biopreparatБИОпрепараты. Профилактика, диагностика, лечениеBiological Products. Prevention, Diagnosis, Treatment2221-996X2619-1156Scientific Centre for Expert Evaluation of Medicinal Products10.30895/2221-996X-2022-22-1-80-93biopreparat-416Research ArticleОРИГИНАЛЬНЫЕ СТАТЬИORIGINAL ARTICLESУвеличение продуктивности клеточной линии-продуцента арилсульфатазы B за счет коэкспрессии формилглицин-генерирующего ферментаIncreasing productivity of arylsulfatase B-producing cell line by coexpression of formylglycine-generating enzymehttps://orcid.org/0000-0003-2733-7458ТимоноваС. С.TimonovaS. S.

Тимонова Софья Сергеевна

ул. Владимирская, д. 14, пос. Вольгинский, Петушинский район, Владимирская область, 601125

Sofia S. Timonova

14 Vladimirskaya St., Volginsky town, Petushinskiy District, Vladimir Region 601125

timonova1993@yandex.ruhttps://orcid.org/0000-0001-9135-9673СмоловаК. А.SmolovaK. A.

Смолова Ксения Александровна, кандидат химических наук

ул. Владимирская, д. 14, пос. Вольгинский, Петушинский район, Владимирская область, 601125

Kseniya A. Smolova, Cand. Sci. (Chem.)

14 Vladimirskaya St., Volginsky town, Petushinskiy District, Vladimir Region 601125

Smolova@ibcgenerium.ruhttps://orcid.org/0000-0001-6104-3352ЗариповаД. Т.ZaripovaD. T.

Зарипова Долия Тагировна

ул. Владимирская, д. 14, пос. Вольгинский, Петушинский район, Владимирская область, 601125

Doliya Т. Zaripova

14 Vladimirskaya St., Volginsky town, Petushinskiy District, Vladimir Region 601125

Zaripova@ibcgenerium.ruПантюшенкоМ. С.PantyushenkoM. S.

Пантюшенко Марина Семеновна, кандидат биологических наук

ул. Владимирская, д. 14, пос. Вольгинский, Петушинский район, Владимирская область, 601125

Marina S. Pantyushenko, Cand. Sci. (Biol.)

14 Vladimirskaya St., Volginsky town, Petushinskiy District, Vladimir Region 601125

Pantyushenko@ibcgenerium.ruhttps://orcid.org/0000-0002-5721-0557КоролеваМ. А.KorolevaM. A.

Королева Мария Александровна

ул. Владимирская, д. 14, пос. Вольгинский, Петушинский район, Владимирская область, 601125

Mariya A. Koroleva

14 Vladimirskaya St., Volginsky town, Petushinskiy District, Vladimir Region 601125

Makoroleva@ibcgenerium.ruАнисимовР. Л.AnisimovR. L.

Анисимов Роман Львович, кандидат биологических наук

ул. Владимирская, д. 14, пос. Вольгинский, Петушинский район, Владимирская область, 601125

Roman L. Anisimov, Cand. Sci. (Biol.)

14 Vladimirskaya St., Volginsky town, Petushinskiy District, Vladimir Region 601125

Anisimov@ibcgenerium.ruhttps://orcid.org/0000-0002-1314-894XХамитовР. А.KhamitovR. A.

Хамитов Равиль Авгатович, доктор медицинских наук, профессор

ул. Владимирская, д. 14, пос. Вольгинский, Петушинский район, Владимирская область, 601125

Ravil A. Khamitov, Dr. Sci. (Med.), Professor

14 Vladimirskaya St., Volginsky town, Petushinskiy District, Vladimir Region 601125

Khamitov@ibcgenerium.ruhttps://orcid.org/0000-0001-5552-5419ПискуновА. А.PiskunovA. A.

Пискунов Александр Александрович, кандидат биологических наук

ул. Владимирская, д. 14, пос. Вольгинский, Петушинский район, Владимирская область, 601125

Aleksandr A. Piskunov, Cand. Sci. (Biol.)

14 Vladimirskaya St., Volginsky town, Petushinskiy District, Vladimir Region 601125

Piskunov@ibcgenerium.ruhttps://orcid.org/0000-0002-9877-5220БадэВ. Н.BadeV. N.

Бадэ Вероника Николаевна, кандидат биологических наук

ул. Владимирская, д. 14, пос. Вольгинский, Петушинский район, Владимирская область, 601125

Veronika N. Bade, Cand. Sci. (Biol.) 

