(173f) Understanding the Effect of Mild Hypothermia on Monoclonal Antibody Glycosylation through Flux Balance Analysis and Mechanistic Modelling

Authors: 
Sou, S. N., Imperial College London
Sellick, C., MedImmune
Lee, K., MedImmune
Mason, A., MedImmune
Polizzi, K. M., Imperial College London
Kontoravdi, C., Imperial College London

The application of mild hypothermic conditions to cell culture is routine industrial practice used to improve recombinant protein (rProtein) production. However, a thorough understanding of the regulation of dynamic cellular processes at lower temperatures is necessary to enhance bioprocess design and optimisation. In this study, with the use of mechanistic modelling, we investigated the impact of mild hypothermia in protein glycosylation usinf a model system of CHO cells expressing a monoclonal antibody (mAb) cultured at 36.5°C and with a temperature shift to 32°C during late exponential/early stationary phase.

It has been widely reported that the composition of N-linked glycans found on rProteins can be influenced by changes in intracellular metabolism brought about by shifts in bioprocess conditions. To better understand CHO cell metabolism at 32°C compared to physiological temperature, flux balance analysis (FBA) was carried out and constrained with exometabolite data from stationary phase of cultures with or without a temperature shift. Estimated fluxomes suggest reduced fluxes of carbon species towards nucleotide and nucleotide sugar donor synthesis for protein glycosylation, and more energy was used for product formation, resulting in a decreased proportion of the more processed glycan structures on the secreted IgG molecules.

To relate the effect of mild hypothermia with mAb glycosylation under the QbD principles, mechanistic modelling was used. The constructed model was capable of describing and estimating parameters for cell culture dynamics including mAb production, the biosynthesis of nucleotide sugars, as well as the process of N-linked glycosylation in the Golgi apparatus, at both 36.5°C and 32°C. By comparing model outputs at both temperatures, the increased mAb titre at 32°C was affected by increased translation rates in mAb heavy and light chains, together with more robust assembly and transport of IgG molecules for secretion during mild hypothermia. Our model estimation also suggested reduced rates of UDP-GlcNAc and UDP-GalNAc synthesis, and decreased turnover rates for some glycosyltrasferases.

The use of the FBA and mathematical models provided a better overview of how product glycosylation can be impacted on changes in culture temperature. Better feeding strategies can therefore be developed based on improved understanding of the metabolic flux distribution.