13C metabolic flux analysis and gene expression analysis of perfusion culture reveal metabolic bottlenecks for stable glycosylation profiles of biologics.

Perfusion Chinese hamster ovary (CHO) cell culture offers potential benefits over fed-batch cultures, including higher productivity and a reduced production footprint. However, maintaining a stable profile of product quality attributes (PQAs) over the entirety of a perfusion culture has proven to be a challenge across the biopharmaceutical industry, which is critical for ensuring lot-to-lot consistency. A deeper understanding of the underlying molecular mechanisms of PQA variability and productivity can allow for more targeted interventions. In this study, 13C metabolic flux analysis (13C MFA) was performed on perfusion bioreactors at both early and late stage timepoints to identify potential metabolic bottlenecks. Additionally, RNA-level expression of transporters and enzymes throughout metabolism was measured using RNAseq analysis. Central carbon metabolism in late stage culture was found to have significantly higher flux throughout glycolysis and the tricarboxylic acid (TCA) cycle. In nucleotide sugar synthesis pathways, the ratio of GDP-Fucose synthesis flux to GDP-Mannose synthesis flux was reduced at later cell culture stages, indicating a potential bottleneck for maintaining stable profiles of fucosylated and mannosylated glycans. This bottleneck could potentially be explained by changes in expression of the enzymes in these synthesis pathways as well as a limited pool of NADPH. This work represents the first report demonstrating the application of 13C MFA in early and late-stage perfusion cultures, elucidating potential molecular bottlenecks contributing to unstable PQA profiles.