A new process
for supercoiled
plasmid DNA purification
G
ene therapy aims to correct disease at the genetic level.
Simple process gives 10 mg of final
Genes are delivered to cells where they express a
therapeutic protein or peptide that combats the disease
product every cycle
in question. Getting the gene to the target cell is the subject
Supercoiled Plasmid Purification Starter Pack provides a
of much research. Gene transfer systems (vectors) currently
complete method for the selective laboratory-scale
under investigation include plasmid DNA. As for any clinical
purification of approximately 10 mg or more of supercoiled
product, quality demands on the final product are high.
plasmid DNA. It comprises three chromatography media plus
For gene therapy studies, the plasmid should be in
a detailed protocol.
supercoiled form and essentially free from bacterial
chromosomal DNA, RNA, proteins and endotoxins, and the
purification media and process should facilitate smooth and • Selectively purifies supercoiled form of plasmid DNA.
cost-effective transfer to large-scale production. As the most • Scalable, RNase-free process for easy transfer to GMP-
common host for plasmid DNA production is E. coli, the compliant production.
real challenge to purification designers is the removal of other
• Reusable after cleaning with NaOH.
similar nucleic acids, endotoxins and trace contaminants.
Our new Supercoiled Plasmid Purification Starter Pack • Easily automated process on ÄKTA™ explorer for
provides purity levels suitable for gene therapy combined evalution, documentation and convenience.
with high reproducibility and reliable scale-up in a
convenient, easy-to-use format.
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4 35
Figure 1 illustrates the flow scheme for the three-step process Table 1. Typical results from non-GMP conditions.
(note that RNase enzyme, organic solvents, detergents, Supercoiled plasmid DNA > 97%
precipitants or animal-derived components are not used). Plasmid DNA up to 850 µg/ml
Figure 2 shows the chromatograms and Table 1 lists typical Endotoxins < 10 EU/mg
results obtained under non-GMP conditions. RNA below fluorescence detection limit
Proteins below BCA-assay detection limit
The three main steps, which start with clarified bacterial Genomic DNA < 0.2% of total DNA
lysate, are:
Step 1. RNA removal and buffer exchange with
Sepharose™ 6 Fast Flow
Step 2. Capture of supercoiled plasmid DNA with
PlasmidSelect Cond
mAU 260 nm 4 (mS/cm)
Step 3. Polishing and concentration with SOURCE™ 30Q 280 nm
Cond
4000 250
Step Flow scheme
3 1. Unbound material
3000 200
Clarified bacterial cell lysate (non-nucleic acid)
2. Open circular plasmid DNA
2 150
3. Supercoiled plasmid DNA
2000 4. RNA
100
1
1 RNA removal and buffer exchange
1000
50
2 3
0 0
0 4 8 12 16 CV
2 Capture of supercoiled plasmid DNA
Fig 2. PlasmidSelect process for the purification of supercoiled plasmid DNA. (Capillary gel electrophoresis performed by PlasmidFactory,
Bielefeld, Germany.)
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35
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