An Accurate and Precise ddPCR-Based Method for Determining the Concentration of Plasmid DNA

Transient transfection is commonly used for the commercial production of adeno-associated viral particles for gene therapy. In this process, packaging cells such as HEK293 cells are transfected with three plasmids, including the Rep/Cap plasmid, the Helper plasmid, and the gene-of-interest plasmid containing the transgene/gene therapy product. The combination of these plasmids allows for the robust production of recombinant adeno-associated viral particles. As a result, the concentration of these plasmids plays a critical role in viral production and must be accurately assessed. Typically, A₂₆₀/A₂₈₀ readings are utilized to measure plasmid titer; however, this approach lacks accuracy and specificity and is susceptible to matrix interference. To address these shortcomings, a digital droplet PCR method was developed to titer plasmids. This method uses a combined restriction digest/PCR protocol to linearize the plasmid template and evaluate copy numbers of a plasmid-specific gene. Qualification demonstrated that the method is highly accurate, specific to plasmid DNA, and impervious to matrix interference. Key features • Digital droplet PCR (ddPCR)-based plasmid quantification using a combined linearization/PCR workflow, enabling highly accurate and precise copy-number measurements across plasmids of varying purity and production stages. • Robust performance in complex sample matrices, with high dilution tolerance that minimizes matrix interference and ensures reliable quantification from crude lysates to highly purified plasmids. • Specific detection of plasmid DNA independent of contaminating nucleic acids, providing reliable readouts when A₂₆₀ measurements are distorted by residual DNA, RNA, or dNTP impurities. • Universally applicable to plasmids sharing common selection cassettes, enabling a single primer-probe set to accurately quantify diverse constructs without plasmid-specific assay redesign.