Plasmid DNA (pDNA) is a critical starting material for DNA vaccines, messenger RNA (mRNA) production, and gene and cell therapies, and frequently serves either as the final drug substance or as a template for therapeutic molecules. In addition, large plasmid constructs are widely used in transient transfection processes for the production of viral vectors and recombinant proteins, which impose stringent requirements on chromatographic capture and polishing steps. Established bead-based chromatography media, originally developed for protein purification, typically exhibit pore sizes of 30–100 nm, limiting intraparticle mass transfer for pDNA whose size is comparable to or larger than the pore dimensions and resulting in diffusion-limited performance. Consequently, continuous beds or convective flow devices such as monoliths and membrane adsorbers, featuring channels or pores larger than 1 μm, have emerged as preferred stationary phases for large biomacromolecules, enabling higher productivity at lower pressure drops. This work addresses the scientific question of whether a monolithic structure can be engineered to deliver equal or superior separation performance for supercoiled pDNA—a critical quality attribute—while operating at substantially reduced backpressure. As a model system, pDNA purification was performed using an AXISFLOW™ Q monolith, which is based on an inverted-flow morphology where hollow spheres are packed into a highly interconnected macroporous network with void volumes up to 80%, yielding pressure drops of roughly one third compared with established polymethacrylate monoliths (CIM® disks) produced by thermal polymerization. Three plasmids, pUC19 (2.7 kbp), pRep2Cap9 (6.9 kbp), and pHelper (12.3 kbp), were purified and benchmarked against CIM® monoliths, cross-linked agarose resins (Capto™ Q, Q ImpRes), cross-linked copolymer resins (POROS™ 50 HQ, POROS™ XQ), and a membrane adsorber (Mustang™ Q XT), focusing on supercoiled yield and product purity. Crude bacterial lysates were processed through two downstream workflows employing either dialysis or tangential flow filtration, and the resulting feed streams were loaded onto the respective anion-exchange devices. Across all conditions and starting materials, the 0.5 mL AXISFLOW™ Q column consistently delivered the highest pDNA yield among the evaluated 1 mL Q-type devices, while meeting GMP-relevant specifications for A260/A280 ratio and supercoiled plasmid content. Levels of process and host-derived impurities, including endotoxin, RNA, and host cell DNA, were at or below required thresholds. These data demonstrate that monolithic materials can be rationally designed to combine low-pressure operation with high-resolution pDNA separation, thereby providing a promising platform for scalable manufacturing of plasmid-based advanced therapies.
Wheelwright et al. (Sun,) studied this question.