Abstract
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.
Highlights
IntroductionMonoclonal antibodies (mAbs) are among the best-selling class of biopharmaceutics [1]
Monoclonal antibodies are among the best-selling class of biopharmaceutics [1]
The impact of different ratios of PB transposase on generation of stable pools To evaluate the impact of PiggyBac transposase/transposon ratio on the generation of Chinese Hamster Ovary (CHO) stable pools containing dual promoter vector, three groups including the N-pBLPCH. 1/ 5-pBLPCH and 1/2.5-pBLPCH cells were defined
Summary
Monoclonal antibodies (mAbs) are among the best-selling class of biopharmaceutics [1]. This rapid growth in demand for mAbs has led to further employ new technologies to increase antibody expression levels in mammalian cells. Chinese Hamster Ovary (CHO) cells are the prominent choice of mammalian expression system [2, 3]. To achieve maximum IgG production in mammalian cells, a balanced expression of both chains is mandatory [4, 5].
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