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

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.

近年来，相较于传统疫苗，病毒样颗粒（virus-like particles, VLPs）凭借其独特优势，其生产的重要性与日俱增。当前生物制药行业正基于VLPs开发新型及现有疫苗的更安全候选方案，并革新生产工艺，以此实现更具可持续性、高效性且物种特异性的生产模式。尽管相较于传统表达平台，哺乳动物细胞的产物得率较低，但其可提供精准的翻译后修饰，因此通过哺乳动物细胞平台强化生物生产过程已成为亟待解决的关键问题。可用于强化生产的手段之一为瞬时基因表达技术，该技术已被证实可高效应用于临床前乃至临床试验阶段的VLP生产。本研究采用多重定量蛋白质组学方法，探究了当细胞密度高于4×10^6 个/mL时HEK293细胞培养物的分子特征，以及细胞转染与VLP生产相关的影响机制。研究结果揭示了HEK293细胞在上述三种不同培养条件下的功能与代谢谱特征，借此可明确VLP生产过程中的生理瓶颈。针对细胞密度效应，本研究提出细胞生物学层面的分子改变，可用于解释高细胞密度下细胞转染效率低下的问题。此外，通过分析发生改变的生物学过程，本研究观察到转染后细胞内稳态整体遭到破坏；在确定可通过代谢工程优化的潜在通路后，研究人员提出了多种可用于提升VLP生产效率的解决方案。