(174q) Why Are You so Sensitive? Clone-Specific Lactate Sensitivity in CHO DG44

In Chinese Hamster Ovary (CHO) cell-based bioprocessing, tight control of lactate metabolism is essential for optimal growth, productivity, and product quality. While many CHO clones exhibit stable and predictable lactate shifts under standard conditions, the mechanisms underlying lactate sensitivity in certain clones remain elusive. Without this understanding, our ability to mitigate disruptions in lactate metabolism and improve process robustness is greatly hindered. Hence, elucidating the factors influencing lactate sensitivity is imperative for advancing bioprocess control strategies.

To address this knowledge gap, we cultivated unique mAb-producing CHO DG-44 cell-line, Clone 6, that has a fickle lactate metabolism. During development, Clone 6 demonstrated high productivity despite not having a lactate shift under our platform process. To improve growth and thus titer, we forced a lactate shift by restricting the feed and nearly starving the cells of glucose. Due to the unique metabolism of Clone 6, we explored the metabolic mechanisms driving the lactate shift sensitivity through cultivation under three conditions: the developed process, our platform process, and excess glucose. We also compared Clone 6 to Clone 7, a fast-growing sibling with stable lactate shift under our platform process. Throughout the 14-day cultivation, we collected daily samples to monitor growth, glucose, lactate, and amino acids. Using the measured data, we fit growth, uptake, and secretion rates and mapped them to a genome-scale metabolic model for CHO DG44 With the condition-specific models, we will characterize each metabolic state through Markov Chain Monte Carlo sampling and Principal Component Analysis. Through comparison of the predicted metabolic states, we will identify the specific perturbations in early exponential phase that differentiate the conditions.

Our focused analysis of a sensitive clone and its stable sibling will provide unique insight in the molecular mechanisms controlling the lactate shift and highlight metabolic markers for improved clone selection. Furthermore, a deeper understanding of the lactate shift mechanism will inspire media formulations and feeding strategies to improve process robustness.