Rapid quantification of supercoiled plasmid deoxyribonucleic acid using a monolithic ion exchanger

Area of Science:

• Biochemistry and Molecular Biology
• Analytical Chemistry
• Pharmaceutical Manufacturing

Background:

• Increasing demand for high-purity supercoiled plasmid DNA for gene therapy and DNA vaccination.
• Need for robust analytical methods to ensure pharmaceutical-grade plasmid DNA quality during production.
• Current methods may not efficiently separate and quantify supercoiled plasmid DNA in complex mixtures.

Purpose of the Study:

• To develop and validate a novel analytical methodology for quantifying and monitoring supercoiled plasmid DNA purity.
• To establish a rapid and accurate method suitable for in-process quality control in biopharmaceutical manufacturing.
• To assess the suitability of anion-exchange chromatography with a monolithic column for plasmid DNA analysis.

Main Methods:

• Development of an anion-exchange chromatography method utilizing a monolithic column.
• Separation of plasmid isoforms using a stepwise sodium chloride (NaCl) gradient and UV detection.
• Validation of the method for specificity, linearity, accuracy, reproducibility, and repeatability.
• Determination of quantification and detection limits.

Main Results:

• The method successfully quantifies supercoiled plasmid DNA purity with high precision and accuracy up to 200μg/mL.
• The analytical technique allows separation of plasmid isoforms within a 4-minute experiment.
• The method demonstrates capability in analyzing supercoiled plasmid DNA in complex sample matrices.
• Validation confirmed the method's suitability according to established guidelines.

Conclusions:

• A validated, rapid, and precise analytical method for supercoiled plasmid DNA quantification has been established.
• The monolithic column-based anion-exchange chromatography offers a significant advantage for in-process quality control.
• This methodology supports the development and manufacturing of plasmid DNA for therapeutic applications, ensuring product quality and safety.