title: Sodium citrate and potassium phosphate as alternative adsorption buffers in hydrophobic and aromatic thiophilic chromatographic purification of plasmid DNA from neutralized lysate.
abstract: 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 1.5 mol/L sodium citrate and 2.0 mol/L potassium phosphate as adsorption buffers. Chromatography with mercaptopyrimidine-agarose adsorbent and 1.5 mol/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 2.0 mol/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.
authors: Faculdade de Engenharia Química, Unicamp, Campinas, SP, Brazil. nebonturi@yahoo.com.br
authors: Nemailla
authors: N
authors: Bonturi
authors: Vanessa Soraia Cortez Oliveira
authors: VS
authors: Radke
authors: Sônia Maria Alves
authors: SM
authors: Bueno
authors: Sindélia
authors: S
authors: Freitas
authors: Adriano Rodrigues
authors: AR
authors: Azzoni
authors: Everson Alves
authors: EA
authors: Miranda
mesh_terms: D000327:Adsorption; D000645:Ammonium Sulfate / Q000737:chemistry; D001709:Biotechnology; D002021:Buffers; D002846:Chromatography, Affinity / Q000379:methods*; D002951:Citrates / Q000737:chemistry*; D004247:DNA / Q000302:isolation & purification*; D004926:Escherichia coli / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D010710:Phosphates / Q000737:chemistry*; D010957:Plasmids / Q000737:chemistry / Q000235:genetics*; D017680:Potassium Compounds / Q000737:chemistry*; D000077559:Sodium Citrate
chemical_list: D002021:Buffers; D002951:Citrates; D010710:Phosphates; D017680:Potassium Compounds; D000077559:Sodium Citrate; D004247:DNA; C013216:potassium phosphate; D000645:Ammonium Sulfate