Enhanced CHO Cell-Based Transient Gene Expression with the Epi-CHO Expression System

Abstract

Transient gene expression systems in mammalian cells continue to grow in popularity due to their capacity to produce significant amounts of recombinant protein in a rapid and scalable manner, without the lengthy time periods and resources required for stable cell line development. Traditionally, production of recombinant monoclonal antibodies for pre-clinical assessment by transient expression in CHO cells has been hampered by low titers. In this report, we demonstrate transient monoclonal antibody titers of 140 mg/l with CHO cells using the episomal-based transient expression system, Epi-CHO. Such titers were achieved by implementing an optimized transfection protocol incorporating mild-hypothermia and through screening of a variety of chemically defined and serum-free media for their ability to support elevated and prolonged viable cell densities post-transfection, and in turn, improve recombinant protein yields. Further evidence supporting Epi-CHO's capacity to enhance transgene expression is provided, where we demonstrate higher transgene mRNA and protein levels of two monoclonal antibodies and a destabilized enhanced green fluorescent protein with Epi-CHO compared to cell lines deficient in plasmid DNA replication and/or retention post-transfection. The results demonstrate the Epi-CHO system's capacity for the rapid production of CHO cell-derived recombinant monoclonal antibodies in serum-free conditions.

Efficient mAb production in CHO cells incorporating PEI‐mediated transfection, mild hypothermia and the co‐expression of XBP‐1

“…However, TGE processes utilizing more costly cationic‐lipid formulations, such as Lipofectamine 2000, have also been shown to give significant protein yields with high transfection efficiencies and capacity to be transferred to large scale 13, 54. In addition, recent work performed by our group demonstrated significant improvements in recombinant protein titers using Lipofectamine 2000 in SF conditions 42…”

“…To investigate this, transfected CHO‐T cultures expressing Ab2 and EGFP, were transferred to 32 °C at 4 (the existing time of transfer used with Epi ‐CHO), 48 and 96 h post‐transfection and diluted with fresh media at the time of transfer (1:1, v:v) and/or immediately post‐transfection (1:2, v:v) (‘double or single feed’) to promote extended culture longevity and viability. In these experiments we utilized ProCHO5 for the dilutions post‐transfection as this media maintains high cell densities over extended culture periods 42…”

“…In addition, it also employs EBNA‐1 and OriP from EBV to facilitate the retention and segregation of plasmid DNA during cell division. Previous studies using the Epi ‐CHO system have demonstrated significant improvements in recombinant protein titers over non‐episomal systems using lipofection in protein containing media 41, 42…”

Improving the secretory capacity of Chinese hamster ovary cells by ectopic expression of effector genes: Lessons learned and future directions

“…Episomal replication can be achieved using, e.g., the Polyomavirus large T gene (PyLT) and its origin of replication (PyOri) (Heffernan and Dennis, 1991), while plasmid maintenance and segregation can be accomplished using Epstein-Barr virus nuclear antigen-1 (EBNA-1) and its origin of replication (OriP) (Lupton and Levine, 1985;Yates et al, 1984). Using these two sets of complementing viral components, the episomal platform was reported to increase and prolong TGE yields of a growth hormone and MAb in CHO cells in comparison to non-replicating plasmid controls (Codamo et al, 2011;Kunaparaju et al, 2005). Without antibiotic-based selection, expression from replication-proficient episomes can be regarded only as a quasi-stable gene expression platform, as episomes eventually will be lost with a half-life of approximately 8-9 days (Silla et al, 2006).…”

Emerging synthetic biology tools for engineering mammalian cell systems and expediting cell line development

“…Improved transient expression was demonstrated in modified HEK293 cells expressing Epstein-Barr virus nuclear antigen 1 [27]. A similar approach in CHO cells using the Epi-CHO transient gene expression (TGE) system showed up to a 64% increase in monoclonal antibody titer when compared to previously reported systems [28,29]. These tools are summarized in Table 1 and collectively provide significant flexibility and precision in genome editing and represent significant improvements over standard practices such as homologous recombination or illegitimate integration.…”