2019 ; Vol.29-1: 55~58
|Author||Juyoung Byun, Sena Yoon, Yunji Jeong, Uitaek Oh, Sujin Cho, Jeongsoo Park, Yongsu Jeong, Kwanghee Baek, Jaeseung Yoon|
|Place of duty||PanGen Biotech Inc, Republic of Korea|
|Title||Efficient Development of Stable Recombinant Chinese Hamster Ovary (rCHO) Cell Lines to Produce Antibodies by Using Dimethyl Sulfoxide (DMSO) in Electroporation|
J. Microbiol. Biotechnol.2019 ;
|Abstract||Development of stable rCHO cell lines is still time consuming and labor intensive, although it
is a critical step in the commercial development of recombinant antibodies. The current work
demonstrates, for the first time, that electroporation of CHO cells with DMSO can enhance
stable expression of recombinant antibodies in rCHO cells. Electroporation with DMSO
resulted in an average 3.7-fold and 2.8-fold increases in expression levels of aflibercept and
pembrolizumab, respectively, in pools of stable rCHO cells. It also resulted in an average of
2.2-fold and 2.6-fold increases in the expression of aflibercept and pembrolizumab,
respectively, in single-cell derived rCHO clones. Simple batch cultures of rCHO cell clones
with the highest expression produced 1.0 g/l for aflibercept and 1.4 g/l for pembrolizumab
without a time-consuming gene amplification process. Electroporation with DMSO also
shortened the development of rCHO cell lines to 2-3 months, allowing rapid establishment of
stable rCHO cell lines with a desirable expression level antibodies.|
|Key_word||Dimethyl sulfoxide, stable cell line, chinese hamster ovary cell, recombinant antibody, electroporation|
Jesus MD, Wurm FM. 2011. Manufacturing recombinant proteins in kg-ton quantities using animal cells in bioreactors. Eur. J. Pharm. Biopharm. 78: 184-188.
Durocher Y, Butler, M. 2009. Expression systems for therapeutic glycoprotein production. Curr. Opin. Biotechnol. 20: 700-707.
Dietmair S, Nielsen LK, Timmins NE. 2010. Engineering a mammalian super producer. J. Chem. Technol. Biotechnol. 86:905-914.
Noh SM, Sathyamurthy M, Lee GM. 2013. Development of recombinant Chinese hamster ovary cell lines for therapeutic protein production. Curr Opin. Chem. Eng. 2: 391-397.
Baldi L, Hacker DL, Adam M, Wurm FM. 2007. Recombinant protein production by large-scale transient gene expression in mammalian cells: state of art and future perspectives. Biotechnol. Lett. 29: 677-684.
Bertschinger M, Schertenleib A, Cavey J, Hacker DL, Wurm FM. 2008. The kinetics of polyethylenimine-mediated transfection in suspension cultures of Chinese hamster ovary cells. Mol Biotechnol. 40: 136-143.
Steger K, Brady J, Wang W, Duskin M, Donato K, Peshwa M. 2015. CHO-S antibody titers >1 gram/liter using flow electroporation-mediated gene expression followed by rapid migration to high-yield stable cell lines. J. Biomol. Screen. 20:545-551.
Cervera L, Fuenmayer J, Gonzalez-Dominguez I, GutierrezGranados S, Segura MM, Godia F. 2015. Selection and optimization enhancer additives for increased virus-like particle production in HEK293 suspension cell cultures. Appl. Microbiol. Biotechnol. 99: 9935-9949.
Melkonyan H, Sorg C, Klempt M. 1996. Electroporation efficiency in mammalian cells is increased by dimethyl sulfoxide (DMSO). Nuc. Acids Res. 24: 4356-4357.
Ye J, Kober V, Tellers M, Naji Z, Salmon P, Markusen JF. 2009. High-level protein expression in scalable CHO transient transfection. Biotechnol. Bioeng. 103: 542-551.
Daly TJ, Fandl JP, Papadopoulos NJ. 2006. Fusion protein capable of binding VEGF. US Patent publication No 7087411B2.
Yoon J, Baek K, Byun T, Park J. 2014. Expression vector animal cells. US Patent Publication No 20140038233.
Ciudad CJ, Urlaub G, Chasin LA. 1988. Deletion analysis of the Chinese hamster dihydrofolate reductase gene promoter. J. Biol. Chem. 263: 16274-16282.
Paredes V, Park JS, Jeong Y, Yoon J, Baek K. 2013. Unstable expression of recombinant antibody during long-term culture of CHO cells is accompanied by histone H3 hypoacetylation. Biotechnol. Lett. 35: 987-993.