title: Preparation of pharmaceutical-grade plasmid DNA using methacrylate monolithic columns.
abstract: Plasmid DNA (pDNA) used in vaccination and gene therapy has to be highly pure and homogenous, which point out necessity to develop efficient, reproducible and scalable downstream process. Convective Interaction Media (CIM) monolithic chromatographic supports being designed for purification of large molecules and nanoparticles seem to be a matrix of choice for pDNA purification. In present work we describe a pDNA purification process designed on two different CIM monolithic columns, based on anion-exchange (AEX) chromatography and hydrophobic interaction chromatography (HIC) chemistry. HIC monolith enabled separation of supercoiled (sc) pDNA from open circular (oc) pDNA, genomic DNA (gDNA) and endotoxins regardless to flow rates in the range at least up to 380cm/h. Dynamic binding capacity of new HIC monolith is up to 4mg of pDNA per milliliter of support. Combination of both chromatographic steps using optimized CaCl(2) precipitation enabled production of pure pDNA, satisfying all regulatory requirements. Process was found to be reproducible, scalable, and exhibits high productivity. In addition, in-line monitoring of pDNA purification process is shown, using CIM DEAE disk monolithic columns.
authors.affiliation: BIA Separations d.o.o., Teslova 30, SI-1000 Ljubljana, Slovenia.
authors.forename: Franc
authors.initials: F
authors.lastname: Smrekar
authors.forename: Ales
authors.initials: A
authors.lastname: Podgornik
authors.forename: Mateja
authors.initials: M
authors.lastname: Ciringer
authors.forename: Sandra
authors.initials: S
authors.lastname: Kontrec
authors.forename: Peter
authors.initials: P
authors.lastname: Raspor
authors.forename: Ales
authors.initials: A
authors.lastname: Strancar
authors.forename: Matjaz
authors.initials: M
authors.lastname: Peterka
mesh_terms: D002122:Calcium Chloride / Q000737:chemistry; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004247:DNA / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D008689:Methacrylates / Q000737:chemistry*; D010957:Plasmids
chemical_list: D008689:Methacrylates; D004247:DNA; D002122:Calcium Chloride