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

Abstract

27As bioprocess intensification has increased over the last 30 years, yields from 28 mammalian cell processes have increased from 10's of milligrams to over 10's of grams per 29 liter. Most of these gains in productivity have been due to increasing cell numbers in the 30 bioreactors, and with those increases in cell numbers, strategies have been developed to 31 minimize metabolite waste accumulation, such as lactate and ammonia. Unfortunately, cell 32 growth cannot occur without some waste metabolite accumulation, as central metabolism is 33 required to produce the biopharmaceutical. Inevitably, metabolic waste accumulation leads to 34 decline and termination of the culture. While it is understood that the accumulation of these 35 unwanted compounds imparts a less than optimal culture environment, little is known about the 36 genotoxic properties and the influence of these compounds on global genome instability. In this 37 study, we examined the effects on Chinese hamster ovary (CHO) cells' genome sequences and 38 physiology due to exposure to elevated ammonia levels. We identified genome-wide de novo 39 mutations, in addition to variants in functional regions of certain genes involved in the mismatch 40 repair (MMR) pathway, such as DNA2, BRCA1 and RAD52, which led to loss-of-function and 41 eventual genome instability. Additionally, we characterized the presence of microsatellites 42 against the most recent Chinese Hamster genome assembly and discovered certain loci are not 43 replicated faithfully in the presence of elevated ammonia, which represents microsatellite 44 instability (MSI). Furthermore, we found 124 candidate loci that may be suitable biomarkers to 45 gauge genome stability in CHO cultures. 46 47