Ultra-high pressure slalom chromatography: Application to the characterization of large DNA and RNA samples relevant in cell and gene therapy

Abstract

Slalom chromatography (SC), initially co-discovered by Boyes and Kasai in the late 1980s, has recently re-emerged as a breakthrough technique to rapidly analyze DNA samples. With the development of cutting-edge ultra-high pressure liquid chromatography (UHPLC) systems and columns, SC now offers enhanced resolution and sensitivity for analyzing large DNA samples. By revisiting the fundamentals of the SC retention mechanism (non-equilibrium separation mode) and considering the physicochemical properties of DNA biopolymers (contour length, extension under shear flow, relaxation time), we provide analytical chemists with a general strategy and framework for selecting the most relevant applications in the expanding field of cell and gene therapy. We then present proof-of-concept data demonstrating the rapid separation (under 2 min) of plasmid digest samples containing linear double-stranded (ds) DNA macromolecules starting from 2 kbp to 25 kbp, as well as the accurate size determination (±6%) of linear dsDNAs. Additionally, we show rapid baseline separation and quantification of extensible linear dsDNAs, along with the more rigid plasmid dsDNA (supercoiled/circular/nicked circular). We also quantify dsRNA impurities present in vitro transcription (IVT) media used for producing new mRNA therapeutics and assess dsRNA structural heterogeneity (conformational isomers). These findings aim to support in a near future application chemists in addressing emerging bioanalytical challenges in cell and gene therapy by offering advanced SC columns and methods.