Combined multivariate statistical and flux balance analyses uncover media bottlenecks to the growth and productivity of Chinese hamster ovary cell cultures

Abstract

Chinese hamster ovary (CHO) cells are widely used for producing recombinant proteins. To enhance their productivity and product quality, media reformulation has been a key strategy, albeit with several technical challenges, due to the myriad of complex molecular mechanisms underlying media effects on culture performance. Thus, it is imperative to characterize metabolic bottlenecks under various media conditions systematically. To do so, we combined partial least square regression (PLS‐R) with the flux balance analysis of a genome‐scale metabolic model to elucidate the physiological states and metabolic behaviors of human alpha‐1 antitrypsin producing CHO‐DG44 cells grown in one commercial and another two in‐house media under development. At the onset, PLS‐R was used to identify metabolite exchanges that were correlated to specific growth and productivity. Then, by comparing metabolic states described by resultant flux distributions under two of the media conditions, we found suboptimal level of four nutrients and two metabolic wastes, which plausibly hindered cellular growth and productivity; mechanistically, lactate and ammonia recycling were modulated by glutamine and asparagine metabolisms in the media conditions, and also by hitherto unsuspected folate and choline supplements. Our work demonstrated how multivariate statistical analysis can be synergistically combined with metabolic modeling to uncover the mechanistic elements underlying differing media performance. It thus paved the way for the systematic identification of nutrient targets for medium reformulation to enhance recombinant protein production in CHO cells.

Data‐driven prediction models for forecasting multistep ahead profiles of mammalian cell culture toward bioprocess digital twins

“…Our study found that ammonia is the most important feature among the others based on its contribution to the model accuracy in predicting one‐step ahead of VCD (Supplementary Figure S1). In fact, ammonia is a waste byproduct of cell metabolism and its accumulation in the culture medium can have a negative impact on cell growth and viability, as an inhibitory and toxic metabolite (Yeo et al, 2022). Additionally, the production and accumulation of ammonia vary depending on the specific cell line and culture conditions (Genzel et al, 2005; Kim et al, 2013; Ozturk et al, 1992).…”

Debottlenecking and reformulating feed media for improved CHO cell growth and titer by data‐driven and model‐guided analyses

“…In this regard, we previously developed a data-driven and in silico model-guided systematic framework to investigate the effect of growth (basal) media on CHO cell culture performance, as such successfully suggesting the new media formulations for the enhanced growth and final titer. [32,33] Similarly, well-designed and tailored targets of nutrients for feed media can be explored using the framework. It should be noted that the composition of the feed media is typically more complex than basal media since many include additional amino acids, vitamins, trace elements, and other supplements that are not present in the basal media.…”

“…In this regard, we previously developed a data‐driven and in silico model‐guided systematic framework to investigate the effect of growth (basal) media on CHO cell culture performance, as such successfully suggesting the new media formulations for the enhanced growth and final titer. [ 32,33 ]…”

Debottlenecking and reformulating feed media for improved CHO cell growth and titer by data-driven and model-guided analyses

“…In this regard, in our previous study, we developed data-driven and in-silico model-guided systematic framework to investigate the effect of growth media on CHO cell culture performance, allowing us to design the new media formulations for enhanced productivity. [33,34] In fact, most of the biologics based on CHO cells are manufactured using fed-batch process. Since basal media may not contain all of the specific nutrients and growth factors required to support high levels of cell growth and productivity, feed media are typically supplemented to the basal media regularly during the culture process to provide additional nutrients and growth factors that are required for improving cell growth, productivity, and viability.…”

“…In this regard, in our previous study, we developed data-driven and in-silico model-guided systematic framework to investigate the effect of growth media on CHO cell culture performance, allowing us to design the new media formulations for enhanced productivity. [33,34]…”