Chromatography on monolithic supports for biological nanoparticles and large biomolecules: Recent developments and state-of-the-art

• Novel monolithic stationary phases increase protein capacity and selectivity. • Ion exchange monoliths resolve AAV empty/full capsids and viral vectors. • Brush-grafted surfaces reduce fouling in pDNA and mRNA purification. • Affinity and ion exchange monoliths improve extracellular vesicle isolation. • Rapid monolith formats accelerate process analytics and quality control. Chromatography on monolithic supports combines convective flow through interconnected macropores with tunable surface chemistries, enabling high-flow, low-shear purification of large biomolecules and biological nanoparticles. Recent developments in stationary-phase design, such as brush-grafted surfaces, together with optimized elution protocols, have additionally advanced both resolution and capacity across a broad range of targets. Demonstrated applications include the high-resolution separation of adeno-associated virus (AAV) empty and full capsids, isolation of supercoiled plasmid DNA, and removal of double-stranded RNA impurities from mRNA therapeutics. Furthermore, the platform’s tolerance for complex matrices enables efficient purification of extracellular vesicles and direct recovery of biotherapeutics from undiluted blood plasma. Miniaturized monolithic columns enable rapid and robust on-line monitoring of industrial processes for current good manufacturing practice (cGMP) control. Ongoing progress in multimodal chemistries, continuous and multicolumn operations, and integrated analytics establishes chromatography on monolithic supports as a versatile and robust platform for the rapid, selective purification of large biomolecules and biological nanoparticles and efficient monitoring of industrial processes.