Integrated Two-Step Membrane Chromatography for Robust and High-Throughput Plasmid DNA Purification

Abstract

Plasmid DNA (pDNA) is a key raw material for advanced therapeutic modalities, including gene therapy, mRNA production, and DNA vaccination, requiring efficient and scalable downstream processing strategies. Increasing demand places stringent requirements on purification processes in terms of yield, impurity removal, preservation of plasmid topology, and robustness at scale. Membrane chromatography offers significant advantages for pDNA purification due to convective mass transfer, enabling high binding capacity at short residence times and straightforward linear scale-up. For the first time, a sequential two-step membrane chromatography platform was developed and optimized for the capture and polishing of pDNA. Capture was performed using Sartobind® Q, a strong anion exchange membrane adsorber, allowing selective binding of negatively charged pDNA through electrostatic interactions with quaternary ammonium ligands under controlled buffer conditions. The capture achieved high plasmid recovery (97%) while enabling substantial removal of RNA and other process-related impurities. Process parameters were optimized to balance dynamic binding capacity, product recovery, and impurity clearance while maintaining plasmid integrity. Polishing was conducted using Sartobind® Phenyl, operated under cosmotropic conditions, enabling further impurity removal and improved separation of plasmid isoforms. This step resulted in enrichment of the supercoiled (sc) pDNA fraction relative to open circular (oc) and linear forms, while maintaining high recovery (94%). The integrated process achieved an overall pDNA purity of 98%, meeting typical regulatory expectations. Process applicability was demonstrated using a second plasmid construct of larger size, highlighting the flexibility of the platform while indicating that optimization may be required for different plasmid formats. Overall, the combination of anion exchange capture and hydrophobic interaction polishing provides a robust, scale-up potential and high-throughput membrane chromatography-based platform to produce high-quality pDNA suitable for advanced therapeutic manufacturing.