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.