Analytical separation of plasmid DNA isoforms using anion exchanging chromatographic monoliths with 6 µm channels

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.

Editorial

“…A part of the published manuscripts was also presented at the last Monolith Summer Symposium that was held last year in Portorož, Slovenia. As a logical consequence of the spirit of this time, the topics of most papers that are summarized here are dealing with purification of nanoparticles like viruses [1][2][3], plasmid DNA isoforms [4,5] and RNA drug substances [6]. As is well known, monolithic stationary phases (including membranes) were firstly developed for fast separation of proteins [7], but only the last manuscript in this issue reports the development of a new monolithic stationary phase for protein separation [8].…”

“…The next three papers are dedicated to fractionation of materials containing different DNA and RNA forms [4][5][6]. Kralj et al reported the optimization of production of large plasmid DNA isoforms with sizes over 10 kbp using anion exchange monoliths.…”

“…Pavlin et al [5] demonstrated again that channel diameter plays a crucial role in production of plasmid DNA (pDNA) purification processes. They demonstrated that pDNA open circular forms can be physically entrapped inside narrow channels of monolith, and demonstrated that this type of monolithic problem can be solved by the use of a column with larger (6 µm) channels with a fine-tuned surface chemistry.…”

Selective hydrophobic interaction chromatography for high purity of supercoiled DNA plasmids

“…Plasmids pHELP (11.6 kbp, 19% OC, 81% SC), pmFIX6 (6.7 kbp, 1% OC, 7% lin, 82% SC, 10% multimers [MM]; 1.8% gDNA), pGMAAV8_Rep-Cap (7.3 kbp, 7% OC, 1% lin, 92% SC; 0.25% gDNA) and pIVTeGFP (3.3 kbp, 4% OC, 67% SC, 28% MM) were stored in 50 mM TRIS, 10 mM EDTA, 0.75 M NaCl, pH 7.2. Isoform purity for each of the plasmids was determined by CIMac pDNA AEX HPLC analysis as described previously (Pavlin et al, 2023) and the gDNA content was determined with qPCR.…”

“…Column (6 µm chan-nels) were performed as previously described (Pavlin et al, 2023). Samples were diluted with binding MP used for analysis to reduce AS concentration below 0.5 M (e.g., samples in 1.55 M AS were diluted three times) and to reduce pDNA concentration, ideally to 10 ng/µL (e.g., sample with 90 ng of pDNA/µL was diluted nine times).…”