biopreparatБИОпрепараты. Профилактика, диагностика, лечениеBiological Products. Prevention, Diagnosis, Treatment2221-996X2619-1156Scientific Centre for Expert Evaluation of Medicinal Productsbiopreparat-71Research ArticleОРИГИНАЛЬНЫЕ СТАТЬИORIGINAL ARTICLESОпределение остаточной ДНК клеток-продуцентов E. coli и CHO в субстанциях рекомбинантых белков методом qPCRDetection of residual E. coli and CHOhost-cellDNA in recombinant proteins by qPCRЁлшинН. Д.YolshinN. D.

Младший научный сотрудник лаборатории иммунофармакологии

Junior research scientist of the Immunopharmacology laboratory

nikita.yolshin@gmail.comГосударственный НИИ особо чистых биопрепаратовРоссияResearch Institute of Highly Pure BiopreparationsRussian Federation201620022018164245252Copyright © Ёлшин Н.Д., 20182018Ёлшин Н.Д.Yolshin N.D.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.biopreparations.ru/jour/article/view/71

Рынок препаратов рекомбинантых белков растет и развивается во всем мире, в том числе и в России. Как продуцент, так и субстанция, должны быть тщательно охарактеризованы и проверены по всем критериям безопасности. Среди обязательных требований, предъявляемых к таким препаратам - содержание остаточной ДНК клеток-продуцентов должно быть не более 10 нг на дозу. Коммерческих наборов для определения остаточной ДНК в мире относительно немного, они основаны на четырех разных методах. В статье представлен краткий обзор этих методов, обозначены их преимущества и недостатки. Общим недостатком всех коммерческих наборов является стоимость. Основными задачами работы было подобрать оптимальный метод выделения ДНК из белковых субстанций и образцов различных этапов очистки белка, отработать определение количества остаточной ДНК Escherichia coli и клеток яичника китайского хомяка (CHO) методом количественной полимеразной цепной реакции (qPCR) без использования коммерческих наборов. Статья также содержит ряд практических рекомендаций по особенностям выделения ДНК и хранению стандарта. Целью исследования был поиск достоверного и недорогого метода определения содержания остаточной ДНК клеток-продуцентов в образцах рекомбинантых белков. В статье приводится обзор методов выделения ДНК для анализа, обоснована необходимость выделения ДНК перед анализом, преимущество отдано методу выделения на спин-колонках. Оптимальный метод выделения ДНК для определения ее количества в субстанции такой, при котором выход ДНК стабилен, в том числе и при различных химических составах исследуемых образцов, а в растворе выделенной ДНК отсутствует белок и другие примеси. Предложенный метод выделения ДНК на спин-колонках является наименее трудозатратным, оптимизирован для выделения ДНК из белковых субстанций и образцов различных стадий очистки белков. Самостоятельное приготовление растворов для выделения ДНК является простой процедурой и может снизить затраты на анализ. Представлен отчет об успешной адаптации методик определения остаточной ДНК E. coli и CHO методом qPCR с использованием флуоресцентных зондов. Продемонстрирована чувствительность метода не менее 1 пг/мл как при анализе количества остаточной ДНК E. coli, так и CHO.

Production of recombinant proteins is a steadily growing industry in Russia and all over the world. Both producer and substance should be properly characterized and tested against all safety criteria. One of the mandatory requirements to the mentioned drugs is the assessment of the content of residual DNA-producing cells, which should be less than 10 ng per dose. There are not many commercial kits for detection of residual DNA and they are based on four different methods. The article provides a brief overview of these methods and highlights their advantages and disadvantages. Common disadvantage of all the commercial kits is their price. The main goal of the research was to choose an optimal method of DNA extraction from protein substances and samples of various protein purification steps, as well as to work over measuring the amount of residual E. coli DNA and CHO by qPCR method not using commercial kits. The article also provides with a number of practical recommendations on specific aspects of DNA extraction and reference standard storage. The aim of the study was to find a reliable and inexpensive method for determining residual DNA-producing cells in recombinant protein samples. The article provides with an overview of DNA extraction methods, stipulates the necessity of DNA extraction prior to the analysis. The advantage was given to the method of spin-column extraction. Optimal DNA extraction method for its assay in a substance is the method, which provides with a stable DNA yield, including different chemical structure of the samples, and upon the condition that no protein and other impurities are detected in the isolated DNA solution. The proposed method for DNA spin-column extraction is the least labor-intensive, is optimized for DNA isolation from protein substances and samples of various protein purification steps. Self preparation of solutions for DNA extraction is a simple procedure and can reduce analysis costs. The report on successful adaptation of the methods of residual E. coli and CHO DNA detection by qPCR using fluorescent probes was provided. The sensitivity of the method was demonstrated at least 1 pg/ml for the analysis of the amount of residual DNA both for E. coli and CHO.

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The authors declare that there are no conflicts of interest present.