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

Abstract

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.

Keywords: Ammonia; Biomarker; CHO; Genome instability; MSI.

Fig. 1

Cell growth, ammonia, titer and metabolic profiles for CHO K-1 VRC01 cells cultured in duplicate in the ambr® 250 bioreactor. The ammonia stresses (10 mM and 30 mM) were added at 12 h. Samples for genomic analysis were harvested at 84 h (3.5 days) as shown by the solid grey line. a Viable cell density (VCD) and viability (filled and hollow symbols, respectively), b ammonia, c alanine, d glucose, e lactate, f titer of recombinant monoclonal antibody, g glutamine, h glutamate, and i Cell specific productivity (qp). Due to low levels of the recombinant protein in culture prior to day 5, the qp value is not shown until a significant titer has been reached. In industry, it is common to only measure titers starting at day 7. Control - 0 mM (blue and purple lines); 10 mM (orange and red lines); 30 mM (green and dark green lines)

Fig. 2

Genome coverage map of genetic variants and MSI loci in the 10 longest CHO scaffolds. A circos plot of the 10 longest CHO genome scaffolds (parsed into 100 kb windows that represents approximately ~ 20% of the CHO genome) that depicts distribution of genetic variants and MSI loci. The innermost track (light purple) depicts candidate MSI loci (23 out of the 124); Indel density and distribution is depicted in light blue; SNP density and distribution is in light red; and gene density is plotted outside of the CHO ideograms in light orange. Ideogram ticks are scaled in megabases

Fig. 3

KEGG enrichment results from over representation analysis of ammonia-sensitive genes linked to genomic instability. a Network plot of most significant enriched KEGG pathways. Enrichment scores (i.e., adjusted p values) and gene counts (the number of genes enriched in a KEGG term) are depicted by dot color and size. b Comparison of enrichment results of KEGG pathways for genes identified with significant variants in the 10 mM Ammonia-stressed culture (111 genes), in the 30 mM culture (102 genes), and in the union of 10 mM and 30 mM cultures (148 genes). Enrichment scores (i.e., adjusted p values) and gene ratios (the percentage of total genes in the given KEGG term) are depicted by dot color and size. The plots are made with clusterProfiler

Fig. 4

Gene Ontology (GO) enrichment results from over representation analysis of ammonia-sensitive genes linked to genomic instability. Terms in the three GO categories are grouped with color bars: biological process (BP, red), cellular component (CC, orange), and molecular function (MF, blue). Fold enrichment is shown in x-axis. Enrichment scores (i.e., adjusted p values) and gene counts (the number of genes enriched in a GO term) are depicted by dot color and size. The plot is made with clusterProfiler

Fig. 5

An Integrated Genome Viewer (IGV) image of a microsatellite located on scaffold NW_020822544.1 at position 4,160,116. a control, b 10 mM, and c 30 mM cultures. This microsatellite contains five repeats of an AAC motif. They grey lines above each nucleotide are indicative of the total read depth at that location; note that it is significantly lower in regions where deletions were detected