Multiple steady states and metabolic switches in continuous cultures of HEK293: Experimental evidences and metabolomics

Abstract

The optimization of the production process in the biotechnology industry needs a deep understanding of the metabolic patterns developed in the bioreactors. In particular, the possibility to induce changes between different metabolic states in these cultures has opened a new path to reach this optimization. The goal is to drift the culture toward a metabolic state of maximum productivity. In this work we experimentally explore and analyze this path using a HEK293 cell line, transfected to produce the HER1 glycoprotein, cultured in continuous mode. We first show that this cell culture exhibits steady state multiplicity, i.e., different cell densities and protein concentrations for the same experimental parameters. We also demonstrate that the switch between these steady states can be triggered manipulating the dilution rate in the bioreactor. Furthermore, we present an extensive metabolic characterization of the steady states measuring metabolic concentrations through Liquid Chromatographic-Mass Spectrometry (LC-MS). The data obtained is processed using Principal Component Analysis (PCA), unveiling a correspondence between the culture multiplicity and the existence of distinctive metabolic states. Our results support the idea that different stationary states, although obtained for the same experimental parameters, are consistent with major metabolic readjustments in the culture. Finally, the comparison of the different steady states demonstrates that, in this case, the state with reduced lactate production benefits volumetric productivity.