As pharmaceutical pipelines tilt increasingly toward gene and RNA-based therapies, analytical scientists are facing new challenges in characterizing complex molecular entities such as mRNA, lipid nanoparticles (LNPs), and viral vectors. A recent review by scientists at Waters outlines a suite of recent innovations in liquid chromatography (LC) that are redefining how these emerging modalities are analyzed, with implications for quality control, regulatory compliance, and therapeutic development.
“Most of emerging modalities are complex biomolecules with both highly charged surfaces (e.g. sugar phosphate backbone of nucleic acids) and hydrophobic regions (e.g. aromatic nucleobases) making them prone to non-specific adsorption and causing problems with recovery and sub-optimal separations,” the authors note.
Several strategies for mitigating these analytical obstacles are discussed. One such approach is the use of low adsorption column hardware, designed to minimize interaction between analytes and reactive surfaces. Hybrid-surface technology (HST), titanium, and polyethylene linings are among the solutions examined. Notably, a hydrophilic-modified hybrid surface was shown to enable “complete recovery of challenging analytes,” suggesting that surface chemistry remains critical when analyzing nucleic acids and proteins under aqueous conditions.
Size-based separations are another cornerstone of analytical workflows for large molecules. The authors highlight advances in ultra-wide pore size exclusion chromatography (SEC) columns, particularly those optimized for analytes up to 1500 Å in hydrodynamic size. Columns packed with 2.5–3 µm particles are presented as a balanced solution offering both resolution and mechanical stability. One example includes a BE-PEO surface-modified material that enabled “high resolution and reproducible separations of DNA fragments up to 1350 bp.”
The review also revives interest in slalom chromatography, a non-equilibrium technique that leverages shear-induced retention for separating large DNA and RNA species. This mode, which operates exclusively in the interparticle space of packed columns, demonstrated clear separation of 4 kbp double-stranded RNA impurities from therapeutic mRNA. According to the authors, slalom LC offers “UHPLC-like speed” and a low limit of detection for nucleic acid analytes (around 1 ng), while maintaining reproducibility and robustness.
Further enhancing size-based separations, the authors evaluate tandem SEC systems – multiple columns with differing average pore diameters – to enable continuous separation across a broader size range. Modeling data supports the idea that such arrangements can simulate restricted access media without complex synthesis.
A theoretical but promising strategy involves gradient SEC columns, where pore sizes vary axially along the column length. Simulated comparisons with traditional and serially coupled columns suggest potential improvements in selectivity for complex mixtures.
Another novel dimension comes from pressure-enhanced liquid chromatography (PELC). Unlike conventional LC, which treats pressure as a byproduct, PELC modulates pressure to fine-tune separation selectivity. When combined with stepwise pressure programming, the method allowed greater resolution among closely related oligonucleotides. “If solute retention increases with pressure,” the authors note, “then a positive pressure gradient will result in band broadening while a negative pressure gradient will yield sharper peaks.”
The study also reconsiders effective column length, particularly in separations involving large biomolecules. Empirical data indicates that ultra-short columns – some as small as 1.5 cm – can achieve optimal performance, provided extra-column dispersion is minimized. “Most of the length of conventional columns […] remains non-utilized,” the authors observe.
Beyond column design, the paper explores procedural innovations such as bracketed injection methods, which improve reproducibility in AEX and HILIC by reducing early-stage non-specific binding. Injecting a high-salt plug alongside the sample was shown to “limit the initial interaction strength at the head of the column,” enhancing analyte recovery.
Lastly, bottom-up RNA characterization through oligo mapping is addressed. The authors describe emerging nucleases like MC1 and Cusativin, which outperform RNase T1 by generating “longer, overlapping fragments” that provide improved sequence resolution. These tools support LC–MS workflows for RNA identity and impurity profiling, including detection of dsRNA or chemical modifications.
The review positions LC as a scalable, modifiable platform that can be adapted for complex molecular entities without relying on less routine methods such as analytical ultracentrifugation or cryo-EM.
“Further extension of the prevalence of LC based assays needs consideration of general challenges troubling these kinds of analyses,” the authors write. Yet the steady progression of column chemistry, instrumentation, and workflow design provides optimism that LC will remain integral in the characterization of next-generation therapeutics.
One of the co-authors of this study, Fabrice Gritti, discussed HPLC innovation and the “Biopharmaceutical Challenge” in a recent interview, which you can read here.
Newsletters
Receive the latest analytical science news, personalities, education, and career development – weekly to your inbox.
