Trace metal optimization in CHO cell culture through statistical design of experiments.

A majority of the biotherapeutics industry today relies on the manufacturing of monoclonal antibodies from Chinese hamster ovary (CHO) cells, yet challenges remain with maintaining consistent product quality from high-producing cell lines. Previous studies report the impact of individual trace metal supplemental on CHO cells, and thus, the combinatorial effects of these metals could be leveraged to improve bioprocesses further. A three-level factorial experimental design was performed in fed-batch shake flasks to evaluate the impact of time wise addition of individual or combined trace metals (zinc and copper) on CHO cell culture performance. Correlations among each factor (experimental parameters) and response variables (changes in cell culture performance) were examined based on their significance and goodness of fit to a partial least square's regression model. The model indicated that zinc concentration and time of addition counter-influence peak viable cell density and antibody production. Meanwhile, early copper supplementation influenced late-stage ROS activity in a dose-dependent manner likely by alleviating cellular oxidative stress. Regression coefficients indicated that combined metal addition had less significant impact on titer and specific productivity compared to zinc addition alone, although titer increased the most under combined metal addition. Glycan analysis showed that combined metal addition reduced galactosylation to a greater extent than single metals when supplemented during the early growth phase. A validation experiment was performed to confirm the validity of the regression model by testing an optimized setpoint of metal supplement time and concentration to improve protein productivity.

Modulation of nutrient precursors for controlling metabolic inhibitors by genome-scale flux balance analysis.

Therapeutic protein productivity and glycosylation pattern highly rely on cell metabolism. Cell culture medium composition and feeding strategy are critical to regulate cell metabolism. In this study, the relationship between toxic metabolic inhibitors and their nutrient precursors was explored to identify the critical medium components toward cell growth and generation of metabolic by-products. Generic CHO metabolic model was tailored and integrated with CHO fed-batch metabolomic data to obtain a cell line- and process-specific model. Flux balance analysis study was conducted on toxic metabolites cytidine monophosphate, guanosine monophosphate and n-acetylputrescine-all of which were previously reported to generate from endogenous cell metabolism-by mapping them to a compartmentalized carbon utilization network. Using this approach, the study projected high level of inhibitory metabolites accumulation when comparing three industrially relevant fed-batch feeding conditions one against another, from which the results were validated via a dose-dependent amino acids spiking study. In the end, a medium optimization design was employed to lower the amount of supplemented nutrients, of which improvements in critical process performance were realized at 40% increase in peak viable cell density (VCD), 15% increase in integral VCD, and 37% increase in growth rate. Tight control of toxic by-products was also achieved, as the study measured decreased inhibitory metabolites accumulation across all conditions. Overall, the study successfully presented a digital twin approach to investigate the intertwined relationship between supplemented medium constituents and downstream toxic metabolites generated through host cell metabolism, further elucidating different control strategies capable of improving cellular phenotypes and regulating toxic inhibitors.

Effect of iron addition on mAb productivity and oxidative stress in Chinese hamster ovary culture.

Trace metals play a critical role in the development of culture media used for the production of therapeutic proteins. Iron has been shown to enhance the productivity of monoclonal antibodies during Chinese hamster ovary (CHO) cell culture. However, the redox activity and pro-oxidant behavior of iron may also contribute toward the production of reactive oxygen species (ROS). In this work, we aim to clarify the influence of trace iron by examining the relationship between iron supplementation to culture media, mAb productivity and glycosylation, and oxidative stress interplay within the cell. Specifically, we assessed the impacts of iron supplementation on (a) mAb production and glycosylation; (b) mitochondria-generated free hydroxyl radicals (ROS); (c) the cells ability to store energy during oxidative phosphorylation; and (d) mitochondrial iron concentration. Upon the increase of iron at inoculation, CHO cells maintained a capacity to rebound from iron-induced viability lapses during exponential growth phase and improved mAb productivity and increased mAb galactosylation. Fluorescent labeling of the mitochondrial hydroxyl radical showed enhanced environments of oxidative stress upon iron supplementation. Additional labeling of active mitochondria indicated that, despite the enhanced production of ROS in the mitochondria, mitochondrial membrane potential was minimally impacted. By replicating iron treatments during seed train passaging, the CHO cells were observed to adapt to the shock of iron supplementation prior to inoculation. Results from these experiments demonstrate that CHO cells have the capacity to adapt to enhanced environments of oxidative stress and improve mAb productivity and mAb galactosylation with minimal perturbations to cell culture.