aim of the study was to assess the possibility of using STR analysis for authentication and genetic stability testing of CLs using U937, WISH, WIL2-S, NK-92, and Jurkat Clone E6-1 CLs as examples.Materials and me­thods: the following human CLs were used in the study: U937 (ECACC), WISH (ATCC), WIL2S (ATCC), NK-92 (ATCC), and Jurkat Clone E6-1 (ATCC). The CL allelic profiles were determined by STR using the COrDIS Plus kit (Gordiz, Russia). The electrophoretic separation was performed using a Genetic Analyzer 3500 Series instrument. The data provided on the websites of the European Collection of Authenticated Cell Cultures and American Type Culture Collection were used to compare the CL profiles.Results: the AuthentiFiler PCR Amplification Kit (Thermo Fisher Scientific, USA) and the GenePrint 10 System (Promega Corporation, USA) intended for CL authentication by STR were compared with the characteristics of the COrDIS plus kit (Gordiz, Russia). The results of the comparison demonstrated that the COrDIS plus kit includes all the loci found in the foreign kits, as well as the loci recommended by the International Cell Line Authentication Committee. The U-937, WIL2S, and NK-92 CLs demonstrated genetic identity with the reference profiles available on the websites of the international collections. The Jurkat Clone E6-1 CL was found to be genetically instable due to the loss of the amelogenin gene.Conclusions: it was demonstrated by the examples of U937, WISH, WIL2-S, NK-92, and Jurkat Clone E6-1 CLs that STR and the COrDIS plus kit could be used for authentication and genetic stability testing. The obtained results suggest the feasibility of using the COrDIS plus kit for the analysis of CLs used in BCPs, for BCP quality control, and biomedical research.

Метод проточной цитометрии -наиболее информативный метод идентификации и количественного определения поверхностных маркеров клеток. Проточная цитометрия дает возможность проводить под-счет клеток, а также характеризацию их типов и подтипов путем мечения клеток моноклональными анти-телами, конъюгированными с флуорохромом. В настоящее время производителями продуктов на основе клеток человека накоплен значительный опыт применения проточной цитометрии, разработано боль-шое количество методик, подлежащих валидации и включению в спецификацию на клеточный продукт. В обзоре авторами рассмотрен опыт применения метода проточной цитометрии для оценки качества клеточных линий человека, используемых, в частности, для создания препаратов с целью применения в клеточной терапии. Учитывая обязательное наличие клеточного компонента в составе биомедицинских клеточных продуктов (БМКП), метод проточной цитометрии будет являться обязательным при подтверж-дении подлинности в ходе экспертизы качества БМКП в Российской Федерации. Про-филактика, диагностика, лечение 2018; 18(1): 16-24. DOI: 10.30895/2221 18(1): 16-24. DOI: 10.30895/ -996Х-2018-24 * Контактное лицо: Трусов Георгий Александрович; trusov@expmed.ru Flow cytometry is the most common method of identification and quantitation of cell surface markers. Flow cytometry can be used for cell counting and characterization of cell types and subtypes by labeling cells with fluorochrome-conjugated monoclonal antibodies. Manufacturers of human cell-based medicinal products have accumulated significant experience in flow cytometry and developed a large number of procedures that can be validated and included into cell products specifications. The present review summarises the experience gained with the use of flow cytometry for characterization of human cell lines used to develop cell therapy products. Since all biomedical cell products (BMCPs) have a cellular component, it will be necessary to use the flow cytometry method for identification testing of BMCPs during evaluation of their quality.

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Preparation of a product file (PF) for a biomedical cell product (BCP) is an important stage in the preparation of documents for marketing authorisation. The PF is the main document of a regulatory submission and is used as the basis for BCP quality control. The requirements for the content of a PF, including appropriate specifications, are laid out in the relevant laws and regulations that support Federal Law No. 180-FZ “On Biomedical Cell Products” of 23.06.2016. However, given the novelty of the Russian legislative framework for innovative products for human use represented by BCPs, the specificity of their composition (i.e., components based on viable human cells) which differs significantly from conventional medicines, and lack of marketing authorisation experience— there is a need to examine specific aspects of a BCP PF. The aim of the study was to formulate methodological approaches to the development and preparation of a BCP PF in accordance with the national legislation and taking into account the experience of foreign regulatory authorities in evaluation of regulatory submissions for BCP analogues. The paper summarises the national regulatory requirements for the description of quality characteristics of cell lines used as components in BCPs, as well as test methods and test procedures used for cell line quality control. These data are required both for quality control of BCP samples, and for preparation of the Expert Commission Conclusion. The paper looks into the content of cellular and process-related impurities in a cell line and a finished BCP, and presents considerations on the description of the viral safety strategy for the finished product and for the cells from the master and working cell banks. The approaches to the presentation of quality characteristics and quality control methods for a finished BCP and for the cell line used in its production could be used by BCP developers for preparation of a PF.