Молекулярно-биологические методы контроля качества субстанций биологических лекарственных препаратов, полученных с использованием технологии рекомбинантной ДНК

Biotechnological products manufactured by recombinant DNA technology are widely used nowadays. According to the national and  international requirements the amount of residual host cell DNA in  such products should not exceed 10 ng per dose. However, for  products intended for frequent or long-term use, this amount must  not exceed 100 pg per dose. This article describes methods most  frequently used for quantification of residual host cell DNA in  biological active substances contained in biotechnological products:  molecular hybridization with biotin- or digoxigenin-labelled DNA- probes (semiquantitative method), Threshold system, real-time PCR, method based on the use of a fluorescent reagent (assays). If  a method based on the use of a fluorescent reagent or real-time PCR are used to replace the current procedure, it is necessary to  demonstrate their validity, e.g. by comparing the results of residual DNA quantification obtained by the two methods — the new one and the current one. The article dwells upon the advantages and  disadvantages of the methods and potential sources of uncertainty.  It highlights the importance of using appropriately certified reference standards and retention samples. The biological active substances  included into the State Register of Medicinal Products conform to the international requirements in terms of the amount of residual host cell DNA.Биотехнологические препараты, полученные с применением технологии рекомбинантных ДНК, широко используются в настоящее время. В соответствии с международными и  отечественными требованиями содержание остаточной ДНК штамма-продуцента в подобных  препаратах не должно превышать 10 нг на 1 дозу, для препаратов, предназначенных для  частого или длительного введения, эта величина не должна превышать 100 пг на 1 дозу. В  статье представлено описание наиболее часто используемых методов определения  содержания остаточной ДНК штамма-продуцента в субстанциях биотехнологических препаратов: молекулярная гибридизация с биотиновой или  дигоксигениновой меткой ДНК-зонда (полуколичественный метод), система Threshold, ПЦР в  режиме реального времени, метод с флуоресцентным реагентом (количественные  методы). В случае использования методов с флуоресцентным реагентом или ПЦР в режиме  реального времени для замены ранее использовавшегося метода требуется подтверждение  их правильности, например с помощью сравнительной оценки содержания остаточной ДНК  двумя методами — вновь вводимым и ранее использовавшимся методом. Отмечены  преимущества и недостатки методов, источники неопределенности результатов при  проведении испытаний, указана необходимость использования стандартных и контрольных  образцов, аттестованных в установленном порядке. В субстанциях, внесенных в  Государственный реестр лекарственных средств, содержание остаточной ДНК штамма-продуцента соответствует международным рекомендациям

Прямые и косвенные методы определения нуклеотидного состава ДНК последовательностей микроорганизмов

Typing of bacterial populations and identification of microorganisms are the overriding priorities in the field of prophylaxis, diagnosis and treatment of bacterial infections. Over the recent two decades rapidly developing approaches to molecular typing of bacterial populations have become an essential tool for the mentioned task. The information encoded in nucleic acids is more complete as compared to other characteristics of an organism. It can be obtained by either direct determination of genome sequences or by using a variety of techniques, that allow to indirectly assess the nucleotide composition in a representative genome locus of a microorganism under study. The article provides an overview of the literature data on direct and indirect methods of determining nucleotide composition of DNA sequences in microorganisms (multiple locus sequence typing, multiple sequeneed sites typing, denaturing gel electrophoresis, high-sensitivity melting curve analysis, DNA macro(micro)arrays).Типирование бактериальных линий и идентификация микроорганизмов - чрезвычайно важные задачи в области профилактики, диагностики и лечения бактериальных инфекций. За последние два десятилетия бурно развивающиеся молекулярные подходы к типированию бактериальных линий стали необходимым инструментом для решения этой задачи. Информация, которую могут дать нуклеиновые кислоты, является наиболее полной по сравнению с другими характеристиками организма. Получить ее можно как непосредственным определением избранных последовательностей генома, так и с помощью различных техник, позволяющих, косвенно оценить нуклеотидный состав репрезентативного участка генома исследуемого микроорагнизма. Статья посвящена обзору литературных данных по прямым и косвенным методам определения нуклеотидного состава ДНК последовательностей микроорганизмов (типирование по мультилокусным сик-венсам, типирование по множеству отсеквенированных участков, электрофорез в денатурирующей системе, высокочувствительный анализ кривых плавления, ДНК макро(микро)чипы)

Использование наборов реагентов для ОТ-ПЦР в реальном времени для оценки подлинности вакцин против кори, паротита и краснухи

