Открытый доступ
Доступ платный или только для подписчиков
Создание стабильной высокопродуктивной платформы на основе клеток CHO, адаптированных к химическому и механическому стрессам
Аннотация
DOI: 10.30906/2073-8099-2020-12-1-15-24
Возрастающий спрос на получение рекомбинантных молекул требует создания не только стабильных, но и высокопроизводительных клеточных линий, пригодных для масштабного производства. В настоящей работе предложен дизайн вектора для эффективной и стабильной экспрессии рекомбинатных моноклональных антител в клетках CHO и схема адаптации линии CHO-S (реципиента) к высокой осмоляльности среды в условиях повышенного содержания хлорида натрия (до 100 мМ), лактата натрия (до 170 мМ) и скорости перемешивания в биореакторе до 240 об/мин. Показана эффективность полученной клеточной платформы на примере экспрессии моноклонального антитела IgG2/4k, позволяющей получать до 2,5 г/л продукта без амплификации гена интереса и оптимизации условий культивирования в режиме fed-batch.Ключевые слова
Ссылки
Walsh G. Biopharmaceutical benchmarks 2018 / Nat. Biotechnol. 2018. V. 36. P. 1136 – 1145.
Grilo A. L., Mantalaris A. The increasingly human and profitable monoclonal antibody market / Trends Biotechnol. 2019. V. 37. No. 1. P. 9 – 16.
Lalonde M. E., Durocher Y. Therapeutic glycoprotein production in mammalian cells / J. Biotechnol. 2017. V. 251. P. 128 – 140.
Gaidukov L. et al. A multi-landing pad DNA integration platform for mammalian cell engineering / Nucl. Acids Res. 2018. V. 46. No. 8. P. 4072 – 4086.
Zhao M. et al. Rapid development of stable transgene CHO cell lines by CRISPR / Cas9-mediated site-specific integration into C12orf35 / Appl. Microbiol. Biotechnol. 2018. V. 102. No. 14. P. 6105 – 6117.
Kostyrko K. et al. MAR-Mediated transgene integration into permissive chromatin and increased expression by recombination pathway engineering / Biotechnol. Bioeng. 2017. V. 114. No. 2. P. 384 – 396.
Orlova N. A. et al. Improved elongation factor-1 alpha-based vectors for stable high-level expression of heterologous proteins in Chinese hamster ovary cells / BMC Biotechnol. 2014. V. 14. No. 1. P. 56.
Van Blokland H. J. M. et al. Methods to create a stringent selection system for mammalian cell lines / Cytotechnology. 2011. V. 63. No. 4. P. 371 – 384.
Kobayashi M. et al. The CMV enhancer stimulates expression of foreign genes from the human EF-1? promoter / Anal. Biochem. 1997. V. 247. No. 1. P. 179 – 181.
Lai T., Yang Y., Ng S. K. Advances in mammalian cell line development technologies for recombinant protein production / Pharmaceuticals. 2013. V. 6. No. 5. P. 579 – 603.
Qin J. Y. et al. Systematic comparison of constitutive promoters and the doxycycline-inducible promoter / PloS One. 2010. V. 5. No. 5. P. e10611.
Osterlehner A., Simmeth S., G?pfert U. Promoter methylation and transgene copy numbers predict unstable protein production in recombinant Chinese hamster ovary cell lines / Biotechnol. Bioeng. 2011. V. 108. No. 11. P. 2670 – 2681.
Brooks A. R. et al. Transcriptional silencing is associated with extensive methylation of the CMV promoter following adenoviral gene delivery to muscle / J. Gene Med.. 2004. V. 6. No. 4. P. 395 – 404.
Saunders F. et al. Chromatin function modifying elements in an industrial antibody production platform-comparison of UCOE, MAR, STAR and cHS4 elements / PloS One. 2015. V. 10. No. 4. P. e0120096.
Moritz B., Becker P. B., G?pfert U. CMV promoter mutants with a reduced propensity to productivity loss in CHO cells / Sci. Rep. 2015. V. 5. P. 16952.
