By Thomas Parker, Process Development Scientist, Vaccines & Viral Therapies, Technology Management; Youness Cherradi, Senior Process Development Scientist, Technology Management; Niddhivinayak Mishra, Process Development Specialist II, Technology Management – MSAT
Plasmid DNA (pDNA) is an important component of viral vector therapies. These circular double helix DNA molecules can be used as the therapeutic transgene, to code for the viral capsid or as the vaccine itself. DNA vaccines have been approved for use in animals and this approach is being leveraged to combat the SARSCoV-2 virus. In addition, pDNA is used as the starting material for mRNA vaccines which have also emerged in response to the pandemic and have other applications such as oncology. Furthermore, Plasmid DNA constructs are used for mammalian cell transfection to produce viral vectors used in gene therapy.
pDNA manufacturing presents several challenges. Production suffers from low productivity of microbial fermentation and the purification process is complicated by the fact that plasmids are quite large and possess a highly negative charge. The bacterial lysate contains contaminants with properties similar to pDNA leading to low resolution separation and can be highly viscous.
A low flow rate is needed for chromatography and it can be difficult to achieve the desired concentrations at the final tangential flow filtration (TFF) step.
Additionally, pDNA is sensitive to shear stress which can lead to changes in its topological form. Plasmid isoforms include supercoiled (fully intact and wound around itself), open circular (one strand is broken and the molecule relaxes) and linear (both strands are broken with free ends). Supercoiled plasmids are recognized as the most therapeutically relevant and regulatory agencies set expectations for the supercoiled percentage in final drug substance for DNA vaccines.
This paper describes an end-to-end platform consisting of single-use technologies and validation and testing services for pDNA manufacturing. Each of these steps is explored below, along with strategies to optimize and streamline the purification workflow.