1 research outputs found
Development of a CHO-Based Cell-Free Platform for Synthesis of Active Monoclonal Antibodies
Chinese
Hamster Ovary (CHO) cells are routinely optimized to stably
express monoclonal antibodies (mAbs) at high titers. At the early
stages of lead isolation and optimization, hundreds of sequences for
the target protein of interest are screened. Typically, cell-based
transient expression technology platforms are used for expression
screening, but these can be time- and resource-intensive. Here, we
have developed a cell-free protein synthesis (CFPS) platform utilizing
a commercially available CHO extract for the rapid <i>in vitro</i> synthesis of active, aglycosylated mAbs. Specifically, we optimized
reaction conditions to maximize protein yields, established an oxidizing
environment to enable disulfide bond formation, and demonstrated the
importance of temporal addition of heavy chain and light chain plasmids
for intact mAb production. Using our optimized platform, we demonstrate
for the first time to our knowledge the cell-free synthesis of biologically
active, intact mAb at >100 mg/L using a eukaryotic-based extract.
We then explored the utility of our system as a tool for ranking yields
of candidate antibodies. Unlike stable or transient transfection-based
screening, which requires a minimum of 7 days for setup and execution,
results using our CHO-based CFPS platform are attained within 2 days
and it is well-suited for automation. Further development would provide
a tool for rapid, high-throughput prediction of mAb expression ranking
to accelerate design–build–test cycles required for
antibody expression and engineering. Looking forward, the CHO-based
CFPS platform could facilitate the synthesis of toxic proteins as
well