Currently, the identity of measles, mumps and rubella virus vaccines is determined during certification testing by a labour-consuming, lengthy and costly neutralisation test using cell cultures. The identity of the virus contained in such vaccines is established based on neutralisation of cytopathic effect of viruses in sensitive RK-13 and Vero cell cultures using a specific immune serum. An urgent challenge is to simplify and reduce the cost of controlling the identity of commercial batches of measles, mumps and rubella vaccines using polymerase chain reaction (PCR) tests. The aim of the study was to determine the possibility of viral ribonucleic acid (RNA) detection in these vaccines using reagent kits from different manufacturers for detection of measles, mumps and rubella viruses in clinical material by real-time reverse transcription-PCR (RT-PCR). The article presents the results of the study, which assessed the possibility of using the real-time RT-PCR for testing the identity of measles, mumps and rubella viruses in vaccines. The authors of the study analysed domestically produced and foreign reagent kits intended for detection of measles, mumps and rubella viruses in clinical material. All the studied reagent kits were able to detect RNAs of the above-mentioned viruses in vaccine products. All the reagent kits demonstrated high specificity and could be used to confirm the identity of the measles, mumps and rubella viruses in all the studied vaccines. Commercial domestic reagent kits can be used to determine the identity of rubella vaccines by RT-PCR. However, it is advisable to develop domestic reagent kits for checking the identity of measles and mumps vaccines by RT-PCR. The acceptability of the test results was assessed using the industry reference standards of measles, mumps and rubella viruses activity with certified stable activity values.В настоящее время подлинность вирусных вакцин против кори, паротита и краснухи при сертификационных испытаниях определяют с помощью трудоемкой, длительной и дорогостоящей реакции нейтрализации на культуре клеток. Подлинность вируса в этих вакцинах устанавливают на основании нейтрализации цитопатогенного действия вирусов на чувствительной культуре клеток RK-13 и Vero специфической иммунной сывороткой. Упростить и удешевить контроль подлинности коммерческих серий вакцин для профилактики кори, паротита и краснухи с помощью ПЦР-тестирования представляется перспективной задачей. Целью работы было определение возможности выявления вирусной РНК в указанных вакцинах с помощью наборов реагентов разных производителей, предназначенных для выявления вирусов кори, паротита и краснухи в клиническом материале методом полимеразной цепной реакции с обратной транскрипцией в реальном времени (ОТ-ПЦР). В статье представлены результаты работы по оценке возможности использования метода ОТ-ПЦР для определения подлинности вирусов кори, паротита и краснухи в вакцинах. Были изучены отечественные и зарубежные наборы реагентов, предназначенные для выявления вирусов кори, паротита и краснухи в клиническом материале. Все изучавшиеся наборы реагентов выявляли рибонуклеиновые кислоты (РНК) названных вирусов в вакцинных препаратах. Показана высокая специфичность использовавшихся наборов реагентов и возможность их применения для подтверждения подлинности вирусов кори, паротита и краснухи во всех исследованных вакцинах. Для оценки подлинности вакцин против краснухи методом ОТ-ПЦР могут быть использованы коммерческие отечественные наборы реагентов. Для оценки подлинности вакцин против кори и паротита методом ОТ-ПЦР целесообразна разработка отечественных наборов реагентов. При оценке приемлемости результатов испытаний использовали отраслевые стандартные образцы активности вирусов кори, паротита и краснухи со стабильной аттестованной активностью

Molecular-Biological Methods of Quality Control of Biological Active Substances Produced by Recombinant DNA Technology

Biotechnological products manufactured by recombinant DNA technology are widely used nowadays. According to the national and  international requirements the amount of residual host cell DNA in  such products should not exceed 10 ng per dose. However, for  products intended for frequent or long-term use, this amount must  not exceed 100 pg per dose. This article describes methods most  frequently used for quantification of residual host cell DNA in  biological active substances contained in biotechnological products:  molecular hybridization with biotin- or digoxigenin-labelled DNA- probes (semiquantitative method), Threshold system, real-time PCR, method based on the use of a fluorescent reagent (assays). If  a method based on the use of a fluorescent reagent or real-time PCR are used to replace the current procedure, it is necessary to  demonstrate their validity, e.g. by comparing the results of residual DNA quantification obtained by the two methods — the new one and the current one. The article dwells upon the advantages and  disadvantages of the methods and potential sources of uncertainty.  It highlights the importance of using appropriately certified reference standards and retention samples. The biological active substances  included into the State Register of Medicinal Products conform to the international requirements in terms of the amount of residual host cell DNA

Direct and indirect methods of determining DNA nucleotide sequences in microorganisms

