Comparison of three transposons for the generation of highly productive recombinant CHO cell pools and cell lines

Abstract

Several naturally occurring vertebrate transposable elements have been genetically modified to enable the transposition of recombinant genes in mammalian cells. We compared three transposons-piggyBac, Tol2, and Sleeping Beauty-for their ability to generate cell pools (polyclonal cultures of recombinant cells) and clonal cell lines for the large-scale production of recombinant proteins using Chinese hamster ovary cells (CHO-DG44) as the host. Transfection with each of the dual-vector transposon systems resulted in cell pools with volumetric yields of tumor necrosis factor receptor-Fc fusion protein (TNFR:Fc) that were about ninefold higher than those from cell pools generated by conventional plasmid transfection. On average, the cell pools had 10-12 integrated copies of the transgene per cell. In the absence of selection, the volumetric productivity of the cell pools decreased by 50% over a 2-month cultivation period and then remained constant. The average volumetric TNFR:Fc productivity of clonal cell lines recovered from cell pools was about 25 times higher than that of cell lines generated by conventional transfection. In 14-day fed-batch cultures, TNFR:Fc levels up to 900 mg/L were obtained from polyclonal cell pools and up to 1.5 g/L from clonal cell lines using any of the three transposons. Biotechnol. Bioeng. 2016;113: 1234-1243. © 2015 Wiley Periodicals, Inc.

Bioreactor scale up and protein product quality characterization of piggyBac transposon derived CHO pools

“…The improved titers were mainly because of the increased specific productivity. This is consistent with previous reports where the use of piggyBac transposon system significantly enhanced cell specific protein productivity . The shake flask titer results were confirmed in an independent experiment (data not shown).…”

“…The use of CHO pools for therapeutic protein generation has gained popularity recently due to improvements in pool generation methodology. The use of transposon systems and other similar technologies has allowed the generation of high expressing pools . Majority of these above referenced reports of piggyBac mediated CHO pools have been in CHO‐DG44 host cell line with antibiotic selection.…”

“…Several researchers have therefore addressed this problem by creating CHO pools capable of yielding high titers. For example, one approach that researchers have used is transposon‐mediated gene integration to obtain higher titer CHO pools . Multiple transposon systems have been used and evaluated including the piggyBac, Sleeping Beauty, Tol2 and Mos1 systems, of which piggyBac is the most widely used.…”

Recombinant protein production from stable mammalian cell lines and pools

“…Since transfection with the PB system yields a high percentage of recombinant cells, the generation of productive cell pools is feasible [4 ,37]. High-yielding CHO cell lines and pools have also been generated with SB and Tol2 [39 ]. Furthermore, both SB and PB have been used to construct cell lines for inducible transgene expression under the control of the Tet-On system [37,38 ,40].…”

Sleeping Beautytransposition: from biology to applications

“…For such a purpose, SB represents an attractive transgene expression vector because of its ability to promote efficient genomic integration in a variety of mammalian cell types. Transposons, including SB, piggyBac and Tol2 are suitable for generating polyclonal cell pools or clonal cell lines for large scale (industrial) production of recombinant proteins in Chinese hamster ovary (CHO) cells (Balasubramanian et al, 2016).…”