Retention mechanism in slalom chromatography: Perspectives on the characterization of large DNA and RNA biopolymers in cell and gene therapy

Abstract

Significant progress has been made in the last two decades in producing small ($<2\mu m$), high-purity, and low-adsorption particles, columns and system hardware, for ultra-high pressure liquid chromatography (UHPLC). Simultaneously, the recent rapid expansion of cell and gene therapies for treating diseases necessitates novel analytical technologies for analyzing large ($>$2 kbp) plasmid double-stranded (ds) DNA (which encodes for the in vitro transcription (IVT) of single-stranded (ss) mRNA therapeutics) and dsRNAs (related to IVT production impurities) biopolymers. In this context, slalom chromatography (SC), a retention mode co-discovered in 1988, is being revitalized using the most advanced column technologies for improved determination of the critical quality attributes (CQAs) of such new therapeutics.

In this review, we first recall non-exhaustively the main currently available analytical techniques (enzyme-linked immunosorbent assay (ELISA), agarose gel electrophoresis (AGE), pulse field gel electrophoresis (PFGE), capillary gel electrophoresis (CGE), mass photometry (MP), anion-exchange chromatography (AEX), ion-pairing reversed-phase liquid chromatography (IP-RPLC), hydrophobic interaction chromatography (HIC), size-exclusion chromatography (SEC), hydrodynamic chromatography (HDC), highly converging flow ultra-filtration (HCF-UF), asymmetrical flow field-flow fractionation (AF4), mass spectrometry (MS), and atomic force microscopy (AFM)) for analyzing mixtures containing large nucleic acid biopolymers, while assessing their strengths and weaknesses.

We then focus comprehensively on the SC technique, report on its past applications since its birth, and review in detail the history and evolution of the proposed retention mechanisms accounting for the observations made in SC. This includes and emphasizes the latest physico-chemical insights (shear rates in packed HPLC columns, entropic elasticity and relaxation of dsDNA, dsRNA, and mRNA biopolymers) governing the retention behavior of such biopolymers in SC.

Finally, based on the recent advancements in understanding the fundamentals of retention in SC, we provide some perspectives and recent proof-of-concept for the analytical characterization by SC of large dsDNAs (plasmid digests, polymerase chain reaction (PCR) verification), the separation of supercoiled/circular and linear dsDNAs (plasmid linearization), the isolation and quantification of large dsRNAs impurities present in mRNA samples produced by IVT, and the differentiation between dsRNA conformers.