Harnessing design of experiments and automation significantly improves transient protein production in Chinese hamster ovary cells and accelerates drug discovery

Chinese Hamster Ovary (CHO) cells produce the majority of approved therapeutic proteins and are the de facto platform for rapid, milligram-scale production via transient expression during early biologics discovery. However, performance is highly sensitive to transfection composition, feed strategy, and execution details; and vendor protocols often underperform without platform-specific optimization. We integrated Design of Experiments (DOE) with liquid-handling automation to develop a rapid, data-driven optimization workflow for CHO transient expression. Key factors and ranges were defined for an IgG1 expression and then extended to additional modalities, encompassing coding DNA, PEI transfection reagent, Feed A/B and glucose, inert filler DNA, and the DNA-PEI pipetting sequence that governs brief pre-complexing. Using Response Surface Methodology executed on an automated liquid handler, we generated structured datasets and fitted predictive models that accurately captured main effects, interactions, and curvature. The optimized conditions increased average volumetric productivity by approximately twofold, improved harvest viability, and reduced coding DNA input by about 70 % without detectable loss of product quality (SEC main-peak purity) within our tests. The settings transferred across multiple biologics modalities with consistent gains in titer. Automation eliminates variable‑volume manual pipetting in complex layouts, reduced handling errors, enabled precise short‑duration pre‑complexing steps, and accelerated model‑driven refinement. This approach shorten iteration cycles and de-risks early-stage process development for candidate therapeutic proteins.