title: Purification of large plasmids with methacrylate monolithic columns.
abstract: The rapid evolution of gene therapy and DNA vaccines results in an increasing interest in producing large quantities of pharmaceutical grade plasmid DNA. Most current clinical trials involve plasmids of 10 kb or smaller in size, however, future requirements for multigene vectors including extensive control regions may require the production of larger plasmids, e. g., 20 kb and bigger. The objective of this study was to examine certain process conditions for purification of large plasmids with the size of up to 93 kb. Since there is a lack of knowledge about production and purification of bigger plasmid DNA, cell lysis and storage conditions were investigated. The impact of chromatographic system and methacrylate monolithic column on the degradation of plasmid molecules under nonbinding conditions at different flow rates was studied. Furthermore, capacity measurements varying salt concentration in loading buffer were performed and the capacities up to 13 mg of plasmid per mL of the monolithic column were obtained. The capacity flow independence in the range from 130 to 370 cm/h was observed. Using high resolution monolithic column the separation of linear and supercoiled isoforms of large plasmids was obtained. Last but not least, since the baseline separation of RNA and pDNA was achieved, the one step purification on larger CIM DEAE 8 mL tube monolithic column was performed and the fractions were analyzed by CIM analytical monolithic columns.
authors: BIA Separations d.o.o., Teslova, Ljubljana, Slovenia.
authors: Nika Lendero
authors: NL
authors: Krajnc
authors: Franci
authors: F
authors: Smrekar
authors: Jasmina
authors: J
authors: Cerne
authors: Peter
authors: P
authors: Raspor
authors: Martina
authors: M
authors: Modic
authors: Danijela
authors: D
authors: Krgovic
authors: Ales
authors: A
authors: Strancar
authors: Ales
authors: A
authors: Podgornik
mesh_terms: D002845:Chromatography / Q000295:instrumentation / Q000379:methods; D004278:DNA, Superhelical / Q000737:chemistry / Q000302:isolation & purification; D008689:Methacrylates / Q000737:chemistry*; D010316:Particle Size; D010957:Plasmids / Q000737:chemistry / Q000302:isolation & purification*; D012313:RNA / Q000302:isolation & purification; D012965:Sodium Chloride / Q000737:chemistry
chemical_list: D004278:DNA, Superhelical; D008689:Methacrylates; D012965:Sodium Chloride; D012313:RNA