Improving baculovirus production at high cell density through manipulation of energy metabolism

The insect cells/baculovirus system is well recognized as a safe and suitable technology to produce heterologous proteins, vaccines and vectors for gene therapy. Efficient and robust production processes, able to deliver higher product concentrations, are however still needed to cope with increased requirements for large-scale manufacture. The work herein presented describes a combined experimental and modelling effort to quantify and environmentally manipulate the metabolism of Spodoptera frugiperda cells, targeting high cell density production of baculovirus vectors with potential application in human gene therapy. Culture medium supplementation with pyruvate or alpha-ketoglutarate at the time of infection resulted in 6-7-fold higher specific baculovirus yields at high cell density when compared to control cultures. This pushed volumetric titers to levels higher than classical low cell density infections. A quantitative description of intracellular pathways is provided using metabolic flux analysis; a direct stimulation of carbon flow through the tricarboxylic acids cycle was observed. Analysis of flux partitioning coefficients at the pyruvate and alpha-ketoglutarate branch-points further revealed a metabolic transition to a more energetically active state, which was confirmed by increased intracellular adenosine triphosphate generation rates. These results represent a cost-efficient and scalable strategy for high cell density production of recombinant baculovirus vectors.