Microsatellite instability as a tool to diagnose genome instability in CHO cell culture

As bioprocess intensification has increased over the last 30 years, yields from mammalian cell processes have increased from 10’s of milligrams to over 10’s of grams per liter.  Most of these gains in productivity have been due to increasing cell numbers in the bioreactors, and with those increases in cell numbers, strategies have been developed to minimize metabolite waste accumulation, such as lactate and ammonia.  Unfortunately, cell growth cannot occur without some waste metabolite accumulation, as central metabolism is required to produce the biopharmaceutical.  Inevitably, metabolic waste accumulation leads to decline and termination of the culture.  While it is understood that the accumulation of these unwanted compounds imparts a less than optimal culture environment, little is known about the genotoxic properties and the influence of these compounds on global genome instability. In this study, we examined the effects on Chinese hamster ovary (CHO) cells’ genome sequences and physiology due to exposure to elevated ammonia levels. We identified genome-wide de novo mutations, in addition to variants in functional regions of certain genes involved in the mismatch repair (MMR) pathway, such as DNA2, BRCA1 and RAD52, which led to loss-of-function and eventual genome instability. Additionally, we characterized the presence of microsatellites against the most recent Chinese Hamster genome assembly and discovered certain loci are not replicated faithfully in the presence of elevated ammonia, which represents microsatellite instability (MSI). Furthermore, we found 124 candidate loci that may be suitable biomarkers to gauge genome stability in CHO cultures.