biopreparatБИОпрепараты. Профилактика, диагностика, лечениеBiological Products. Prevention, Diagnosis, Treatment2221-996X2619-1156Scientific Centre for Expert Evaluation of Medicinal Products10.30895/2221-996X-2022-22-3-279-292biopreparat-425Research ArticleОРИГИНАЛЬНЫЕ СТАТЬИORIGINAL ARTICLESОптимизация условий культивирования клона-продуцента, коэкспрессирующего арилсульфатазу B и формилглицин-генерирующий фермент, с целью повышения выхода фермента арилсульфатазы BOptimisation of culture conditions for a producer clone coexpressing arylsulfatase B and a formylglycine-generating enzyme in order to increase the yield of arylsulfatase Bhttps://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/0000-0002-0364-8107КирикИ. А.KirikI. A.

Кирик Инесса Анатольевна, кандидат биологических наук

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

Inessa A. Kirik, Cand. Sci. (Biol.)

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

Kirik@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.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.ruГЕНЕРИУМ, АОРоссияGENERIUM, JSCRussian Federation202210102022223279292Copyright © Тимонова С.С., Смолова К.А., Кирик И.А., Пантюшенко М.С., Анисимов Р.Л., Хамитов Р.А., Пискунов А.А., Бадэ В.Н., 20222022Тимонова С.С., Смолова К.А., Кирик И.А., Пантюшенко М.С., Анисимов Р.Л., Хамитов Р.А., Пискунов А.А., Бадэ В.Н.Timonova S.S., Smolova K.A., Kirik I.A., Pantyushenko M.S., 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/425

Синдром Марото—Лами (мукополисахаридоз VI типа) — орфанное генетическое заболевание, вызванное мутациями гена ARSB, который кодирует лизосомальный фермент арилсульфатазу В. Актуальность исследования заключается в необходимости разработки отечественного препарата рекомбинантной арилсульфатазы В для лечения пациентов с данным заболеванием в Российской Федерации. Ранее были получены клеточные линии-продуценты, коэкспрессирующие целевой фермент арилсульфатазу В и вспомогательный формилглицин-генерирующий фермент на основе клеточной линии СНО. Однако для дальнейшей разработки препарата рекомбинантной арилсульфатазы В представляется важным повышение выхода фермента. Цель работы: увеличение продуктивности клонов-продуцентов за счет оптимизации процесса культивирования и добавления в культуральную среду хлорида кальция и сульфата меди. Материалы и методы: использовали суспензионную клеточную линию СНО. Моноклональные клеточные линии получали с использованием систем Cell Metric и Clone Pix FL. Концентрацию арилсульфатазы В в культуральной жидкости определяли методом иммуноферментного анализа. Использовали периодическое культивирование (batch culture) и/или периодическое культивирование с подпиткой (fedbatch culture) в среде с добавлением различных концентраций сульфата меди и хлорида кальция. Результаты: продемонстрировано, что одновременное добавление сульфата меди и хлорида кальция в концентрации 300 мкM при периодическом культивировании клонов-продуцентов, коэкспрессирующих арилсульфатазу В и формилглицин-генерирующий фермент, увеличивает жизнеспособность культур и повышает удельную продуктивность клеток до 4,58±1,62 пг/(клетка×сут). При культивировании лидерного клона-продуцента, коэкспрессирующего арилсульфатазу В и формилглицин-генерирующий фермент, в условиях периодического культивирования с подпиткой длительностью 12 сут достигнуто увеличение выхода активного лизосомального фермента арилсульфатазы В до 420 мг/л при добавлении в ростовую среду сульфата меди в концентрации 300 мкM. Выводы: культивирование клонов-продуцентов, коэкспрессирующих арилсульфатазу В и формилглицин-генерирующий фермент, в условиях периодического культивирования с подпиткой и с добавлением в среду сульфата меди приводит к значительному улучшению ростовых свойств клеточной линии и выхода целевого фермента. Данный подход в подборе условий культивирования продуцентов можно применять к другим ферментам подкласса сульфатаз.

Maroteaux—Lamy syndrome (mucopolysaccharidosis type VI) is an orphan genetic disease caused by mutations in the arylsulfatase B gene (ARSB), which encodes the lysosomal enzyme arylsulfatase B (ASB). The relevance of the study lies in the need of a Russian recombinant ASB product for patients with the disease in the Russian Federation. Previously, the authors have developed producer lines coexpressing the target ASB enzyme with an auxiliary formylglycine-generating enzyme (FGE), based on Chinese hamster ovary (CHO) cells. Further development of the recombinant ASB preparation places priority on increasing the enzyme yield. The aim of this study was to increase the productivity of producer clones by optimising the culture process and adding calcium chloride and copper sulfate to the culture medium. Materials and methods: a suspension-adapted CHO cell line was used. Monoclonal cell lines were developed using Cell Metric and ClonePix FL systems. The concentration of ASB in the culture liquid was determined using the enzyme-linked immunosorbent assay (ELISA). The authors analysed batch culture and/or fed-batch culture in media supplemented with various concentrations of copper sulfate and calcium chloride. Results: the combined addition of copper sulfate and calcium chloride at concentrations of 300 μM during batch culture of producer clones coexpressing ASB and FGE increases viability and specific productivity of the cells up to 4.58±1.62 pg/ (cell×day). The cultivation of the lead producer clone coexpressing ASB and FGE under fed-batch conditions for 12 days and the addition of copper sulfate to the growth medium at the concentration of 300 μM allow for increasing the yield of the active lysosomal enzyme, arylsulfatase B, to 420 mg/L. Conclusions: the cultivation of producer clones coexpressing ASB and FGE under fed-batch conditions with copper sulfate added to the medium significantly improves cell line growth properties and the ASB yield. This approach to the selection of culture conditions for producer cell lines can be applied to other enzymes of the sulfatase family.

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