biopreparatБИОпрепараты. Профилактика, диагностика, лечениеBiological Products. Prevention, Diagnosis, Treatment2221-996X2619-1156Scientific Centre for Expert Evaluation of Medicinal Products10.30895/2221-996X-2023-23-2-219-230biopreparat-438Research ArticleСтатьиОптимизация условий индукции синтеза проинсулина аспарт в клетках бактериального штамма-продуцентаOptimisation of induction conditions for a bacterial strain producing proinsulin asparthttps://orcid.org/0000-0003-0207-5244КорнаковИ. А.KornakovI. A.

Корнаков Игорь Александрович

ул. Связи, д. 34, лит. А, пос. Стрельна, Санкт-Петербург, 198515

Igor A. Kornakov

Svyazi St., 34, Strelna, Saint Petersburg 198515

Igor.Kornakov@geropharm.comhttps://orcid.org/0000-0002-0065-1853ХасаншинаЗ. Р.KhasanshinaZ. R.

Хасаншина Зухра Рамилевна

ул. Связи, д. 34, лит. А, пос. Стрельна, Санкт-Петербург, 198515

Zukhra R. Khasanshina

Svyazi St., 34, Strelna, Saint Petersburg 198515

Zuhra.hasanshina@geropharm.comhttps://orcid.org/0000-0003-2220-0591СеничкинаД. А.SenichkinaD. A.

Сеничкина Дина Александровна

ул. Связи, д. 34, лит. А, пос. Стрельна, Санкт-Петербург, 198515

Dina A. Senichkina

Svyazi St., 34, Strelna, Saint Petersburg 198515

Dina.Senichkina@geropharm.comhttps://orcid.org/0000-0002-9985-9573ФилипенкоА. А.FilipenkoA. A.

Филипенко Антон Александрович

ул. Связи, д. 34, лит. А, пос. Стрельна, Санкт-Петербург, 198515

Anton A. Filipenko

Svyazi St., 34, Strelna, Saint Petersburg 198515

Anton.Filipenko@geropharm.comЛуневИ. С.LunevI. S.

Лунев Иван Сергеевич

ул. Связи, д. 34, лит. А, пос. Стрельна, Санкт-Петербург, 198515

Ivan S. Lunev

Svyazi St., 34, Strelna, Saint Petersburg 198515

inav.lunev96@gmail.comhttps://orcid.org/0000-0003-4594-6097ДрайР. В.DraiR. V.

Драй Роман Васильевич, канд. мед. наук

ул. Связи, д. 34, лит. А, пос. Стрельна, Санкт-Петербург, 198515

Roman V. Drai, Cand. Sci. (Med.)

Svyazi St., 34, Strelna, Saint Petersburg 198515

roman.drai@geropharm.comЗакрытое акционерное общество «Фарм-Холдинг»РоссияPharmholding CJSCRussian Federation202305062023232От традиционных биологических к высокотехнологичным лекарственным препаратам: вопросы разработки и применения219230Copyright © Корнаков И.А., Хасаншина З.Р., Сеничкина Д.А., Филипенко А.А., Лунев И.С., Драй Р.В., 20232023Корнаков И.А., Хасаншина З.Р., Сеничкина Д.А., Филипенко А.А., Лунев И.С., Драй Р.В.Kornakov I.A., Khasanshina Z.R., Senichkina D.A., Filipenko A.A., Lunev I.S., Drai R.V.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/438

Сахарный диабет несет серьезную угрозу здоровью людей во всем мире, в связи с чем в 2021 г. Всемирная организация здравоохранения учредила Глобальный пакт по борьбе с диабетом — инициативу, направленную на обеспечение улучшений в области лечения и профилактики диабета. Быстрый рост числа больных диабетом увеличивает потребность в инсулине. Применение аналогов инсулина человека ультракороткого действия, в том числе инсулина аспарт, способствует повышению эффективности инсулинотерапии. Одним из способов получения инсулина аспарт является его биосинтез в виде проинсулина в клетках Escherichia coli, однако выход рекомбинантного белка в значительной степени определяется оптимизацией процесса производства.

