Context-dependent genomic locus effects on antibody production in recombinant Chinese hamster ovary cells generated through random integration
High-yield production of therapeutic protein using Chinese hamster ovary (CHO) cells requires stable cell line development (CLD). CLD typically uses random integration of transgenes; however, this results in clonal variation and subsequent laborious clone screening. Therefore, site-specific integrat...
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Elsevier
2024-12-01
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| Series: | Computational and Structural Biotechnology Journal |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2001037024001016 |
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| author | Hyun Jee Woo Jaehoon Kim Seul Mi Kim Dongwoo Kim Jae Yun Moon Daechan Park Jae Seong Lee |
| author_facet | Hyun Jee Woo Jaehoon Kim Seul Mi Kim Dongwoo Kim Jae Yun Moon Daechan Park Jae Seong Lee |
| author_sort | Hyun Jee Woo |
| collection | DOAJ |
| description | High-yield production of therapeutic protein using Chinese hamster ovary (CHO) cells requires stable cell line development (CLD). CLD typically uses random integration of transgenes; however, this results in clonal variation and subsequent laborious clone screening. Therefore, site-specific integration of a protein expression cassette into a desired chromosomal locus showing high transcriptional activity and stability, referred to as a hot spot, is emerging. Although positional effects are important for therapeutic protein expression, the sequence-specific mechanisms by which hotspots work are not well understood. In this study, we performed whole-genome sequencing (WGS) to locate randomly inserted vectors in the genome of recombinant CHO cells expressing high levels of monoclonal antibodies (mAbs) and experimentally validated these locations and vector compositions. The integration site was characterized by active histone marks and potential enhancer activities, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mediated indel mutations in the region upstream of the integration site led to a significant reduction in specific antibody productivity by up to 30%. Notably, the integration site and its core region did not function equivalently outside the native genomic context, showing a minimal effect on the increase in exogenous protein expression in the host cell line. We also observed a superior production capacity of the mAb expressing cell line compared to that of the host cell line. Collectively, this study demonstrates that developing recombinant CHO cell lines to produce therapeutic proteins at high levels requires a balance of factors including transgene configuration, genomic locus landscape, and host cell properties. |
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| institution | Directory Open Access Journal |
| issn | 2001-0370 |
| language | English |
| last_indexed | 2024-04-24T07:05:18Z |
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| series | Computational and Structural Biotechnology Journal |
| spelling | doaj.art-4adeb295d6c04db89f5a92993605699c2024-04-22T04:11:37ZengElsevierComputational and Structural Biotechnology Journal2001-03702024-12-012316541665Context-dependent genomic locus effects on antibody production in recombinant Chinese hamster ovary cells generated through random integrationHyun Jee Woo0Jaehoon Kim1Seul Mi Kim2Dongwoo Kim3Jae Yun Moon4Daechan Park5Jae Seong Lee6Department of Molecular Science and Technology, Ajou University, Suwon 16499, Republic of KoreaDepartment of Molecular Science and Technology, Ajou University, Suwon 16499, Republic of Korea; Molecular Science and Technology Research Center, Ajou University, Suwon 16499, Republic of KoreaDepartment of Molecular Science and Technology, Ajou University, Suwon 16499, Republic of KoreaDepartment of Molecular Science and Technology, Ajou University, Suwon 16499, Republic of KoreaDepartment of Molecular Science and Technology, Ajou University, Suwon 16499, Republic of KoreaDepartment of Molecular Science and Technology, Ajou University, Suwon 16499, Republic of Korea; Department of Biological Sciences, Ajou University, Suwon 16499, Republic of Korea; Corresponding authors at: Department of Molecular Science and Technology, Ajou University, Suwon 16499, Republic of Korea.Department of Molecular Science and Technology, Ajou University, Suwon 16499, Republic of Korea; Department of Applied Chemistry and Biological Engineering, Ajou University, Suwon 16499, Republic of Korea; Corresponding authors at: Department of Molecular Science and Technology, Ajou University, Suwon 16499, Republic of Korea.High-yield production of therapeutic protein using Chinese hamster ovary (CHO) cells requires stable cell line development (CLD). CLD typically uses random integration of transgenes; however, this results in clonal variation and subsequent laborious clone screening. Therefore, site-specific integration of a protein expression cassette into a desired chromosomal locus showing high transcriptional activity and stability, referred to as a hot spot, is emerging. Although positional effects are important for therapeutic protein expression, the sequence-specific mechanisms by which hotspots work are not well understood. In this study, we performed whole-genome sequencing (WGS) to locate randomly inserted vectors in the genome of recombinant CHO cells expressing high levels of monoclonal antibodies (mAbs) and experimentally validated these locations and vector compositions. The integration site was characterized by active histone marks and potential enhancer activities, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mediated indel mutations in the region upstream of the integration site led to a significant reduction in specific antibody productivity by up to 30%. Notably, the integration site and its core region did not function equivalently outside the native genomic context, showing a minimal effect on the increase in exogenous protein expression in the host cell line. We also observed a superior production capacity of the mAb expressing cell line compared to that of the host cell line. Collectively, this study demonstrates that developing recombinant CHO cell lines to produce therapeutic proteins at high levels requires a balance of factors including transgene configuration, genomic locus landscape, and host cell properties.http://www.sciencedirect.com/science/article/pii/S2001037024001016Chinese hamster ovary (CHO)Cell line developmentHot spotRandom integration |
| spellingShingle | Hyun Jee Woo Jaehoon Kim Seul Mi Kim Dongwoo Kim Jae Yun Moon Daechan Park Jae Seong Lee Context-dependent genomic locus effects on antibody production in recombinant Chinese hamster ovary cells generated through random integration Computational and Structural Biotechnology Journal Chinese hamster ovary (CHO) Cell line development Hot spot Random integration |
| title | Context-dependent genomic locus effects on antibody production in recombinant Chinese hamster ovary cells generated through random integration |
| title_full | Context-dependent genomic locus effects on antibody production in recombinant Chinese hamster ovary cells generated through random integration |
| title_fullStr | Context-dependent genomic locus effects on antibody production in recombinant Chinese hamster ovary cells generated through random integration |
| title_full_unstemmed | Context-dependent genomic locus effects on antibody production in recombinant Chinese hamster ovary cells generated through random integration |
| title_short | Context-dependent genomic locus effects on antibody production in recombinant Chinese hamster ovary cells generated through random integration |
| title_sort | context dependent genomic locus effects on antibody production in recombinant chinese hamster ovary cells generated through random integration |
| topic | Chinese hamster ovary (CHO) Cell line development Hot spot Random integration |
| url | http://www.sciencedirect.com/science/article/pii/S2001037024001016 |
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