Purification of large plasmids with methacrylate monolithic columns

Area of Science:

• Biotechnology
• Molecular Biology
• Process Chemistry

Background:

• Gene therapy and DNA vaccines increasingly require large quantities of pharmaceutical-grade plasmid DNA (pDNA).
• Future applications may necessitate pDNA vectors exceeding 20 kb, demanding scalable purification methods.
• Current knowledge gaps exist regarding the production and purification of large pDNA molecules.

Purpose of the Study:

• To investigate process conditions for purifying large pDNA molecules (up to 93 kb).
• To evaluate cell lysis and storage conditions impacting large pDNA integrity.
• To assess the performance of methacrylate monolithic columns in large pDNA purification.

Main Methods:

• Investigated cell lysis and storage conditions for large pDNA.
• Studied the impact of chromatographic systems and methacrylate monolithic columns on pDNA degradation at various flow rates.
• Performed capacity measurements by varying salt concentration in the loading buffer.
• Utilized high-resolution monolithic columns for isoform separation and RNA/pDNA separation.

Main Results:

• Achieved high capacities of up to 13 mg of pDNA per mL of monolithic column.
• Demonstrated flow-independent capacity in the range of 130–370 cm/h.
• Successfully separated linear and supercoiled pDNA isoforms.
• Obtained baseline separation of RNA and pDNA, enabling one-step purification.

Conclusions:

• Developed and optimized a purification process for large pDNA using methacrylate monolithic columns.
• The process demonstrates high capacity, flow independence, and excellent resolution for pDNA isoforms and contaminants.
• This method supports the production of high-quality, large pDNA for advanced therapeutic applications.