Unique DNA impacts on transient Expi293TMPRO expression for enhancing biotherapeutic protein production

Introduction: Mammalian transient gene expression technology is an essential tool for biotherapeutics engineering and production. Various reported transient expression systems in human embryonic kidney (HEK) 293 or Chinese hamster ovary (CHO) cells display different expression patterns and production yields, yet the mechanisms to which these patterns and yields can be attributed remain unknown. Methods: PRO, Expi293F™, ExpiCHO-S™, and CHO4Tx®. A novel surface-display molecule fused with a CD4 transmembrane domain was designed to utilize flow cytometry to monitor transient expression stages of reaching cell surfaces prior to release into the extracellular medium. A cytosolic green fluorescence protein (GFP) was transfected as a readout for transfection efficiency, DNA transcription, and protein translation. The final stage of transient expression was monitored by expression titers for antibody proteins, which reflected the secretion and accumulation of protein products in culture media. Results: PRO system. Conclusion: These results together suggested that the efficiency in handling transfected DNAs for protein synthesis (i.e., the ability to make intracellularly up-taken plasmid DNA complexes competent for DNA transcription in the nucleus) is likely a key limiting factor for effective transient production.