The use of antioxidants in Chinese hamster ovary (CHO) cell cultures to improve monoclonal antibody production has been a topic of great interest. Nevertheless, the mechanisms by which antioxidant pathways are regulated in CHO cells and their effect on metabolism are not fully understood. In this work, we investigated how treatment with the antioxidant rosmarinic acid (RA) improved viable cell density and titer in CHO cell cultures, and attempted to explore the underlying mechanism(s) using transcriptomics and metabolomics. In particular, transcriptomics analysis indicated that RA treatment modified gene expression and strongly affected the MAPK and Akt signaling pathways which regulate cell survival and cell death. Moreover, we observed that these effects did not appear related to an intracellular metabolism change. In summary, this integrated ‘omics analysis has important implications for the role of the antioxidant RA in industrial cell culture processes. The current study also represents an example in the industry of how multi-omics can be applied to gain an in‐depth understanding of CHO cell biology and to identify critical pathways that can contribute to cell culture process improvement and cell line engineering.

The biopharmaceutical industry is transitioning from currently deployed batch-mode bioprocessing to a highly efficient and agile next generation bioprocessing with the adaptation of continuous bioprocessing, which reduces the capital investment and operational costs. Continuous bioprocessing, aligned with FDA’s quality-by-design (QbD) platform, is designed to develop robust processes to deliver safe and effective drugs. With the deployment of knowledge based operations, product quality can be built into the process to achieve desired critical quality attributes (CQAs) with reduced variability. To facilitate next generation continuous bio-processing, it is essential to embrace a fundamental shift-in-paradigm from “quality-by-testing” to “quality-by-design”, which requires the deployment of process analytical technologies (PAT). With the adaptation of PAT, a systematic approach of process and product understanding and timely process control are feasible. Deployment of PAT tools for real-time monitoring of CQAs and feedback control is critical for continuous bioprocessing. Given the current deficiency in PAT tools to support continuous bioprocessing, we have integrated Agilent 2D-LC with a post-flow-splitter in conjunction with the SegFlow automated sampler to the bioreactors. With this integrated system, we have established a platform for online measurements of titer and CQAs of monoclonal antibodies (mAbs) as well as amino acid concentrations of bioreactor cell culture.

Process analytical technology (PAT) has been defined by the Food and Drug Administration (FDA) as a system for designing, analyzing, and controlling manufacturing through timely measurements to ensure final product quality. Based on quality-by-design (QbD) principles, real-time data monitoring is essential for timely control of critical quality attributes (CQAs) to keep the process in a state of control. To facilitate next-generation continuous bioprocessing, deployment of PAT tools for real-time monitoring is integral for process understanding and control. Real-time monitoring and control of CQAs is essential to keep the process within the design space, which is in alignment with the guiding principles of QbD concepts. The contents of this manuscript are pertinent to the online/at-line monitoring of upstream titer and downstream product quality. We have demonstrated that Waters’ PATROL system can be utilized to measure product titer and CQAs directly from bioreactors and downstream unit operations, respectively. We have established that online titer assay results from fed-batch and perfusion-based alternating tangential flow (ATF) bioreactors are comparable to at-line and conventional offline results. The utility of the PATROL system for online product quality measurements of down-steam unit operations for real-time peak pooling has been demonstrated.