Addressing amino acid-derived inhibitory metabolites and enhancing CHO cell culture performance through DOE-guided media modifications.

Previously, we identified six inhibitory metabolites (IMs) accumulating in Chinese hamster ovary (CHO) cultures using AMBIC 1.0 community reference medium that negatively impacted culture performance. The goal of the current study was to modify the medium to control IM accumulation through design of experiments (DOE). Initial over-supplementation of precursor amino acids (AAs) by 100% to 200% in the culture medium revealed positive correlations between initial AA concentrations and IM levels. A screening design identified 5 AA targets, Lys, Ile, Trp, Leu, Arg, as key contributors to IMs. Response surface design analysis was used to reduce initial AA levels between 13% and 33%, and these were then evaluated in batch and fed-batch cultures. Lowering AAs in basal and feed medium and reducing feed rate from 10% to 5% reduced inhibitory metabolites HICA and NAP by up to 50%, MSA by 30%, and CMP by 15%. These reductions were accompanied by a 13% to 40% improvement in peak viable cell densities and 7% to 50% enhancement in IgG production in batch and fed-batch processes, respectively. This study demonstrates the value of tuning specific AA levels in reference basal and feed media using statistical design methodologies to lower problematic IMs.

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.

Identification of novel inhibitory metabolites and impact verification on growth and protein synthesis in mammalian cells.

Mammalian cells consume large amount of nutrients during growth and production. However, endogenous metabolic inefficiencies often prevent cells to fully utilize nutrients to support growth and protein production. Instead, significant fraction of fed nutrients is diverted into extracellular accumulation of waste by-products and metabolites, further inhibiting proliferation and protein synthesis. In this study, an LC-MS/MS based metabolomics pipeline was used to screen Chinese hamster ovary (CHO) extracellular metabolites. Six out of eight identified inhibitory metabolites, caused by the inefficient cell metabolism, were not previously studied in CHO cells: aconitic acid, 2-hydroxyisocaproic acid, methylsuccinic acid, cytidine monophosphate, trigonelline, and n-acetyl putrescine. When supplemented back into a fed-batch culture, significant reduction in cellular growth was observed in the presence of each metabolite and all the identified metabolites were shown to impact the glycosylation of a model secreted antibody, with seven of these also reducing CHO cellular productivity (titer) and all eight inhibiting the formation of mono-galactosylated biantennary (G1F) and biantennary galactosylated (G2F) N-glycans. These inhibitory metabolites further impact the metabolism of cells, leading to a significant reduction in CHO cellular growth and specific productivity in fed-batch culture (maximum reductions of 27.2% and 40.6% respectively). In-depth pathway analysis revealed that these metabolites are produced when cells utilize major energy sources such as glucose and select amino acids (tryptophan, arginine, isoleucine, and leucine) for growth, maintenance, and protein production. Furthermore, these novel inhibitory metabolites were observed to accumulate in multiple CHO cell lines (CHO-K1 and CHO-GS) as well as HEK293 cell line. This study provides a robust and holistic methodology to incorporate global metabolomic analysis into cell culture studies for elucidation and structural verification of novel metabolites that participate in key metabolic pathways to growth, production, and post-translational modification in biopharmaceutical production.

Characterization of Chinese hamster ovary cell culture feed media precipitate.

Process intensification of monoclonal antibody production is leading to more concentrated feed media causing issues with precipitation of solids from the media solution. This results in processing problems since components in the precipitate are no longer in solution, changing the media composition and leading to variability in cell culture performance. The goal of this work is to characterize the feed media precipitate, and in particular to identify the precipitated components so that mitigation strategies can be developed. From the conducted analysis, the precipitate was predominately found to be organic and was analyzed with liquid chromatography-mass spectrometry and inductively coupled plasma-optical emission spectroscopy (ICP-OES) to identify the constituent components. Up to ten amino acids were identified with tyrosine (approximately 77 wt.%) and phenylalanine (approximately 4 wt.%) being the most prevalent amino acids. Elemental analysis with ICP-OES revealed that inorganic components were accounted for less than one weight percentage of the solid precipitate with metal sulfates being the predominant inorganic components.

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.

Bioprocess Technologies that Preserve the Quality of iPSCs.

Large-scale production of induced pluripotent stem cells (iPSCs) is essential for the treatment of a variety of clinical indications. However, culturing enough iPSCs for clinical applications is problematic due to their sensitive pluripotent state and dependence on a supporting matrix. Developing stem cell bioprocessing strategies that are scalable and meet clinical needs requires incorporating methods that measure and monitor intrinsic markers of cell differentiation state, developmental status, and viability in real time. In addition, proper cell culture modalities that nurture the growth of high-quality stem cells in suspension are critical for industrial scale-up. In this review, we present an overview of cell culture media, suspension modalities, and monitoring techniques that preserve the quality and pluripotency of iPSCs during initiation, expansion, and manufacturing.