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

Currently, 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 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. SXC is a size-based separation mode whereby retention is achieved through mutual steric exclusion of polyethylene glycol and a large target solute in a hydrophilic solid phase. SXC shares common mechanistic roots with PEG precipitation, with the main operational difference being that SXC employs a hydrophilic solid phase as a nucleation centre on which biomolecules accrete instead of forming precipitates. The experimental workflow included cultivation of Escherichia coli cells, harboring the model plasmids pUC18 (2,686 bp), pVAX-eGFP (3,685 bp), or pCEP4 (10,410) bp, alkaline lysis, and the use of SXC for pDNA capture and purification on 3D printed hydrophilic supports. The process concludes with concentration of the plasmid-containing fractions through precipitation with high concentrations of PEG. After resuspension, the fractions were analyzed by HIC-HPLC and the protein content was determined by BCA. Applying SXC on 0.70 mL 3D-printed monolithic columns successfully purified and isolated the desired sc pDNA isoform with high yield in a single chromatographic step. The SXC operation was tested with PEGs of different molecular weights (1500 to 8000 Da) and concentration (5 to 15%). Total pDNA retention was achieved when employing short PEG chains at high concentrations, or longer PEGs at lower concentrations. This had the spill over benefit of mitigating backpressures and shear stress effects. Most importantly, under the optimised conditions tested, the total purity of sc pDNA was consistently above 97%, with an 80% HCP reduction.