Effect of plasmid DNA isoforms on preparative anion exchange chromatography

Abstract

Increased need for plasmid DNA (pDNA) with sizes above 10 kbp (large pDNA) in gene therapy and vaccination brings the need for its large‐scale production with high purity. Chromatographic purification of large pDNA is often challenging due to low process yields and column clogging, especially using anion‐exchanging columns. The goal of our investigation was to evaluate the mass balance and pDNA isoform composition at column outlet for plasmids of different sizes in combination with weak anion exchange (AEX) monolith columns of varying channel size (2, 3 and 6 µm channel size). We have proven that open circular pDNA (OC pDNA) isoform is an important driver of reduced chromatographic performance in AEX chromatography. The main reason for the behaviour is the entrapment of OC pDNA in chromatographic supports with smaller channel sizes. Entrapment of individual isoforms was characterised for porous beads and convective monolithic columns. Convective entrapment of OC pDNA isoform was confirmed on both types of stationary phases. Porous beads in addition showed a reduced recovery of supercoiled pDNA (on an 11.6 kbp plasmid) caused by diffusional entrapment within the porous structure. Use of convective AEX monoliths or membranes with channel diameter >3.5 µm has been shown to increase yields and prevent irreversible pressure build‐up and column clogging during purification of plasmids at least up to 16 kbp in size.

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.…”