A platform method for plasmid isoforms analysis by capillary gel electrophoresis

Area of Science:

• Biotechnology and Molecular Biology
• Analytical Chemistry
• Pharmaceutical Sciences

Background:

• Plasmids are crucial vectors for gene/cell therapies and vaccines, with supercoiled form being essential for transfection efficiency.
• Assessing plasmid topological forms is vital for quality control in biological product manufacturing.
• Existing methods may lack the resolution or sensitivity required for comprehensive plasmid analysis.

Purpose of the Study:

• To establish an optimized capillary gel electrophoresis (CGE) method for separating and quantifying different plasmid topological forms.
• To develop a platform-based analytical tool for plasmid manufacturers and regulatory agencies.
• To evaluate the performance of CGE with laser-induced fluorescence (LIF) detection against other separation techniques.

Main Methods:

• Optimization of CGE separation conditions, including buffer concentration (e.g., 10× gel buffer).
• Utilized laser-induced fluorescence (LIF) detection for enhanced sensitivity and resolution compared to ultraviolet detection.
• Compared CGE-LIF with ion exchange chromatography and agarose gel electrophoresis using plasmids of various sizes (5.9–15.4 kb).

Main Results:

• Optimal CGE conditions achieved baseline separation of supercoiled, linear, and open circular plasmid forms and impurities across different sizes.
• CGE-LIF demonstrated superior resolution and quantitation accuracy compared to ion exchange chromatography and agarose gel electrophoresis.
• LIF detection provided significant advantages in sensitivity and resolution over UV detection for plasmid analysis.

Conclusions:

• CGE-LIF is a highly sensitive, high-resolution, and accurate method for separating and quantifying plasmid DNA topological forms.
• This platform-based method is ideal for monitoring plasmid purity and stability for various plasmid sizes.
• CGE-LIF serves as a valuable tool for quality control in the production of plasmid-based biological products.