Evaluating Apoptotic Gene Efficiency for CHO Culture Performance Using Targeted Integration

Chinese hamster ovary
(CHO) cells have long been the favored platform
for producing complex biopharmaceuticals, such as monoclonal antibodies.
Cell death is a critical factor in all CHO cultures, dictating the
duration until harvest in batch cultures and viable cell density in
perfusion. The programmed cell death, or apoptosis, pathway has been
widely studied due to its relevance in affecting cell culture performance
and the extensive knowledge about its protein-to-protein interaction
network. However, clonal variation seen with random integration has
confounded results, and it remains unclear which effector genes should
be overexpressed. Here, we employed the recombinase-mediated cassette
exchange strategy to develop isogenic cell lines expressing one copy
of erythropoietin, as a model protein product, and various antiapoptotic
genes: bcl-2 from CHO and human origin, bcl-xL from CHO and human
origin, mcl-1, and bhrf-1. We tested the generated isogenic cell lines
in the presence of sodium butyrate, a well-known apoptotic initiator,
in a batch culture. The most promising candidates were cultured in
fed-batch in the microbioreactor ambr15 system. The observed phenotype
varied significantly depending on the overexpressed gene; therefore,
the metabolic differences were further characterized using multiplexed
quantitative proteomics. We showed that overexpressing bcl-2 from
the CHO origin significantly improved productivity and established
a methodology to successfully test candidate genes via targeted integration.
This will enable future metabolic engineering strategies to be more
comparable and overcome the challenges faced thus far.