Coacervate-Based Monitoring of Oxidative Stress in Chinese Hamster Ovary (CHO) Cell Biomanufacturing

Introduction: Chinese hamster ovary (CHO) cells are the industry's primary host for the biomanufacturing of biologics, including monoclonal antibodies and therapeutic proteins for the treatment of numerous diseases¹. However, the accumulation of excess reactive oxygen species (ROS) during production can significantly impair CHO cell viability and productivity². Elevated ROS levels compromise product yield and increase the risk of batch failure, resulting in substantial material waste, production delays, and increased manufacturing costs². Despite these challenges, current methods for monitoring extracellular ROS are limited by slow response times and retrospective data analysis³. We compare the performance of a novel coacervate-based fluorescent sensor to the industry standard Amplex Red assay for real-time monitoring of oxidative stress in CHO cell biomanufacturing

Materials & Methods: CHOZN host CHO cells were cultured in batch-mode shake flasks, providing a controlled environment for evaluating extracellular ROS dynamics. The polyamine spermine was modified with a patent-pending reaction to instill ROS responsivity to coacervates made from the modified spermine and AlexaFluor568-tagged polyuridylic acid (polyU) (Figure 1). The coacervates were stabilized using polyethylene glycol (PEG) diamine at varying weight percentages to optimize their performance in CHO culture media-.. Amplex Red assay and coacervate-based measurements were taken during each phase of culture. Measurement sensitivity, precision, consistency, and speed were compared between the two methods.

Results: Our batch-mode shake flask setup enabled reproducible cell culture conditions representative of industry standards. Coacervates exhibited enhanced stability in CHO media with increasing PEG-DIAMINE concentration (Figure 2). However, these changes also impacted ROS responsiveness, indicating a balance between structural robustness and sensing performance. Optimized formulations-maintained ROS sensitivity while improving physical stability in culture conditions. Comparisons with Amplex Red Assay measurements suggest that our novel coacervate-based sensor can deliver ROS measurements with similar accuracy and precision, but with significantly faster response times, highlighting its potential for real-time monitoring applications in biomanufacturing.

Conclusions: This study demonstrates the potential of our novel coacervate-based fluorescent sensor, stabilized with PEG-diamine, as a rapid and reliable platform for real-time monitoring of extracellular ROS in CHO cell cultures. The batch-mode culture system provided a consistent and industry-relevant environment to evaluate coacervate performance. Adjustments to PEG-diamine molecular weight and concentration significantly impacted coacervate stability and ROS responsiveness, highlighting the importance of tuning formulation parameters. Optimized coacervates showed comparable accuracy to standard ROS assays (Amplex Red), with the added advantage of faster response times. These findings support the integration of coacervate-based sensors into upstream bioprocessing workflows, offering a promising tool for earlier intervention, improved yields, and reduced manufacturing costs in biomanufacturing