Kinetic characterization of vero cell metabolism in a serum‐free batch culture process

Abstract

A global kinetic study of the central metabolism of Vero cells cultivated in a serum-free medium is proposed in the present work. Central metabolism including glycolysis, glutaminolysis, and tricarboxylic acid cycle (TCA) was demonstrated to be saturated by high flow rates of consumption of the two major substrates, glucose, and glutamine. Saturation was reavealed by an accumulation of metabolic intermediates and amino acids, by a high production of lactate needed to balance the redox pathway, and by a low participation of the carbon flow to the TCA cycle supply. Different culture conditions were set up to reduce the central metabolism saturation and to better balance the metabolic flow rates between lactate production and energetic pathways. From these culture conditions, substitutions of glutamine by other carbon sources, which have lower transport rates such as asparagine, or pyruvate in order to shunt the glycolysis pathway, were successful to better balance the central metabolism. As a result, an increase of the cell growth with a concomitant decrease of cell death and a better distribution of the carbon flow between TCA cycle and lactate production occurred. We also demonstrated that glutamine was a major carbon source to supply the TCA cycle in Vero cells and that a reduction of lactate production did not necessary improve the efficiency of the Vero cell metabolism. Thus, to adapt the formulation of the medium to the Vero cell needs, it is important to provide carbon substrates inducing a regulated supply of carbon in the TCA cycle either through the glycolysis or through other pathways such as glutaminolysis. Finally, this study allowed to better understand the Vero cell behavior in serum-free medium which is a valuable help for the implementation of this cell line in serum-free industrial production processes.

Improved poliovirus d-antigen yields by application of different Vero cell cultivation methods

“…These cell concentrations were at comparable levels to those previously reported for recirculation cultures [15]. In addition, the cell densities reached using perfusion, semi-batch and batch cultures were comparable to those reported by others [8], [16], [17].…”

Real-time monitoring of adherent Vero cell density and apoptosis in bioreactor processes

“…5). Taking into account that the usual cell densities targeted for virus infection of Vero cells are mostly below 1 9 10 6 cell mL -1 , such a method for in situ monitoring the Vero cell density could be attractive for vaccine manufacturers (Liu et al 2007;Petiot et al 2010b;Toriniwa and Komiya 2008;Butler et al 2000). In the case of higher Vero cell densities, additional studies would be needed to quantify more precisely the cell size and to modelize the cell morphological modifications appearing after confluency on a spherical surface.…”

“…The lysed cells were quantified in culture supernatant by the analysis of the lactate dehydrogenase (LDH) activity according to the protocol previously described Petiot et al (2010b). The activity of the LDH released in the cell culture supernatant was analyzed with the enzymatic LDH PAP kit (Ellitech, Salon-de-Provence, France).…”

Influence of HEK293 metabolism on the production of viral vectors and vaccine

“…Media defined in the literature as non-ammoniagenic, have substitution of glutamine by other AA (glutamate, asparagine), alanine-glutamine dipeptide (Glutamax ® ) or tricarboxylic acids (pyruvate, oxoglutarate), all having either none, or lower, degradation to ammonia. This strategy has been applied successfully to a number of different cell lines (Vero cells and MDCK cells [86,87]) and for HEK293 cells [61]. Glutamate was evaluated for the production of adenovirus [61], and such non-ammoniagenic medium strategy resulted in a 5-fold increase in adenovirus productivity.…”