Generation of stable Chinese hamster ovary pools yielding antibody titers of up to 7.6 g/L using the piggyBac transposon system

Abstract

Chinese hamster ovary (CHO) cells remain the default production host for many biopharmaceutical drugs, particularly monoclonal antibodies (mAb). Production of gram and kilogram quantities of protein typically requires the generation of stable CHO clones. Unfortunately, this process takes several months, significantly slowing down the drug discovery and development process. Therefore, improved technologies are needed to accelerate biopharmaceutical drug discovery and final drug substance manufacturing. In this study, we describe the generation of stable CHO pools using the piggyBac transposon system. We evaluated the system using four model antibody molecules (3 mAbs and 1 bispecific Ab). Stable CHO pools were isolated in 7-12 days. Using a simple 16-day fed-batch process, we measured titers ranging from 2.3 to 7.6 g/L for the four model antibodies. This represented a 4- to 12-fold increase relative to the controls. Additionally, we isolated stable CHO clones. We found that the stable CHO clones isolated from the piggyBac transposon pools yielded titers two to threefold higher relative to the control clones. Taken together, these results suggest that stable CHO pool and clone generation can be significantly improved by using the piggyBac transposon system. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1301-1307, 2016.

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

“…Once the control pools and piggyBac pools were fully recovered (viability >95%), all the pools were subjected to a 14–17 day fed batch process. As previously reported, piggyBac pools recovered 4–5 days faster than the control pools (data not shown) . The expression results are summarized in Table .…”

“…We have previously reported the use of the piggyBac transposon system for the generation of CHO cell pools yielding titers up to 7.6 g/L from a fed‐batch process . There were three key objectives for this current study.…”

Limiting the metabolic burden of recombinant protein expression during selection yields pools with higher expression levels

“…9G1 cells require glutamine supplementation in the media for survival and thus, cells that have stably integrated the target protein (Fc‐A) and GS can be selected in media lacking glutamine. To increase stringency of selection and isolate high‐producing cells, 75 or 100 μM MSX was added to the media during stable pool generation . Additionally, transfectants were treated with 0.5 μg/mL Dox to induce Fc‐A expression or without Dox, as a control, during the selection process.…”

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

“…This leads to a generally higher rate of transgene transcription compared to the random integration of plasmids (Ding et al, 2005;Galvan et al, 2009;Wilson et al, 2007). Consequently, higher q p and stability compared to stable producer clones obtained by random integration have been achieved with MAb titers of up to 7.6 g/L Rajendra et al, 2016).…”