TAAV-manufactured enzymatic DNA¹ is a double stranded, linear molecule with a covalently closed structure. The process consists of Rolling Circle Amplification of the precursor template by Phi29 polymerase, precursor backbone processing by restriction enzymes, covalent closure of the sequence of interest by protelomerase TelN, and exonucleases treatment to remove residual DNA. Furthermore, the process includes chromatographic and filtration steps to eliminate enzymes and DNA residuals.

To monitor the manufacturing process and ensure the quality of final enzymatic DNA product, a set of analytical, physical and biochemical techniques are utilized. Currently, in-process controls are analyzed by agarose gel electrophoresis (AGE) and Ultraviolet.  Specifications of enzymatic DNA final product include process related impurities analyzed by ELISA for TelN, purity by AGE and densitometry, and identity by Sanger sequencing, among others.

This work aims to monitor the TAAV-manufactured enzymatic DNA process and final product analyses, using Ultra-High-Pressure Liquid Chromatography (HPLC) with accuracy and higher resolution. Results obtained with HPLC indicate that the technique resolves the peak of enzymatic DNA final product, which elutes at <5 minutes with a high degree of purity (>95%). Moreover, HPLC can be used to identify and separate TAAV-manufactured enzymatic DNA final product from other components present at different manufacturing stages.

In summary, HPLC technique is accurate, sensitive, and has higher resolution compared to AGE. Potentially, the technique can be used for both monitoring the process and measuring purity of TAAV-manufactured enzymatic DNA final product.

1. Technology for making TAAV-manufactured enzymatic DNA technology is licensed from Touchlight IP Ltd.