A compendium of stable hotspots in the CHO genome

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Author(s)Hilliard, William
Author(s)Lee, Kelvin H.
Date Accessioned2023-06-20T19:18:20Z
Date Available2023-06-20T19:18:20Z
Publication Date2023-04-04
DescriptionThis article was originally published in Biotechnology and Bioengineering. The version of record is available at: https://doi.org/10.1002/bit.28390. © 2023 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.
AbstractThe use of targeted integration for industrial CHO cell line development currently requires significant upfront effort to identify genomic loci capable of supporting multigram per liter therapeutic protein production from a limited number of transgene copies. To address this barrier to widespread adoption, we characterized transgene expression from thousands of stable hotspots in the CHO genome using the Thousands of Reporters Integrated in Parallel high-throughput screening method. This genome-scale data set was used to define a limited set of epigenetic properties of hotspot regions with sizes on the order of 10 kb. Cell lines with landing pad integrations at eight retargeted hotspot candidates consistently exhibited higher transgene mRNA expression than a commercially viable hotspot in equivalent culture conditions. Initial benchmarking of NISTmAb and trastuzumab productivity from one of these hotspots yielded mAb productivities of approximately 0.7–2 g/L (qP range: 2.9–8.2 pg/cell/day) in small-scale fed-batches. These findings indicate the list of hotspot candidates identified here will be a valuable resource for targeted integration platform development within the CHO community.
SponsorThe authors would like to thank the Eleftherios T. Papoutsakis lab for allowing for the use of their GenePulser XCell for electrocompetent E. coli transformations, the University of Delaware DNA Sequencing and Genotyping Center for their Sanger sequencing and Illumina NGS services, and the NIH (NIAID) for sharing of cell lines. Computational work was performed on the BIOMIX compute cluster with support from the University of Delaware Center for Bioinformatics and Computational Biology Core Facility funded through Delaware INBRE (NIGMS P20GM103446), the State of Delaware, and the Delaware Biotechnology Institute. This work was supported by funding from the National Science Foundation under the grant number 1736123. This work was supported, in part, by financial assistance award 70NANB17H002 from U.S. Department of Commerce, National Institute of Standards and Technology. Figures 1a, 1b, 1c, 3a, and 4a were created using Biorender.
CitationHilliard, W., & Lee, K. H. (2023). A compendium of stable hotspots in the CHO genome. Biotechnology and Bioengineering, 1– 11. https://doi.org/10.1002/bit.28390
ISSN1097-0290
URLhttps://udspace.udel.edu/handle/19716/32934
Languageen_US
PublisherBiotechnology and Bioengineering
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/4.0/
Keywordsbioprocessing
KeywordsCHO cell
Keywordsgenomic hotspot
Keywordssite-specific integration
KeywordsTRIP
TitleA compendium of stable hotspots in the CHO genome
TypeArticle
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