Matilde Costa

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.

Background/Objectives: Cervical cancer is a leading cause of cancer-related mortality among women, primarily driven by persistent infections with high-risk human papillomavirus (HPV), particularly HPV-16. Vaccines based on plasmid DNA encoding the viral oncogenes E6 and E7 represent a promising immunotherapeutic strategy, but their efficacy remains limited due to poor cellular uptake. Cell-penetrating peptides such as RALA improve intracellular delivery, and functionalization with octa-arginine peptide conjugated to mannose (R8M) further enhances targeting of antigen-presenting cells (APCs). This study aimed to obtain the minicircle DNA (mcDNA) encoding mutant HPV-16 E6 and/or E7 antigens, and optimize its complexation with mannosylated RALA-based nanoparticles to improve vector delivery and consequently antigen presentation. Methods: Nanoparticles were formulated at different concentrations of RALA, with and without R8M functionalization. Their characterization included hydrodynamic diameter, polydispersity index, zeta potential, complexation efficiency (CE), stability, morphology, and Fourier-Transform Infrared Spectroscopy. In vitro assays in JAWS II dendritic cells (DCs) assessed biocompatibility, transfection efficiency and target gene expression. Results: Optimal conditions were obtained at 72.5 µg/mL of RALA, producing nanoparticles smaller than 150 nm with high CE (>97%) and uniform size distribution. Functionalization with R8M at 58 µg/mL preserved these characteristics when complexed with all mcDNA vectors. The formulations were biocompatible and effectively transfected DCs. Mannosylated formulations enhanced antigenic expression compared to non-mannosylated counterparts, evidencing a mannose-receptor-mediated uptake, while increasing the production of pro-inflammatory cytokines. Conclusions: Nanoparticles based on the RALA peptide and functionalized with R8M significantly improved mcDNA transfection and gene expression in APCs. These findings support further investigation of this system as a targeted DNA vector delivery platform against HPV-16.