Characterization of a topologically aberrant plasmid population from pilot-scale production of clinical-grade DNA

Separation of topological forms of plasmid DNA by anion-exchange HPLC: Shifts in elution order of linear DNA

“…By AEHPLC three monomeric topoisomers were detected, as well as a trailing peak that consisted predominantly of plasmid multimers. Using a previously characterized 5.5 kb plasmid [18] we demonstrated by TEM that both concatemers and catenanes were present (Fig. 2).…”

“…All supercoiled plasmids used in this study were estimated to be less than 0.4 m in length and the 7.6 kb open circular plasmid 0.81 m, according to the equation of Boles et al [22]. The supercoiled plasmids were demonstrated by TEM to approach rod form [18]. The length of the linear forms was calculated to be 1.00, 1.86 and 2.56 m for the 3.0, 5.5 and 7.6 kb plasmids, respectively.…”

“…Plasmid pVax-1 (Invitrogen, Carlsbad, CA; 2999 bp, GC content 53%) pWRG728 [18] (5554 bp, GC content 49%) and pmpSmpCc, a derivative of pWRG728 containing also the core antigen [19], (7629 bp, GC content 49%) were transformed into competent E. coli HB101 (Gibco/BRL, Rockville, MD, USA) according to the manufacturer's instructions. Plasmids were produced by the GSK BioResources group.…”

HPLC for Separation and Quantification of Deoxyribonucleic Acid Fragments and Measurement of Deoxyribonucleic Acid Degradation

“…Because the molecular weight of an oligonucleotide is larger than 1,000, few applications of HPLC for DNA analysis have been reported. In those reports, size exclusion [7,8], ion-pair reversed-phase [9,10], and anion-exchange columns [1,[10][11][12][13][14] were used. DNA was separated according to molecular weight by using a sizeexclusion column, but the resolution was poor compared with an anion-exchange column or an ion-pair reversed-phase column.…”

“…HPLC with an ion-pair reversed-phase column is applicable to only small DNAs, because the increase of hydrophobicity due to the ion-pair reagent has a relatively small effect on large DNA molecules. An anion-exchange column has been used to separate plasmid DNA of different conformations [1,11,12] and mixed DNA samples in a molecular weight marker [10,13,14]. In a report on the quality control of plasmid DNA by Quaak et al [11], plasmid DNAs having different conformations were well separated and a good quantitative accuracy of 1.6 % was obtained.…”