Multiplexed Quantitative Proteomic Analysis of HEK293 Provides Insights into Molecular Changes Associated with the Cell Density Effect, Transient Transfection, and Virus-Like Particle Production.,Journal of Proteome Research

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Multiplexed Quantitative Proteomic Analysis of HEK293 Provides Insights into Molecular Changes Associated with the Cell Density Effect, Transient Transfection, and Virus-Like Particle Production.
Journal of Proteome Research ( IF 3.8 ) Pub Date : 2020-02-07 , DOI: 10.1021/acs.jproteome.9b00601
Jesús Lavado-García ₁ , Inmaculada Jorge _{2,

3} , Laura Cervera ₁ , Jesús Vázquez _{2,

3} , Francesc Gòdia ₁

Affiliation

The production of virus-like particles (VLPs) has gained importance over the last few years owing to the benefits they provide compared to conventional vaccines. The biopharmaceutical industry is currently searching for safer candidates based on VLPs for new and existing vaccines and implementing new methods of manufacturing, thus allowing a more sustainable, effective, and species-specific production. Despite achieving lower yields compared to traditional platforms, the use of mammalian cells provides the right post-translational modifications, and consequently, the intensification of bioprocesses using mammalian cell platforms has become a matter of pressing concern. One of the methods subjected to intensification is transient gene expression, which has been proven to be highly effective regarding VLP production for preclinical or even clinical trials. In this work, a multiplexed quantitative proteomic approach has been applied to study the molecular characteristics of HEK293 cell cultures when growing at cell densities higher than 4 × 106 cells/mL and to study the effects related to cell transfection and VLP production. The obtained results revealed a set of functional and metabolic profiles of HEK293 under these three different conditions that allowed the identification of physiological bottlenecks regarding VLP production. Regarding the cell density effect, molecular alterations in the cell biology were proposed to help explain the difficulty for the cells to be transfected at higher densities. In addition, an overall disruption of cellular homeostasis after transfection was observed based on altered biological processes, and after identifying potential pathways liable to be optimized via metabolic engineering, different solutions were proposed to improve VLP production.

中文翻译：

HEK293的多重定量蛋白质组学分析提供了与细胞密度效应，瞬时转染和病毒样颗粒产生相关的分子变化的见解。

由于病毒样颗粒（VLP）与常规疫苗相比具有优势，因此近几年来，这种产品的重要性日益提高。生物制药行业目前正在寻找基于VLP的更安全的候选药物，以用于新疫苗和现有疫苗，并实施新的生产方法，从而实现更可持续，更有效且针对特定物种的生产。尽管与传统平台相比产量较低，但使用哺乳动物细胞仍可提供正确的翻译后修饰，因此，使用哺乳动物细胞平台加强生物过程已成为迫切关注的问题。进行强化的方法之一是瞬时基因表达，已被证明对于临床前乃至临床试验的VLP生产非常有效。在这项工作中，已经采用了多重定量蛋白质组学方法来研究HEK293细胞培养物在高于4×106细胞/ mL的细胞密度下生长时的分子特征，并研究与细胞转染和VLP产生有关的影响。所获得的结果揭示了在这三种不同条件下，HEK293的一组功能和代谢谱，从而可以鉴定出有关VLP产生的生理瓶颈。关于细胞密度效应，提出了细胞生物学中的分子改变，以帮助解释以更高密度转染细胞的困难。此外，

更新日期：2020-02-07

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