biopreparatБИОпрепараты. Профилактика, диагностика, лечениеBiological Products. Prevention, Diagnosis, Treatment2221-996X2619-1156Scientific Centre for Expert Evaluation of Medicinal Products10.30895/2221-996X-2025-553biopreparat-553Research ArticleТЕМА НОМЕРА: РЕКОМБИНАНТНЫЕ ТЕРАПЕВТИЧЕСКИЕ БЕЛКИISSUE TOPIC RECOMBINANT THERAPEUTIC PROTEINSРазработка клеточных линий-продуцентов рекомбинантных терапевтических белков на примере деносумабаDevelopment of cell lines producing recombinant therapeutic proteins, with denosumab as a case studyhttps://orcid.org/0000-0003-2733-7458ТимоноваС. С.TimonovaS. S.

Тимонова Софья Сергеевна, канд. биол. наук 

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

Sofia S. Timonova, Cand. Sci. (Biol.)

14 Vladimirskaya St., Volginsky, Petushinsky District, Vladimir Region 601125

timonova1993@yandex.ruhttps://orcid.org/0000-0002-0364-8107КирикИ. А.KirikI. A.

Кирик Инесса Анатольевна, канд. биол. наук 

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

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

14 Vladimirskaya St., Volginsky, Petushinsky District, Vladimir Region 601125

https://orcid.org/0009-0006-1542-3905ФилатоваМ. А.FilatovaM. A.

Филатова Мария Александровна, канд. вет. наук 

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

Maria A. Filatova, Cand. Sci. (Vet.)

14 Vladimirskaya St., Volginsky, Petushinsky District, Vladimir Region 601125

https://orcid.org/0009-0003-9518-2942АнфилатоваА. И.AnfilatovaA. I.

Анфилатова Анастасия Ивановна

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

Anastasia I. Anfilatova

14 Vladimirskaya St., Volginsky, Petushinsky District, Vladimir Region 601125

https://orcid.org/0009-0003-5762-2193ШубинаС. С.ShubinaS. S.

Шубина Софья Сергеевна

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

Sofia S. Shubina

14 Vladimirskaya St., Volginsky, Petushinsky District, Vladimir Region 601125

https://orcid.org/0009-0005-2975-9111ПолупановаА. А.PolupanovaA. A.

Полупанова Анна Андреевна

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

Anna A. Polupanova

14 Vladimirskaya St., Volginsky, Petushinsky District, Vladimir Region 601125

https://orcid.org/0000-0001-6022-0863ГабдрахмановаА. Ф.GabdrakhmanovaA. F.

Габдрахманова Алина Фаритовна

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

Alina F. Gabdrakhmanova

14 Vladimirskaya St., Volginsky, Petushinsky District, Vladimir Region 601125

https://orcid.org/0009-0005-0441-4918ИвановЕ. С.IvanovE. S.

Иванов Евгений Сергеевич

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

Evgeny S. Ivanov

14 Vladimirskaya St., Volginsky, Petushinsky District, Vladimir Region 601125

https://orcid.org/0000-0002-9877-5220БадэВ. Н.BadeV. N.

Бадэ Вероника Николаевна, канд. биол. наук 

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

Veronika N. Bade, Cand. Sci. (Biol.)

14 Vladimirskaya St., Volginsky, Petushinsky District, Vladimir Region 601125

https://orcid.org/0000-0001-5552-5419ПискуновА. А.PiskunovA. A.

Пискунов Александр Александрович, канд. биол. наук 

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

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

14 Vladimirskaya St., Volginsky, Petushinsky District, Vladimir Region 601125

https://orcid.org/0000-0002-1314-894XХамитовР. А.KhamitovR. A.

Хамитов Равиль Авгатович, д-р мед. наук, проф. 

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

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

14 Vladimirskaya St., Volginsky, Petushinsky District, Vladimir Region 601125

Акционерное общество «ГЕНЕРИУМ»РоссияGENERIUM JSCRussian Federation202529092025253245257Copyright © Тимонова С.С., Кирик И.А., Филатова М.А., Анфилатова А.И., Шубина С.С., Полупанова А.А., Габдрахманова А.Ф., Иванов Е.С., Бадэ В.Н., Пискунов А.А., Хамитов Р.А., 20252025Тимонова С.С., Кирик И.А., Филатова М.А., Анфилатова А.И., Шубина С.С., Полупанова А.А., Габдрахманова А.Ф., Иванов Е.С., Бадэ В.Н., Пискунов А.А., Хамитов Р.А.Timonova S.S., Kirik I.A., Filatova M.A., Anfilatova A.I., Shubina S.S., Polupanova A.A., Gabdrakhmanova A.F., Ivanov E.S., Bade V.N., Piskunov A.A., Khamitov R.A.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/553ВВЕДЕНИЕ

ВВЕДЕНИЕ. При создании биотехнологических лекарственных препаратов ключевое значение имеет разработка высокопродуктивных клеточных линий-продуцентов рекомбинантных белков. Отработка протоколов селекции и подходов для эффективного скрининга линий-продуцентов целевых белков — необходимый этап разработки технологии производства рекомбинантных белков с высоким выходом целевого продукта.

