Evaluation and characterisation of phenotypic heterogeneity in Chinese Hamster Ovary cell populations during long-term culture

The selection of CHO cell lines for manufacturing therapeutic proteins involves multiple screening steps of transfected cells that would meet industrial standards such as productivity and stability. This study is a starting point for the idea that cell line development could be improved if a reversed approach involving the selection of un-transfected cell with desirable growth characteristics is first implemented, followed by an accelerated genetic drift in a long-term sub-cultivation. To address this, the inherent cellular heterogeneity within an un-transfected parental CHO-S population was exploited by isolating 22 clonal CHO-S populations through two rounds of the limiting dilution cloning, followed by an accelerated genetic drift and directed evolution resulted from a continuous sub-cultivation (up to 220 generations), along with the cryopreservation of subpopulations approximately every 40 generations. The initial growth compassion exhibited positive correlations between the specific growth rate and generation number and between the peak of viable cell density and generation number. The fed-batch studies also showed that the integral of viable cell density (IVCD) performance can be enhanced along the long-term cultivation, but this was not necessarily improved with increasing generation number because clones were evolved to enhance the rate of biomass production and not to withstand severe environmental conditions typically found at mid- and late- stages of fed-batch cultivation.
Metabolic analyses showed that glucose and glutamine were rapidly metabolised to provide energy and intermediates for cell division, also showing that glutamine availability defines the duration of the exponential growth phase and that its depletion promotes a switch to a more efficient glucose usage tightly coupled to oxidative phosphorylation (OXPHOS) this in turn reduced glucose uptake and lactate production, and even switched to net lactate consumption. The lactate: glucose ratio in proliferating populations showed that glutamine was also metabolised to provide enough levels of NADPH for fatty acid biosynthesis and redox homeostasis, thus producing lactate given the down-regulation of pyruvic acid flux towards the tricarboxylic acid (TCA) cycle. The mitochondrial and glycolytic analysis along the long-term cultivation showed that clones reduced both metabolisms, whereas the analysis at exponential growth phase showed that populations with high proliferation rates present an elevated OXPHOS activity -supported by glutamine metabolism- and strong aerobic glycolysis -supported by glucose uptake-. Contrary, populations at stationary growth phase with high global IVCD performance presented a low respiratory metabolism, but efficiently couple to ATP production, to reduce a potential mitochondrial damage resulted from increments in the proton leakage across the inner mitochondrial membrane.
In conclusion, the work presented in this thesis exhibited the dynamic nature of CHO cell and revealed metabolic characteristics which enabled a cell to reach growth improvements. In the same context, the reversed strategy presented here generated a panel of 132 CHO cell variants with enhanced functional characteristic that meet industrial standards and therefore open a huge potential to increase our understanding of the nature of relevant cell lines with desirable metabolic and growth phenotypes.