Retention mechanism in combined hydrodynamic and slalom chromatography for analyzing large nucleic acid biopolymers relevant to cell and gene therapies

Abstract

Slalom chromatography (SC) was discovered in 1988 for analyzing double-stranded (ds) DNA. However, its progress was impeded by practical issues such as low-purity particles, sample loss, and lack of a clear retention mechanism. With the rise of cell and gene therapies and the availability today of bio-inert ultra-high-pressure liquid chromatography (UHPLC) columns and systems, SC has regained interest. In SC, the elution order is opposite to that observed in hydrodynamic chromatography (HDC): larger DNA molecules are more retained than small ones. Yet, the underlying SC retention mechanism remains elusive.We provide the physicochemical background necessary to explain, at a microscopic scale, the full transition from a HDC to a SC retention mechanism. This includes the persistence length of the DNA macromolecule (representing DNA stiffness), their relaxation time (τR) from the non-equilibrium contour length to the equilibrium entropic configuration, and the relationship between the mobile phase shear rate (〈γ̇〉) in packed columns and the DNA extended length. We propose a relevant retention model to account for the simultaneous impact of hydrodynamic chromatography (HDC) and SC on the retention factors of a series of large and linear dsDNAs (ranging from 2 to 48 kbp). SC data were acquired using bio-inert MaxPeakTM Columns packed with 1.7μm BEHTM 45 Å, 1.8μm BEH 125 Å, 2.4μm BEH 125 Å, 5.3μm BEH 125 Å, and 11.3μm BEH 125 Å Particles, an ACQUITYTM UPLCTM I-class PLUS System, and either 1 × PBS (pH 7.4) or 100 mM phosphate buffer (pH 8) as the mobile phase. SC is a non-equilibrium retention mode that is dominant when the Weissenberg number (Wi=〈γ̇〉τR) is much larger than 10 and the average extended length of DNA exceeds the particle diameter. HDC, on the other hand, is an equilibrium retention mode that dominates when Wi<1 (DNA chains remaining in their non-extended configuration). Maximum dsDNA resolution is observed in a mixed HDC-SC retention mode when the extended length of the DNA is approximately half the particle diameter. This work facilitates the development of methods for characterizing various plasmid DNA mixtures, containing linear, supercoiled, and relaxed circular dsDNAs which all have different degree of molecular stiffness.