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Plasmid pVAX1-NH36 purification by membrane and bead perfusion chromatography
Diana Ivonne Franco-Medrano1, Patricia Guerrero-Germán2, Rosa María Montesinos-Cisneros3
1Departamento de Ingeniería Química y Metalurgia, Universidad de Sonora, Blvd. Luis Encinas y Rosales s/n, 83000, Hermosillo, SON, México.
Bioprocess and Biosystems Engineering
|December 4, 2016
Summary
This study compared Mustang Q membranes and POROS 50 HQ perfusion beads for purifying plasmid DNA (pDNA). Mustang Q membranes yielded higher plasmid DNA recovery (88%) than perfusion beads (63%) for vaccine applications.
Area of Science:
- Biotechnology
- Bioprocess Engineering
- Molecular Biology
Background:
- Rising demand for plasmid DNA (pDNA) in vaccine development necessitates efficient purification methods.
- Current bioprocessing challenges include isolating supercoiled plasmid DNA (sc-pDNA) from large volumes of bacterial lysates.
- Ion-exchange chromatography is a critical step in pDNA purification due to its molecular properties.
Purpose of the Study:
- To evaluate and compare the efficacy of Mustang Q membranes versus POROS 50 HQ perfusion beads for the final purification of pDNA.
- To assess the resolution, purification factor, and recovery rates of each chromatographic matrix.
- To enhance understanding of perfusion-based matrices in pDNA purification.
Main Methods:
- Ion-exchange chromatography was employed for the final purification step.
- Two chromatographic matrices were tested: Mustang Q membranes and POROS 50 HQ perfusion beads.
- The separation of plasmid pVAX1-NH36 from clarified Escherichia coli lysates was analyzed.
Main Results:
- Both Mustang Q membranes and POROS 50 HQ perfusion beads demonstrated adequate resolution for separating pDNA from impurities.
- A global purification factor of 24-fold was achieved with Mustang Q membranes and 21-fold with POROS 50 HQ beads.
- Plasmid recovery rates were 88% for Mustang Q membranes and 63% for POROS 50 HQ perfusion beads.
Conclusions:
- Mustang Q membranes are a highly effective matrix for the final purification of plasmid DNA, offering superior recovery rates.
- Perfusion-based matrices like POROS 50 HQ also show promise but require further optimization for pDNA purification.
- This research contributes to optimizing bioprocesses for large-scale pDNA production for therapeutic and vaccine applications.
