Plasmids, along with other DNA vectors such as minicircles and linear derivatives, play a central role as starting materials, intermediates, drug substances, and drug products in the manufacturing of various therapeutics. These applications encompass DNA and mRNA vaccines, gene-editing templates, and gene therapies employing both viral and non-viral vectors.

The quality of starting material plays a pivotal role in determining final product quality. The Mediphage team recently published a study examining the fidelity of DNA replication using enzymatic methods ( in vitro ) compared to plasmid DNA produced in vivo in E. coli , using a novel assay with greater sensitivity than next-generation sequencing.

Our findings show that DNA production in E. coli results in significantly fewer loss-of-function (LOF) mutations (80- to 3,000-fold less) compared to enzymatic ( in vitro ) DNA replication methods, meaning DNA synthesized in vitro has a significantly higher mutation rate than DNA produced traditionally in E. coli. Therefore, utilizing in vitro enzymatically produced DNA in biotechnology and biomanufacturing may introduce a substantial number of mutation impurities, potentially affecting the quality and yield of final pharmaceutical products, while using DNA starting material derived from E. coli substantially mitigates this risk.

Using high-quality materials in the manufacture of genetic medicines is critical in reducing patient risks due to mutations, but can further increase potency (lower doses) and improve production yield and efficiency (reduce cost).