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Sample displacement chromatography of plasmid DNA isoforms.
Urh Černigoj1, Urška Martinuč1, Sara Cardoso2
1BIA Separations d.o.o, Mirce 21, SI-5270 Ajdovščina, Slovenia.
Journal of Chromatography. A
|August 31, 2015
Summary
Sample displacement chromatography (SDC) effectively separates plasmid DNA (pDNA) isoforms using convective interaction media (CIM) monoliths. This method achieves high purity pDNA, offering a promising approach for large-scale purification and increased production efficiency.
Area of Science:
- Biotechnology
- Chromatography
- Molecular Biology
Background:
- Sample displacement chromatography (SDC) is a technique for separating mixtures based on binding affinities, commonly used for peptide and protein purification.
- Plasmid DNA (pDNA) exists in various isoforms, including supercoiled (sc), open circular (oc), and linear forms, necessitating effective purification methods for biotechnological applications.
Purpose of the Study:
- To investigate the application of SDC for separating plasmid DNA (pDNA) isoforms.
- To evaluate the use of convective interaction media (CIM) monoliths as stationary phases for pDNA isoform separation under overloading conditions.
- To optimize SDC conditions for high-purity sc pDNA enrichment and assess its suitability for large-scale purification.
Main Methods:
- Utilized SDC with convective interaction media (CIM) monoliths (CIM C4 HLD, CIM-histamine, CIM-pyridine) under hydrophobic interaction chromatography (HIC) conditions.
- Investigated the influence of column selectivity, efficiency, and ammonium sulfate (AS) concentration on SDC performance for pDNA isoform separation.
- Assessed the impact of negative mode elution on the overall purification efficiency.
Main Results:
- Achieved 98% supercoiled (sc) pDNA homogeneity with a dynamic binding capacity exceeding 1 mg/mL at low ammonium sulfate (AS) concentrations.
- Demonstrated successful enrichment of sc pDNA for various plasmid vector sizes and effective separation of linear and sc isoforms, irrespective of oc:sc isoform ratio or flow rate.
- SDC efficiency was found to be dependent on column selectivity, column efficiency, and AS concentration.
Conclusions:
- SDC is identified as a promising technique for large-scale plasmid DNA (pDNA) purification, offering high purity and efficiency.
- The use of CIM monoliths provides suitable stationary phases for pDNA isoform separation via SDC.
- This approach is compatible with continuous, multicolumn chromatography systems, potentially increasing pDNA production productivity.
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