Цель работы

Цель работы: оптимизация условий индукции синтеза рекомбинантного проинсулина аспарт в клетках штамма-продуцента E. coli с помощью подхода математического планирования эксперимента (Design of Experiment, DoE) для повышения продуктивности.

Материалы и методы

Материалы и методы: использовали штамм-продуцент проинсулина аспарт на основе клеток E. coli. Эксперимент планировали с помощью программного обеспечения MODDE с использованием дизайна, который позволяет оценить взаимодействие факторов и в дальнейшем построить проектные поля (reduced central composite design, face-centred, CCF). Культивирование штамма-продуцента проводили в биореакторе Biostat® объемом 5,0 л. Определение концентрации проинсулина аспарт осуществляли методом капиллярного гель-электрофореза. Обработку результатов выполняли с помощью программы GraphPad Prism 6.

Результаты

Результаты: проведена оптимизация условий роста штамма-продуцента и биосинтеза проинсулина аспарт с применением подхода DoE. На основании данных графиков поверхности отклика (для параметров — концентрация влажной биомассы, удельная продуктивность и объемная продуктивность) и построенных моделей были определены проектные поля процесса индукции проинсулина аспарт в клетках штамма-продуцента E. coli. Показано, что построенные модели характеризуются высокой прогностической способностью и высокой воспроизводимостью полученных результатов. Проведена успешная валидация процесса индукции синтеза проинсулина аспарт в оптимизированных условиях в биореакторе. Значение показателя объемной продуктивности штамма-продуцента проинсулина аспарт увеличено с 3,06±0,16 г/л (стандартные условия) до 4,93±0,80 г/л (оптимизированные условия).

Выводы

Выводы: достигнуто увеличение объемной продуктивности штамма-продуцента проинсулина аспарт на 60%. Полученные результаты исследования могут быть использованы для интенсификации промышленного производства инсулина аспарт.

Diabetes poses a serious threat to the health of people around the world. Therefore, in 2021, the World Health Organisation launched the Global Diabetes Compact, an initiative aimed at improving the management and prevention of diabetes. The rapid growth in the number of diabetic patients has increased the need for insulin. Rapid-acting human insulin analogues, including insulin aspart, improve the efficacy of insulin therapy. Methods for insulin aspart production include its biosynthesis in the proinsulin form in Escherichia coli. However, the yield of the recombinant protein largely depends on the optimisation of the production process.

The aim of the study was to optimise the induction conditions for an E. coli strain expressing recombinant proinsulin aspart through applying the Design of Experiment (DoE) approach to enhance bacterial cell productivity.

Materials and methods

Materials and methods. The study focused on a strain of E. coli producing proinsulin aspart. The authors planned the experiment using MODDE software and the reduced face-centred central composite design (CCF) enabling the assessment of factor interactions and the creation of design spaces. The authors carried out fermentations of the producing strain in a 5 L Biostat® B bioreactor and measured proinsulin aspart concentrations by capillary gel electrophoresis. The results were analysed using GraphPad Prism 6.

Results

Results. Using the DoE approach, the authors optimised the conditions for the growth of the producer strain and the biosynthesis of proinsulin aspart. Based on data from response surface plots for wet biomass concentration, specific productivity, and volumetric productivity, as well as plotted models, the authors established design spaces for the induction of proinsulin aspart expression in E. coli. The plotted models demonstrated high predictive power and high reproducibility of the results. The authors successfully validated the induction process for the synthesis of proinsulin aspart in a bioreactor under optimised conditions. The volumetric productivity of the strain producing proinsulin aspart increased from 3.06±0.16 g/L (conventional conditions) to 4.93±0.80 g/L (optimised conditions).

Conclusions

Conclusions. The authors achieved a 60% increase in the volumetric yield of proinsulin aspart. The study results may be used to intensify the industrial production of insulin aspart.

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