Resilient immortals, characterizing and utilizing Bax/Bak deficient Chinese hamster ovary (CHO) cells for high titer antibody production

Abstract

Cell death due to apoptosis is frequently observed in large-scale manufacturing of therapeutic proteins, and can reduce product accumulation in bioreactors. Several different strategies that involve overexpression of antiapoptotic or downregulation of proapoptotic proteins have been designed in attempt to curb this problem in Chinese hamster ovary (CHO) cell culture. However, each of these designs has their own shortcomings and limits, rendering them ineffective for large-scale protein production. Recently, we have reported generation of a Bax and Bak deficient dhfr(-/-) CHO cell line using zinc-finger nucleases. Here we demonstrate that puromycin, but not methotrexate, selection can be used to generate antibody-expressing Bax and Bak deficient clones that are not only resistant to apoptosis, but that can also achieve higher titers relative to parental CHO cells due to higher cell density. Additionally, we show that Bax and Bak deficient cells have more mitochondria with healthy membrane potential, an attribute that perhaps contributes to their more potent growth compared to parental cells. Bax and Bak deficient cells do not readily apoptose, as shown by the ability to withstand high concentrations of apoptosis inducing agents, such as sodium butyrate, without a reduction in viability, growth, or titer. These traits render Bax and Bak deficient cells a potentially attractive host for production of therapeutic proteins at industrial scale.