Abstract Chinese hamster ovary (CHO) cell lines are widely used to manufacture biopharmaceuticals. However, CHO cells are not an optimal expression host due to the intrinsic plasticity of the CHO genome. Genome plasticity can lead to chromosomal rearrangements, transgene exclusion, and phenotypic drift. A poorly understood genomic element of CHO cell line instability is extrachromosomal circular DNA (eccDNA) in gene expression and regulation. EccDNA can facilitate ultra-high gene expression and are found within many eukaryotes including humans, yeast, and plants. EccDNA confers genetic heterogeneity, providing selective advantages to individual cells in response to dynamic environments. In CHO cell cultures, maintaining genetic homogeneity is critical to ensuring consistent productivity and product quality. Understanding eccDNA structure, function, and microevolutionary dynamics under various culture conditions could reveal potential engineering targets for cell line optimization. In this study, eccDNA sequences were investigated at the beginning and end of two-week fed-batch cultures in an ambr ® 250 bioreactor under control and lactate-stressed conditions. This work characterized structure and function of eccDNA in a CHO-K1 clone. Gene annotation identified 1551 unique eccDNA genes including cancer driver genes and genes involved in protein production. Furthermore, RNA-seq data is integrated to identify transcriptionally active eccDNA genes.
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Systematic identification of safe harbor regions in the CHO genome through a comprehensive epigenome analysis
The Chinese hamster ovary (CHO) cell lines that are used to produce commercial quantities of therapeutic proteins commonly exhibit a decrease in productivity over time in culture, a phenomenon termed production instability. Random integration of the transgenes encoding the protein of interest into locations in the CHO genome that are vulnerable to genetic and epigenetic instability often causes production instability through copy number loss and silencing of expression. Several recent publications have shown that these cell line development challenges can be overcome by using site‐specific integration (SSI) technology to insert the transgenes at genomic loci, often called “hotspots,” that are transcriptionally permissive and have enhanced stability relative to the rest of the genome. However, extensive characterization of the CHO epigenome is needed to identify hotspots that maintain their desirable epigenetic properties in an industrial bioprocess environment and maximize transcription from a single integrated transgene copy. To this end, the epigenomes and transcriptomes of two distantly related cell lines, an industrially relevant monoclonal antibody‐producing cell line and its parental CHO‐K1 host, were characterized using high throughput chromosome conformation capture and RNAseq to analyze changes in the epigenome that occur during cell line development and associated changes in system‐wide gene expression. In total, 10.9% of the CHO genome contained transcriptionally permissive three‐dimensional chromatin structures with enhanced genetic and epigenetic stability relative to the rest of the genome. These safe harbor regions also showed good agreement with published CHO epigenome data, demonstrating that this method was suitable for finding genomic regions with epigenetic markers of active and stable gene expression. These regions significantly reduce the genomic search space when looking for CHO hotspots with widespread applicability and can guide future studies with the goal of maximizing the potential of SSI technology in industrial production CHO cell lines.
