A platform method for plasmid isoforms analysis by capillary gel electrophoresis.

ABSTRACT

In the production of novel biological products, plasmids are often engineered into delivery vectors for target genes, which can be used directly as vaccines or as intermediate products for gene/cell therapy. Plasmid DNA exists in several topological forms such as supercoiled, linear, and open circular. As supercoiled plasmid shows the highest efficiency in transfecting eukaryotic cells, the content of supercoiled plasmids becomes an important indicator of plasmid quality. CGE is an effective analysis method for separating different topological structures of plasmids. For the purpose of providing plasmid manufacturers and regulatory agencies with an efficient and readily used tool for monitoring the quality of plasmids, this article identifies the optimal separation and detection conditions of CGE, presents a platform-based plasmid analytical method, and uses plasmid of different sizes to verify the feasibility of this method. In terms of detector, the LIF detector has obvious advantages over the ultraviolet detector in sensitivity and resolution. Using the optimal CE condition (10× gel buffer), baseline separation of different topological forms and impurities can be achieved for different plasmid sizes (5.9, 7.8, 15.4 kb). In addition, 6.5 kb plasmid was used to compare the different separation technologies such as CGE-LIF, ion exchange chromatography and agarose gel electrophoresis. The result shows that CGE-LIF can provide better resolution and quantitation accuracy than ion exchange chromatography and agarose gel electrophoresis. CGE-LIF, as a quick and convenient method to separate and quantify plasmids, has the advantages of high sensitivity, high resolution, and high quantitative accuracy. Therefore, it is ideal for analysis of plasmids with different sizes, and it can also be used as a platform method for manufacturers and regulatory agencies to monitor the purity and stability of plasmids.