Optimization of PEI-mediated transient transfection for antibody production in Expi293F cells by iron ion supplementation.

Polyethylenimine (PEI)-mediated transient transfection represents a pivotal methodology for recombinant protein production in HEK293 and derived cell lines. Despite extensive application, substantial potential for enhancement in transfection efficiency and exogenous protein expression persists, with the role of metal ions remaining inadequately characterized. We hypothesized that iron ions exert a concentration-dependent, biphasic regulation on PEI-mediated transfection, affecting both extracellular complex formation and intracellular gene expression. To test this hypothesis, we systematically investigated the impact of ferric ammonium citrate (FAC) on transient transfection efficiency and anti-PD-1 antibody production in Expi293F cells. Supplementation of iron-depleted basal medium with 10 μM FAC was identified as optimal, achieving 63.7% transfection efficiency for enhanced green fluorescent protein (eGFP) and an anti-PD-1 antibody yield of 55.0 mg/L. Transmission electron microscopy analysis revealed that FAC modulates transfection efficiency by altering PEI/DNA complex morphology. Quantitative real-time PCR demonstrated significant enhancement of intracellular DNA transcription by both FAC and calcium ions. The histone deacetylase inhibitor valproic acid further augmented transcription efficiency by 2.8-fold. Ultimately, anti-PD-1 monoclonal antibody production reached 171.0 mg/L in a 3-L bioreactor, representing a 10.6-fold improvement over initial conditions. This study provides preliminary evidence for establishing an efficient transient expression platform while offering initial mechanistic insights into how iron ions influence PEI-mediated transfection via complex morphology and transcriptional regulation.