Clarification and Concentration

Filtration

A clarification and concentration step is required after cell lysis to remove cell debris and structurally unrelated impurities, while simultaneously concentrating and conditioning the plasmid DNA preparations for the purification step.

For large-scale production of plasmid DNA, centrifugation can cause shear stress and damage the precipitates. Flocculation can be appied, but the flloculating agents tend to interfere with product. Thus, filtration is the most suitable choice in general. Filter aids should be used to reduce the shearing of the precipitate and redissolution of genomic DNA.

Purification

Anion Exchange Chromatography (AEC)

Anion exchange chromatography (AEC) is particularly well suited to the capture of plasmid DNA due to the polyanionic structure of DNA. Supercoiled DNA is more compact, stretched and bent than linear or relaxed plasmid DNA, and so presents higher charge densities and more able to interact with the stationary phase.

Although high protein and nucleic acid clearances can be achieved by AEC, it always ended up with contamination of plasmid DNA with genomic DNA, endotoxins and RNA. Thus to purify large amount of plasmid, AEC can be used as an initial capturing and concentrating process. This problem can be addressed by performing a final size-exclusion chromatography.

It is followed by a ultrafiltration/diafiltration, for buffer changing and extra-purification purposes.

Size-exclusion chromatography (SEC)

Supercoiling reduces the hydrodynamic radius of the plasmid DNA. Thus size-exclusion chromatography (SEC) allows the separation of supercoiled plasmid DNA from its topoisomers and from genomic DNA. Also, SEC works well for endotoxin, RNA and protein removal. Another advantage of SEC is its ability to enable a process buffer to be exchanged for a formulation or storage buffer.