Abstract
To date, the production of antibodies (mAbs) usually faces the risks of transgene expression reduction and instability, especially after long-time culture. The inclusion of ubiquitous chromatin opening element (UCOE) into expression vectors was reported to enhance protein production and maintain transgene expression stability in CHO cell lines. Thus, we investigate the effects of UCOE on recombinant monoclonal anti-TNFα antibody (mAbTNFα) production and expression stability in CHO-DG44 cells. In our study, non-UCOE and UCOE-based vectors encoding mAbTNFα were constructed and introduced into the CHO-DG44 cells. Cell pools and single-cell clones were obtained by selecting transfected cells with G418, amplifying them by treatment with methotrexate (MTX), and isolating them by limiting dilution. The effects of UCOE on mAb production and stable transgene expression in transfected cells were analyzed via the correlation between mAb yields and mRNA expression level variations, and gene copy number changes. The UCOE pool exhibited higher mAb yield compared to non-UCOE pool. The UCOE was associated with higher transgene transcriptional activity, leading to improvement of mAb production after MTX-mediated gene amplification. The incorporation of UCOE generated cells allowed isolation of greater numbers of positive clones with higher expression. Despite the slightly decreased mAb yield, UCOE clones still retain stable long-term expression in the absence of selective pressure, which was explained by the loss of transgene copies rather than due to the decline of transcriptional activity. In addition, the purified mAb had primary chemical and biological characteristics similar to those of adalimumab. The results showed that the incorporation of UCOE within vectors provides significant advantages in the generation of high-producing clones, enhancement of mAb production, and improvement of gene expression stability.
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Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Code availability
The code used during the current study are available from the corresponding author.
Abbreviations
- ADCC:
-
Antibody dependent cell-mediated cytotoxicity
- CDC:
-
Complement-dependent cytotoxicity
- CHO:
-
Chinese hamster ovary cells
- CMV:
-
Cytomegalovirus
- DHFR:
-
Dihydrofolatereductase
- ELISA:
-
Enzyme linked immunosorbent assay
- GOI:
-
Gene of interest
- HC:
-
Heavy chain
- HRP:
-
Horseradish peroxidase
- IRES:
-
Internal ribosome entry site
- LC:
-
Light chain
- mAbTNFα:
-
Anti-TNFα monoclonal antibody
- MTX:
-
Methotrexate
- PAGE:
-
Polyacrylamide gel
- PCR:
-
Polymerase chain reaction
- SDS:
-
Sodium dodecyl sulfate
- SV40:
-
Simian virus 40
- TMB:
-
3,3′,5,5′-Tetramethylbenzidine
- UCOEs:
-
Ubiquitous chromatin opening elements
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Acknowledgements
We would like to express gratitude to Dr. Bruce May, The University of British Columbia, Vancouver, Canada for pN plasmid as a gift.
Funding
This research was supported by Vietnam Academy of Science and Technology, Ha Noi, Vietnam under grant number: VAST02.01/20-21.
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All of the experiments were conducted by the authors. CCD, TLL and NQCH performed the experiments. CCD and TLL designed experiments, analyzed data. NSH supervised the studies. CCD prepared and revised the manuscript. All authors read and approved the manuscript.
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Doan, C.C., Le, T.L., Ho, N.Q.C. et al. Effects of ubiquitous chromatin opening element (UCOE) on recombinant anti-TNFα antibody production and expression stability in CHO-DG44 cells. Cytotechnology 74, 31–49 (2022). https://doi.org/10.1007/s10616-021-00503-1
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DOI: https://doi.org/10.1007/s10616-021-00503-1