Effect of plasmid DNA isoforms on preparative anion exchange chromatography.

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.

Multi-modal imaging probe for assessing the efficiency of stem cell delivery to orthotopic breast tumours.

Stem cells have been utilised as anti-cancer agents due to their ability to home to and integrate within tumours. Methods to augment stem cell homing to tumours are being investigated with the goal of enhancing treatment efficacy. However, it is currently not possible to evaluate both cell localisation and cell viability after engraftment, hindering optimisation of therapy. In this study, luciferase-expressing human adipocyte-derived stem cells (ADSCs) were incubated with Indium-111 radiolabelled iron oxide nanoparticles to produce cells with tri-modal imaging capabilities. ADSCs were administered intravenously (IV) or intracardially (IC) to mice bearing orthotopic breast tumours. Cell fate was monitored using bioluminescence imaging (BLI) as a measure of cell viability, magnetic resonance imaging (MRI) for cell localisation and single photon emission computer tomography (SPECT) for cell quantification. Serial monitoring with multi-modal imaging showed the presence of viable ADSCs within tumours as early as 1-hour post IC injection and the percentage of ADSCs within tumours to be 2-fold higher after IC than IV. Finally, histological analysis was used to validate engraftment of ADSC within tumour tissue. These findings demonstrate that multi-modal imaging can be used to evaluate the efficiency of stem cell delivery to tumours and that IC cell administration is more effective for tumour targeting.