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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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Bioinformatic analysis of Chinese hamster ovary host cell protein lipases
Complete, accurate genome assemblies are necessary to design targets for genetic engineering strategies. Successful gene knockdowns and knockouts in Chinese hamster ovary (CHO) cells may prevent the expression of difficult‐to‐remove host cell proteins (HCPs). HCPs, if not removed, can cause problems in stability, safety, and efficacy of the biotherapeutic. A significantly improved Chinese hamster (CH) reference genome was used to identify new knockout targets with similar predicted functions and characteristics as the difficult‐to‐remove host cell lipases, LPL, PLBL2, and LPLA2. The CHO‐K1 gene and protein sequences of several of these lipases were corrected using the updated CH genome. Sequence alignments were then used to identify conserved regions that may serve as possible targets for multiple simultaneous gene knockouts. Finally, the comparison of the CHO‐K1 lipase protein sequences to their human orthologs provided insight into which lipases, if persistent in the drug product, could possibly cause immunogenic responses in patients. Topical heading: Biomolecular Engineering, Bioengineering, Biochemicals, Biofuels, and Food. © 2018 American Institute of Chemical EngineersAIChE J, 64: 4247–4254, 2018
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- Award ID(s):
- 1736123
- PAR ID:
- 10075019
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- AIChE Journal
- Volume:
- 64
- Issue:
- 12
- ISSN:
- 0001-1541
- Format(s):
- Medium: X Size: p. 4247-4254
- Size(s):
- p. 4247-4254
- Sponsoring Org:
- National Science Foundation
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