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 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. 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. 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. 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. 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.
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