Investigating the Influence of Dissolved Oxygen and pH Dynamics on Cell Growth, Metabolism and Recombinant Protein Production in High-Throughput Bioreactors

Abstract

Dissolved oxygen (DO) concentration and culture pH are two of the most influential process parameters governing Chinese Hamster Ovary (CHO) cell physiology, metabolic activity, and recombinant protein production during upstream bioprocessing. Precise regulation of these parameters is essential for maximizing cell growth, maintaining viability, minimizing the accumulation of inhibitory metabolites, and ensuring consistent product quality. Despite advances in bioprocess control, the dynamic interactions between DO, pH, cellular metabolism, and recombinant protein expression remain incompletely understood, particularly in high-throughput process development platforms. This study investigates the combined effects of varying dissolved oxygen and pH conditions on CHO cell growth, metabolism, and recombinant protein productivity using the Ambr® 15 high-throughput bioreactor system. Parallel fed-batch cultures will be established under systematically controlled DO and pH setpoints using a factorial experimental design to evaluate their individual and interactive effects on bioprocess performance. Cell growth kinetics, viable cell density, cell viability, glucose consumption, lactate production and consumption, glutamine utilization, ammonia accumulation, osmolality, and recombinant protein titer will be monitored throughout the cultivation period using standard analytical techniques. Where applicable, critical quality attributes, including glycosylation profiles and product integrity, will also be assessed. Multivariate statistical analysis and correlation modeling will be employed to identify significant relationships between environmental conditions, metabolic responses, and productivity outcomes. It is anticipated that optimized DO and pH control strategies will reduce metabolic stress, promote favorable metabolic shifts, extend culture longevity, improve nutrient utilization efficiency, and significantly enhance recombinant protein yield without compromising product quality. The findings will provide mechanistic insights into the influence of microenvironmental conditions on CHO cell metabolism and contribute to the development of more robust, scalable, and data-driven upstream bioprocesses. Ultimately, this research will support Quality by Design (QbD) principles, accelerate process development, improve manufacturing consistency, and provide practical guidance for optimizing industrial production of recombinant therapeutic proteins in mammalian cell culture systems.