Co-amplification of EBNA-1 and PyLT through dhfr-mediated gene amplification for improving foreign protein production in transient gene expression in CHO cells

• Published in: Applied microbiology and biotechnology Vol. 102; no. 11; pp. 4729 - 4739
• Main Authors: Lee, Joo-Hyoung, Park, Jong-Ho, Park, Sun-Hye, Kim, Sun-Hong, Kim, Jee Yon, Min, Jeong-Ki, Lee, Gyun Min, Kim, Yeon-Gu
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.06.2018; Springer; Springer Nature B.V
• Subjects: Amplification; Animals; Antigen T (large); Antigens; Antigens, Polyomavirus Transforming - genetics; Biomedical and Life Sciences; Biotechnological Products and Process Engineering; Biotechnology; Cell culture; Cell lines; Chinese hamsters; CHO Cells; Cricetinae; Cricetulus; Deoxyribonucleic acid; Dihydrofolate reductase; DNA; EBNA-1 protein; Epstein-Barr virus; Epstein-Barr Virus Nuclear Antigens - genetics; Fc receptors; Fusion protein; Gene Amplification; Gene expression; Genetic aspects; Glycan; Human gammaherpesvirus 4; Life Sciences; messenger RNA; Methods; Methotrexate; Microbial Genetics and Genomics; Microbiology; Nucleic Acid Amplification Techniques - methods; Physiological aspects; production technology; Protein synthesis; Proteins; R&D; recombinant proteins; Recombinant Proteins - genetics; Reductases; Research & development; research and development; Transfection; Viruses; EBNA-1; Gene amplification; TGE; CHO cells; Fc-fusion protein; PyLT
• ISSN: 0175-7598; 1432-0614; 1432-0614
• DOI: 10.1007/s00253-018-8977-6

Despite the relatively low transfection efficiency and low specific foreign protein productivity ( q p ) of Chinese hamster ovary (CHO) cell-based transient gene expression (TGE) systems, TGE-based recombinant protein production technology predominantly employs CHO cells for pre-clinical research and development purposes. To improve TGE in CHO cells, Epstein-Barr virus nuclear antigen-1 (EBNA-1)/polyoma virus large T antigen (PyLT)-co-amplified recombinant CHO (rCHO) cells stably expressing EBNA-1 and PyLT were established using dihydrofolate reductase/methotrexate-mediated gene amplification. The level of transiently expressed Fc-fusion protein was significantly higher in the EBNA-1/PyLT-co-amplified pools compared to control cultures. Increased Fc-fusion protein production by EBNA-1/PyLT-co-amplification resulted from a higher q p attributable to EBNA-1 but not PyLT expression. The q p for TGE-based production with EBNA-1/PyLT-co-amplified rCHO cells (EP-amp-20) was approximately 22.9-fold that of the control culture with CHO-DG44 cells. Rather than improved transfection efficiency, this cell line demonstrated increased levels of mRNA expression and replicated DNA, contributing to an increased q p . Furthermore, there was no significant difference in N -glycan profiles in Fc-fusion proteins produced in the TGE system. Taken together, these results showed that the use of rCHO cells with co-amplified expression of the viral elements EBNA-1 and PyLT improves TGE-based therapeutic protein production dramatically. Therefore, EBNA-1/PyLT-co-amplified rCHO cells will likely be useful as host cells in CHO cell-based TGE systems.