ЦЕЛЬ

ЦЕЛЬ. Получение высокопродуктивных клеточных линий-продуцентов рекомбинантного белка деносумаба на основе суспензионной клеточной линии СНО.

МАТЕРИАЛЫ И МЕТОДЫ

МАТЕРИАЛЫ И МЕТОДЫ. Использовалась суспензионная клеточная линия СНО-К1. Для культивирования применялись среды и подпитки без использования сыворотки или других компонентов животного происхождения. Клетки линии СНО трансфицировали плазмидами, содержащими гены легкой и тяжелой цепей деносумаба, с использованием метода электропорации с помощью системы MaxCyte STX. Селекцию трансфицированных клеток проводили с добавлением антибиотиков (гигромицин, генетицин). Моноклональные клеточные линии получали с использованием системы ClonePix FL. Проводили периодическое культивирование с добавлением подпитки и определение концентрации деносумаба методом иммуноферментного анализа (ИФА) в культуральной жидкости для выявления лидерных моноклональных клеточных линий.

РЕЗУЛЬТАТЫ

РЕЗУЛЬТАТЫ. Оптимальная концентрация антибиотиков для селекции клеточных линий-продуцентов на основе клеток СНО составила 600 мг/л гигромицина и 600 мг/л генетицина. После проведения трансфекции из 1920 минипулов селекцию прошел 1041 (около 54%). Отбор минипулов-продуцентов деносумаба проводили путем скрининга проб культуральной жидкости с использованием ИФА из 96-, 24- и 6-луночных планшетов. Отобранные 23 лидерных минипула были адаптированы к условиям шейкерного культивирования. С учетом показателей ростовых и продукционных характеристик минипулов определен лидерный минипул (mp-19) с продуктивностью 1,92 г/л (на 7 сут периодического культивирования). На основе mp-19 получены моноклональные клеточные линии-продуценты деносумаба с продуктивностью до 6,5 г/л на 9 сут культивирования с подпиткой.

ВЫВОДЫ

ВЫВОДЫ. Получены высокопродуктивные моноклональные клеточные линии-продуценты деносумаба. Предложенный подход к созданию клеток-продуцентов может быть применен для получения различных рекомбинантных белков, включая моноклональные антитела, ферменты, факторы свертывания крови.

INTRODUCTION

INTRODUCTION. A key factor in the creation of biotechnological medicinal products is to establish cell lines for high-yield production of recombinant proteins. The development of selection protocols and highly efficient screening approaches for cell lines producing target proteins is a necessary step in the development of recombinant technology for high-yield target protein production.

AIM

AIM. This study aimed to derive producer cell lines from a CHO suspension cell line for high-yield production of the recombinant monoclonal antibody denosumab.

MATERIALS AND METHODS

MATERIALS AND METHODS. A CHO-K1 suspension cell line was cultured using serum- and animal component-free media and feeds. The cells were transfected with plasmids containing light and heavy chains of denosumab by electroporation using a MaxCyte STX system. The transfected cells were selected under antibiotic pressure (hygromycin and geneticin). Monoclonal cell lines were obtained using a ClonePix FL system. Leader monoclonal cell lines were identified by determining denosumab concentrations by enzyme-linked immunosorbent assay (ELISA) following fed-batch culture.

RESULTS

RESULTS. The optimum concentrations of antibiotics for the selection of CHO-derived denosumab-producing cell lines were 600 mg/L for hygromycin and 600 mg/L for geneticin. The selection process following transfection was successful in 1041 (about 54%) of 1920 minipools. Denosumab-producing minipools were identified by screening culture fluid samples from 96-, 24-, and 6-well plates using ELISA. Then, 23 leader minipools were chosen and adapted to suspension culture in shaker flasks. The growth and production characteristics of these 23 minipools indicated the leader minipool for cloning (mp-19). This minipool provided a denosumab yield of 1.92 g/L on day 7 of fed-batch culture. Using mp-19, the authors obtained monoclonal cell lines providing up to 6.5 g/L denosumab yields on day 9 of fed-batch culture.

CONCLUSIONS

CONCLUSIONS. The authors obtained monoclonal cell lines for high-yield denosumab production. The offered approach to producer cell line development can be applied to the production of various recombinant proteins, including monoclonal antibodies, enzymes, and blood coagulation factors.

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