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Current Molecular Medicine


ISSN (Print): 1566-5240
ISSN (Online): 1875-5666

Letter Article

Vaccine Plasmid Topology Monitoring by Capillary Gel Electrophoresis

Author(s): K. Steven Cook, Jane Luo, Andras Guttman and Lawrence Thompson*

Volume 20 , Issue 10 , 2020

Page: [798 - 805] Pages: 8

DOI: 10.2174/1566524020666200427110452

Price: $65


Background: Plasmid DNA has been widely used in vaccination as well as in cell and gene therapy. It exists in multiple isoforms, including supercoiled, nicked or open circular and linear forms. Regulatory agencies recommend having more than 80% of the supercoiled isoform for the bulk release of plasmid products; thus, it should be analyzed accordingly.

Methods and Results: The traditional analysis method for plasmid DNA is agarose gel electrophoresis. However, due to time-consuming manual sample loading, visualization, and data analysis, it has limitations in obtaining consistently quantitative results. In this short communication, we introduced a fast, sensitive, and robust plasmid analysis method using capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF). CGE-LIF analysis of the supercoiled isoform and its open circular counterpart was completed in 20 minutes with excellent sensitivity by using a common fluorescent DNA binding dye. The advantage of the method was demonstrated by the purity analysis of two large plasmids (7 kb and 10 kb). The fully automated sample loading, separation and data analysis featured enhanced assay repeatability and ease of quantitation over agarose gel electrophoresis.

Conclusion: As a worked example, analysis of plasmid samples treated at elevated temperature during an accelerated stability test also demonstrated the applicability of CGE-LIF to monitor plasmid topology and possible degradation.

Keywords: CGE-LIF, plasmid, topology, purity, stability, vaccine.

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