more » « less- Award ID(s):
- 1736123
- NSF-PAR ID:
- 10236245
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Biotechnology and Bioengineering
- Volume:
- 118
- Issue:
- 2
- ISSN:
- 0006-3592
- Page Range / eLocation ID:
- p. 659-675
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Chinese hamster ovary (CHO) cells are essential to biopharmaceutical manufacturing and production instability, the loss of productivity over time, is a long‐standing challenge in the industry. Accurate prediction of cell line stability could enable efficient screening to identify clones suitable for manufacturing saving significant time and costs. DNA repair genes may offer biomarkers to address this need. In this study, over 40 cell lines representing various host lineages from three companies/organizations were evaluated for expression of five DNA repair genes (
Fam35a ,Lig4 ,Palb2 ,Pari , andXrcc6 ). Expression measured in cells with less than 30 population doubling levels (PDLs) was correlated to stability profiles at 60+ PDL. Principal component analysis identified markers which separate stable and unstable CHO‐DG44 cell lines. Notably, two genes,Lig4 andXrcc6 , showed higher expression in unstable CHO‐DG44 cell lines with copy number loss identified as the mechanism of production instability. Expression levels across all cell ages showed lower DNA repair gene expression was associated with increased cell age. Collectively, DNA repair genes provide critical insight into long‐term behavior of CHO cells and their expression levels have potential to predict cell line stability in certain cases. -
Chinese hamster ovary (CHO) cells are the most commonly used cell lines in biopharmaceutical manufacturing. Genome-scale metabolic models have become a valuable tool to study cellular metabolism. Despite the presence of reference global genome-scale CHO model, context-specific metabolic models may still be required for specific cell lines (for example, CHO-K1, CHO-S, and CHO-DG44), and for specific process conditions. Many integration algorithms have been available to reconstruct specific genome-scale models. These methods are mainly based on integrating omics data (i.e., transcriptomics, proteomics, and metabolomics) into reference genome-scale models. In the present study, we aimed to investigate the impact of time points of transcriptomics integration on the genome-scale CHO model by assessing the prediction of growth rates with each reconstructed model. We also evaluated the feasibility of applying extracted models to different cell lines (generated from the same parental cell line). Our findings illustrate that gene expression at various stages of culture slightly impacts the reconstructed models. However, the prediction capability is robust enough on cell growth prediction not only across different growth phases but also in expansion to other cell lines.more » « less
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Abstract Targeted gene knockout and site‐specific integration (SSI) are powerful genome editing techniques to improve the development of industrially relevant Chinese hamster ovary (CHO) cell lines. However, past efforts to perform SSI in CHO cells are characterized by low efficiencies. Moreover, numerous strategies proposed to boost SSI efficiency in mammalian cell types have yet to be evaluated head to head or in combination to appreciably boost efficiencies in CHO. To enable systematic and rapid optimization of genome editing methods, the SSIGNAL (
s ite‐s pecifici ntegration andg en omeal teration) reporter system is developed. This tool can analyze CRISPR (clustered regularly interspaced palindromic repeats)/Cas9 (CRISPR‐associated protein 9)‐mediated disruption activity alone or in conjunction with SSI efficiency. The reporter system uses green and red dual‐fluorescence signals to indicate genotype states within four days following transfection, facilitating rapid data acquisition via standard flow cytometry instrumentation. In addition to describing the design and development of the system, two of its applications are demonstrated by first comparing transfection conditions to maximize CRISPR/Cas9 activity and subsequently assessing the efficiency of several promising SSI strategies. Due to its sensitivity and versatility, the SSIGNAL reporter system may serve as a tool to advance genome editing technology. -
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Abstract Secretion levels required of industrial Chinese hamster ovary (CHO) cell lines can challenge endoplasmic reticulum (ER) homeostasis, and ER stress caused by accumulation of misfolded proteins can be a bottleneck in biomanufacturing. The unfolded protein response (UPR) is initiated to restore homeostasis in response to ER stress, and optimization of the UPR can improve CHO cell production of therapeutic proteins. We compared the fed-batch growth, production characteristics, and transcriptomic response of an immunoglobulin G1(IgG1) producer to its parental, non-producing host cell line. We conducted differential gene expression analysis using high throughput RNA sequencing (RNASeq) and quantitative polymerase chain reaction (qPCR) to study the ER stress response of each cell line during fed-batch culture. The UPR was activated in the IgG1producer compared to the host cell line and our analysis of differential expression profiles indicated transient upregulation of ATF6α target mRNAs in the IgG1producer, suggesting two upstream regulators of the ATF6 arm of the UPR, ATF6β and WFS1, are rational engineering targets. Although both ATF6β and WFS1 have been reported to negatively regulate ATF6α, this study shows knockdown of either target elicits different effects in an IgG1-producing CHO cell line. Stable knockdown of
ATF6β decreased cell growth without decreasing titer; however, knockdown ofWFS1 decreased titer without affecting growth. Relative expression measured by qPCR indicated no direct relationship betweenATF6β andWFS1 expression, but upregulation ofWFS1 in one pool was correlated with decreased growth and upregulation of ER chaperone mRNAs. While knockdown ofWFS1 had negative impacts on UPR activation and product mRNA expression, knockdown ofATF6β improved the UPR specifically later in fed-batch leading to increased overall productivity.
