Characterization of a topologically aberrant plasmid population from pilot-scale production of clinical-grade DNA.
As part of a program to develop DNA vaccines for pharmaceutical applications, we recently established a manufacturing process for the production of clinical grade plasmid DNA. In an evaluation of two cell separation methods, the cell culture experienced a temperature spike in a new tangential flow filtration rig, resulting in an aberrant plasmid HPLC peak. Analysis by agarose gel electrophoresis and HPLC demonstrated that the aberrant plasmid material's overall primary structure, methylation pattern and topological integrity was indistinguishable from that of reference material. Transmission electron microscopy and high-resolution agarose gel electrophoresis revealed that the unknown plasmid form exhibited a very low level of supercoiling, whereas the normal supercoiled fraction contained highly twisted DNA. We hypothesized that an enzymatic process, induced by stress during the temperature spike, caused the distinct plasmid topology. This idea was supported by a lab-scale fermentation experiment, where plasmid topology was shown to be similarly altered by conditions designed to induce metabolic stress.
Weigl
Debra
D
BioPharmaceutical Product Development-US, GlaxoSmithKline, 5 Moore Drive, Research Triangle Park, NC 27709, USA.
Molloy
Mike J
MJ
Clayton
Tim M
TM
Griffith
Jack
J
Smith
Clara R
CR
Steward
Terry
T
Merrill
Barbara M
BM
Deprince
Randolph B
RB
Jone
Carl S
CS
Persmark
Magnus
M
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D019175:DNA Methylation; D004278:DNA, Superhelical / Q000032:analysis* / Q000302:isolation & purification* / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission / Q000379:methods; D010957:Plasmids / Q000032:analysis* / Q000302:isolation & purification* / Q000648:ultrastructure; D012015:Reference Standards; D014462:Ultrafiltration / Q000379:methods
D004278:DNA, Superhelical