14 Vladimirskaya St., Volginsky town, Petushinskiy District, Vladimir Region 601125

Bade@ibcgenerium.ruАО «ГЕНЕРИУМ»РоссияJSC “GENERIUM”Russian Federation2022090420222218093Copyright © Тимонова С.С., Смолова К.А., Зарипова Д.Т., Пантюшенко М.С., Королева М.А., Анисимов Р.Л., Хамитов Р.А., Пискунов А.А., Бадэ В.Н., 20222022Тимонова С.С., Смолова К.А., Зарипова Д.Т., Пантюшенко М.С., Королева М.А., Анисимов Р.Л., Хамитов Р.А., Пискунов А.А., Бадэ В.Н.Timonova S.S., Smolova K.A., Zaripova D.T., Pantyushenko M.S., Koroleva M.A., Anisimov R.L., Khamitov R.A., Piskunov A.A., Bade V.N.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/416

Мукополисахаридоз VI типа (синдром Марото–Лами) — орфанное генетическое заболевание, которое связано с дефицитом лизосомального фермента арилсульфатазы В. Актуальность исследования связана с необходимостью разработки высокопродуктивной клеточной линии-продуцента рекомбинантного фермента арилсульфатазы В. Наиболее перспективным подходом представляется создание клеточных линий-продуцентов, коэкспрессирующих целевой фермент арилсульфатазу В и вспомогательный формилглицин-генерирующий фермент на основе клеточной линии СНО. При этом большое практическое значение имеет возможность культивирования клеточных линий-продуцентов в виде суспензии, без использования сыворотки или других компонентов животного происхождения. Цель работы: разработка высокопродуктивных клеточных линий-продуцентов рекомбинантного фермента арилсульфатазы В за счет коэкспрессии вспомогательного формилглицин-генерирующего фермента. Материалы и методы: использовали суспензионную клеточную линию СНО. Трансфекцию клеток СНО проводили методом электропорации с использованием системы MaxCyte STX. Моноклональные клеточные линии получали с использованием системы Cell Metric. Концентрацию арилсульфатазы В в культуральной жидкости определяли методом иммуноферментного анализа. Образцы культуральной жидкости анализировали с применением электрофореза в полиакриламидном геле и вестерн-блота. Уровень мРНК измеряли методом полимеразной цепной реакции в режиме реального времени. Результаты: получены клеточные линии-продуценты, коэкспрессирующие целевой фермент арилсульфатазу В и вспомогательный формилглицин-генерирующий фермент. Достигнуто увеличение выхода активного целевого фермента арилсульфатазы В с 2 до 100 мг/л за счет подбора оптимального соотношения плазмид во время трансфекции. Наибольший выход целевого фермента арилсульфатазы В наблюдался при соотношении плазмид, кодирующих гены арилсульфатазы В и формилглицин-генерирующего фермента, равном 90:10 (%). Выводы: разработаны высокопродуктивные клеточные линии-продуценты рекомбинантного фермента арилсульфатазы В, коэкспрессирующие целевой и вспомогательный ферменты. Коэкспрессия арилсульфатазы В и формилглицин-генерирующего фермента приводит к улучшению ростовых и продукционных характеристик клеточной линии, что, по-видимому, обусловлено модификацией активного центра целевого фермента арилсульфатазы В. Полученные результаты позволят решить проблему низкого выхода фермента, характерную для препаратов подобного класса.

Mucopolysaccharidosis type VI (Maroteaux–Lamy syndrome) is an orphan genetic disease caused by deficiency of the lysosomal enzyme arylsulfatase B (ASB). The need to develop a highly productive cell line for the production of recombinant ASB, is behind the concept and relevance of this study. The most promising approach seems to be the development of CHO producer cell lines coexpressing the target ASB enzyme and an auxiliary formylglycine-generating enzyme (FGE). At the same time, it is important from a practical perspective to have the possibility of cultivating producer cell lines as suspensions free of serum or other components of animal origin. The aim of the study was to develop highly productive cell lines for the production of recombinant ASB by coexpression of the auxiliary FGE. Materials and methods: a suspension CHO cell line was used in the study. CHO cells were transfected by electroporation using the MaxCyte STX system. Monoclonal cell lines were obtained with the help of the Cell Metric system. Enzyme-linked immunosorbent assay was used for determination of ASB concentration in the culture fluid. Culture fluid samples were analysed using polyacrylamide gel electrophoresis and Western blotting. The mRNA level was measured by real-time polymerase chain reaction. Results: producer cell lines coexpressing the target ASB enzyme and auxiliary FGE were obtained. An increase in the yield of the active target ASB enzyme from 2 to 100 mg/L was achieved by selecting the optimal ratio of plasmids during transfection. The highest yield of the target ASB enzyme was achieved at the 90:10 ratio (%) of plasmids encoding the ASB and FGE genes, respectively. Conclusions: the authors developed highly productive cell lines for the production of recombinant ASB, which coexpress the target and auxiliary enzymes. The coexpression of ASB and FGE improves the growth and production characteristics of the cell line, probably due to the modification of the ASB active site. The obtained results will help resolve the problem of low enzyme yield, which is typical of this class of medicines.

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