Multicopy Targeted Integration for Accelerated Development of High-Producing Chinese Hamster Ovary Cells

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The ever-growing biopharmaceutical industry relies on the production of recombinant therapeutic proteins in Chinese hamster ovary (CHO) cells. The traditional timelines of CHO cell line development can be significantly shortened by the use of targeted gene integration (TI). However, broad use of TI has been limited due to the low specific productivity (q(P)) of TI-generated clones. Here, we show a 10-fold increase in the q(P) of therapeutic glycoproteins in CHO cells through the development and optimization of a multicopy TI method. We used a recombinase-mediated cassette exchange (RMCE) platform to investigate the effect of gene copy number, 5' and 3' gene regulatory elements, and landing pad features on q(P). We evaluated the limitations of multicopy expression from a single genomic site as well as multiple genomic sites and found that a transcriptional bottleneck can appear with an increase in gene dosage. We created a dual-RMCE system for simultaneous multicopy TI in two genomic sites and generated isogenic high-producing clones with q(P) of 12-14 pg/cell/day and product titer close to 1 g/L in fed-batch. Our study provides an extensive characterization of the multicopy TI method and elucidates the relationship between gene copy number and protein expression in mammalian cells. Moreover, it demonstrates that TI-generated CHO cells are capable of producing therapeutic proteins at levels that can support their industrial manufacture.
Publisher
AMER CHEMICAL SOC
Issue Date
2020-09
Language
English
Article Type
Article
Citation

ACS SYNTHETIC BIOLOGY, v.9, no.9, pp.2546 - 2561

ISSN
2161-5063
DOI
10.1021/acssynbio.0c00322
URI
http://hdl.handle.net/10203/276920
Appears in Collection
BS-Journal Papers(저널논문)
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