Wang W. et al. Enhanced transgene expression using cis-acting elements combined with the EF1 promoter in a mammalian expression system / Eur. J. Pharm. Sci. 2018. V. 123. P. 539 – 545.
Kim S. Y. et al. The human elongation factor 1 alpha (EF-1?) first intron highly enhances expression of foreign genes from the murine cytomegalovirus promoter / J. Biotechnol. 2002. V. 93. No. 2. P. 183 – 187.
Wang X. Y. et al. Impact of different promoters on episomal vectors harbouring characteristic motifs of matrix attachment regions / Sci. Rep. 2016. V. 6. P. 26446.
Fan L. et al. Development of a highly-efficient CHO cell line generation system with engineered SV40E promoter / J. Biotechnol. 2013. V. 168. No. 4. P. 652 – 658.
Lanza A. M., Kim D. S., Alper H. S. Evaluating the influence of selection markers on obtaining selected pools and stable cell lines in human cells / Biotechnol. J. 2013. V. 8. No. 7. P. 811 – 821.
Schorpp M. et al. The human ubiquitin C promoter directs high ubiquitous expression of transgenes in mice / Nucl. Acids Res. 1996. V. 24. No. 9. P. 1787 – 1788.
Galbete J. L., Buceta M., Mermod N. MAR elements regulate the probability of epigenetic switching between active and inactive gene expression / Mol. BioSyst. 2009. V. 5. No. 2. P. 143 – 150.
Harraghy N. et al. Using matrix attachment regions to improve recombinant protein production / Protein Expression in Mammalian Cells. — NY: Humana Press, 2012. P. 93 – 110.
Zhao C. P. et al. Matrix attachment region combinations increase transgene expression in transfected Chinese hamster ovary cells / Sci. Rep. 2017. V. 7. P. 42805.
Lin Y. et al. MAR characteristic motifs mediate episomal vector in CHO cells / Gene. 2015. V. 559. No. 2. P. 137 – 143.
Cho M. S., Chan S. Y. Vectors having terminal repeat sequence of Epstein-Barr virus. US Patent No. 6180108. 2001.
Voronina E. V. Design of a stable cell line producing a recombinant monoclonal anti-TNF? antibody based on a CHO cell line producing recombinant monoclonal antibody to tumor necrosis factor alfa based on CHO-DG44 / SpringerPlus. 2016. V. 5(1). P. 1584.
Cacciatore J. J. Gene amplification and vector engineering to achieve rapid and high-level therapeutic protein production using the DHFR-based CHO cell selection system / Biotechnol. Adv 2010. V. 28 (6). P. 673–681.
Al-Rubeai M., Singh R. P., Goldman M. H., Emery A. N. Death mechanisms of animal cells in conditions of intensive agitation / Biotechnol. Bioeng. 1995. V. 45(6). P. 463 – 472.
Michaels J. D., Nowak J. E., Mallik A. K., Koczo K., Wasan D. T., Papoutsakis E. T. Analysis of cell-¬to-bubble attachment in sparged bioreactors in the presence of cell-protecting additives. / Biotechnol Bioeng. 1995. V. 47(4). P. 407 – 419.
Senger R. S. Effect of shear stress on intrinsic CHO culture state and glycosylation of recombinant tissue-type plasminogen activator protein / Biotechnol. Prog. 2003. V. 19(4). P. 1199 – 1209.
Shukurov R. R. et al. Optimization of genetic constructs for high-level expression of the darbepoetin gene in mammalian cells / Appl. Biochem. Microbiol. 2014. V. 50(9). P. 802 – 811.
Rose S. / Mammalian cell culture: Process development considerations. Handbook of industrial cell culture. — NY: Humana Press; Totowa,2003. P. 546.
Ссылки
- Ссылки не определены.
** ** ** ** ** **
ISSN: 2073-8099
** ** ** ** ** **
Подписаться на наши издания Вы можете через почтовые каталоги Объединенный каталог «Пресса России» «Урал Пресс», «Ивис», «Прессинформ» и «Профиздат».
Наши партнеры:
iIPhEB - Международная выставка и форум по фармацевтике и биотехнологиям, 2–4 апреля 2024