Assessment of pDNA isoforms using microfluidic electrophoresis.

Area of Science:

• Biotechnology and Bioengineering
• Analytical Chemistry
• Molecular Biology

Background:

• Increasing demand for plasmid DNA (pDNA) in nucleic acid-based vaccines and therapies necessitates streamlined manufacturing processes.
• Current plasmid DNA design and construction are manufacturing bottlenecks, lacking high-throughput analytical methods for optimization and quality control.
• Plasmid DNA exists as supercoiled, linear, and open circular isoforms, with specific isoforms required for different applications like cell transfection or mRNA synthesis.

Purpose of the Study:

• To develop a high-throughput microfluidic electrophoresis method for rapid analysis of plasmid DNA isoforms.
• To enable detection and quantification of supercoiled, linear, and open circular plasmid DNA isoforms.
• To support process optimization and batch-to-batch purity monitoring in plasmid DNA manufacturing.

Main Methods:

• A novel high-throughput microfluidic electrophoresis system was employed.
• The method was optimized for detecting and determining the size and concentration of plasmid DNA isoforms.
• Analysis included assessment of detection limits, loading capacity, sample volume, and turnaround time.

Main Results:

• The microfluidic electrophoresis method successfully detected three plasmid DNA isoforms (supercoiled, linear, open circular).
• It accurately determined the size and concentration of supercoiled and linear isoforms (2-7 kb).
• Achieved limits of detection of 0.1 ng/µL (supercoiled/linear) and 0.5 ng/µL (open circular), with a 1-minute/sample turnaround time and 10 µL volume requirement.

Conclusions:

• The developed high-throughput microfluidic electrophoresis method is suitable for rapid plasmid DNA isoform analysis.
• This technique addresses the need for efficient analytical tools in plasmid DNA manufacturing and quality control.
• Findings contribute to understanding microfluidic electrophoretic transport and advance microfluidic device technology.