Typing of bacterial populations and identification of microorganisms are the overriding priorities in the field of prophylaxis, diagnosis and treatment of bacterial infections. Over the recent two decades rapidly developing approaches to molecular typing of bacterial populations have become an essential tool for the mentioned task. The information encoded in nucleic acids is more complete as compared to other characteristics of an organism. It can be obtained by either direct determination of genome sequences or by using a variety of techniques, that allow to indirectly assess the nucleotide composition in a representative genome locus of a microorganism under study. The article provides an overview of the literature data on direct and indirect methods of determining nucleotide composition of DNA sequences in microorganisms (multiple locus sequence typing, multiple sequeneed sites typing, denaturing gel electrophoresis, high-sensitivity melting curve analysis, DNA macro(micro)arrays)

Genotyping problems of microorganisms

Genotyping efficiency depends on resolution of methods which provided by the target sequence of genome, efficiency of enzymes, primers and conditions of DNA restriction and PCR amplification. The most differential tools are methods involving the high evolutionary markers. They are methods MST, MLVA, DGE, HRM against to the method MLST based on the slow evolutionary marker. Methods PFGE, AFLP, DNA chips exploit the preliminary information of genome and have the better resolution than ribotyping and RFLP-PCR limited by certain loci. Monitoring of local outbreaks may be carried out using a rapidly evolving markers (methods RAPD-PCR, MST, or MLVA, DGE, HRM). The most suitable methods for prolongated epidemiological or population researches are MLST, PFGE, ribotyping and pyrosequencing analysis. Polymicrobial samples are investigated with DNA specific methods PCR-RFLP, MLVA, DGE, HRM, MLST, MST, DNA chips. The metagenomic analysis is the most informative to identify the species of bacterium from the polymicrobial sample. It is essential to use the conservative DNA sequences, for example 16S rRNA. The modern genotyping assays provide the informative and technological platform for phylogenetic studies of bacterial strains even through the several generations. The important results are figures of genetic distances between strains involving in complex of epidemiological characteristics: virulence, antibiotic resistance and the others. The computing tools for integration the phenotyping and sequencing data have developed. Whole-genome sequencing is the optimal method for bacterial genotyping. But there are several crucial restrictions of this method. It is still rather expensive and needs to operate with high quality DNA

Development and certification of an industrial reference standard for determination of filgrastim activity

The article provides with the information on certification of an industrial reference standard (IRS) for determination of filgrastim activity. In order to confirm the quality of the IRS candidate, the following tests have been performed: biological activity, description, identification, clarity, colority, sterility, pH, foreign impurities, bacterial endotoxins, as well as the assay of: Filgrastim, Acetate ion, Polysorbate 80, residual Host Cell Proteins, residual Host Cell DNA. The results fully meet the requirements for filgrastim samples. The reference standard is certified for biological activity. The purpose of the IRS for determination of filgrastim activity is the assessment of acceptability of the results of biological activity tests in the quality control of substances and drugs based on filgrastim. Biological activity of the IRS candidate has been assessed by interlaboratory studies as compared against the second international standard NIBSC-09/136. The certified value for «Biological activity» of the IRS for determination of filgrastim activity has been set as 32.2±5.35 million IU/ml. The shelf-life under the storage conditions of 2 to 8oC has been set as not less than 2 years. The results of long-term stability studies the IRS confirm its stability during the monitored period

Using Real-Time RT-PCR Reagent Kits for Identity Testing of Measles, Mumps and Rubella Vaccines

Currently, the identity of measles, mumps and rubella virus vaccines is determined during certification testing by a labour-consuming, lengthy and costly neutralisation test using cell cultures. The identity of the virus contained in such vaccines is established based on neutralisation of cytopathic effect of viruses in sensitive RK-13 and Vero cell cultures using a specific immune serum. An urgent challenge is to simplify and reduce the cost of controlling the identity of commercial batches of measles, mumps and rubella vaccines using polymerase chain reaction (PCR) tests. The aim of the study was to determine the possibility of viral ribonucleic acid (RNA) detection in these vaccines using reagent kits from different manufacturers for detection of measles, mumps and rubella viruses in clinical material by real-time reverse transcription-PCR (RT-PCR). The article presents the results of the study, which assessed the possibility of using the real-time RT-PCR for testing the identity of measles, mumps and rubella viruses in vaccines. The authors of the study analysed domestically produced and foreign reagent kits intended for detection of measles, mumps and rubella viruses in clinical material. All the studied reagent kits were able to detect RNAs of the above-mentioned viruses in vaccine products. All the reagent kits demonstrated high specificity and could be used to confirm the identity of the measles, mumps and rubella viruses in all the studied vaccines. Commercial domestic reagent kits can be used to determine the identity of rubella vaccines by RT-PCR. However, it is advisable to develop domestic reagent kits for checking the identity of measles and mumps vaccines by RT-PCR. The acceptability of the test results was assessed using the industry reference standards of measles, mumps and rubella viruses activity with certified stable activity values

Mycoplasma - contamination of cell cultures

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