Metabolomic profiling of CHO fed‐batch growth phases at 10, 100, and 1,000 L

Abstract

Established bioprocess monitoring is based on quick and reliable methods, including cell count and viability measurement, extracellular metabolite measurement, and the measurement of physicochemical qualities of the cultivation medium. These methods are sufficient for monitoring of process performance, but rarely give insight into the actual physiological states of the cell culture. However, understanding of the latter is essential for optimization of bioprocess development. Our study used LC-MS metabolomics as a tool for additional resolution of bioprocess monitoring and was designed at three bioreactors scales (10 L, 100 L, and 1,000 L) to gain insight into the basal metabolic states of the Chinese hamster ovary (CHO) cell culture during fed-batch. Metabolites characteristics of the four growth stages (early and late exponential phase, stationary phase, and the phase of decline) were identified by multivariate analysis. Enriched metabolic pathways were then established for each growth phase using the CHO metabolic network model. Biomass generation and nucleotide synthesis were enriched in early exponential phase, followed by increased protein production and imbalanced glutathione metabolism in late exponential phase.Glycolysis became downregulated in stationary phase and amino-acid metabolism increased. Phase of culture decline resulted in rise of oxidized glutathione and fatty acid concentrations. Intracellular metabolic profiles of the CHO fed-batch culture were also shown to be consistent with scale and thus demonstrate metabolomic profiling as an informative method to gain physiological insight into the cell culture states during bioprocess regardless of scale. K E Y W O R D SChinese hamster ovary, CHO, growth phase, metabolomic, profile, scale-up

Quantification of the dynamics of population heterogeneities in CHO cultures with stably integrated fluorescent markers

“…Different growth phases are characterized generally by different bulk growth behaviors and varying metabolic fluxes and regulations [45][46][47][48]. The transition of the cultures into the stationary growth phase occurs along with apoptosis and necrosis [49,50].…”

Small-Scale Fluidized Bed Bioreactor for Long-Term Dynamic Culture of 3D Cell Constructs and in vitro Testing

“…It yielded comparable viable cell numbers to the clinical scale FBB as viability and cell density were within the intended interval. Minor differences between scales have also been reported in other scale-down models, for example in viable cell number or metabolic activity either due to uncontrolled parts of the process or just a consequence of inherent biomass variability (Zeilinger et al, 2011;Vodopivec et al, 2019). Furthermore, hydrogel beads maintaining their integrity and sphericity throughout 12 days (and equivalent to those under static conditions) indicated that the hydrodynamic shear forces were low and not detrimental to their structure with the hydrogel still supporting and shielding the cell spheroids from direct contact with shear stress.…”

Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

“…The PAT framework was also used during the production of Lethal Toxin-Neutralizing Factor (LTNF) by E. coli, which was controlled by a decoupled input-output linearizing controller (DIOLC) (Dalal et al, 2019). LC-MS metabolomics at three bioreactor scales (10 l, 100 l, and 1,000 l) were utilized to gain insight into the basal metabolic states of the CHO cell culture during fed-batch, and this was demonstrated as a useful technique to obtain physiological information on the cell culture state during a bioprocess, regardless of scale (Vodopivec et al, 2019).…”