title: Sample displacement chromatography of plasmid DNA isoforms.
abstract: Sample displacement chromatography (SDC) is a chromatographic technique that utilises different relative binding affinities of components in a sample mixture and has been widely studied in the context of peptide and protein purification. Here, we report a use of SDC to separate plasmid DNA (pDNA) isoforms under overloading conditions, where supercoiled (sc) isoform acts as a displacer of open circular (oc) or linear isoform. Since displacement is more efficient when mass transfer between stationary and mobile chromatographic phases is not limited by diffusion, we investigated convective interaction media (CIM) monoliths as stationary phases for pDNA isoform separation. CIM monoliths with different hydrophobicities and thus different binding affinities for pDNA (CIM C4 HLD, CIM-histamine and CIM-pyridine) were tested under hydrophobic interaction chromatography (HIC) conditions. SD efficiency for pDNA isoform separation was shown to be dependent on column selectivity for individual isoform, column efficiency and on ammonium sulfate (AS) concentration in loading buffer (binding strength). SD and negative mode elution often operate in parallel, therefore negative mode elution additionally influences the efficiency of the overall purification process. Optimisation of chromatographic conditions achieved 98% sc pDNA homogeneity and a dynamic binding capacity of over 1mg/mL at a relatively low concentration of AS. SDC was successfully implemented for the enrichment of sc pDNA for plasmid vectors of different sizes, and for separation of linear and and sc isoforms, independently of oc:sc isoform ratio, and flow-rate used. This study therefore identifies SDC as a promising new approach to large-scale pDNA purification, which is compatible with continuous, multicolumn chromatography systems, and could therefore be used to increase productivity of pDNA production in the future.
authors: BIA Separations d.o.o, Mirce 21, SI-5270 Ajdovščina, Slovenia. Electronic address: urh.cernigoj@monoliths.com.
authors: Urh
authors: U
authors: Černigoj
authors: BIA Separations d.o.o, Mirce 21, SI-5270 Ajdovščina, Slovenia.
authors: Urška
authors: U
authors: Martinuč
authors: BIA Separations d.o.o, Mirce 21, SI-5270 Ajdovščina, Slovenia; University of São Paulo, Escola Politécnica da USP - Department of Chemical Engineering, Av. Prof. 7 Lineu Prestes, 580, Conjunto das Químicas, Bloco 18, 61548, CEP 05424-970 São Paulo, Brazil.
authors: Sara
authors: S
authors: Cardoso
authors: BIA Separations d.o.o, Mirce 21, SI-5270 Ajdovščina, Slovenia.
authors: Rok
authors: R
authors: Sekirnik
authors: BIA Separations d.o.o, Mirce 21, SI-5270 Ajdovščina, Slovenia.
authors: Nika Lendero
authors: NL
authors: Krajnc
authors: BIA Separations d.o.o, Mirce 21, SI-5270 Ajdovščina, Slovenia; The Centre of Excellence for Biosensors, Instrumentation and Process Control - COBIK, Velika pot 22, SI-5250 Solkan, Slovenia.
authors: Aleš
authors: A
authors: Štrancar
mesh_terms: D000645:Ammonium Sulfate; D002021:Buffers; D002846:Chromatography, Affinity / Q000379:methods; D004270:DNA, Circular / Q000302:isolation & purification*; D004278:DNA, Superhelical / Q000302:isolation & purification; D057927:Hydrophobic and Hydrophilic Interactions; D010957:Plasmids
chemical_list: D002021:Buffers; D004270:DNA, Circular; D004278:DNA, Superhelical; D000645:Ammonium Sulfate
keywords: Chromatographic monoliths; Hydrophobic interaction chromatography; Plasmid DNA; Preparative chromatography