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Plasmid DNA intended for clinical applications


must meet stringent purity requirements. If it
is then to be used commercially, the purification
method employed must also be suitable for large-
scale GMP-compliant production. Our new
Supercoiled Plasmid Purification Starter Pack,
containing PlasmidSelect, fulfills both demands.
PlasmidSelect is a new thiophilic adsorption
chromatography medium that selectively captures
native supercoiled plasmid DNA.

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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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 3. The selectivity of PlasmidSelect. Clarified alkaline lysate


was loaded on a 4.6 x 150 mm column packed with PlasmidSelect.
Polishing and concentration Elution was initiated with 3 M NaCl in 2.25 M (NH4)2SO4, 10 mM
3 EDTA, 100 mM Tris-HCl, pH 7.0 at 120 cm/h, and after 5 CV
completed with a gradient to water. Open circular plasmid DNA is
separated from supercoiled plasmid DNA in a salt gradient, while
Fig 1. The purification flow scheme. RNA and other contaminants such as proteins and endotoxins only
elute in water.

Fig 2. PlasmidSelect process for the purification of supercoiled plasmid DNA. (Capillary gel electrophoresis performed by PlasmidFactory,
Bielefeld, Germany.)

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PlasmidSelect the key to successful Only standard equipment is required


purification The Starter Pack contains 500 ml Sepharose 6 Fast Flow,
Sepharose 6 Fast Flow (a stable, highly cross-linked 90 µm, 30 ml PlasmidSelect and 25 ml SOURCE 30Q. Neither
6% agarose for group separation) and SOURCE 30Q (a columns nor buffers are supplied. Recommended columns
rigid, monosized 30 µm polystyrene/divinyl benzene bead for for laboratory-scale work are XK 50/30 (Sepharose 6 Fast
high performance at large-scale) are familiar to researchers Flow) and XK 16/20 (PlasmidSelect and SOURCE 30Q). The
and process operators the world over. columns/media are easily cleaned and can be reused following
PlasmidSelect is new and the key member of the CIP. Recommended chromatography systems include the
purification strategy. The medium interacts differentially with ÄKTAdesign platform.
nucleic acids by thiophilic aromatic adsorption in the
presence of water structuring salts. Simple adjustment of the Full support for large-scale production
chromatographic conditions enables the topoisomere-
selective purification of native supercoiled plasmid DNA and All three media can also be ordered separately in process scale
removal of damaged, nicked or open circular DNA. Figure 3 quantities. Furthermore, all are members of the BioProcess™
demonstrates this selectively. Table 2 lists the characteristics family, which means that they enjoy the extensive support,
of PlasmidSelect. e.g. Regulatory Support Files and validation help, required to
meet the demands of GMP-compliant production. The
Table 2. Characteristics of PlasmidSelect. process itself is robust, reliable and easily automated. It does
Matrix Highly cross-linked agarose 6% not involve precipitating agents, detergents, organic solvents
Ligand 2-mercaptopyridine or other components that require time-consuming handling
Ligand concentration 4 mg/ml
or removal.
Capacity for
supercoiled pDNA (6125 bp) > 0.4 mg/ml
Mean particle size 90 µm
For gene therapy applications large and
pH stability
Regular use 3–11 small
Cleaning 2–13
Combining the high selectivity of new PlasmidSelect with the
Cleaning-in-place 0.5 M NaOH
well-known performances of Sepharose 6 Fast Flow and
Linear flow rate at 25 °C
SOURCE 30Q media has resulted in a simple, three-step
General ≤400 cm/h at 100 kPa (1 bar, 14.5 psi)
For supercoiled
procedure for the purification of native supercoiled plasmid
plasmid purification ≤120 cm/h, XK 16/20, 15 cm bed height DNA suitable for gene therapy. In addition, the robustness of
Operating temperature 15–30 ºC the process, plus the extensive support available, allows
Storage temperature 4–30 ºC trouble-free transfer to large-scale production.
Delivery conditions 20% ethanol

Group separation with Sepharose 6 Fast Flow is strongly


recommended prior to application on the column. It greatly
reduces the risk of precipitation when ammonium sulfate is
For more details, including ordering information,
added to elute the supercoiled plasmid DNA, and it limits the
please ask for the following Data File:
variation in initial salt concentration which may influence
selectivity, thereby making the process robust. This means PlasmidSelect, Supercoiled Plasmid Purification
that the capacity of PlasmidSelect for binding the supercoiled Starter Pack, Code No. 18-1161-73
form is fully utilized, and that the process is robust enough to
scale up.

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