Analytical Separation of Plasmid DNA Isoforms Using Anion Exchanging Chromatographic Monoliths with 6 µm Channels

Nejc Pavlin, Urh Černigoj, Mojca Bavčar, Tjaša Plesničar, Jan Mavri, Martin Zidar, Matevž Bone, Urška Kralj Savič, Tadej Sever, and Aleš Štrancar

Electrophoresis. 2023; 1– 11

Abstract:
High-performance liquid chromatography (HPLC)-based analytical assays are used to effectively monitor purity and quantity of plasmid DNA (pDNA) throughout the purification process. However, the phenomenon of physical entrapment of open circular (OC) isoforms pDNA inside narrow channels of chromatographic support decreases its accuracy and precision and the effect increases with pDNA size. The purpose of the study was to develop a chromatographic method for accurate analytical separation between isoforms of <16 kbp pDNA using weak anion exchanging monolithic column with large (6 µm) convective channels. Purified samples of 4.7 and 15.4 kbp large pDNA with known isoform composition were prepared and their isoforms separated in ascending salt gradient. Both OC and supercoiled (SC) isoforms were baseline separated at a flow rate below 0.5 mL min−1 in a guanidinium chloride (GdnCl) gradient with a ≥95% OC pDNA elution recovery. However, these chromatographic conditions increased 2 times the peak width for linear (LIN) pDNA isoform compared to the results using monoliths with 1.4 µm channel size. If other chaotropic agents, such as urea or thiocyanate (SCN), were added to Gdn ions, the elution volume for LIN isoform decreased. Optimization of combined GdnCl/GdnSCN gradient for pDNA elution resulted in a simple and robust chromatographic method, where OC–LIN and LIN–SC pDNA (up to 15 kbp size) were separated with resolution above 1.0 and above 2.0, respectively. The accessibility and general acceptance of anion exchange chromatography for pDNA analytics give the newly developed method a great potential for in-process control monitoring of pDNA production processes.

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