Rapid recombinant protein production from piggyBac transposon-mediated stable CHO cell pools

Balasubramanian, Sowmya; Matasci, Mattia; Kadlecova, Zuzana; Baldi, Lucia; Hacker, David L.; Wurm, Florian M.

doi:10.1016/j.jbiotec.2015.03.001

Cited by 54 publications

(48 citation statements)

References 37 publications

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“…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).…”

Section: Resultssupporting

confidence: 93%

“…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.…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Rajendra

Balasubramanian

Peery

et al. 2017

Biotechnology Progress

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Chinese hamster ovary (CHO) cells remain the most popular host for the production of biopharmaceutical drugs, particularly monoclonal antibodies (mAbs), bispecific antibodies, and Fc-fusion proteins. Creating and characterizing the stable CHO clonally-derived cell lines (CDCLs) needed to manufacture these therapeutic proteins is a lengthy and laborious process. Therefore, CHO pools have increasingly been used to rapidly produce protein to support and enable preclinical drug development. We recently described the generation of CHO pools yielding mAb titers as high as 7.6 g/L in a 16 day bioprocess using piggyBac transposon-mediated gene integration. In this study, we wanted to understand why the piggyBac pool titers were significantly higher (2-10 fold) than the control CHO pools. Higher titers were the result of a combination of increased average gene copy number, significantly higher messenger RNA levels and the homogeneity (i.e. less diverse population distribution) of the piggyBac pools, relative to the control pools. In order to validate the use of piggyBac pools to support preclinical drug development, we then performed an in-depth product quality analysis of purified protein. The product quality of protein obtained from the piggyBac pools was very similar to the product quality profile of protein obtained from the control pools. Finally, we demonstrated the scalability of these pools from shake flasks to 36L bioreactors. Overall, these results suggest that gram quantities of therapeutic protein can be rapidly obtained from piggyBac CHO pools without significantly changing product quality attributes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:534-540, 2017.

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Section: Resultssupporting

confidence: 93%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Rajendra

Balasubramanian

Peery

et al. 2017

Biotechnology Progress

Self Cite

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“…Investigators have demonstrated the ability of piggyBac to generate stable cell lines for large-scale protein production [25, 26]. Engineered doxycycline inducible stable cells lines have proven capable of reproducible protein production in the absence of selection, even the ability to produce 750 mg of an endoplasmic-reticulum-resident fucosyultransferase.…”

Section: Engineering Stable Cell Linesmentioning

confidence: 99%

piggyBac-ing models and new therapeutic strategies

Woodard

Wilson

2015

Trends in Biotechnology

111

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DNA transposons offer an efficient non-viral method of permanently modifying the genomes of mammalian cells. The piggyBac transposon system has proven effective in genomic engineering of mammalian cells for pre-clinical applications including gene discovery, simultaneous multiplexed genome modification, animal transgenesis, gene transfer in vivo achieving long-term gene expression in animals, and for genetic modification of clinically relevant cell types including induced pluripotent stem cells and human T lymphocytes. piggyBac has many desirable features including seamless excision of transposons from the genomic DNA and the potential to target integration events to desired DNA sequences. This review explores these recent applications and also highlights the unique advantages of using piggyBac for developing new molecular therapeutic strategies.

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“…The PB transposon system has one or more transposon donor vectors, which express the transgene(s) and a helper vector encoding the PBase enzyme [19]. PB targets the tetranucleotides TTAA in the genome [20, 21].…”

Section: Introductionmentioning

confidence: 99%

Monoclonal antibodies expression improvement in CHO cells by PiggyBac transposition regarding vectors ratios and design

et al. 2017

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Establishing stable Chinese Hamster Ovary (CHO) cells producing monoclonal antibodies (mAbs) usually pass through the random integration of vectors to the cell genome, which is sensitive to gene silencing. One approach to overcome this issue is to target a highly transcribed region in the genome. Transposons are useful devices to target active parts of genomes, and PiggyBac (PB) transposon can be considered as a good option. In the present study, three PB transposon donor vectors containing both heavy and light chains were constructed, one contained independent expression cassettes while the others utilized either an Internal Ribosome Entry Site (IRES) or 2A element to express mAb. Conventional cell pools were created by transferring donor vectors into the CHO cells, whereas transposon-based cells were generated by transfecting the cells with donor vectors with a companion of a transposase-encoding helper vector, with 1:2.5 helper/donor vectors ratio. To evaluate the influence of helper/donor vectors ratio on expression, the second transposon-based cell pools were generated with 1:5 helper/donor ratio. Expression levels in the transposon-based cells were two to five -folds more than those created by conventional method except for the IRES-mediated ones, in which the observed difference increased more than 100-fold. The results were dependent on both donor vector design and vectors ratios.

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Rapid recombinant protein production from piggyBac transposon-mediated stable CHO cell pools

Cited by 54 publications

References 37 publications

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

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

piggyBac-ing models and new therapeutic strategies

Monoclonal antibodies expression improvement in CHO cells by PiggyBac transposition regarding vectors ratios and design

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