Sodium citrate and potassium phosphate as alternative adsorption buffers in hydrophobic and aromatic thiophilic chromatographic purification of plasmid DNA from neutralized lysate

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2013-03, Vol.919-920, p.67-74
• Main Authors: Bonturi, Nemailla, Radke, Vanessa Soraia Cortez Oliveira, Bueno, Sônia Maria Alves, Freitas, Sindélia, Azzoni, Adriano Rodrigues, Miranda, Everson Alves
• Format: Article
• Language: English
• Subjects: adsorbents; Adsorption; ammonium sulfate; Ammonium Sulfate - chemistry; Aromatic thiophilic chromatography; Biotechnology; Buffers; Chromatography, Affinity - methods; Citrates - chemistry; compliance; DNA - isolation & purification; endotoxins; Escherichia coli; Escherichia coli - chemistry; fermentation; gene therapy; Hydrophobic and Hydrophilic Interactions; Hydrophobic interaction chromatography; hydrophobicity; neutralization; Neutralized lysate; pDNA; Phosphates - chemistry; plasmid vectors; plasmids; Plasmids - chemistry; Plasmids - genetics; Potassium Compounds - chemistry; potassium phosphates; RNA; sodium citrate
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2013.01.010

► Alternative salts can be used in pDNA HIC and TAC as buffer systems. ► pDNA purification with HIC and TAC can be done directly with neutralized lysate. ► Mercaptopyrimidine agarose/sodium citrate system recovered 91.1% of the pDNA. ► Mercaptopyrimidine agarose/potassium phosphate system resulted in 98.8% pDNA purity. The number of studies on gene therapy using plasmid vectors (pDNA) has increased in recent years. As a result, the demand for preparations of pDNA in compliance with recommendations of regulatory agencies (EMEA, FDA) has also increased. Plasmid DNA is often obtained through fermentation of transformed Escherichia coli and purification by a series of unit operations, including chromatography. Hydrophobic interaction chromatography (HIC) and thiophilic aromatic chromatography (TAC), both using ammonium sulfate buffers, are commonly employed with success. This work was aimed at studying the feasibility of utilizing alternative salts in the purification of pDNA from neutralized lysate with phenyl-agarose (HIC) and mercaptopyrimidine-agarose (TAC) adsorbents. Their selectivity toward sc pDNA was evaluated through adsorption studies using 1.5mol/L sodium citrate and 2.0mol/L potassium phosphate as adsorption buffers. Chromatography with mercaptopyrimidine-agarose adsorbent and 1.5mol/L sodium citrate was able to recover 91.1% of the pDNA with over 99.0% removal of gDNA and endotoxin. This represents a potential alternative for the primary recovery of sc pDNA. However, the most promising result was obtained using 2.0mol/L potassium phosphate buffer and a mercaptopyrimidine-agarose column. In a single chromatographic step, this latter buffer/adsorbent system recovered 68.5% of the pDNA with 98.8% purity in accordance with the recommendations of regulatory agencies with regard to RNA and endotoxin impurity.