Glycosylation-related genes in NS0 cells are insensitive to moderately elevated ammonium concentrations

Brodsky, Arthur Nathan; Caldwell, M. L.; Bae, Sooneon; Harcum, Sarah W.

doi:10.1016/j.jbiotec.2014.07.018

Cited by 6 publications

(3 citation statements)

References 70 publications

(101 reference statements)

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“…The most highly changed species was G2FS1 ( p value ≤ 0.05) (Figure 4A), which also is consistent with previous work. [ 11,12,14,31,33,75,76 ] Overall, culture age affected sialylation under ammonia stress ( p value ≤ 0.05), but this was less pronounced compared to the galactosylation changes. By Day 8.5, there was no significant difference in sialylation for either young‐ or standard‐aged cultures ( p value > 0.05) (Figure 4A).…”

Section: Resultsmentioning

confidence: 99%

Transient ammonia stress on Chinese hamster ovary (CHO) cells yield alterations to alanine metabolism and IgG glycosylation profiles

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McGraw

Elliott

et al. 2021

Biotechnology Journal

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Background Ammonia concentrations typically increase during mammalian cell cultures, mainly due to glutamine and other amino acid consumption. An early ammonia stress indicator is a metabolic shift with respect to alanine. To determine the underlying mechanisms of this metabolic shift, a Chinese hamster ovary (CHO) cell line with two distinct ages (standard and young) was cultured in parallel fed‐batch bioreactors with 0 mM or 10 mM ammonia added at 12 h. Reduced viable cell densities were observed for the stressed cells, while viability was not significantly affected. The stressed cultures had higher alanine, lactate, and glutamate accumulation. Interestingly, the ammonia concentrations were similar by Day 8.5 for all cultures. We hypothesized the ammonia was converted to alanine as a coping mechanism. Interestingly, no significant differences were observed for metabolite profiles due to cell age. Glycosylation analysis showed the ammonia stress reduced galactosylation, sialylation, and fucosylation. Transcriptome analysis of the standard‐aged cultures indicated the ammonia stress had a limited impact on the transcriptome, where few of the significant changes were directly related metabolite or glycosylation reactions. These results indicate that mechanisms used to alleviate ammonia stress are most likely controlled post‐transcriptionally, and this is where future research should focus.

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Section: Resultsmentioning

confidence: 99%

Transient ammonia stress on Chinese hamster ovary (CHO) cells yield alterations to alanine metabolism and IgG glycosylation profiles

Synoground

McGraw

Elliott

et al. 2021

Biotechnology Journal

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“…Until now, efforts to characterize ammonia stress effects on CHO cells have mainly focused on transcriptome, proteome, and product characteristic changes [ 14 , 16 , 17 , 40 – 44 ]. In this study, the effects of ammonia stress were further characterized by examining variants within functional genes and microsatellites.…”

Section: Discussionmentioning

confidence: 99%

Characterization of metabolic responses, genetic variations, and microsatellite instability in ammonia-stressed CHO cells grown in fed-batch cultures

et al. 2021

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Background 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 can be attributed to increasing cell densities within bioreactors. As such, strategies have been developed to minimize accumulation of metabolic wastes, such as lactate and ammonia. Unfortunately, neither cell growth nor biopharmaceutical production can occur without some waste metabolite accumulation. 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 suboptimal culture environment, little is known about the genotoxic properties of these compounds that may lead to global genome instability. In this study, we examined the effects of high and moderate extracellular ammonia on the physiology and genomic integrity of Chinese hamster ovary (CHO) cells. Results Through whole genome sequencing, we discovered 2394 variant sites within functional genes comprised of both single nucleotide polymorphisms and insertion/deletion mutations as a result of ammonia stress with high or moderate impact on functional genes. Furthermore, several of these de novo mutations were found in genes whose functions are to maintain genome stability, such as Tp53, Tnfsf11, Brca1, as well as Nfkb1. Furthermore, we characterized microsatellite content of the cultures using the CriGri-PICR Chinese hamster genome assembly and discovered an abundance of microsatellite loci that are not replicated faithfully in the ammonia-stressed cultures. Unfaithful replication of these loci is a signature of microsatellite instability. With rigorous filtering, we found 124 candidate microsatellite loci that may be suitable for further investigation to determine whether these loci may be reliable biomarkers to predict genome instability in CHO cultures. Conclusion This study advances our knowledge with regards to the effects of ammonia accumulation on CHO cell culture performance by identifying ammonia-sensitive genes linked to genome stability and lays the foundation for the development of a new diagnostic tool for assessing genome stability.

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“…These culture profiles indicate that ammonia impacts CHO cells in a dose dependent manner, and even mild ammonia stress has an influence on culture productivity. Until now, efforts have mainly focused on transcriptome and proteome effects of ammonia stress [11,43,44].…”

Section: Discussionmentioning

confidence: 99%

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

Wang

Elliott

et al. 2019

Preprint

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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

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Glycosylation-related genes in NS0 cells are insensitive to moderately elevated ammonium concentrations

Cited by 6 publications

References 70 publications

Transient ammonia stress on Chinese hamster ovary (CHO) cells yield alterations to alanine metabolism and IgG glycosylation profiles

Transient ammonia stress on Chinese hamster ovary (CHO) cells yield alterations to alanine metabolism and IgG glycosylation profiles

Characterization of metabolic responses, genetic variations, and microsatellite instability in ammonia-stressed CHO cells grown in fed-batch cultures

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

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