Selected new approaches and future perspectives in liquid chromatography for the analysis of emerging modalities

Abstract

Emerging biopharmaceutical modalities, such as genetic medicines and RNA therapies, offer transformative potential for treating previously intractable diseases. However, these complex drugs present unique analytical challenges due to their intricate structures, sophisticated manufacturing processes, and modality-specific product quality attributes. Liquid chromatography (LC) has emerged as a versatile tool for addressing these challenges, enabling precise characterization and quality control strategies. This review highlights recent advancements in LC technologies, including low-adsorption hardware, ultra-wide pore size exclusion chromatography (SEC) columns, and innovative separation modes such as slalom chromatography and pressure-enhanced liquid chromatography (PELC). These developments tackle issues such as non-specific adsorption, carryover, and inadequate selectivity while improving resolution and robustness for large biomolecules like mRNA, adeno-associated viruses (AAVs), and lipid nanoparticles (LNPs). Novel approaches, such as tandem SEC systems, gradient SEC columns, and dual stationary phase gradients, further expand the scope of LC techniques by enhancing separations for diverse analyte sizes and complexities. Additionally, practical innovations like bracketed injection methods and new enzymatic tools for oligo-mapping improve reproducibility, efficiency, and confidence in RNA sequence analysis. These advancements not only address current analytical limitations but also pave the way for regulatory-compliant approaches, which will support the broader adoption of LC in both discovery and quality control settings. As the field continues to evolve, these innovations are poised to play a pivotal role in ensuring the safety, efficacy, and consistency of next-generation therapeutics.