Autor: |
Nina B. Reese, Sherman Ku |
Jazyk: |
angličtina |
Rok vydání: |
2021 |
Předmět: |
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Zdroj: |
Current Research in Biotechnology, Vol 3, Iss , Pp 269-280 (2021) |
Druh dokumentu: |
article |
ISSN: |
2590-2628 |
DOI: |
10.1016/j.crbiot.2021.10.001 |
Popis: |
Current methods for stably expressing recombinant protein therapeutics in CHO cells often rely on random or semi-random genomic integration events which result in a widely heterogeneous cell population. Consequently, a significant portion of cell line development efforts involves extensive pool and clone screening to identify clones with high expression, growth, and product quality. In this study, we developed two targeted integration systems that express high levels of recombinant protein in CHO cells. We first generated two clonal cell lines stably expressing enhanced green fluorescent protein (eGFP) reporter landing pads in genomic hot spots. We then demonstrated functional integration of several donor vectors encoding both monoclonal antibodies and a Fc-fusion molecule using Cre or PhiC31 recombinase mediated integration. TLA and PCR characterization of integrated cell lines showed correct targeting of landing pads. Post-integration enrichment for fully saturated landing pads using GCV increased recombinant protein titer by 2–2.5-fold and specific productivity by ∼3.4-fold with observed potential off-target random integration detected only following GCV enrichment. By targeting predefined genomic locations that are known to support high expression of an exogenous protein, several cell lines expressing different recombinant protein therapeutics can easily be established with a high degree of specificity and reproducibility. |
Databáze: |
Directory of Open Access Journals |
Externí odkaz: |
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