13C metabolic flux analysis identifies limitations to increasing specific productivity in fed-batch and perfusion

- The stationary phase does not imply negligible gross growth rates or death rates.
- Enhanced cell turnover increased HCP impurities and correlated with reduced Qp.
- Magnitude of specific biomass protein productivity comparable to Qp.
- Enhanced BCAA catabolic rate associated with enhanced death rate.

Industrial cell culture requires substantial energy to generate protein. The protein generated is not only the product of interest (IgG in this case), but also the protein associated with biomass. Here, 13C-Metabolic Flux Analysis (13C-MFA) was utilized to compare the stationary phase of a fed-batch process to a perfusion process producing the same product by the same clone. The fed-batch process achieved significantly higher specific productivity, approximately 60% greater than the perfusion process. In spite of this, a general lack of difference was observed when globally comparing glycolysis, pentose phosphate pathway, and TCA cycle fluxes. In contrast, gross growth rate was significantly different, approximately 80% greater in the perfusion process. This difference was concealed by a significantly greater death rate in the perfusion process, such that net growth rates were both similar and near-zero. When considering gross growth rate and IgG specific productivity, total protein specific productivity (Biomass+ IgG) differed little, offering rationale for the observed central carbon pathway similarities. Significant differences were identified in anaplerotic branched-chain amino acid (BCAA) contributions by 13C-MFA. The perfusion process exhibited markedly higher (up to three times) BCAA catabolism, an observation often associated with increased death.