Due to stringent requirements for the purity and efficacy of the plasmid DNA (pDNA) as a pharmaceutical product, chromatography is often used in the downstream process. Monolithic stationary phases exhibit several advantages over the conventional beaded supports mainly due to the convective flow and higher surface accessibility and are efficient chromatographic supports for separation of large biomolecules such as pDNA. High ligand density butyl-modified (C4 HLD) monolithic support is currently used in a polishing step of a pDNA purification process in CIM (TM) HiP(2) Plasmid Process Pack and the goal of the present work was a development of a hydrophobic methacrylate monolith with improved resolution for pDNA isoforms separation and removal of host contaminants. After a detailed search for an appropriate ligand, a pyridine-modified monolithic support was chosen and tested under descending ammonium sulfate linear gradient. The purification process was optimized according to the most efficient pDNA isoforms separation and the quantification of the main impurities during the purification steps was performed, as well as the purity and the recovery of eluted supercoiled (sc) pDNA isoform. Usage of pyridine-modified monoliths resulted in a more efficient separation between pDNA isoforms, with a similar dynamic binding capacity and recovery as C4 HLD monoliths (over 3 mg/mL and 90%, respectively). Pyridine and C4 HLD monoliths were equally efficient for the removal of the main process impurities, but pyridine exhibited higher purity in terms of sc pDNA homogeneity (98%) comparing with C4 HLD (95%), showing to be a suitable alternative to C4 HLD for this polishing step. (C) 2015 Elsevier B.V. All rights reserved.