PATfix^®pDNAPlatformandPCR Methodsas OrthogonalAnalytical Toolsfor Quantificationof pDNA

Accurate characterisation and quantification of plasmid DNA (pDNA) is critical for gene therapy, biotechnology, and vaccine production. Ensuring pDNA quality and consistency is especially important for mRNA, AAV, and other vector-based modalities, where it directly impacts therapeutic efficacy and safety. pDNA exists in multiple topological isoforms: supercoiled (sc), open circular (oc), multimeric forms (multi), and linear (lin). In this study, we evaluated three orthogonal analytical approaches across complex Escherichia coli lysates, process intermediates, and purified fractions using:

▪ PATfix^®pDNAPlatform

▪ quantitative PCR (qPCR)

▪ and digital PCR (dPCR). 

Sample matrices contained typical impurities, including host cell proteins (HCP), host cell RNA (hcRNA), other cellular components, and residual process chemicals. Selected samples were obtained from representative downstream process steps (Fig. 1) employing monolithic columns (DEAE, C4 HLD), and sample-preparation effects were assessed by comparing restriction-digested versus non-digested plasmids for PCR-based quantification.