Transient transfections were performed at a 1 mL working volume in 2 mL 96 deep well plate, at a 30 mL working volume in 50 mL spin tubes, or at a 100 mL working volume in 250 mL shake flasks. For resuspension transfections, CHO-K1 cells were seeded at 12 x 10^6 cells/mL and transfected with 3 mg/L of DNA and PEIpro at 4.5 mg/L, they were sequentially mixed with the resuspended cells without forming DNA:PEIpro pre-complex.

Biopharmaceuticals like therapeutic monoclonal antibodies (mAbs) and other derived proteins are popular for treating various diseases. Transient gene expression (TGE) is typically used as a fast yet efficient method to generate moderate amounts of material. It has been used to support early stage research and discovery processes. Introduction of a robust high yielding and predictive TGE platform in Chinese hamster ovary (CHO) is crucial. It maintains the consistency in cell lines and processes throughout the early drug discovery and downstream manufacturing processes. This helps researchers to identify the issues at an early stage for timely resolution. In this study, we have demonstrated a simple high-titer platform for TGE in CHO based on a dilution process of seeding cells. We achieved titers ranging from 0.8 to 1.9 g/L for eight model mAbs at three scales (1, 30, 100 mL) in 10 days using our new platform. The ability to seed by dilution significantly streamlined the process and dramatically enhanced platform throughput. We observed a modest reduction in titer ranging from 11% to 28% when cells were seeded using dilution compared to when cells were seeded using medium exchange. Further studies revealed that carry over of spent medium into transfection negatively affected the DNA uptake and transcription processes, while the translation and secretion was minimally impacted. In summary, our transient CHO platform using cells prepared by dilution at high densities can achieve high titers of up to 1.9 g/L, which can be further improved by targeting the bottlenecks of transfection and transcription.