3D-printed matrices for the purification of plasmid DNA via steric exclusion chromatography

Plasmid DNA (pDNA) holds great promise not only for gene therapy and DNA vaccination, but also as a raw material to produce mRNA and viral vaccines. However, achieving high-quality pDNA purification remains a significant challenge. Current purification methods, often involving multiple precipitation and chromatographic steps, can compromise the structural integrity and stability of the desired supercoiled (sc) pDNA isoform, which is crucial for effective gene transfer. This work aims to explore the potential of steric exclusion chromatography (SXC) as a novel approach for capturing pDNA, immediately after cell lysis, and using 3D-printed chromatographic matrices. The experimental workflow includes cultivation of Escherichia coli cells, harboring the model plasmid pVAX-eGFP (3696 bp), product release by alkaline lysis, and pDNA capture and purification in a single chromatographic step by SXC on 3D-printed hydrophilic supports. The influence of polyethylene glycol (PEG) molecular weights (1500 to 8000 Da) and concentrations (5 to 15 %) on pDNA retention was systematically explored. Total pDNA capture was achieved either employing short PEG chains at high concentrations or longer PEG chains at lower concentrations. Under optimized conditions, the process consistently achieved >97 % supercoiled (sc) pDNA total purity and allowed an 80 % reduction in host cell proteins (HCP). In conclusion, this study demonstrates that SXC, implemented on 3D-printed hydrophilic monoliths, significantly mitigates the relaxation effects in plasmids observed in current purification procedures required for isolating sc pDNA, making it a promising and innovative approach for the development of a fast, high-capacity, and cost-effective pDNA capture step. • SXC allows a one-step capture of sc pDNA directly from clarified lysate. • High supercoiled pDNA purity (>97 %) under optimized PEG conditions. • High protein removal efficiency up to 90 %, ensuring selective purification. • Shorter PEGs improved separation and reduced viscosity and backpressure. • 3D-printed monoliths enable scalable and cost-effective SXC pDNA purification.