Comparison of Internal Ribosome Entry Site (IRES) and Furin-2A (F2A) for monoclonal antibody expression level and quality in CHO cells
Four versions of tricistronic vectors expressing IgG1 light chain (LC), IgG1 heavy chain (HC), and dihydrofolate reductase (DHFR) in one transcript were designed to compare internal ribosome entry site (IRES) and furin-2A (F2A) for their influence on monoclonal antibody (mAb) expression level and qu...
| Main Authors: | , , , , , , , |
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| Format: | Journal Article |
| Language: | English |
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2013
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| Online Access: | https://hdl.handle.net/10356/97890 http://hdl.handle.net/10220/13223 |
| _version_ | 1811686726052085760 |
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| author | Bardor, Muriel Li, Bin Lee, Jia Juan Song, Zhiwei Tong, Yen Wah Goh, Lin-Tang Yang, Yuansheng Ho, Steven C. L. |
| author2 | School of Chemical and Biomedical Engineering |
| author_facet | School of Chemical and Biomedical Engineering Bardor, Muriel Li, Bin Lee, Jia Juan Song, Zhiwei Tong, Yen Wah Goh, Lin-Tang Yang, Yuansheng Ho, Steven C. L. |
| author_sort | Bardor, Muriel |
| collection | NTU |
| description | Four versions of tricistronic vectors expressing IgG1 light chain (LC), IgG1 heavy chain (HC), and dihydrofolate reductase (DHFR) in one transcript were designed to compare internal ribosome entry site (IRES) and furin-2A (F2A) for their influence on monoclonal antibody (mAb) expression level and quality in CHO DG44 cells. LC and HC genes are arranged as either the first or the second cistron. When using mAb quantification methods based on the detection antibodies against HC Fc region, F2A-mediated tricistronic vectors appeared to express mAb at higher levels than the IRES-mediated tricistronic vectors in both transient and stable transfections. Further analysis revealed that more than 40% of products detected in stably transfected pools generated using the two F2A-mediated tricistronic vectors were aggregates. LC and HC from the F2A stably transfected pools were not properly processed, giving rise to LC+F2A+HC or HC+F2A+LC fusion proteins, LC and HC polypeptides with F2A remnants, and incorrectly cleaved signal peptides. Both IRES-mediated tricistronic vectors express mAb with correct sizes and signal peptide cleavage. Arrangement of LC as the first cistron in the IRES-mediated tricistronic vectors exhibits increased mAb expression level, better growth, and minimized product aggregation, while arrangement of HC as first cistron results in low expression, slower growth, and high aggregation. The results obtained will be beneficial for designing vectors that enhance mAb expression level and quality in mammalian cells |
| first_indexed | 2024-10-01T05:04:59Z |
| format | Journal Article |
| id | ntu-10356/97890 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2024-10-01T05:04:59Z |
| publishDate | 2013 |
| record_format | dspace |
| spelling | ntu-10356/978902023-12-29T06:48:44Z Comparison of Internal Ribosome Entry Site (IRES) and Furin-2A (F2A) for monoclonal antibody expression level and quality in CHO cells Bardor, Muriel Li, Bin Lee, Jia Juan Song, Zhiwei Tong, Yen Wah Goh, Lin-Tang Yang, Yuansheng Ho, Steven C. L. School of Chemical and Biomedical Engineering Four versions of tricistronic vectors expressing IgG1 light chain (LC), IgG1 heavy chain (HC), and dihydrofolate reductase (DHFR) in one transcript were designed to compare internal ribosome entry site (IRES) and furin-2A (F2A) for their influence on monoclonal antibody (mAb) expression level and quality in CHO DG44 cells. LC and HC genes are arranged as either the first or the second cistron. When using mAb quantification methods based on the detection antibodies against HC Fc region, F2A-mediated tricistronic vectors appeared to express mAb at higher levels than the IRES-mediated tricistronic vectors in both transient and stable transfections. Further analysis revealed that more than 40% of products detected in stably transfected pools generated using the two F2A-mediated tricistronic vectors were aggregates. LC and HC from the F2A stably transfected pools were not properly processed, giving rise to LC+F2A+HC or HC+F2A+LC fusion proteins, LC and HC polypeptides with F2A remnants, and incorrectly cleaved signal peptides. Both IRES-mediated tricistronic vectors express mAb with correct sizes and signal peptide cleavage. Arrangement of LC as the first cistron in the IRES-mediated tricistronic vectors exhibits increased mAb expression level, better growth, and minimized product aggregation, while arrangement of HC as first cistron results in low expression, slower growth, and high aggregation. The results obtained will be beneficial for designing vectors that enhance mAb expression level and quality in mammalian cells Published version 2013-08-26T06:31:00Z 2019-12-06T19:47:48Z 2013-08-26T06:31:00Z 2019-12-06T19:47:48Z 2013 2013 Journal Article Ho, S. C. L., Bardor, M., Li, B., Lee, J. J., Song, Z., Tong, Y. W., Goh, L. T.,& Yang, Y. (2013). Comparison of Internal Ribosome Entry Site (IRES) and Furin-2A (F2A) for Monoclonal Antibody Expression Level and Quality in CHO Cells. PLoS ONE, 8(5). 1932-6203 https://hdl.handle.net/10356/97890 http://hdl.handle.net/10220/13223 10.1371/journal.pone.0063247 23704898 en PLoS ONE © 2013 Ho et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. application/pdf |
| spellingShingle | Bardor, Muriel Li, Bin Lee, Jia Juan Song, Zhiwei Tong, Yen Wah Goh, Lin-Tang Yang, Yuansheng Ho, Steven C. L. Comparison of Internal Ribosome Entry Site (IRES) and Furin-2A (F2A) for monoclonal antibody expression level and quality in CHO cells |
| title | Comparison of Internal Ribosome Entry Site (IRES) and Furin-2A (F2A) for monoclonal antibody expression level and quality in CHO cells |
| title_full | Comparison of Internal Ribosome Entry Site (IRES) and Furin-2A (F2A) for monoclonal antibody expression level and quality in CHO cells |
| title_fullStr | Comparison of Internal Ribosome Entry Site (IRES) and Furin-2A (F2A) for monoclonal antibody expression level and quality in CHO cells |
| title_full_unstemmed | Comparison of Internal Ribosome Entry Site (IRES) and Furin-2A (F2A) for monoclonal antibody expression level and quality in CHO cells |
| title_short | Comparison of Internal Ribosome Entry Site (IRES) and Furin-2A (F2A) for monoclonal antibody expression level and quality in CHO cells |
| title_sort | comparison of internal ribosome entry site ires and furin 2a f2a for monoclonal antibody expression level and quality in cho cells |
| url | https://hdl.handle.net/10356/97890 http://hdl.handle.net/10220/13223 |
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